Skip to main content

Full text of "USPTO Patents Application 10555896"

See other formats


(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(19) World Intellectual Property Organization 

International Bureau 

(43) International Publication Date 
25 July 2002 (25.07.2002) 




PCT 



t mil inimi n iiihi iiiii mi i n m urn urn tun urn urn mi iiiiid iiii mi mi 

(10) International Publication Number 

WO 02/057792 A2 



(51) International Patent Classification 7 : G01N 33/68, 

33/53, C12N 15/00 



(74) Agents: MCISAAC, Robert et al.; Hale and Don LLP, 60 
State Street, Boston, MA 02109 (US). 



(21) International Application Number: PCT/US0 1/50088 

(22) International Filing Date: 

19 December 2001 (19.12.2001) 



(25) Filing Language: 



(26) Publication Language: 



English 



English 



(30) Priority Data: 

60/258,970 



29 December 2000 (29.12.2000) US 



(71) Applicant: NEOGENESIS PHARMACEUTICALS, 
INC. [US/US]; 840 Memorial Drive, Cambridge, MA 
02139 (US). 

(72) Inventors: FELSCH, Jason, S.; 11 Chase Road, 
Waltham, MA 02452-6401 (US). ANNIS, David, Allen, " 
Jr.; 14 Remington Street, Cambridge, MA 02138 (US). 
KALGHATGI, Krishna; 25 Jacob Amsden Road, West- 
boro, MA 01581 (US). NASH, Huw, M.; 109 River Street 
3-B, Cambridge, MA 02139 (US). 



(81) Designated States (national): AE, AG, AL, AM, AT, AU, 
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, 
CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, 
HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, 
LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, 
MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, 
TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW. 

(84) Designated States (regional): ARIPO patent (GH, GM, 
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW), 
Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), 
European patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, 
GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent 
(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, 
NE, SN, TD, TG). 

Published: 

— without international search report and to be republished 
upon receipt of that report 

For two-letter codes and other abbreviations, refer to the "Guid- 
ance Notes on Codes and Abbreviations" appearing at the begin- 
ning of each regular issue of the PCT Gazette. 



= (54) Title: AFFINITY SELECTION-BASED SCREENING OF HYDROPHOBIC PROTEINS 



< 

OS 

t> 

IT) 
© 



2 pM M2R1 + 10 pM pirenzepine 
2 pM M2R1 + 10 pM atropine 
2pMM2R1 + 10 pM QNB + NGM-66 
2 pM M2R1 + 10 pM pirenzepine + NGM-66 
2 pM M2R1 + 10 pM pirenzepine + NGM-340 
2 pM M2R1 + 10 pM pirenzepine + NGL-10-A-41 
2 pM M2R1 + 10 pM pirenzepine + NGL-1 16-A-470 
2 MM M2R1 + 10 pM atropine + NGM-66 
2 pM M2R1 + 10 pM atropine + NGM-340 
2 pM M2R1 + 10 pM atropine ♦ NGL-1 0-A-41 
2 pM M2R1 + 1 0 pM atropine ♦ NGL-1 16-A-470 
2 pM M2R1 + 10 pM QNB + NGM-66 
2 pM M2R1 + 10 pM QQN8 + NGM-340 
2 pM M2R1 + 10 pM QNB + NGL-10-A-41 
2 pM M2R1 + 10 pM QNB + NGL-116-A^70 




^ (57) Abstract: The invention relates to methods based on affinity selection for the identification of ligands for hydrophobic proteins 
bound by amphiphile. The invention also provides hydrophobic proteins and methods of isolation of hydrophobic proteins that are 
^ suitable for ligand screening. 



WO 02/057792 



PCT/US01/50088 



10 



15 



AFFINITY SELECTION-BASED 
SCREENING OF HYDROPHOBIC PROTEINS 

BACKGROTJND OF THE INVENTION 

Tf^ld of t ha Tnvantion. 

, , Ba to th e fields of pharmacology and 
The invention relates to tne riex v 

medicine. More specifically, the 

screening of hydrophobic proteins for the 

the respective ligand molecules with particular relevance 

Z development or novel medicines and medical diagnostics. 

^■ m -.—y ~f «-he Related Art 

Hydrophobic proteins (HPS. present a unigue 
the pharmaceutical industry in the development o agomsts 
, ,-„ n onists of hydrophobic protein function. The 
and antagonists or ny p easily 
difficulty arises fro. the fact that HPS ™ 
purified and are difficult to worK with ^ 
, „ solubility difficulties, etc.). Given tne y 
I!-; rtdrjhobic protei ^ 

organelles therein, ay intearal 
hy drophobic protein includes , -t g 

nrn) . P i nS transmembrane proteins, 
Benfcrane po l y topic membrane proteins, pump 

; membrane proteins, polytop prot eins, receptor 

proteins (a subclass of enzymes), channel p 
kinase proteins, G protein-coupled receptor P 
k m rrane- P associated enzymes, transporter proteins t . 
Frequently, these proteins play an important role in intr 
. Intercellular signaling and the V-^T -s 
cell to its environment, e.g. solute movement et. 
hydrophobic proteins are important targets 

development. „ rov ide an enormous 

The human geneome project will provade ^ 

35 amount of information about the structure 

1 



WO 02/057792 



PCT/US01/50088 



hydrophilic and hydrophobic proteins encoded therein. For 
example, it is estimated that 1,700-5,000 G protein-coupled 
receptor proteins (GPCRs) will be discovered in the human 
genome (Marchese, A., et al. (1999) Trends Pharmacol. Sci. 
5 20:370; Henikoff, S., et al. (1997) Science 278:609). 
However, given the lack of suitable screening methodologies 
for the identification of ligands that bind hydrophobic 
proteins, hundreds of. the GPCRs identified by the human 
genome project will be classified as orphan receptors, 

10 having no known ligand to advance their study. GPCRs are so 
important to the medical sciences that a separate database 
has been established to provide information on sequence 
data, mutant data, and ligand binding data, for example 
(Horn, F. et al. (1998) Nucleic Acids Research 26: 227-281). 

15 Thus, there is a need in the art for the development of 
screening methodologies, particularly high throughput 
methodologies, for HP ligand identification. 

In the prior art, there is no record of affinity 
selection-based screening of HPs. Instead, these targets 

20 are screened in functional assays or ligand displacement 
assays. All ligand displacement assays and most functional 
assays used to screen HPs are either performed in cell -based 
formats (for example, see Jayawickreme, C.K. and Kost, T.A., 
(1997) Current Opinion in Biotechnology 8: 629-634 and Chen, 

25 G., et al. (1999) Molecular Pharmacology 57: 125-124, which 
both dislcose cell-based melanophore assays; Mere, L. et al. 
(1999) Drug Discovery Today 4:363-369, which discloses a 
cell-based fluorescence resonance engery transfer (FRET) - 
based. assay; and Schaeffer, M.T., et al. (1999) J. Receptor 
30 & Signal Transduction Research 19: 927-938, which discloses 
a cell-based aequorin assay) or use impure cell membrane 
preparations (for example, see Cromlish et al. US Patent No. 
5,543,297, which discloses a microsome-based assay; and see 
Labella, F.S., et al. Fed. Proc. (1985) 44: 2806-2811, which 
35 discloses a radioligand displacement assay using membrane 



WO 02/057792 



PCT/US01/50088 



15 



20 



25 



30 



35 



preparations.). These screening formats are poorly defined 
at the molecular level and suffer from low signal-to-noise 
ratios, false positives, and variability in the degree to 
which the target protein is expressed and wide variability 
in gene expression parameters. 

More rarely, HP targets are purified for screening. 
For example, COX-2 purified in a detergent- solubilized form 
can be screened by monitoring its enzymatic activity in a 
homogeneous solution phase assay wherein small molecule 
inhibitors of enzymatic activity can be identified as drug 
leads (see Song, Y. , et al. (1999) J. Med. Chem. 42: 1151- 
1160 and Barnett, J., et al. (1994) Biochimica et Biophysica 
Acta 1209: 130-139 . Alternatively, the HP may be bound to 
a carrier for screening purposes (see Sklar, L.A. et al. 
(2000) Biotechniques 28: 976-985; Bieri, C. et al. (1999) 
Nature Biotechnology 17: 1105-1108; and Schmid, E.L., et al. 
(1998) Anal. Chem. 70: 1331-1338). However, screening 
assays that use functional readouts presume foreknowledge of 
the target's function. Also, as in the case of many imaging 
agents used for medical diagnosis, many desired protein 
ligands do not modulate an assayable function and only bind 

to the protein. 

Affinity selection to identify ligands to water-soluble 
proteins is known in the art. For example, International 
Publication No. WO 99/35109 by Nash et al. describes a 
method for producing mass-coded combinatorial libraries, 
which are useful in combination with affinity selection and 
the identification of the bound ligand by mass spectroscopy, 
international Patent Application No. WO 97/01755 by Jindal 
et al. describes the affinity selection of ligands bound to 
a target molecule combined with the subsequent isolation of 
the ligand molecule by multidimensional chromatographic 
methodology. And U.S. Patent No. 6,020,141 by Pantoliano et 
al. describes a' method of affinity selection combined with 
. ligand identification by thermal shift assay. 



1 

WO 02/057792 PCT/US0 1/50088 



Regardless of these advances with affinity ligand 
selection and ligand identification, there still remains a 
fundamental challenge to apply affinity selection to non- 
water- soluble HP targets because of the hindering presence 
5 of excess amphiphile, which is required to maintain the pure 
HP in a biologically active conformation. 

It is important to recognize the difference between a 
preparation of -a water-soluble protein and a preparation of 
pure HP. The HP is solvated through hydrophobic 

10 interactions between the hydrophobic parts of the HP and the 
hydrophobic moiety of the amphiphile. In a preparation of 
pure water-soluble protein, all buffer components are 
hydrophilic and solvate the protein either through hydration 
or by participating in electrostatic or ionic bonds* By 

15 contrast the amphiphile in preparations of pure HP imparts a 
colloidal characteristic to the solution. Typically, HPs 
are purified in 100 to 10,000-fold molar excess of 
detergent. These amphiphilic detergent molecules interact 
with both the HP and the drug molecules being screened. In 

20 addition, amphiphiles form macromolecular assemblies, like 
micelles or liposomes, that are just as large as most 
proteins. These macromolecular assemblies impart a 

colloidal characteristic to solutions of amphiphile- 
solublized HP, further distinguishing HP's from soluble 

25 proteins. 

Compared to soluble protein targets, the extra 
complexity of HP-amphiphile preparations hampers the 
detection of the bound ligands, lowers screening 
sensitivity, and yields high rate of false positive. In a 

30 typical preparation, the molecular entities responsible for 
these complications could be identified as: HP-amphiphile 
complexes (20 pM, HP: amphiphile :: 1 : 5-250 ; MW=50-500 kD) , 
micelles (5000 ]M MW=60 kD) , monomeric amphiphile (500 }iM; 
MW=1200) . In an analogous water-soluble protein 

35 preparation, one would have only 20 ]M protein. In both 



4 



PCT/CS01/50088 



WO 02/057792 



10 



caS es buffers (e.g. trie - ^ "„ 

-CX or KC1> would -o be P-- rio - a ;J pMle en tities 
proteins, the present - the ta ^ protei n 

presents extra complexity 

preparations. contimling need in the art tor an 

T tionbased HP screening method that can 
affinity -^-^ an ampniphil e without regard to 
operate in the presence 01 target, 
tl specific biological function of the HP targ 

BRIEF STJMMRE1 OF THE INVENTION 

an affinity selection-based HP 
The invention provides an affin y ^ ^ ^ 

screening method that can operat : , biolos i^ 
amphiphile without regard to 

function of the HP target. a8socia ted with 

T he present by enabling detection of 

af£init y selection o f H ^ ^ ^ leB t o an HP 

the specific binding ot addit ion, the present 

in the presence of an ^ compositions of 
0 invention also provides novel ««» use£ul £or 

matter for the production J**^ _ ^ited 

rCiTT' — " hich includes 

compositions and -<**.. n ides a method for 

X» a firs, ■^■J a l ^ obic prot ein, the method 
identifying a ligand for a y P lecule by affinity 

comprising (a) selecting a ^^t p rot.in bound by 
selection by -osing a h^ro <^J^J tQ promot e the 
an amphiphile to * between the hydrophobic 

M formation of at least ^ separatlng the 

target protein and the lig identifying the 

complex from the unbound molecules, and (c) 

ligand molecule. aspect, exposure of 

In certain embo.m^nts - he firs ^ P^^ rf 

35 the hydrophobic target: y 



WO 02/057792 



PCT/US01/50088 



molecules occurs under homogeneous solution phase 
conditions. In certain embodiments of the first aspect, 
exposure of the hydrophobic target protein to a multiplicity 
of molecules occurs under heterogeneous solution phase 
conditions. In certain embodiments of the first aspect, 
selection of the ligand molecule is done using multi- 
dimensional chromatography . 

In certain embodiments of the first aspect, the 
hydrophobic target protein is selected from the group 
consisting of a membrane protein, an integral membrane 
protein, a transmembrane protein, a monotopic membrane 
protein, a polytopic membrane protein, a pump protein, a 
channel protein, a receptor kinase protein, a G protein- 
coupled receptor protein, a membrane-associated enzyme, and 
a transporter protein. 

In certain embodiments of the first aspect, the 
multiplicity of molecules is a mass coded library of 
molecules. In certain embodiments of the first aspect, the 
multiplicity of molecules is a library of molecules that is 
not mass coded. In certain embodiments of the first aspect, 
the amphiphile is selected from the group consisting of (a) 
a polar lipid, (b) an amphiphilic macromolecular polymer, 
(c) a surfactant or detergent, and (d) an amphiphilic 
polypeptide. In certain embodiments of the first aspect, 
ligand molecule identification is done by mass spectral 
analysis. In certain embodiments of the first aspect, the 
ligand molecule is deconvoluted by mass spectral analysis. 
In certain embodiments of the first aspect, separation of 
the complex from the unbound molecules is accomplished with 
solid phase chromatography media. 

In certain embodiments of the first aspect, the 
hydrophobic target protein comprises (a) at least one 
transmembrane domain sequence, (b) at least two tag 
sequences useful for affinity purification, and (c) a 
hydrophobic protein (HP) sequence. In certain embodiments 



(d) 



10 



15 



WO 02/057792 

PCT7US01/50088 

integral me ^ raae ^ "> • ■"-»- protein. (k) „ 
. • ™>noto pl c membrane ^ J » " transmembrane protein, , 
' P-tein. ,f , . ^ •> * Polyopic membrane 

*•« Protern I,' 9 3 ™ * 

P«tein, (J) a »e*rane-al 0^2" OUPUd 
transporter protein i„ " ated en2 »^ and ( k , a 
tag essences comprise epulTt 'h«eof, the 

">e group consisting of m T****' 
'^IDNoa,, (b) an EE tag (who Ep« 9 

N0 ==). (a) a hemagglutinin I (,iH2 " KEEE ™™-C00H, , SEQ 1D 

<•> - hsv tag rrr* (seq m 

a rhodopsin tag (MH2 C °° H ' ,SK 0 ™ «0:S) and (£) 

In certain embodiments of th 7 T ' ,SE ° ID »°^) . 
hydrophobic target protein » ■ ' the 

—> terminus to ^JT* a ~e with an 
'roup consisting of ( a , T aal T ° rder s ^«ed from the 
.'«> HP- T a gl . Tag l ^^:^~ d . «=> ^I-HP- T ag 2 , Md 
invention provides a metho, Z^T^ " 
P»tein is selected from th. hydrophobic target 

tag- EE tag . Human c-sisting of (a) ^ 

25 tachR, (SEQi DH0:7)> (b) S ° F !"7 -etylcholine receptor 
*eceptor- EE tag (SEQ ^ « a J tag-Human Beta 2 

teceptor-HSV tag-My. t ^ (c > H »»" Neurokinin 3 

" «°»- tag (SEa ID 9 ^ ° « • <« Piag tag-Human 

„ ' a3 - 0c taHis tag , SEQ „ ' <"> "at m3 mSChR-HSV 

30 thereof, tha j •»■ m certain embodiments 

hydrophobic target protein Lr he r * ^ «- 
"anal seguence ( Ss) at .^T «■»»*— a heterologous 
Pediments thereof, the ^JIT In ca "ain 

Elected from the group Z*?*? 0 " -ana! seguence is 
35 Clonal seguence of * ff Z 3 ° f W tha *llitin 

NH2-KPLVNVALVEMWYlfiYlYA_cooH (SEQ ID 



20 



WO 02/057792 



PCT/US01/50088 



NO: 12), (b) the GP signal sequence of NH2-VRTAVLILLLVRFSEP- 
COOH (SEQ ID NO: 13) , (c) the Hemagglutinin signal sequence 
of NH2 - KTI I ALS YI FCLVFA- COOH (SEQ ID NO: 14), (d) the 
rhodopsin tag 1 signal sequence of NH2- 
MNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEP - COOH ( SEQ ID NO: 15) , and 
(e) the rhodopsin tag ID4 signal sequence of NH2- 
GKNPLGVRKTETSQVAPA- COOH (SEQ ID NO: 16). In certain 

embodiments thereof the tag sequences further comprise a 
hexahistidine sequence (SEQ ID NO: 17) and a decahistidine 
sequence (SEQ ID NO: 18) . In yet certain embodiments thereof 
the hydrophobic target protein is selected from the group 
consisting of (a) GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ 
ID NO: 19) , (b) Mellitin SS-Flag tag-Human Beta 2 Adrenergic 
Receptor-EE tag(SEQ ID NO:20), (c) Hemagglutinin SS-Human 
Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID NO: 21), (d) 
Mellitin SS-Flag tag-Human ml mAChR-EE tag (SEQ ID NO-.22) , 
and (e) Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHis tag 
(SEQ ID NO: 23) . 

In a second aspect, the invention provides a method of 
isolating a hydrophobic protein, the method comprising (a) 
purifying the hydrophobic protein by sucrose gradient 
ultracentrifugation, (b) purifying the hydrophobic protein 
by antibody affinity purification, and (c) purifying the 
hydrophobic protein by immobilized metal affinity 
chromatography . 

In certain embodiments of the second aspect, the 
hydrophobic protein comprises (a) at least one transmembrane 
domain sequence, (b) at least two tag sequences useful for 
affinity selection, and (c) a hydrophobic protein (HP) 
sequence. In certain embodiments thereof, the hydrophobic 
protein sequence is selected from the group consisting of 
(a) a membrane protein, (b) an integral membrane protein, 
(c) a transmembrane protein, (d) a monotopic membrane 
protein, (e) a polytopic membrane protein, (f) a pump 
protein, (g) a channel protein, (h) a receptor kinase 



8 



WO 02/057792 



PCT/US01/50088 



10 



15 



protein, (i) a 0 protein- coupled receptor protein, ( D ) a 
membrane-associated enzyme, and (k) a transporter protein, 
in certain embodiments of the second aspect, the tag 
sequences of the hydrophobic protein comprise epitope tag 
sequences selected from the group consisting of (a) a FLAG 
tag (NH2 - DYKDDDDK- COOH ) (SEQ ID 80:1) , (b) an EE tag (NH2- 
EEEE YMPME - COOH ) (SEQIDNO:2), (O a hemagglutinin tag (NH2- 
YPYDVPDYA-COOH) (SEQ ID NO: 3), (d) a myc tag NH2- 
KHKLEQLRNSGA- COOH) (SEQ ID N0:4) , (e) an HSV tag (NH2- 
QPELAPEDPED - COOH ) (SEQ ID NO: 5) and (f) a rhodopsin tag 
(NH2 MNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEPWQFSM-COOH) (SEQ ID 
NO- 6) in certain embodiments of the second aspect, the 
hydrophobic protein comprises a sequence with an ammo 
terminus to carboxy terminus order selected from the group 
consisting of (a) TagI-Tag2-HP, (b) Tagl-HP-Tag2, and (c) 
HP-Tagl-Tag2. 

in certain embodiments of the second aspect, the 
hydrophobic protein is selected from the group f 
(a) M yc tag-EE tag-Human m2 mAChR (SEQ ID NO:7) , (b) Flag 
tag-Human Beta 2 Adrenergic Receptor-EE tag (SEQ ID NO: 8) 
(e) Human Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID 
NO-.9), (d) Flag tag-Human ml mAChR -EE tag (SEQ ID BOH0] , 
and (e) Rat m3 mAChR-HSV tag-OctaHis tag (SEQ ID NO:ll) In 
embodiments thereof, the hydrophobic protein further 
comprises a heterologous signal sequence (88) at the ammo 
terminus. In certain embodiments thereof, the heterologous 
signal sequence is selected from the group consisting of (a) 
the Mellitin signal sequence of NH2 -KFLVNVALVFMVVYISYIYA- 
COOH (SEQ ID N0-.12), (b) the GP signal sequence of NH2- 
30 VRTAVLILLLVRFSEP- COOH (SEQ ID NO:13) , (c) the Hemagglutinin 
signal sequence of NH2-KTIIALSYIFCLVFA-C00H (SEQ *> *>'">' 
(d , the rhodopsin tag 1 signal sequence of NH2- 
MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEP - COOH (SEQ ID N0:15), and 
(e) the rhodopsin tag ID4 signal sequence of NH2- 
35 GKNPLGVRKTETSQVAPA- COOH (SEQ ID NO:16). In certain 



20 



25 



9 



WO 02/057792 



PCT/US01/50088 



embodiments of the second aspect, the tag sequences of the 
hydrophobic protein further comprise a hexahistidine 
sequence (SEQ ID NO: 17) and a decahistidine sequence (SEQ ID 
N0:18) . 

In certain embodiments thereof, the hydrophobic target 
protein is selected from the group consisting of (a) GP67 
SS-Myc tag-EE tag-Human m2 mAChR (SEQ ID NO:19) , (b) 
Mellitin SS-Flag tag-Human Beta 2 Adrenergic Receptor-EE 
tag(SEQ ID NO:20), (c) Hemagglutinin SS-Human Neurokinin 3 
Receptor-HSV tag-Myc tag (SEQ ID NO:21), (d) Mellitin SS- 
Flag tag-Human ml mAChR-EE tag (SEQ ID NO:22), and (e) 
Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHis tag (SEQ ID 
NO:23) . 

In a third aspect, the invention provides an isolated 
nucleic acid molecule suitable for hydrophobic protein 
expression, comprising (a) a vector polynucleotide sequence 
for protein expression in a eukaryotic cell, and (b) a 
polynucleotide sequence encoding an engineered hydrophobic 
protein comprising the following elements (i) an N- terminal 
methionine residue, (ii) a heterologous signal sequence 
(SS) , (iii) at least one transmembrane domain sequence, (iv) 
at least two tag sequences useful for affinity purification, 
and (v) a hydrophobic protein (HP) sequence. In certain 
embodiments thereof, the N- terminal methionine sequence and 
the heterologous signal sequence are selected from the group 
consisting of (a) MKFLVNVAL VFMVVYI S Y I YA (SEQ ID NO:24), (b) 
MVRTAVLILLLVRFSEP (SEQ ID N0:25) , (c) MKTIIALSYIFCLVFA (SEQ 
ID NO: 26), (d) MMNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEP-COOH (SEQ 
ID NO: 27), and (e) MGKNPLGVRKTETSQVAPA- COOH (SEQ ID NO: 28) . 

In certain embodiments thereof, the tag sequences 
comprise epitope tag sequences selected from the group 
consisting of (a) a FLAG tag (NH2 -DYKDDDDK-COOH) (SEQ ID 
NO:l), (b) an EE tag ( NH2 - EEEE YMPME - COOH ) (SEQ ID NO:2), (c) 
a hemagglutinin tag ( NH2 - YPYDVPD YA - COOH ) (SEQ ID NO:3), (d) 



10 



WO 02/057792 



PC17TJS01/50088 



10 



a "rye tag (NH2-KHKLEQLKNSGA-COOH) (SEQ ID 220:4), and (a) an 
HSV tag (NH2 -QPELAPEDPED- COOH) (SEQ ID H 0:5) . 

in certain embodiments of the third aapaot the 
elements of tha engineered hydrophobic protein are arrayed 
from an amxno to carboxy terminus ordar aalactad from tha 

tnereof 1 ( °' SS - HP -^l- T ag 2 . Jn oertain enbodiments 
tha orl Sn9 r ered hydr ° ph0bi0 P-tai„ i 3 se lactad fro. 

of (b) < ; ) ™: ss - m - — - 

AdT . e . 9) ' (b) Melllt ^ SS-Flag tag-Human Beta 2 

Adrenergic Keceptor-E* tag (SEQ ID N0:20) , and 

27TT^ri: rrr 3 Receptor - Hsv *™ ^ 

aspect th!'! ^ embodiment °f the third 

aspect, the tag sequences further comprise a hexahistidine 

15 sequence (SEQ ID MO-171 „ „ t . . tl<uns 

NO: 18) . decahistidiaa sequence (SEQ ID 

identic 3 ^ aSPeCt ' 1 — «» "as a method for 

xdent fy lng a Ugand for . hydrophobic 

ompr^ng (selecting a hydrophobic target protein from 
the group oons ls ti„g of ,i) a „, erabrane protein> 
Integra! membrane protain, (iii, . transmembrane protain, 

pro einT^f " Pr ° tein ' W 3 — >«— 

prote ln , , vll , a pump protein, (viii, a channel protein, 

UX) a receptor kinase protein, (X) a G protein-coupled 

rece^or protein, xii, a membrane-associated enzyme, and 

(Xllr) a transporter protain, wherein the hydrophobic 

protein ia bound by amphiphile selected fro* tha group 

cons ls ting of (i, . polar lipid, (U) an amphiphilic 

macromoiecular poller, .(iii, a surfactant or detergent, and 

UV) an amphxphilic polypeptide; ft,, selecting a ligand 

ZllZLT 9 mUUi - di " ensl °- 1 ^atcgraphy by affinity 
selection by exposmg under homogenous or heterogeneous 

bounlT PhaSS °° ndltl0nS protein 

lsTcld an iT 1Phlle " 3 - "lecules fro* a 

".ass coded Ubrary to promote tha formation of at least one 



20 



11 



WO 02/057792 



PCTYUS01/50088 



complex between the hydrophobic target protein and the 
ligand molecule, (c) separating the complex from the unbound 
molecules, and (d) identifying the ligand molecule by mass 
spectral analysis. 
5 In a fifth aspect, the invention provides a method for 

identifying a ligand for a hydrophobic protein, the method 
comprising (a) selecting a hydrophobic target protein from 
the group consisting of(i) a membrane protein, (ii) an 
integral membrane protein, (iii) a transmembrane protein, 

10 (iv) a monotopic membrane protein, (v) a polytopic membrane 
protein, (vii) a pump protein, (viii) a channel protein, 
(iX)a receptor kinase protein, (X) a G protein-coupled 
receptor protein, Xii) a membrane- associated enzyme, and 
(Xiii) a transporter protein, wherein the hydrophobic 

15 protein is bound by amphiphile selected from the group 
consisting of (i) a polar lipid, (ii) an amphiphilic 
macromolecular polymer, (iii) a surfactant or detergent, and 
(iV) an amphiphilic polypeptide; (b) selecting a ligand 
molecule using multi-dimensional chromatography by affinity 

20 selection by exposing under homogenous or heterogeneous 
solution phase conditions the hydrophobic target protein 
bound by an amphiphile to a multiplicity of molecules from a 
library that is not mass -coded to promote the formation of 
at least one complex between the hydrophobic target protein 

25 and the ligand molecule, (c) separating the complex from the 
unbound molecules, and (d) identifying the ligand molecule 
by mass spectral analysis. 

In a sixth aspect, the invention provides a method of 
isolating a hydrophobic protein, the method comprising: (a) 

30 selecting a hydrophobic protein comprising: (i) at least one 
transmembrane domain sequence, (ii) at least two tag 
sequences useful for affinity selection selected from the 
group consisting of: (A) a FLAG tag (NH2 -DYKDDDDK-COOH) (SEQ 
ID NO: 29), (B) an EE tag (NH2-EEEEYMPME-COOH) (SEQ ID 
35 N0:30), (C) a hemagglutinin tag (NH2-YPYDVPDYA-C00H) (SEQ ID 

12 



WO (12/057792 



PCT/US01/50088 



10 



15 



NO: 31), (D) a myc tag ( NH2 - KHKLEQLRNSGA- COOH ) (SEQ ID 
NO: 32)', and (E) an HSV tag ( NH2 - QPELAPEDPED - COOH ) (SEQ ID 
NO:33); (iii) a hydrophobic protein (HP) sequence selected 
from the group consisting of: (A) a membrane protein, (B) an 
integral membrane protein, (C) a transmembrane protein, (D) 
a monotopic membrane protein, (E) a polytopic membrane 
protein, (F) a pump protein, (S) a channel protein, (H) a 
receptor kinase protein, (I) a G protein-coupled receptor 
protein, (J) a membrane-associated enzyme, and (K) a 
transporter protein; (b) purifying the hydrophobic protein 
by sucrose gradient ultracentrifugation; (c) purifying the 
hydrophobic protein by antibody affinity purification; and 
(d) purifying the hydrophobic protein by immobilized metal 
affinity chromatography. 

in a seventh aspect, the invention provides, an 
isolated nucleic acid molecule suitable for hydrophobic 
protein expression, comprising: (a) a vector polynucleotide 
sequence for protein expression in a eukaryotic cell, and 
(b) a polynucleotide sequence encoding an engineered 
hydrophobic protein comprising the following elements (i) an 
N-terminal methionine residue, (ii) a heterologous signal 
sequence (SS) , wherein the N-terminal methionine sequence 
and the heterologous signal sequence are selected from the 
group consisting of (1) MKFLVNVALVFMWYISYIYA (SEQ ID 
25 NO:24), (2) MVRTAVLILLLVRFSEP (SEQ ID NO: 25) , (3) 
MKTIIALSYIFCLVFA (SEQ ID NO: 26) , (4) 

MMNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEP-COOH (SEQ ID NO:27) and 
(5) MGKNPLGVRKTETSQVAPA- COOH (SEQ ID NO: 28); (iii) at least 
one transmembrane domain sequence, (iv) at least two tag 
30 sequences useful for affinity selection selected from the 

group consisting of (1) a FLAG tag ( NH2 - D YKDDDDK- COOH ) (SEQ 
ID N0:1), (2) an EE tag ( NH2 - EEEE YMPME - COOH ) (SEQ ID NO:2), 

(3) a hemagglutinin tag (NH2 -YPYDVPDYA-COOH) (SEQIDNO:3), 

(4) a myc tag (NH2- KHKLEQLRNSGA- COOH) (SEQ ID NO:4), and (5) 
35 an HSV tag (NH2 -QPELAPEDPED- COOH) (SEQ ID NO: 5)., and (v) a 



20 



13 



i 



WO 02/057792 



PCT/US01/50088 



hydrophobic protein (HP) sequence selected from the group 
consisting of (1) a membrane protein, (2) an integral 
membrane protein, (3) a transmembrane protein, (4) a 
monotopic membrane protein, (5) a polytopic membrane 
protein, (6) a pump protein, (7) a channel protein, (8) a 
receptor kinase protein, (9) a G protein-coupled receptor 
protein, (10) a membrane -associated enzyme, and (11) a 
transporter protein. 

BRIEF DESCRIPTION OF THE DRAWINGS 

Figure 1 presents the amino acid sequence of the HP 
protein GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ ID 
MO:19) . 

Figure 2 presents the amino acid sequence of the HP 
protein Melli tin- Flag Tag-Human Beta 2 Adrenergic Receptor- 
EE (SEQ ID NO:20) . 

Figure 3 presents the amino acid sequence of the HP 
protein Hemagglutinin SS -Human Neurokinin 3 Receptor-HSV-Myc 
(SEQ ID NO:21) . 

Figure 4 presents the amino acid sequence of the HP 
protein Mellitin-Flag Tag-Human ml mAChR- EE (SEQ ID NO: 22) . 

Figure 5 presents the amino acid sequence of the HP 
protein Hemagglutinin SS-Rat m3 mAChR-HSV-OctaHis (SEQ ID 
NO:23) . 

Figure 6 presents SEC chromatograms represented by a 
screenshot from a computer interface developed to monitor 
the performance of the ALIS screening system. Relative 
absorbance at 230 nm is plotted on the vertical axis versus 
elution time following sample injection after injection onto 
the SEC column. The view compiles separation profiles for 

14 



WO 02/057792 



PCT/US01/50U88 



the analysis of six different binding reaction mixtures. In 
each case the mixture is composed of 25 JAM each indomethacm 
and meclofenamate, 10 pN COX-1, and 1 HM each for 
approximately 2500 individual screening compounds The 
5 peaks eluting between 12-15 seconds correspond to the COX-1 
containing SEC fractions that are sent to the mass 
spectrometer for analysis. The peaks elutxng after 
seconds corresponds to unbound library members. 

J 

>0 Figure 7 present, mass spectral analysis showing the 

estimatfd recovery of two Known COX-1 Uganda (HSAID «. 
composed of inclomethacin and meclof enamate) extracted from 
test libraries as described in Fig. 1. Different COX-1 
preparations from different days (10/15 and 10/18) bxnd the 

,5 Known Uganda in the absence and presence of competing 
libraries (NGL-15-X-137, KGL-10-A-41. HGL-11S-A-470, Mt-H. 
HGM-108, NGM-177). By comparison to standard curve., the 
mass spectral analysis permits estimation of the pmol of 
each ligand recovered. Estimates were performed m 

20 triplicate for both indomethacin and meclof enamate . 

Figure 8 presents the structure of an example COX-1 
ligand identified by Alls. This compound, termed ^-177-A- 
1128-A-2a, is one example of a compound identified as a COX- 
25 1 ligand by ALIS screening. 

Figure 9 presents a bar graph demonstrating competition 
with meclofenamate for COX-1 binding. Selected COX-1 hit 
compounds, each present at approximately 1 ^ and identxfxed 

30 by ten character names prefixed with NGL-x, were 
individually tested to determine whether they compete wxth 
25 UM meclofenamate for binding to COX-1. The mass spectral 
response corresponding to the mass of either «^f«n«te 
or the test ligand was quantified. For test ligands that 

35 are competitive with meclofenamate for COX-1 bindxng, the 

15 



i 



WO 02/057792 



PCT7US01/50088 



"ligand + competitor" response will be lower that the 
"ligand - competitor" response. Also, the meclof enamate 
response will be lower for that "ligand + competitor" trial 
than in the "COX1 + 25 |IM Meclof enajnate trial," For 
example, the test compound represented by NGL-169-A-1151-A-4 
is competitive with meclof enamate while the test compound 
represented by NGL-175-A-1127-A-1 is not significantly 
competitive. 

Figure 10 presents a bar graph demonstrating the extent 
of M2R1 ligand recovery quantified by the signal strength of 
the mass spectral analysis in accordance with the ALIS 
procedure. The x-axis is in relative units of mass 
spectrometric signal response for the respective masses of 
pirenzepine, QNB, and atropine. 

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 

The invention relates to the fields of pharmacology and 
medicine. More specifically, the invention relates to the 
screening of hydrophobic proteins for the identification of 
the respective ligand molecules with particular relevance to 
the development of novel 'medicines and medical diagnostics. 

The patent and scientific literature cited herein 
establishes the knowledge that is available to those with 
skill in the art. The issued U.S. patents, allowed 
applications, published foreign applications, and references 
cited herein are hereby incorporated by reference. Any 
conflicts between these sources and the present 
specification shall be resolved in favor of the latter. 

The invention provides an affinity selection-based HP 
screening method that can operate in the presence of an 
amphiphile without regard to the specific biological 
function of the HP target. 

Aspects of the invention utilize techniques and methods 
common to the fields of molecular biology, cell biology and 

16 



10 



PCT/CS01/50088 

WO 02/057792 

f these types of 

i-unology. i r at «i 1 " e thca 0 I BKiUed in the 

.athodologies are readUy a ^^ a2I „ B9 ^^ Siat2 « 

art . sea, for ^^ft^^TS^ 6 - *' 
„i 2nd edition, edited by i» „,_ hor Laboratory 

Banaa1 ' „ rio«9) Cold spring Harbor " 

and Maniatis, T.. (1989). ^^ s] ^ hlsi uaS-S^^ 

Press,- ^r^^f*-*^^ 

*°*°s° 1 *-- i *-T?t^ cold sprin9 

Harlow * Lane, ^at^^— ^XW^ 
Harbor Press, Cold Sprmg Harbor ^ preparatlon an d 

invention herein relate £or the 

purification o £ ^^/^r bind speoif i=ally » 
ld anti«ication of ^J^, the tar. .bydropbobic 
hyd rophobic proteins. As used ^ u p d 

protein- refers to any te „ TOy also refer to 

; Lh a„pbiphile. »"n-t»-y^t than X t 

any protein for which, when pur ^ 
purity or purified to greater than r than 50% 

greater than 2 S% purity OTP- presenoe o£ „ 

'purity, aitber reguires or benefit* ^ 
» aa^bipbile for function^ ^ asW . £reeze . t haw cycles, , or 
(S nelf-life or ability to withst by 
to retain conformational rntagr y ^ ligand 

moratory n^ouynaaic — ^ 

binding assays, crrcular drcb lntera ction with 

25 of mean size, shape, _« In a preferre d embodiment 

conformation-specific antrbodres- ^ ^ % 

the hydrophobic protein of tn ^ ^.^^ preferred 
^ ^phobic Protein of the invention is a 

» — —^0 protein; .aso meant ~ 

ptcteins identified by ^es of algorithms 

P th a use of the « ^ h > ic proteins, «., 

desi gne d for the region s in promotes 

35 DAS - Prediction of transm 

17 



WO 02/057792 



PCT/US01/50088 



using the Dense Alignment Surface method (Stockholm 
University) M. Cserzo. E. Wallin. I. Simon , G. von Heijne 
and A. Elofs son: Predi ction of transmembrane alpha-helices 
in prokaryotic membrane proteins: the Dense Alignment 
5 Surface method: Prot. Eng. vol. 10, no, 6. 673-676,1997 : (b) 
HMMTOP - Prediction of transmembrane helices and topology of 
proteins (Hungarian Academy of Sciences) G . E Tusnady and I . 
Simon (1998) Principles Governing Amino Acid Composition of 
Integral Membrane Proteins: Applications to Topology 

10 Prediction. J. Mol. Biol. 283, 489-506; (c) Hidden Markov 
Model Predictions ELL Sormh ammer, G . von Hei jne, and A. 
Krogh: A hidden Markov model for predicting transmembrane 
helices in protein sequences. Proc. of the Sixth Intern 
Conf . on Intelligent Systems for Molecular Biology (ISMB98) . 

15 175-182. 1998 : (D) TMAP - Transmembrane detection based on 
multiple sequence alignment (Karolinska Ins ti tut; Sweden) No 
reference available: see URL at http//www.mbb.ki . se/tmap/ ; 
and (e) TopPred 2 - Topology prediction of membrane proteins 
(Stockholm University) . "Membrane Protein Structure 

20 Prediction. Hydrophobicity Analysis and the Positive- inside 
Rule". Gunnar von Heijne. J. Mol. Biol. (1992) 225. 487-494 
and M. Cserzo, E. Wallin, I. Simon. G. von Heijne and A. 
Elofsson: Prediction of transmembrane alpha-helices in 
prokaryotic membrane proteins: the Dense Alignment Surface 

25 method: Prot. Eng. vol. 10, no. 6, 673-676.1997 . 

For a comparison of these methods see "Prediction of 
transmembrane alpha-helices in prokaryotic membrane 
proteins: the dense alignment surface method". Miklos 
Cserzo. Erik Wallin, Istvan Simon. Gunnar von Heijne. and 

30 Arne Elofsson, to appear in Protein Engineering, vol. 10, 
no. 6. (1997) . 



18 



WO 02/057792 



PCT/USO 1/50088 



For exemplary purposes only, non-limiting examples of 
hydrophobic proteins (as the term is used herein) are 
presented in Table 1. These proteins are listed in GenBank, 
as indicated by the locus designations from the National 
Center for Biotechnology Information (NCBI) . 



Table 1: Non-Limitincj Exam] 
Common Namp 

Kvl.3 

Shaker Family K* Channel 
Poly topic 


?les of Hydrophobic Proteins 
NCBI Locus 

LiOCUS NP 002223 523 aa PRI 
3 1 - OCT- 2 000 DEFINITION 
potassiumvoltag e-gated 
channel , shaker-related 
subfamily, member 3 Funm^ 
sapiens] . ACCESSION 
NP__002223 
PID g4504815 
VERSION NP 002223.1 
GI:4504815 


m2 Muscarinic Acetylcholine 
Receptor 

G Protein- Coupled Receptor 
Class A, Polytopic 


LOCUS NP 000730 466 aa PRI 
3 1 -OCT-2 0 0 0 DEFINITION 
cholinergic receptor, 
muscarinic 2; muscarinic 
acetylcholine receptor M2 
[Homo sapiens] . ACCESSION 
NP_000730 
PID g4502817 
VERSION NP 00073 0.1 
GI:4502817 

DBSOURCE REFSEQ: accession 
NM 000739.1 


Secretin Receptor 

G Protein-Coupled Receptor 

Class B, Polytopic 


LOCUS NP 002971 440 aa PRI 
31-OCT-2000 

DEFINITION secretin receptor 
[Homo sapiens] . 
ACCESSION NP 002971 
PID g4506825 
VERSION NP 002971.1 
GI:4506825 

DBSOURCE REFSEQ: accession 
NM 002980.1 



19 



WO 02/057792 



PCT/USO 1/50088 



1 Table 1: Non- Limit ina Examples of Hydrophobic Proteins 


Common Name 


NCBI Locus 


Metabotropic Glutatmate 
Receptor . Tvds 4 
G Protein-Coupled Receptor 
Class C, Polytopic 


LOCUS NP_000832 912 aa PRI 
31-OCT-2000 
DEFINITION glutamate 
receptor, metabotropic 4 
[Homo sapiens] . ACCESSION 
NP 000832 
PID g4504141 
VERSION NP 000832.1 
GI:4504141 

DBSOURCE REFSEQ: accession 
NM 000841.1 


Epidermal Growth Factor 
Transmembrane Receptor Kinase 


LOCUS AAB19486 10 aa PRI 
pcj-iTTJN-2 00 0 

DEFINITION epidermal growth 
factor receptor; EGFR [Homo 
sapiens] . ACCESSION AAB19486 
PID g8815559 
VERSION AAB19486.2 
GI:8815559 

DBSOURCE locus S51343 

accession 

S51343.1 


Cyc looxygenas e - 2 ( COX- 1 ) 
Integral Membrane Enzyme 
Monotopic 


LOCUS PGH2 HUMAN 604 aa 

PRI 15-DEC-1998 

DEFINITION PROSTAGLANDIN G/H 

SYNTHASE 2 PRECURSOR 
( nVfT ,OOY YfiFMA CIT? - 7 ) ( COX- 1 ) 
( PROSTAGLANDIN- ENDOPEROXIDE 

SYNTHASE 2) (PROSTAGLANDIN H2 

SYNTHASE 2) (PGH SYNTHASE 2) 
(PGHS-2) (PHS II) . 

ACCESSION P35354 

PID g3915797 

VERSION P35354 GI: 3915797 
DBSOURCE swissprot: locus 
PGH2 HUMAN, accession P35354 


Ca ++ ATPase 

Integral Membrane Enzyme 
Polytopic 


LOCUS NP 001675 1205 aa 
PRI 31-OCT-2000 
DEFINITION ATPase, Ca++ 
transporting, plasma membrane 
4 [Homo sapiens] . ACCESSION 
NP 001675 
PID g4502289 
VERSION NP 001675.1 
GI:4502289 

DBSOURCE REFSEQ: accession 
NM 001684.1 
EC 3.6.1.38 



20 



WO 02/057792 



PCT/US01/50088 



tpKIa i< wnn-r.imifcincr ExamDles of Hydrophobic Proteins. 


Common Name 


NCBI Locus 


Cytochrome c Oxidase 
Integral Membrane Enzyme 
Polytopic 


13 Distinct Polypeptide 
Subunits 

See Protein Data Bank #10CC 
for details. 

http : //www . rcsb . org/pdb/cgi/e 
xplore . cgi? job= chains &pdbld=l 
OCC&page=&pid=4725 
EC 1.9.3.1 


Arm^nnTi n Tvoe 3 

Channel, Polytopic 


LOCUS NP 004916 292 aa PRI 
Ol-NOV-2000 

DEFINITION aquaporin 3 [Homo 

sapiens] . ACCESSION 

NP 004916 

PID g4826645 

VERSION NP 004916.1 

GI:4826645 

DBSOURCE REFSEQ: accession 
NM 004925.2 


Outer Membrane Phospholipase 
A 

Integral Membrane Enzyme 
Polytopic (J-Barrel 


See Protein Data Bank #1QD5 
for details. EC 3.1.1.32 


Serotonin Transporter 
Transporter, Topology Unknown 


LOCUS NP 00103 6 630 aa PRI 
31-OCT-2000 

DEFINITION solute carrier 
family 6 (neurotransmitter 
transporter, serotonin) , 
member 4; Solute carrier 
family 6 (neurotransmitter 
transporter, serotonin) , 
[Homo sapiens] . 
ACCE S S I ON NP J) 0103 6 
PID g4507043 
VERSION NP 001036.1 
61:4507043 

DBSOURCE REFSEQ: accession 
NM 001045.1 


Erythropoietin Receptor 
Non- Enzymatic Transmembrane 
Receptor 


LOCUS NP 058698 507 aa ROD 
Ol-NOV-2000 

DEFINITION erythropoietin 
receptor [Rattus norvegicus] . 
ACCESSION NP_0 58698 
PID g8393319 
VERSION NP 058698.1 
GI: 8393319 

DBSOURCE REFSEQ: accession 
NM 017002.1 



21 



WO 02/057792 



PCT/US01/50088 



As will be understood by those in the art, a method of 
identifying a ligand for a hydrophobic protein is synonymous 
with a method of screening for a ligand that binds a small 
molecule. Furthermore, as used herein, the term "screening" 
refers to a procedure used to detect the interaction between 
polypeptide, for example a hydrophobic protein, and a small 
molecule; it is useful for discriminating between ligands 
that bind to proteins with a Kd < 200pM from large ensembles 
of ligands that either do not bind to the protein or bind 
only weakly with a Kd > 200pM. 

The present invention utilizes mass spectrometry (MS) 
in the identification of hydrophobic protein ligands. The 
MS technique is only rarely performed to analyze samples 
containing hydrophobic proteins because these samples 
contain detergent amphiphiles. Detergents suppress analyte 
ion formation, a critical phenomenon for MS, and so 
significantly hamper MS, that reports of successful MS 
analysis of hydrophobic proteins are few. Nevertheless, 
several labs have tried to perform MS analysis of purified 
hydrophobic proteins by identifying methods to remove the 
detergent prior to MS analysis. All of these labs use 
matrix-assisted laser desorption ionization (MALDI) to 
present the protein sample to the detector. 

However, all of the known methods for sample 
preparation of hydrophobic proteins use organic solvents 
and/or acid to extract the detergent from the polypeptide 
prior to MS analysis. Such treatment denatures the 
polypeptide, a fact that precludes the binding of ligands to 
the analyte hydrophobic protein. For researchers interested 
in using MS for the study of hydrophobic protein- ligand 
interactions, denaturing preparation methods are not 
suitable. Moreover, the preparation of a membrane protein 
for analysis by MALDI -MS is laborious compared to the method 
provided by the present invention. 



22 



PCTmSOl/50088 



WO 02/057792 



10 



15 



• retain preferred embodiments the 

present invention uses eie * mem brane protein 

Ich permits the fluid har^ of a ^ ^ ^ ^ 

reasons why detergents must - remove^ ^ 

samples prior to mass -—r.o" fences, .a, 
and are provided, e.g., Vissers, J--P 

M997) 22:244-250; J. PC Vlb 
BioTechmques (b) Prot ein Science 

K. Sanhorn. and J.-P Salzmann^ CM » 

toa rews, (o) J. Mass. Spectre. (It95) 1 • 270: 519-5D 

► v et al.) Methods Enzymol. 1199b) 
Strupat, K, et al . ^ Acad. Sc. US* 

Beavis, *C and Chait, BT. ) Faarnley, I.M. 

(U9 „ 81,6873-7) Beavis, *C and Ch , 
et a;., Biochem. Soc. Trans., (1996) 

In a nrst aspect, the invention provides . .ethod^r 
identifying a ligand for a hvdrophohr ^protein ^ 
, comprising (a) selecting pro tein bound by 

selection by ^-^iTo /^ee to promote the 

^ amPhiPW1 ; at leaT one coile* between the hydrophobic 
formation of at least o (b) separating the 

target protein and the ligand identifying the 

5 complex from the unbound molecules, and 

Ugand /noXecul" ^ o£ ^ . mention are to 

The affinity screen! g „ lect ion methodologies . 

be distinguished from functional sele selection 
actional selection «thodol°gies^ -™ protein . liga nd 
30 hased on criteria that identify ^ ^ ^ 

or protein-protein interaction as sign ^ ^ 

selection depends on either an acti ^ 

/ „ chemical catalysis 1 
protein e.g., <** . nteraotion tetween the protein and 
identification of some 



WO 02/057792 



PCT/US01/50088 



some other molecule (e.g., interaction between a protein and 
a known small molecule ligand in the case of non-enzymes) . 
In summary, these screening methodologies are generally 
based on enzymatic or biofunctional assays or ligand 
displacement assays. 

Various embodiments of the method of the invention may 
utilize an automated ligand identification system (ALIS) for 
the discovery of novel drug leads. ALIS selects ligands 
based on the affinity of the compound for its target protein 
and identifies the ligands by mass spectrometry (see 
International Patent Application WO 99/35109) . The 
invention herein provides for the application of ALIS to 
amphiphile complexed HPs. 

As used herein, the term "affinity selection" means a 
ligand selection based on the affinity of one molecule for a 
selected protein target; such ligand selection is 
independent of the functional activity of the protein of 
interest other than for the fact the protein binds the small 
molecule. 

As used herein, the term "amphiphile" is used to mean 
any molecule generally with the properties of a detergent, 
phospholipid, or surfactant that enhances the water 
solubility of hydrophobic polypeptides; specifically any 
molecule known to assume an association colloid in aqueous 
solution; non-limiting examples of such amphiphiles would 
include phospholipids and other polar lipids (exemplified by 
phosphatidylcholines, lysophospholipids, cholesterols, 
lecithins, ceramides, etc) ; amphiphilic macromolecular 
polymers (exemplified by the work of Chris tophe Tribe t and 
Jean-Luc Popot (Tribet, C. et al. J.L. Natl. Acad.' Sci. USA 
(1996) 93:15047-50); surfactants including alkyl 
saccharides, alkyl thioglycosides, alkyl dimethylamine 
oxides, bile acid derivatives like cholate and the CHAPS 
series, FOS-CHOLINE™ series, CYMAL™ or CYGLU™ series, 
glucamides, and alkyl polyoxyethylenes, etc; or polypeptides 



24 



WO 02/057792 



PCT/US01/50088 



kno wn to adopt antipathic structures (exemplified by work 
of C.E. Schafmeister and K.M. Stroud (Schafmeister, C.E. 
al , RM Science (1993) 262:734-8). ^ rules « 
As used herein, the term "multiplicity of molecuies 
* \« a Plurality of molecules to be tested for the 
refers to a plurality hydrop hobic target 

property of specific binding to hyd p 
protein. By the term "molecule" is -ant «y «-P 
the size range of 150 to 5000 atomic mass unite amuK 

k ^nerated by any means known in the art. 
compounds may be generated Dy a y mil itir>iicity 

or co^ounds which are formed W-^^ 

possible way for a given compound length, a set 

or biochemical building Ho*. which may or may not 

related in structure. Alternatively, the term can refer to 

'/plurality of chemical or ^^^J^Z 

t ormed by selectively combining • P£^£ set 

building blocks. ,cr example twenty a^ino ^ ^ 

combined into hexamenc peptides wall p library" 

6 4 million compounds. With the "^" e . male, 

approach, as many ^^'^CU them 
and then candidate compounds are eelecte y ^ ^ 

for binding activity against the targ 

~ e ie- ; 0 :c"ru ^ in «. - « 

1 6 147 344 • (WO 99/35109; 6,114,309; 6,025,371, b,l> 

59 62 337 5,919,955; and 5,856,496, to name a few. As used 
hlrein the term ..multiplicity of molecules- may also refer 
herein, the te m „ leou i e s or compounds, obtained 

to a natural plurality of molecules r 
for example from body fluids, tissues or cells. Thes 



25 



WO 02/057792 



PCT/US01/50088 



samples may be manipulated, e.g., proteolytically digested, 
in vitro prior to their use in a ligand screening protocol. 

In certain embodiments of the first aspect, exposure of 
the hydrophobic target protein to a multiplicity of 
5 molecules occurs under homogeneous solution phase 
conditions. In certain embodiments of the first aspect, 
exposure of the hydrophobic target protein to a multiplicity 
of molecules occurs under heterogeneous solution phase 
conditions. In certain embodiments of the first aspect, 

10 selection of the ligand molecule is done using multi- 
dimensional chromatography. 

As used herein, the term "homogeneous solution phase" 
means a protein preparations whereby a protein is combined 
with (a) ligand (s) with the intent to facilitate possible 

15 protein-ligand interactions; such preparations are found as 
sols in the temperature range of -40 to 60 °C such that 
neither protein nor ligand are bound to a supporting 
element; these preparations would either pass through a 
semipermeable membrane with a size cut-off of 5.0 ]M or 

20 behave as though they a sedimentation coefficient of less 
than 500 Svedbergs or both; examples of such preparations 
would include combinations of ligand (s) with proteins that 
are solubilized in amphiphile, proteins incorporated into 
proteoliposomes, proteins incorporated into cell -derived 

25 virus-like particles, etc. 

As used herein, the term "heterogeneous solution phase" 
means a protein preparations whereby a protein is combined 
with (a) ligand (s) with the intent to facilitate possible 
protein-ligand interactions; such preparations are found as 

30 mixtures in the temperature range of -40 to 60 °C such that 
either the protein or the ligand is bound to a supporting 
element; these preparations would either fail to pass 
through a semipermeable membrane with a size cut-off of 5.0 
]M or they would behave as though they have a sedimentation 

35 coefficient of greater than 500 Svedbergs or both; examples 



26 



WO 02/057792 



PCT/USO 1/50088 



of such preparations would include combinations of ligandfs) 
With Proteins that are presented on the surface of a 
bead/stationary element whereby the protein is attached to 
the bead/stationary element through either a covalent or 
non-covalent linkage or a linkage dependent upon the self- 

uZTlr ^ ampMphi ^ 8 ° r combinations of proteins with 
ligands that are fixed to a stationary phase. 

chron^t hereln ' ^ tSZm "^ti-di^ensional 

10 ZZ Y " ' Pr °° edUre f « * -mple 

Z in than "graphic method in tandem. 

Representative types of chromatographic methods include: (1) 
solid phase chromatography media: any of a variety of 
-terxals eluding small particles ,<5 pM> , solid porous 
castings, Enters, or semipermeable membranes that may 
15 commonly be referrprf t-« , Y 
artifioL referred to " resins, ge i S/ immobilized 
artxf lclal membraneSf stationary phage 

™' ^ ™ USSd "i* the intent of priding 
aLlvtes " OV6r WhiCh ° r thr ° Ugh WhiGh -lubili 2 ed 



20 i„l " PaSSed ° r " Uh " Mch solubiUzed anaiytes 

s" art 0 ' 38 \" tomographic or electrophone 
separations or fractionations; and ,2) solution phase 
chromatography media: solutions or fluids suitable for use 
in electrophoretic separations or fractionations when used 
in combination with stationary phase chromatography media. 

hvdr n J ertain eWboii ^ Ms ° f tha first aspect, the 
hydrophobic target protein is selected from the group 
consisting o a membrane protein, an integral membrane 
ZT2' 3 t i ranSM * ra - P-tein, a monotopic membrane 
channel protein, a receptor kinase protein, a G prctein- 

aTansnT^" Pr ° teln ' * -*-™°ciated «ryme, and 
a transporter protein. 

In certain embodiments of the first aspect the 

35 :: ; p icity , of m ° ieouies is * — — »»4 ° 

molecules. In certain embodiments of the first aspect the 



27 



WO 02/057792 



PCT/US01/50088 



multiplicity of molecules is a library of molecules that is 
not mass coded. 

As used herein, the term "mass-coded library" refers to 
a mass coded combinatorial library. The compounds of the 
mass -coded combinatorial library are of the general formula 
X(Y) n , wherein X is a scaffold, each Y is a peripheral 
moiety and n is an integer greater than 1, typically from 2 
to about 6. The term "scaffold", as used herein, refers to 
a molecular fragment to which two or more peripheral 
moieties are attached via a covalent bond. The scaffold is 
a molecular fragment which is common to each member of the 
mass-coded set of compounds. The term "peripheral moiety", 
as used herein, refers to a molecular fragment which is 
bonded to a scaffold. Each member of the set of mass -coded 
compounds will include a combination of n peripheral 
moieties bonded to the scaffold and this set of compounds 
forms a mass -coded combinatorial library. More details of 
mass-coded libraries are provided in the patent application 
WO9935109A1, which is incorporated herein by reference. 

As used herein, the phrase "a library of molecules that 
is not mass-coded" means any plurality of molecules or 
compounds that are not produced by a mass -coded 
combinatorial process. Thus, the term includes any and all 
other methods of producing a combinatorial library. In 
addition, the term also includes compounds constructed by 
"Structure Based Drug Design" methodology, which seeks to 
design a drug based on the structure of the target protein, 
and natural libraries of compounds obtained from body 
fluids, tissues or cells. 

In certain embodiments of the first aspect, the 
amphiphile is selected from the group consisting of (a) a 
polar lipid, (b) an amphiphilic macromolecular polymer, (c) 
a surfactant or detergent, and (d) an amphiphilic 
polypeptide. In certain embodiments of the first aspect, 
ligand identification is done by mass spectral analysis. In 



28 



PCT/CSOl/5008* 



WO 02/057792 



10 



- of the first aspect, the ligand molecule 
certain embodiments of the fir analygis . In certain 

iB deconvoluted by mass spect ^ Qf ^ coraplex 

events of the ^V-coZ^ ^ 
from the unbound molecules is 

chromatography media. v . ficat ion« as used herein is 

meant any process that can d in a sor een. 

composition c£ a email -^J^ aspect , the 

In certain events of th ^ ^ ^ 

hydrophobic target proton comprr < ^ ^ 

transmembrane domain se^ence, ^ ^ . 

sequences useful for at tin y cert ain embodiments 

hydr ophobic protein (HP) is se lected from 

. thereof, the hyd^bic protein , W an 

the group consisting of (a) ^ transmetnbrane pro tein, (d) 
integral membrane protein, v po iytopic membrane 

a .onotopic me^rane protein, I a 
protein, (£) a pump protein, W n _ coupled reC eptor 

receptor kinase protein U> ^ (k) a 

0 protein, (J) a n thereof , the 

transporter protein. In ce ceS selected from 

tag sequences W ri8 % e ^°f ^ tag (NH2 -DYKDDDDK- COOH) 
tne group consisting of (a) ^.cooH) (SEQ ID 

(SE Q ID NO:l), (b) an SB tag ^.^^-COOH) (SEQ B> 

.-^.'1--=^=- — — 

COOH) (SEQ 10 MO = 6) . f lrst aspe ct, the 

30 In certain embodiments ^ ^ an 

hydrophobic target » r0t " n orfer .elected from the 

mlno terminus to carboy ^ <b> Tagl-HP-,ag 2 , and 

group consisting of (a) Tag! rf thereof , the 

HP-Ta gl -Tag2. In cert bydroph obic target 
invention provides a method whe 



35 

29 



WO 02/057792 



PCT/US01/50088 



protein is selected from the group consisting of (a) Myc 
tag-EE tag-Human m2 mAChR (SEQ ID NO: 6) , (b) Flag tag-Human 
Beta 2 Adrenergic Receptor-EE tag (SEQ ID NO: 7), (c) Human 
Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID NO: 9), (d) 
Flag tag-Human ml mAChR- EE tag (SEQ ID NO: 10) , and (e) Rat 
m3 mAChR-HSV tag-OctaHis tag (SEQ ID NO: 11) . In certain 
embodiments thereof, the invention provides a method wherein 
the hydrophobic target protein further comprises a 
heterologous signal sequence (SS) at the amino terminus. In 
certain embodiments thereof, the heterologous signal 
sequence is selected from the group consisting of (a) the 
Mellitin signal sequence of NH 2 - KFLVNVALVFMWYI SYIYA- COOH 
(SEQ ID NO: 12), (b) the GP signal sequence of NH 2 - 
VRTAVL I LLLVRFSE P - COOH (SEQ ID NO: 13), (c) the Hemagglutinin 
signal sequence of NH 2 -KTIIALSYIFCLVFA-COOH (SEQ ID NO:14), 

(d) the rhodopsin tag 1 signal sequence of NH 2 - 
MNGTEGPNFYVPFSNKTGVVRS PFEAPQYYLAE P - COOH (SEQ ID NO: 15), and 

(e) the rhodopsin tag ID4 signal sequence of NH 2 - 
GKNPLGVRKTETSQVAPA- COOH (SEQ ID NO: 16). In certain 
embodiments thereof the tag sequences further comprise a 
hexahistidine sequence (SEQ ID NO: 17) and a decahistidine 
sequence (SEQ ID NO: 18). In yet certain embodiments thereof 
the hydrophobic target protein is selected from the group 
consisting of (a) GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ 
ID NO: 19), (b) Mellitin SS-Flag tag-Human Beta 2 Adrenergic 
Receptor-EE tag(SEQ ID NO:20), (c) Hemagglutinin SS-Human 
Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID NO:21), (d) 
Mellitin SS-Flag tag-Human ml mAChR-EE tag (SEQ ID NO:22), 
and (e) Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHis tag 
(SEQ ID NO:23) . 

In a second aspect, the invention provides a method of 
isolating a hydrophobic protein, the method comprising (a) 
purifying the hydrophobic protein by sucrose gradient 
ultracentrifugation, (b) purifying the hydrophobic protein 
by antibody affinity purification, and (c) purifying the 



30 



WO 02/057792 



PCT/US01/50088 



hydrophobic protein by immobilized metal affinity 
chromatography . 

In certain embodiments of the second aspect, the 
hydrophobic protein comprises (a) at least one transmembrane 
5 domain sequence, (b) at least two tag sequences useful for 
affinity selection, and (c) a hydrophobic protein (HP) 
sequence. In certain embodiments thereof, the hydrophobic 
protein sequence is selected from the group consisting of 
(a) a membrane protein, (b) an integral membrane protein, 

10 (c) a transmembrane protein, (d) a monotopic membrane 
protein, (e) a polytopic membrane protein, (f) a pump 
protein, (g) a channel protein, (h) a receptor kinase 
protein, (i) a G protein- coupled receptor protein, (j) a 
membrane -associated enzyme, and (k) a transporter protein. 

15 In certain embodiments of the second aspect, the tag 
sequences of the hydrophobic protein comprise epitope tag 
sequences selected from the group consisting of (a) a FLAG 
tag ( NH2 - D YKDDDDK- C00H ) (SEQ ID N0:1), (b) an EE tag (NH2- 
EEEEYMPME - C00H ) (SEQ ID NO:2), (c) a hemagglutinin tag (NH2- 

20 YPYDVPDYA-C00H) (SEQ ID NO: 3), (d) a myc tag (NH2- 
KHKLEQLRNSGA - C00H ) (SEQ ID NO: 4), (e) an HSV tag (NH2- 
QPELAPEDPED - COOH ) (SEQ ID NO: 5) and (f) a rhodopsin tag 
(NH2 MNGTEGPNFWPFSNKTGVWSPFEAPQYYLAEPWQFSM-COOH) (SEQ ID 
NO: 6). In certain embodiments of the second aspect, the 

25 hydrophobic protein comprises a sequence with an amino 
terminus to carboxy terminus order selected from the group 
consisting of (a) Tagl-Tag2-HP, (b) Tagl-HP-Tag2 , and (c) 
HP-Tagl-Tag2. 

In certain embodiments of the second ■ aspect, the 
30 hydrophobic protein is selected from the group consisting of 
(a) Myc tag-EE tag-Human m2 mAChR (SEQ ID NO: 7), (b) Flag 
tag-Human Beta 2 Adrenergic Receptor-EE tag (SEQ ID NO: 8), 
(c) Human Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID 
N0:9), (d) Flag tag-Human ml mAChR-EE tag (SEQ ID NO:10), 
35 and (e) Rat m3 mAChR -HSV tag-OctaHis tag (SEQ ID NO:ll) . In 

31 



L 



WO 02/057792 



PCT/US01/50088 



embodiments thereof, the hydrophobic protein further 
comprises a heterologous signal sequence (SS) at the amino 
terminus. In certain embodiments thereof, the heterologous 
signal sequence is selected from the group consisting of (a) 
the Mellitin signal sequence of NH2-KFLVNVALVFMVVYISYIYA- 
COOH (SEQ ID NO: 12) , (b) the GP signal sequence of NH2- 
VRTAVLILLLVRFSEP-COOH (SEQ ID NO: 13), (c) the Hemagglutinin 
signal sequence of NH2-KTIIALSYIFCLVFA-COOH (SEQ ID NO: 14), 

(d) the rhodopsin tag 1 signal sequence of NH2- 
MNGTEGPNFYVPFSNKTGWRS PFEAPQYYLAEP - COOH (SEQ ID NO: 15), and 

(e) the rhodopsin tag ID4 signal sequence of NH2- 
GKNPLGVRKTETSQVAPA- COOH (SEQ ID NO: 16). In certain 
embodiments of the second aspect, the tag sequences of the 
hydrophobic protein further comprise a hexahistidine 
sequence (SEQ ID NO: 17) and a decahistidine sequence (SEQ ID 
NO:18) . 

In certain embodiments of the second aspect, the 
hydrophobic target protein is selected from the group 
consisting of (a) GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ 
ID NO:19), (b) Mellitin SS-Flag tag-Human Beta 2 Adrenergic 
Receptor-EE tag(SEQ ID NO:20), (c) Hemagglutinin SS-Human 
Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ ID NO: 21), (d) 
Mellitin SS-Flag tag-Human ml mAChR-EE tag (SEQ ID NO:22) , 
and (e) Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHis tag 
(SEQ ID NO: 23) . 

In a third aspect, the invention provides an isolated 
nucleic acid molecule suitable for hydrophobic protein 
expression, comprising (a) a vector polynucleotide sequence 
for protein expression in a eukaryotic cell, and (b) a 
polynucleotide sequence encoding an engineered hydrophobic 
protein comprising the following elements (i) an N- terminal 
methionine residue, (ii) a heterologous signal sequence 
(SS) , (iii) at least one transmembrane domain sequence, (iv) 



32 



WO 02/057792 



PCT/US01/50088 



at least two tag sequences useful for affinity purification, 
and (v) a hydrophobic protein (HP) sequence. 

By the phrase "a vector polynucleotide sequence for 
protein expression in a eukaryotic cell" is meant any 
polynucleotide sequence comprising an origin of replication 
allowing replication in a eukaryotic cell, a selectable 
marker, e.g., antibiotic resistance marker, and a promoter 
sequence element to promote transcription of the structural 
gene, which may be viral, prokaryotic or eukaryotic in 
origin. The origin of the vector polynucleotide sequence 
may be viral, prokaryotic or eukaryotic or a combination 
thereof. As will be understood in the art, the vector 
sequence while being designed for expression in a eukaryotic 
cell may optionally contain a prokaryotic origin of 
replication. Non-limiting examples of suitable vector 
polynucleotide sequences include the following: baculovirus 
vectors such as pVL1392 (Pharmingen, San Diego, CA) and 
pBAC-1 (Novagen, Madison, WI) and mammalian expression 
vectors such as pcDNA 3.1 (Invitrogen, San Diego, CA) and 
pTriEx-1 (Novagen, Madison, WI) . 

The appropriate DNA sequence may be inserted into the 
vector by a variety of procedures . In general , the DNA 
sequence is inserted into an appropriate restriction 
-endonuclease site(s) by procedures known in the art. Such 
procedures and others are deemed to be within the scope of 
those skilled in the art. 

In certain embodiments, the present invention relates 
to host cells containing the above -described constructs. The 
host cell can be a higher eukaryotic cell, such as a 
mammalian cell, or a lower eukaryotic cell, such as a yeast 
cell, or the host cell can be a prokaryotic cell, such as a 
bacterial cell. Introduction of the construct into the host 
cell can be effected by any means known in the art, 
including but not limited to transduction or transformation 
or transfection or electroporation. 



33 



WO 02/057792 



PCT7US01/50088 



i of a suitable heterologous signal sequences 

— — r°i ""x^..-- 

Z'c t ,%e 98 — . gp, ss — : 

I— 1 Method, » • » ^ > Q0H) (SEQ ID 

hemagglutml .88 W m _ 7M)i the rhoa opsin 

n> «,,«), or the « ™ eral ly, such 

^PLOVRKTE^Q^-COOH) <8E Q ID «0 .!«• ' 

m. sss villi be less that 75 aa long. mes a 
suitable SSs will the protein u pon 

fences -ay or may not be cle ^ ^ 

5 expression in animal cells by ^ 

1 is not cleaved off of the p rhodopsin tag ID* 

cell to the plasma membrane, whereas rno p 
signal sequence is cleaved off. affinity 
Non-limiting examples of a suitable epitop 

, ^ m 6G (HH2-DYKDDDDK-COOH) (SEQ ID HO ; 29) , 
!0 tags include FIAG (HH2 v*» hemagglutinin (HH2- 

YPYEV PDYA-COOH) (8BQ ID HO.31 . ^ < «- 
(SEQ ID HO:32), or herpes simplex virus tag 

Q P E1A P E DPED-COOH, (SEQ » »-»>^ hydrophobio protein are 

These design "J^^I in one of the 

arrayed from ammo to carbox, _ sS-Tagl-HP- 
following permutations: (1) SS Tagl g 

Tag2; (3) SS-HP-Tagl-Tag2 N . te rminal 

In certain embodiment* , ■ _ 

30 methionine sequence and the heterol g^^ ^ (>j 

35 (e) MGKNPLGVRKTETSQVAPA-COOH (SEQ ID N0:28) . 

34 



WO 02/057792 



PCT7US01/50088 



In certain embodiments thereof, the tag sequences 
comprise epitope tag sequences selected from the group 
consisting of (a) a FLAG tag (NH2 -DYKDDDDK-COOH) (SEQ ID 
NO:l), (b) an EE tag (NH2 - EEEE YMPME - COOH ) (SEQ ID NO:2) , (c) 
a hemagglutinin tag (NH2 - YPYDVPDYA- COOH ) (SEQ ID NO: 3) , (d) 
a myc tag (NH2 - KHKLEQLRNSGA- COOH ) (SEQ ID NO:4), (e) an HSV 
tag ( NH2 - QPELAPEDPED - COOH ) (SEQ ID NO : 5 ) , and (f) a 
rhodopsin tag (NH2 MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSM- 
COOH) (SEQ ID NO: 6) . 

In certain embodiments of the third aspect, the 
elements of the engineered hydrophobic protein are arrayed 
from an amino to carboxy terminus order selected from the 
group consisting of (a) SS-Tagl-Tag2-HP, (b) SS-Tagl-HP- 
Tag2, and (c) SS-HP-Tagl-Tag2 . In embodiments thereof, the 
engineered hydrophobic protein is selected from the group 
consisting of (a) GP67-Myc-EE-Human m2 mAChR (SEQ ID 
NO: 19), (b) Mellitin-Flag Tag-Human Beta 2 Adrenergic 
Receptor-EE (SEQ ID NO:20), and (c) Hemagglutinin SS-Human 
Neurokinin 3 Receptor-HSV-Myc (SEQ ID NO: 21). In a further 
embodiment of the third aspect, the tag sequences further 
comprise a hexahistidine sequence (SEQ ID NO: 17) and a 
decahistidine sequence (SEQ ID NO: 18) . In certain 

embodiments of the third aspect, the engineered hydrophobic 
protein is selected from the group consisting of (a) GP67- 
Myc-EE-Human m2 mAChR (SEQ ID NO: 19), (b) Mellitin-Flag Tag- 
Human ml mAChR -EE (SEQ ID NO: 20), and (c) Hemagglutinin SS- 
Rat m3 mAChR-HSV-OctaHis (SEQ ID NO:21) . 

The HP sequence of the isolated polynucleotide may be 
any polynucleotide encoding a protein that is isolated with 
amphiphile present. Alternatively, the term may also refer 
to any polynucleotide encoding a protein for which, when 
purified to greater than 1% purity or purified to greater 
than 10% purity or purified to greater than 25% purity or 
purified to greater than 50-99% purity, either requires or 
benefits from the presence of an amphiphile for functional 



35 



WO 02/057792 



PCT/USO 1/50088 



assays, to enhance stability (shelf-life or ability to 
withstand freeze-thaw cycles, , or to retain conformational 
integrity as observed by common Moratory techniques 

5 nvdr^ 19and Wndin9 aSSayS " =i ~ Ular 

hydrodynamic assessments of mean size, shape, or density, 

interacts with conformation-specific antibodies. In a 

preferred embodiment the hydrophobic protein of the 

invention is a mammalian hydrophobic protein. In a 

.0 of r tL CUlarly , Preferred tb. hydrophobic protein 

of the invention is a human hydrophobic protein 

i„,l T " P , Se9Uen0e ° f thS iS ° lated P°lyhUcleotide may 
include polynucleotides encoding proteins that are 
identif.ed by bioinformatice-assisted means through the use 
of the following non-limiting examples of algorithms 
BaT p T r - «obic proteins: U 

ustna" tT n 10n * tJ ""— -9*°- in prokaryotee 
using the Dense Alignment Surface method (Stockholm 
University, Cserzo, M. et al. <„„, Prot . ^ 10;6 , 3 . 

'0 till " Predlction ° f transmembrane helices and 

topology of proteins (Hungarian Academy of Sciences) G . 
Tusnady and I. Simon („,„ j. w . BioJ . 283s 4 „_ 
Hidden Markov Model Predictions Sonnhammer, ell et al 
«»8) A hidden Markov model for predicting transmembrane 
helices an protein sequences. Proc. of the sixth Intern 

175-182; o» TMAP - Transmembrane detection based on 
multiple sequence alignment (Karolinska Institut, Sweden, No 
reference available: see URL at http//www.mj*. ki.se/tmap/, 

ZJ! r p r d 2 " Topolo9y prediction ° f — ««- P"f£» 

25^1 Eity> - ^ HeiJne ' °' (1992 » J - «• ««!. 

225,487-494 and Cserzo, M. et al. ( 1SS7) p rot . ^ 10 .^_ 

Representative non-limiting examples of proteins that 

ZlVn ^ HP of the invention are 

presented h<=r-«=H-n ^ m„ L1 _ , 



30 

676. 



35 presented herein in Table l. 

36 



WO 02/057792 



PCT/US01/50088 



All nucleic acid sequences and the respective amino 
acid sequences encoded thereby identified above by the 
appropriate GenBank accession numbers are herein 
incorporated by reference. 
5 In a fourth aspect, the invention provides a method for 

identifying a ligand for a hydrophobic protein, the method 
comprising (a) selecting a hydrophobic target protein from 
the group consisting of (i) a membrane protein, (ii) an 
integral membrane protein, (iii) a transmembrane protein, 

10 (iv) a monotopic membrane protein, (v) a polytopic membrane 
protein, (vii) a pump protein, (viii) a channel protein, 
(iX)a receptor kinase protein, (X) a G protein-coupled 
receptor protein, Xii) a membrane-associated enzyme, and 
(Xiii) a transporter protein, wherein the hydrophobic 

15 protein is bound by amphiphile selected from the group 
consisting of (i) a polar lipid, (ii) an amphiphilic 
macromolecular polymer, (iii) a surfactant or detergent, and 
(iV) an amphiphilic polypeptide; (b) selecting a ligand 
molecule using multi -dimensional chromatography by affinity 

20 selection by exposing under homogenous or heterogeneous 
solution phase conditions the hydrophobic target protein 
bound by an amphiphile to a multiplicity of molecules from a 
mass-coded library to promote the formation of at least one 
complex between the hydrophobic target protein and the 

25 ligand molecule, (c) separating the complex from the unbound 
molecules, and (d) identifying the ligand molecule by mass 
spectral analysis. 

In a fifth aspect, the invention provides a method for 
identifying a ligand for a hydrophobic protein, the method 

30 comprising (a) selecting a hydrophobic target protein from 
the group consisting of (i) a membrane protein, (ii) an 
integral membrane protein, (iii) a transmembrane protein, 
(iv) a monotopic membrane protein, (v) a polytopic membrane 
' protein, (vii) a pump protein, (viii) a channel protein, 

35 (iX)a receptor kinase protein, (X) a G protein-coupled 

37 



WO 02/057792 



PCT/US01/50088 



10 



receptor protein, Xii) a membrane -associated enzyme, and 
(Xiii) a transporter protein, wherein the hydrophobic 
protein is bound by amphiphile selected from the group 
consisting of (i) a polar lipid, (ii) an amphiphilic 
macromolecular polymer, (iii) a surfactant or detergent, and 
(iV) an amphiphilic polypeptide; (b) selecting a ligand 
molecule using multi -dimensional chromatography by affinity 
selection by exposing under homogenous or heterogeneous 
solution phase conditions the hydrophobic target protein 
bound by an amphiphile to a multiplicity of molecules from a 
library that is not mass-coded to promote the formation of 
at least one complex between the hydrophobic target protein 
and the ligand molecule, (c) separating the complex from the 
unbound molecules, and (d) identifying the ligand molecule 
15 by mass spectral analysis. 

In a sixth aspect, the invention provides a method of 
isolating a hydrophobic protein, the method comprising: (a) 
selecting a hydrophobic protein comprising: (i) at least one 
transmembrane domain sequence, (ii) at least two tag 
sequences useful for affinity selection selected from the 
group consisting of: (A) a FLAG tag (NH2 -DYKDDDDK- COOH) (SEQ 
ID N0:29), <B) an EE tag (NH2-EEEEYMPME-COOH) (SEQ ID 
NO: 30), (C) a hemagglutinin tag ( NH2 - YPYDVPDYA- COOH ) (SEQ ID 
NO:31), (D) a myc tag (NH2-KHKLEQLRNSGA-C00H) (SEQ ID 
25 NO: 32), and (E) an HSV tag (NH2 -QPELAPEDPED-COOH) (SEQ ID 
NO:33); (iii) a hydrophobic protein (HP) sequence selected 
from the group consisting of: (A) a membrane protein, (B) an 
integral membrane protein, (C) a transmembrane protein, (D) 
a monotopic membrane protein, (E) a polytopic membrane 
30 protein, (F) a pump protein, (G) a channel protein, (H) a 
receptor kinase protein, (I) a G protein-coupled receptor 
protein, (J) a membrane-associated enzyme, and (K) a 
transporter protein; (b) purifying the hydrophobic protein 
by sucrose gradient ultracentrifugation; (c) purifying the 
35 hydrophobic protein by antibody affinity purification; and 



20 



38 



WO 02/057792 



PCT/US01/50088 



(d) purifying the hydrophobic protein by immobilized metal 
affinity chromatography. 

In a seventh aspect, the invention provides, an 
isolated nucleic acid molecule suitable for hydrophobic 
5 protein expression, comprising: (a) a vector polynucleotide 
sequence for protein expression in a eukaryotic cell, and 
(b) a polynucleotide sequence encoding an engineered 
hydrophobic protein comprising the following elements (i) an 
N-terminal methionine residue, (ii) a heterologous signal 
10 sequence (SS) , wherein the N-terminal methionine sequence 
and the heterologous signal sequence are selected from the 
group consisting of (1) MKPLVWALVFMVVYISYIYA (SEQ ID 
N0:24), (2) MVRTAVLILLLVRFSEP (SEQ IDNO:25), (3) 
MKTI I ALSYI FCLVFA (SEQ ID NO:26) , (4) 

15 MMNGTEGPNFYVPFSNKTGWRS PFEAPQYYLAE P - COOH (SEQ ID NO:27) and 
(5) MGKNPLGVRKTETSQVAPA- COOH (SEQ ID NO:28); (iii) at least 
one transmembrane domain sequence, (iv) at least two tag 
sequences useful for affinity selection selected from the 
group consisting of (1) a FLAG tag (NH2-DYKDDDDK-C00H) (SEQ 

20 ID NO:l) 7 (2) an EE tag ( NH2 - EEEE YMPME - COOH ) (SEQ ID NO:2), 
(3) a hemagglutinin tag (NH2-YPYDVPDYA-COOH) (SEQ IDNO:3) 7 
• (4) a myc tag ( NH2 - KHKLEQLRNSGA - COOH ) (SEQ ID NO:4) , and (5) 
an HSV tag (NH2 - QPELAPEDPED - COOH) (SEQ ID NO: 5), and (v) a 
hydrophobic protein (HP) sequence selected from the group 

25 consisting of (1) a membrane protein, (2) an integral 
membrane protein, (3) a transmembrane protein, (4) a 
monotopic membrane protein, (5) a polytopic membrane 
protein, (6) a pump protein, (7) a channel protein, (8) a 
receptor kinase protein, (9) a G protein-coupled receptor 

30 protein, (10) a membrane-associated enzyme, and (11) a 
transporter protein. 

The following examples are intended to further 
illustrate certain preferred preferred embodiments of the 
invention but are not meant to limit the scope of the 

35 invention in any way. 

39 



WO 02/057792 



PCT/US01/50088 



EXAMPLES 

EXAMPLE It AFFINITY SELECTION OF COX-1 LIGANDS AND 

IDENTIFICATION BY ALIS 

Purified ovine COX-1 (>95% by SDS-PAGE) from Cayman 
Chemical Company (Ann Arbor, MI) was prepared for screening 
by exchanging the detergent. To remove the detergent m 
which the protein was supplied, Tween-20, ion exchange 
chromatography was conducted. Approximately 6 mL of 0.27 
mg/mL COX-1 in a buffer of 80 mM Tris-8.0, 0.09% Tween-20, 
270 jjM DDC, and 240 pM dodecyl-p-D-maltoside (Dpi), which 
would provide a theoretical yield of 1.8 mg, was applied to 
a Poros HQ column with a buffer of 80 mM tris, pH 8.0, 240 
pM DpM (TBS-AGD) . The column was developed with a linear 
gradient from 0 to 0.5 M NaCl over 10 minutes with a flow 
rate of 5 mL/minute. The eluted protein fractions were 
identified by monitoring absorbance at 280 nm. 

After this treatment, 18 mL of protein-containing 
material were - pooled and concentrated in an Amicon-30 
centricon according to the manufacturer's instructions 
(Millipore, inc.; Bedford, MA). This yielded a 

concentration of approximately 1.8 mg/ml of COX-1 which was 
diluted to 1.3 mg/mL (20 pM COX-1) for screening. It is 
estimated that the buffer in the final protein preparation 
consisted of 2.4 mM DpM, 80 mM tris-8.0, about 50 mM NaCl . 
This COX-1 solution was promptly supplemented with 20 UM 
hemin, and 300 pM diethyldithiocarbamate (DDC) in accordance 
with the handling procedures used by Cayman Chemical 
company . 

The COX-1 protein preparation was then used for sample 
preparation according to the appended sample preparation 
standard operating procedure (SOP, Table 2) . 



40 



WO 02/057792 



PCT/US01/50088 



•d 

0) 

I 

•H 
4-> 

I 



0) 
P 
3 

•a 

0) 

o 
o 

P 

bi 
cs 

fi 

0) 
0) 
M 

u 
w 

CM ^ 
H 5 

1 s 

a 

-H 
Q) 
P 

2 



in p. 

O • CM 

I— I CM I - I !— 1 



M 
U 
O 
P 



T3 
C 

rd 



51 

o 

o • 



p 3. 



^ a. 



o 



G 
S) 
H 

I 

U 



•d 

0) 
N 

•H 
C 
o 
o 

■H 



3 

0J 
P 
d 
P 

s 

dl 
c 

TJ 
-H 

m 



c ~ 
o c 

•H O 
■P -H 

w o 

P 0) 
„ C -o 

) O -rl 

1 c 



M 

■P "d _ 

a a -h 
© <d « 

c «h p 
oho 

u <M 

<t> w 
>i 4J 
M ID Q) 

id m i 
p o 3 

fQ 0) H 
-H -H 0 
H -O > 

H H H 

to (0 A3 

d c v 

t*t tn H 



P 0) 
tn Q) 
0) p 

•d d> 

0) 

^ -d 
p o 

10 — . OD 
W I 
•O 0) 
0)0)4-) 
P P cd 
O Oft 
■M 0) *d 
0) TJ CD 
M 

wO O 

8"S 

— P 
(0 * 

0) 0) o< 
•P P w 

o o 

P P W 
CM tQ "d 

H * <0 

o x & 

P O -H 
P <r >-} 

g W o) 

O >i*i 
p a> 
« <d P 
u p d 

H tI i-l 

m hi a 



o 

CO 
0) 

•H 
P 
H 



d 
O 
P 

co 
p 

0) 
<H 
«H 
0 

03 P 

0) 

O P 
w ft 
CO s 



0) 

p 

d 
O 
P 

cu 

di 
c 

*H 
C 

o 
a 

CO 



~ a 

' x to 

d 0) 
O 0) 

p p 

0) 

d *d 
o 

-H CM 
P ^ 

d 

H O 
O U 

to 

0) 

•as 

S3 

an 

si 

co a) 
i 

0) 0) 

p p 
td a 
o* 

o) -d 

2S 



O 

a 



J2 

u 



o 
p 



•d 

p 

0} 0) 
H H 

a) a 
(0 I 
O 

d p 

S3 



a 

O O 

o 

in to 
a) 

to a> 
p 

O CP 
4J a) 
XJ 

M 

u o 
O co 

4-> I 

CD 

4J 
•d <t» 
0 

cd p 
tn^: 

■H Cn 
H -H 

>i P 
P CD 

2 § 
5 • 

H P 
O 

X -P 
U 03 
<ti 

o - 
a) 

•d X3 

S3 



41 



WO 02/057792 



PCT/US01/50088 



x 

1 

u 



g 

-P 
C 

o 



o 

I 

3- 



3 




42 



WO 02/057792 



PCT/US01/50088 



This sample prep SOP yields binding assays that combine COX- 
1 with mass-encoded combinatorial libraries made of 
approximately 2500 small drug-like molecules, each member at 
5 a concentration of 1 JJM. The sample was incubated 30- 

minutes at 4°C. As the total volume was 12 |i,L, the sample 
thus contained 24 0 pmol protein and 12 pmol of each library 
component. As a control test, an unrelated membrane 
protein, diacyl glycerol kinase (Calbiochem, Inc.; San 
10 Diego, CA) , was also prepared in the same buffer at 20 (iM 
and incubated with the same 2500 -member library and treated 
similarly. 

Then the mixtures were individually subjected to ALIS 
Analysis. If any ligand of suitably high affinity was bound 

15 to the COX at the time its fraction was collected, the mass 
spectral analysis would identify its mass. By virtue of the 
mass-coding, the precise combination of building blocks and 
core molecule can be identified (see U.S. Patent No. 
6,147,344). If the same compound failed to appear in the 

20 diacyl glycerol kinase control experiment, the compound may 
then be identified as a specific ligand of the COX-1. 

The mixtures were then individually subjected to 
modified ALIS analysis as follows. The large detergent- 
solubilized protein was separated from the small drug-like 

25 molecules by size exclusion chromatography (SEC) over a 
4.6mm x 50 rain x 5 [im SEC column at 0°C using a running 
buffer of TBS (80 mM tris," pH 8.0, 150 mM NaCl, 2.5% DMSO) 
at a flowrate of 2 mL/minute. The eluting SEC fraction- 
containing protein was identified by UV-VIS detection 

30 monitoring at 230 nm and transferred by way of a sample loop 
to a low-flow (100 |LtL/minute) reverse -phase chromatography 
(RPC) system. The RPC column (Higgins C-18; 1 mm x 50 mm x 
5 \lm) is maintained at 60°C to promote dissociation of 
ligands from the complex. From this RPC column, the ligand 

43 



WO 02/057792 



PCT/US01/50088 



is eluted into a high-resolution mass spectrometer for 
analysis using a gradient of 5%-95% acetonitrile (0,1% 
formic acid counterion) in water (w/ 0.1% formic acid) over 
5 minute. If any ligand of suitably high affinity was bound 

5 to the COX at the time its fraction was collected, the mass 
spectral analysis will identify its mass. By virtue of the 
mass encoding the precise combination of building blocks and 
core can be identified (see U.S. Patent No. 6,147,344 by 
Annis et al.) . If the same compound failed to appear in the 

10 diacyl glycerol kinase control experiment, the compound 
would be identified as a specific ligand of the COX-1. 
-Figure 6 illustrates the separation of protein from unbound 
small molecules using ALIS. 

Control experiments demonstrated that COX-1 screened in 

15 this manner enabled known COX-1 ligands to be extracted from 
large mixtures of small molecules (Figure 7) . When a test 
library composed of 25 fJK meclofenamate, 25 |iM indomethacin, 
1 |iM each of various test libraries, these known COX-1 
ligands are recovered and identified by the ALIS screening 

20 method . 

This experiment demonstrated that after screening over 
330,000 small drug-like molecules, 41 small molecules were 
identified as COX-1 ligands, one example of which is shown 
in Figure 8. These COX-1 ligand molecules were identified 
25 in two screens against small libraries, and their single 
molecule formulations are in preparation for further 
testing. Furthermore, many of these hits are observed to 
compete with known COX-1 ligand, meclofenamate, for binding 
(Figure 9) . 

30 

EXAMPLE 2: IDENTIFICATION OF LIGAND BINDING TO m2 mAChR 

PROTEIN BY MASS SPECTROSCOPY 

A gene construct encoding the m2 subtype of the 

muscarinic acetylcholine receptor (m2R) was cloned into a 

35 baculovirus expression vector according to conventional 



WO 02/057792 



PCT/US0i;50088 



cloning methods (see e.g., Baculovirus Expression Vector 
System , 6th Edition, 1999, Pharmingen, San Diego, CA) . The 
gene construct encoded a polypeptide with an amino terminal 
methionine followed immediately in frame by the melittin 
signal sequence (SEQ ID NO: 12) followed immediately in frame 
by the FLAG Ml epitope tag (SEQ ID NO:l) followed 
immediately in frame by the sequence for the m2 muscarinic 
acetylcholine receptor (NCBI Accession No. X04708) . The 
full-length polypeptide sequence therefore was: 

NH2- 

MKFLVNVALVFMVVYI S YI YADYKDDDDKMMNNSTNS SNSGLALTS PYKT 

FEVVPIVLVAGSLSLOTIIGNILVIW^ 

I IGVFSMNLYTLYTVIGYWPLGPWCDLWLALDYW 

R YFCVTKPLT YP VKRTTKMAGMM I AAAWVLS F I LWAPA I LFWQF I VGVRT 

VEDGECYIQFFSNAAVTFGTAIAAFYLPVIIMTVLYWHISRASKBRIKKD 

KKEPVANQEPVS PSLVQGRI VKPNNNNMPGSDEALEHNKIQNGKAPRDAV 

TENCVQGEEKESSNDSTSVSAVASNMRDDEITQDENTVSTSLGHSKDENS 

KQTCIKIVTKTQKSDSCTPANTTVELVGSSGQNGDEKQNIVARKIVK^ 

QPAJKKKPPPSREKKVTRTILAILLA 

WTIGYl^CYINSTINPACYALCNATFKKTFKHLLMCHYKNIGATR- 
COOH (SEQ ID NO: 34) 

Upon expression, the mellitin signal sequence is cleaved after Ala(21) revealing 
an amino terminal FLAG epitope which is bound specifically by the FLAG Ml antibody 
resin (Sigma; St. Louis, MO). This baculovirus expression vector was used to generate 
baculovirus that directed the expression of the above polypeptide in insect cells according 
the conventional methods (Baculovirus Expression Vector System, 6th Edition, 1999, 
Pharmingen, San Diego, CA). 

To purify FLAG-tagged m2R, 60 g of insect cells expressing the above 
polypeptide were suspended in 0.6 L of TBS [50 mM Tris-CL, pH 7.4, 100 mM NaCl), 
and the sample was homogenized by nitrogen cavitation. The homogenate was subjected 

to centrifugation at 500 x g for 30 minutes at 4° C to removed non-homogenized cells. 

The pellet was discarded and the supernatant was subjected to ultracentrifugation at 

45 

i 



WO 02/057792 



PCT/US01/50088 



.00,000 * g for 45 minne* „ 4 o c ne n sapamm wffl ^ 

-to peUeted ceU membranes were fc ^ 

Ap-onm OBS-D buffer) to . protein of 2 5 ^ ^ 

mcubated, sfei ng for 60 « 4 ° C before mh.centrifcgation at .00,000 x g for 45 

5 *Wc ra pene^ llIbIemleiial rhesombresupernaUntwasappHedtoaJ 

ot '^7 "** "ding fte solume mamria, 
andth^ 6 antibody resin, the column was washed with 50 mL of TBS-D buffer 

and to the FLACagged m2R protein was eiuted front to cohtmn with TBS-D buffer 
10 ^^"tS '00 pg/mL of FLAG peptide (Sigma; St- Louis, MO). Biumd com™ 
ffacuons contanung purified FLAG-tagged m2R were identified by SDS-PAGE 

capable* b^I " "* ^ «- « 

1* ° f "T 8 ^ *- «- filter-binding assays we* perfomred 

I5 ^ " — »' - - <-> X Bil Cfcem 

K d preparauon consist of 6 pM m2R in TBS-D with .00 pg/mL of 
^AG pept.de. Each m2R polypeptide is reversibly associated with a mulupUciry of 
moieculea, creating . ^ ^ ' 

-chromeny of m^tonin of .,-500. » ^ was ^ „ ^ 

srr 

Cher amdc reagents were prepared. Stcc* cyclooxygenaae , (COX) was 
^ accordmg ,0 me memo, in Exampie , to yi d d a concenhation of 6 pM COX in 

QNB). and ahopme wore prepared m 400 pM in- TBS. Pom combinatorial chemkal 

2 ? " "* * 3 — " *» UK n. four drug 

stoTlT G " 6S ' NGM " 41,NOL - 1,>A - 41 ' Four 

stock test hbranea were prepared containing 400 uM atmnin. aa- 



WO 02/057792 



PCT/US01/50088 



NMG-66 Plus Atropine, Stock NGM-41 Plus Atropine, Stock NGL-10-A-41 Plus 
Atropine, and Stock NGL-116-A-470 Plus Atropine. Four stock test libraries were 
prepared containing 400 pM QNB by adding QNB dissolved in DMSO to the individual 
drug libraries mentioned above to yield Stock NMG-66 Plus QNB, Stock NGM-41 Plus 
5 QNB, Stock NGL-10-A-41 Plus QNB, and Stock NGL-116-A-470 Plus QNB. Premix 
Buffer was prepared by combining 100 tjlL of 5% digitonin, with 4.6 mL water, and 400 

UL 1 M Tris-Cl, pH 7.5, then equilibrated at 42°C. 

Binding reactions were prepared that combined protein (either m2R test protein or 
COX control protein) with ligand (either QNB alone, atropine alone, pirenzepine alone, 

10 atropine plus drug library, or QNB plus drug library) or protein with DMSO as a control. 
In each case, 38 ]iL of Premix Buffer was dispensed into polypropylene tubes containing 
2 pL of DMSO or DMSO-solubilized ligands, mixed by vortexing, and centrifuged at 
8,000 x g for 10 minutes at room temperature to remove insoluble material. The clarified 
supernatants (2 ]iL) containing either aqueous DMSO alone or aqueous DMSO- 

15 solubilized ligands (with or without drug libraries) were transferred to polypropylene 
tubes at 4° C. Target protein (8 yL) m2R or control (8 iaL) protein COX was added to 

the supernatants, mixed well by pipetting, and incubated at 4° C for 60 minutes. 

These binding reaction preparations were then subjected to ALIS analysis. The 
binding reaction preparations combine 4.8 iiM target membrane protein (m2R) or 4.8 yM 

20 control membrane protein (COX) with a multiplicity of approximately 2500 small drug 
like molecules each at a concentration of 1-10 yM in a manner that established 
equilibrium binding conditions. High affinity ligands (IQd <100 pM) to the proteins are 
then identified by ALIS. Small molecule ligands of the target protein m2R that do not 
also bind to the control protein COX are considered as specific ligands of the target 

25 protein. Separately, for comparison to experiments with a multiplicity of drug molecules, 
binding reactions were also prepared combining m2R or COX with individual (discrete) 
m2R ligands. This ALIS Analysis proceeded as described below with a series of size 
exclusion chromatography (SEC), followed by reverse phase chromatography (RPC), 
followed by mass spectrometric (MS) analysis. 



47 



WO 02/057792 



PCT/US01/50088 



Tbese ptepared binding reaction — • — *** * 

SBC fiacfion - Mow, The 6 
<ift mm x 5 urn SEC column at 4"C using a running buffer of 50 mM Tns-Cl, pH 



230 nm. 



10 



The pro.ein.ontaining fraction was tiansfetred by wa, of a sample loop to a to. 
fio^a^HPCa^ *«-«-<"»-^ 
^ is maintained a. 60 ° C to promote ligand diss.ia.iou torn the compfe, firom Ihrs 
v r /4 in eluted into a high-resolution mass spectrometer for analysis 

IlCerSntinutes. Mass analysed Uganda ^wim^-— 
« *J. by virme of Its »» affinity for «he pnotein^eu, m* complex were 
identifiedbyfoK-knowledgeoftbeirpreciaemass. 

BxpLmenla dominated ft* «2R sereeued by ALIS Analysrs » tins u^ne 

0 ^ ^AUS-torm^se^— n^tigands^gU^- 
J m it recovered m2R figands bound » the m2R-«ergen t complex . tire absence 

^<^— 

B ^ONB-ora^m^ab^^pceaencoof^al^^ 

mOM-66, NOM-340, NGL-10-A-41, ** »«— 

ST Jl M — each at a concennation of 1-10 pM. The bmdmg 
subiected ,0 AUS Analysis and the extent of ligand recovery w, 
^tifiedbytiresignals.reugthof.hemaaaspectromete, Tue x-axrs ts . 
30 of mass ****** ** response fo, fte respective masses o, pmrnaeprne. QNB, and 
atropine. ^ 



WO 02/057792 



PCTYUS01/50088 



EXAMPLE 3: DISPLACEMENT ASSAY IN AN AFFINITY- BASED 

SELECTION AND ALIS IDENTIFICATION OF HP 
LIGANDS 

5 As a representative HP, m2 mAChR is purified according 

to the method of Peterson et al . (Peterson, G.L. et al. 
(1995) J. Biol. Chem. 270: 17808). The target protein is 
adjusted to a concentration of 20 p,M in a buffer of TBS-AGD 
and is incubated with a known muscarinic ligand, pirenzepine 

10 (MW = 424.3), which is at a concentration of 1 |iM. As a 
control test, an unrelated membrane protein, glycophorin, is 
also prepared in TBS-AGD at 20 |1M and is incubated with the 
compound pirenzepine (MW = 424.3), which is at a 
concentration of 1 |1M. 

15 A library of mass -coded compounds is added to the m2 

mAChR/pirenzepine mixture, and the sample is analyzed to 
determine if a library compound displaces pirenzepine from 
the HP protein. Displacement of pirenzepine indicates that 
the library compound binds more tightly and at the same site 

20 as pirenzepine itself. 

The displacement assay is conducted as follows. The 
mixture of m2 mAChR, 1 \im pirenzepine, and 1 |J,M of each 
library compound are subjected to ALIS Analysis. In this 
analysis MS signal corresponding to the mass of pirenzepine 

25 is monitored, while the mass of the library compounds are 
ignored. If the library compound quantitatively reduces the 
MS signal corresponding to the mass of pirenzepine, it is 
inferred that the library compound displaced pirenzepine 
from the HP protein, in this case mAChR protein. If 

30 incubation with the library compounds does not alter the MS 
signal corresponding to the mass of pirenzepine, the library 
compounds are considered not to contain an m2 mAChR ligand. 
By comparing pirenzepine MS signal with and without 
incubation with the library compounds, one can assess 



49 



WO 02/057792 



PCT/TS01/50088 



whether the library compound displaces pirenzepine and thus 
binds to the mAChR protein. 

EXAMPLE 4: AFFINITY SELECTION OF m2 mACHR MASS -CODED 

LIGANDS AND IDENTIFICATION BY ALIS 

As a representative HP, m2 mAChR is purified according 
to the method of Peterson et al. (Peterson, G.L. (1995) J. 
Biol. Chem 270: 17808). The protein is adjusted to 20 \M 
in a buffer of TBS-AGD is incubated with a 2500 -member 
library of mass-coded compounds, each member at a 
concentration of 1 |1M. After a 30 minute incubation at 
22°C, the sample was chilled at 4°C pending ALIS analysis. 
As a control test, an unrelated membrane protein, 
glycophorin, is also prepared in TBS-AGD at 20 \M and 
incubated with the mass-coded library. To determine if the 
compound specifically binds to the mAChR, the mAChR- compound 
mixture is analyzed by ALIS Analysis. If a mass 
corresponding to one of the members of the mass -coded 
library appears when the protein peak is collected and 
surveyed by MS, that compound may be identified as a binding 
ligand. If the same compound fails to appear in the 
glycophorin control experiment, the compound may then be 
identified as a specific ligand of the mAChR. By virtue of 
the mass encoding the precise combination of building blocks 
and core can be identified (see U.S. Patent No. 6,147,344 by 
Annis et al.). Using MS-MS analysis (see U.S. Patent No. 
6,147,344 by Annis et al.) , the exact structure of the core 
plus building block combination can also be pinpointed. 

EXAMPLE 5: AFFINITY SELECTION OF COX-1 LIGANDS AND 

IDENTIFICATION BY MODIFIED ALIS WITH ON-LINE 
FLUORESCENCE DETECTION 

As a representative HP, COX-1 protein was purified from 
ram seminal vesicles according to the method of Johnson et 
al. (Johnson, J.L. (1995) Arch. Biochem. Biophys. 324:26- 

34) . The COX-1 sample is adjusted to 20 uM in TBS-AGD (50 

50 



WO 02/057792 



PCT/US01/50088 



mM tris, pH 8.0, 150 mM NaCl, 800 mM dodecyl P-D-maltoside, 
2.5% DMSO) is mixed and incubated with a 2500-member library 
of mass -coded compounds, each member at a concentration of 1 
|J,M. After a 3 0 minute incubation at 22°C, the sample was 
chilled at 4°C pending ALIS analysis. As the total volume 
is 12 \ih, the sample thus contains 240 pmol protein and 12 
pmol of each library component. As a control test, an 
unrelated membrane protein, glycophorin, is also prepared in 
TBS-AGD at 20 \iM and incubated with the same 2500-member 
library and treated similarly. 

Then the mixtures are individually subjected to 
modified ALIS analysis as follows. The large detergent - 
solubilized protein is separated from the small drug-like 
molecules by size exclusion chromatography (SEC) over a 
4.6mm x 50 mm x 5 \im SEC column at 0°C using a running 
buffer of TBS (50 mM tris, pH 8.0, 150 mM NaCl, 2.5% DMSO) 
at a flowrate of 2 mL/minutes. The eluting SEC fraction 
containing protein is identified by on-line fluorescence 
detection exciting at 240-250 nm and monitoring emission at 
340 nm and transferred by way of a sample loop to a low-flow 
(100 jiL/minute) reverse-phase chromatography (RPC) system. 
The RPC column (Higgins C-18; 1 mm x 50 mm x 5 ym) is 
maintained at 60°C to promote dissociation of ligands from 
the complex. From the RPC column, the ligand is eluted into 
a high-resolution mass spectrometer for analysis using a 
gradient of 5%-95% acetonitrile (0.1% formic acid 
counterion) in water (w/ 0.1% formic acid) over 5 minutes. 
Ligand of suitably high affinity bound to the COX-1 at the 
time its fraction is collected, and the mass of the ligand 
is identified by mass spectral analysis. 

Through the mass coding, the precise combination of 
building blocks and core molecule are identified (see U.S. 
Patent No. 6,147,344 by Annis et al.) . If the same compound 
fails to appear in the glycophorin control experiment, the 

51 



WO 02/057792 



PCT/CS01/50088 



al 
10 34) 



compound may then he identic as a specifio 1^ of the 
COX-1 protein. 

LIGHT SCATTERING DETECTION 

A a a representative HP, COX-1 protein was purged J* om 
ram s^allesioles accord to the metho, - - 

(Johnson, J.L. (19.5) Arch. Biochem. Baopbys. 3 
,„ The COX-1 sarnie is adjusted to 20 rn TBS-AGD (50 
„ ' . 150 mM NaCl, 800 mM dodecyl p-D-maltoside, 

r 5 1 i Med and incubated wit, a 2500-me.fcer ™ 
Welded, pounds, eao^er at a —a ^n -X 
u „. After a 30 minute incubatron at 22 C, the p 
15 lined at « pending modified ALIS analyers. Aa the total 
volume ia 12 * the sample covins « » 
pm ol of each library component. As a 
related membrane protein, ^ al- 
TBS-AGD at 20 |>M and incubated with the same 

m iib Trre^:r rte. ----^ 

aubjected to modified AUS analyse ^ * ^ 

aru9 - iik ; r: ; - r — - « - in9 * 

25 over a 4.6mm x 50 mm p 2 5% 

running buffer of TBS (50 mM trr ^ pH .... 1 ^ 
D „SO, at a flowrate of 2 * llght 

30 to a loJflo. ,100 (— —phase oh^raphy 

(RPC , system The « ~ «^»^ \Lciation of 
5 urn, is maxntarnsd at 60 C to p 

Uganda from the complex. Prom the RPC =o^ , ^ 
is elnted into a high-resolution mass sp 

52 



WO 02/057792 



PCT/US01/50088 



analysis using a gradient of 5%-95% acetonitrile (0.1% 
formic acid counterion) in water (w/ 0.1% formic acid) over 
5 minutes. Ligand of suitably high affinity bound to the 
COX-1 protein at the time its fraction is collected, and the 
5 mass of the ligand is identified by mass spectral analysis. 

Through the mass coding, the precise combination of 
building blocks and core molecule are identified (see U.S. 
Patent No. 6, 147 , 344 by Annis efc al.). If the same compound 
fails to appear in the glycophorin control experiment, the 
10 compound may then be identified as a specific ligand of the 
COX-1 protein. 



EXAMPLE 7: HETEROGENEOUS SOLUTION PHASE SCREENING FOR 

LIGANDS THAT BIND m2 mACHR: IDENTIFICATION 
15 USING AFFINITY SELECTION AND ALIS ANALYSIS 

HP ligands may also be screened by utilizing a 

heterogeneous solution phase screening method in which a 

tagged target sequence is immobilized on a solid support. 

For example, anti-flag antibody- loaded protein A agarose 

20 beads ( ant i- flag beads) are prepared for use as a 
sedimentable stationary element in an immunoprecipitation 
(IP) -based screening protocol. 

Briefly, using a buffer of TBS -AG (50 mM tris, pH 8.0, 
150 mM NaCl, 800 mM dodecyl p-D-maltoside) , 100 \ih of 50% 

25 v/v slurry of protein A agarose (Santa Cruz Biotechnology, 
St. Louis, MO.) are washed in a 1.5 mL eppendorf tube with 
three room temperature cycles of: (1) combining the beads 
with 1 mL TBS -AG; (2) mixing the sample by tumbling for 2 0 
minutes; and (3) centrifugation at lOOOOxg, followed by a 

30 careful removal of the supernatant that leaves the pelleted 
agarose beads in the bottom of the tube. 

After the beads have been washed, the 50 |IL pellet of 
beads is brought up in 1.0 mL TBS -AG. To that mixture, 50 
(XL of 1 mg/mL P-galactosidase in TBS -AG buffer is added and 

35 the mixture is incubated at 4°C for 60 minutes to block non- 



WO 02/057792 



PCT/USO 1/50088 



specific binding of protein to the beads. To that mixture 
is added 10 [iL of 1.0 jlg/mL anti-flag antibody (Sigma, St. 
Louis, Montana), and the mixture is incubated at 4°C for 60 
minutes. In parallel, another preparation of control 
agarose beads handled similarly is treated with 10 |1L of 
TBS -AG instead of the anti-flag antibody. The anti-flag 
loaded beads and the control beads are then washed to remove 
excess antibody and protein and, finally, resuspended in 
0.10 mL of TBS -AG buffer and transferred to 0.5 mL eppendorf 
tubes. 

CHO cells expressing an m2 mAChR-flag tag-His tag 
protein are cultured, lysed, and homogenized. The m2 mAChR- 
containing membranes of the cells are purified by sucrose 
step-gradient ultracentrifugation. This sucrose step- 
gradient ultracentrifugation step removes most non-membrane 
proteins and cell debris. The ultracentrifugation step also 
has the potential of isolating specific populations of 
membranous cellular substructures. The mAChR-enriched 
membranes are solubilized with detergent (dodecyl-p- 
maltoside, DpM or CYMAL-7; (Anatrace; Maumee, OH)) and 
subjected to three steps of affinity purification. 

First, metal chelate affinity chromatography (MCAQ will 
be used to take advantage of the polyhistidine-tagged C- 
terminus, followed by a second affinity purification, an 
antibody affinity purification based on the FLAG- tagged N- 
terminus (Kobilka, B.K. (1995) Anal. Biochem. 231: 269). 
Third, ligand affinity purification over a column of 
immobilized mAChR ligand 3- (2 ' -aminobenzhydryloxy) -tropane 
(ABT) , followed by a desalting step will yield a final 
enrichment of active m2 mAChR protein. 

The sample is adjusted to 20 |IM m2 mAChR protein in a 
buffer of TBS-AGD and is incubated with a 2500-member 
library of mass -coded compounds, each member at a 
concentration of 1 |!M. The reaction is done in a volume of 



54 



WO 02/057792 



PCT7US01/50088 



40 \ih. After a 30 minute incubation at 22°C, the sample is 
chilled at 4°C pending MS analysis. As a comparison test, 
P-galactosidase is similarly prepared in TBS-AGD at 20 |iM 
and incubated with the mass-coded library. 
5 The m2 mAChR protein- compound mixtures are prepared for 

analysis by MS analysis with the following procedure. The 
purified protein-library mixtures, either the m2 mAChR 
protein or p-galactosidase protein trials, are each split 
into two 2 0 [ih volumes. For the m2 mAChR-library mixture, 

10 one volume is combined with the anti-flag beads (anti- 
flag/protein A agarose ) for IP and the other volume is 
subjected to a mock IP by combination with the control 
agarose beads (buffer/protein A agarose beads) . Similarly, 
for the p-galactosidase mixture, one 20 [ih volume is 

15 combined with the anti-flag beads for a control IP and the 
other 20 |XL volume is subjected to a mock IP by combination 
with the control agarose beads. These IPs proceed, mixed by 
tumbling, for 60 minutes at 4°C. Afterwards, each IP is 
.washed with three room temperature cycles of: (1) combining 

20 the beads with 1 mL TBS -AG, (2) mixing by tumbling for 2 
minutes, and (3) centrifugation at 10, 000 g, followed by a 
careful removal of the supernatant that leaves the pelleted 
agarose beads in the bottom of the tube. Finally, each 50 
\ih bed volume bead preparation is then resuspended in an 

25 additional 50 |iL of TBS (50 mM tris, pH 8.0, 150 mM NaCl) 
and kept at 4°C. At the completion of this process, four 
heterogeneous bead preparations are made: (1) m2 mAChR/anti- 
f lag/protein A agarose (m2 beads) , (2) m2 
inAChR/buf fer/protein A agarose beads (m2 control beads) , (3) 

30 P-galactosidase/anti-f lag/protein A agarose beads (P~lac 
beads) , and (4) p-galactosidase/buf fer/protein A agarose 
beads (p-galactosidase control beads) . 



55 



PCT/CS01/50088 

WO 02/057792 

, „arations constitute heterogeneous 
Tb ese bead-based P«*~ in the followrng 

solution phase system usef ^ ^ 50 ^ 

manner. Kaoh 100 uL bead P"*^ ^ £or 5 minu tes to 
o£ 30% aoetonitrile in water he ^ ^ mi:tture 

dissociate bound Uganda to. the , ^ ^ the 

U contrived at 10 00 J ^ ^ trans£erred to a 
dissociated protern m the sup 

new tube. . „. „ a££ inity selection of a 

The IP procedure allows bd « *« ^ m2mACBt . U gand 

„ ligand and the ^f^^rs. Tbese supernatant* 
co^le* from unbound library ^ ^ ^ ^ p . D . 
contain 50 mM tris, pH 8. , imat ely 60 ub of 

^Itoside, and 10% acetonrtrrle^ .^.^ into a 
these supernatant samples - urography (RP=> 

15 xow-flow (100 Ub/^inutes, -verse ph ^ ^ ^ = ^ 

sys tem. The kpc coiu« (Higg- c-^ , ^ ugana u 

i. maintained at ^Spectrometer for analysis 

elu ted into ^^-"\tt% Xitrile .0.1% 
using a 

gradient or > ^ over 5 min utes. 

20 counterion) in water fcy ob9erving a mass 

m 2 mRChR-ligsnds are ^ 250 o-member mass- 

corresponding to one o£ the m2 maChP protein 

oodB d library in the «*«-"^ o£ the m2 mfcCn* protein 
heads and not from the supernatant ^ 



25 



30 



beaas fi laC con troi 

control, Mac Ubrsry, « 3elec ted in the 

struo ture of the (see D . S . Pstent Ho. 
procedure are easily ^ OTa lysis (see U.S. 

6,147,344 by Annis et al.l- exact 6tru cture 
Patent Ho. e,147,344 by ^ - ca n also be 
o£ ^ core plus buxldrng block 

pinpointed . 

pHASB SCREENING FOR 



EXAMPLE 9: 

56 



WO 02/057792 



PCT/US01/50088 



USING A DISPLACEMENT ASSAY COMBINED WITH 
AFFINITY SELECTION AND MS ANALYSIS 

Flag-tagged m2 mAChR protein is purified as outlined 
herein above and resuspended at a concentration of 20 [iM in 
a buffer of TBS-AGD. The protein is incubated with a 2500- 
member library of mass -coded compounds, each member at a 
concentration of 1 \iM and with 1 [J.M pirezepine, a known 
ligand for m2 mAChR protein, in a volume of 40 [XL. After a 
30 minute incubation at 22°C, the sample was chilled at 4°C 
pending MS analysis. As a comparison test, p- galactosidase 
is similarly prepared in TBS-AGD at 2 0 JIM and incubated with 
the mass-coded library and pirezepine. 

The purified protein-library-pirenzepine mixtures, from 
either the m2 mAChR protein or p-galactosidase trials, are 
each split into two 20 \ih volumes. For the m2 mAChR - 
library-pirenzepine mixture, one volume is combined with the 
anti-flag beads (anti- flag/protein A agarose) for IP and the 
other volume is subjected to a mock IP by combination with 
the control agarose beads (buffer/protein A agarose beads) . 
Protein A agarose beads are prepared as previously described 
herein. Similarly, for the p-galactosidase mixture, one 20 
volume is combined with the anti -flag beads for a control 
IP and the other 2 0 \ih volume is subjected to a mock IP by 
combination with the control agarose beads. These IPs 
proceed, mixed by tumbling, for 60 minutes at 4°C. Then 
each IP is washed with three room temperature cycles of: (1) 
combining the beads with 1 mL TBS -AG; (2) mixing by tumbling 
for 2 minutes; and (3) centrifugation at 10,000 x g, 
followed by a careful removal of the supernatant that leaves 
the pelleted agarose beads in the bottom of the tube. 
Finally, each 50 |iL bed volume bead preparation is then 
resuspended in an additional 50 \xh of TBS (50 mM tris, pH 
8.0, 150 mM NaCl) and kept at 4°C. At the completion of 



57 



WO 02/057792 



PCTATS01/50088 



10 



15 



20 



25 



30 



this process, four heterogeneous bead preparations are made: 
(1) m2 mAChR/anti-flag/protein A agarose (m2 beads); (2) m2 
mAChR/buffer/protein A agarose beads (u2 control beads) , (3) 
P-galactosidase/anti-flag/protein A agarose beads (p- 
galactosidase beads) , and (4) p-galactosidase/buf f er/protein 
A agarose beads (p-galactosidase control beads) . 

' These bead-based preparations constitute heterogeneous 
solution phase systems useful for screening in the following 
manner. Each 100 |J.L bead preparation is combined with 50 mL 
of 30% acetonitrile in water heated to 60°C for 5 minutes to 
dissociate any bound pirenzepine from the protein. Then the 
mixture is centrifuged at 10,000 x g to pellet the beads, 
and dissociated protein in the supernatant is transferred to 
a new tube. The IP procedure allows both an affinity 
selection of the ligand and the physical separation of the 
m2 mAChR-ligand complex from unbound library members. These 
supernatants contain 50 mM tris, P H 8.0, 150 mM NaCl, 80 mM 
dodecyl p-D-maltoside, 10% acetonitrile, and <50 pmol of 
pirenzepine. Approximately 60 UL of these supernatants are 
injected into low-flow (100 |iL/minute) reverse-phase 
chromatography (RPC) system. The RPC column (Higgins C-18; 
1 mm x 50 mm x 5 (in) is maintained at 60«C. Prom this RPC 
column, the ligand is eluted into a high- resolution mass 
spectrometer for analysis using a gradient of 5%-95% 
acetonitrile (0.1% formic acid counterion) in water (w/ 0.1% 
formic acid) over 5 minutes. 

The mass corresponding to pirenzepine from the 
supernatant of the m2 mAChR protein control, 0- 
galactosidase library test, or p-galactosidase control beads 
should be higher than the pirenzepine mass response in the 
MS analysis of the m2 mAChR protein beads. It is then 
inferred that the 2500-member mass-coded library contains a 
ligand that binds to the m2 mAChR protein with an affinity 



58 



WO 02/057792 PCT/US0 1/50088 



greater than that of pirenzepine. Libraries that contain 
hits can thus be detected and selected from among other 
libraries that do not contain hits. 

5 EXAMPLE 10: ENHANCING THE HYDROPHOBIC PROTEIN 

SCREENING SUCCESS BY MULTIPLEXING 

To maintain the amphiphile-solubilized HP in a three- 
dimensional conformation that enhances the success of 
screening, HPs are multiplexed in the preparation of 

10 screening samples. Multiplexing is defined to mean any 
method of preparation wherein the target protein is combined 
with some known molar equivalent of one or more accessory 
proteins (APs) . Five independent criteria are identified to 
guide the selection of most favorable screening conditions 

15 with regard to the use of APs: (1) in the presence of the 
AP(s), the target HP is observed to bind a known agonist 
with greater affinity than without the AP(s) present; (2) in 
the presence of the AP(s) , the target HP is observed to bind 
a known antagonist with greater affinity than without the 

20 AP(s) present; (3) in the presence of the AP(s), the HP is 
shown to have greater functional activity as assayed by 
enzymatic, in vitro, or cell-based assays; (4) in the 
presence of the AP(s), the HP is shown to alter its state of 
multimerization; and (5) in the presence of the AP(s), the 

25 HP is shown to have greater conformational stability . or 
uniformity. 

As a first example, a method for multiplexed screening 
of m2 mAChR with G ail p lY2 - Guanos ine- diphosphate (GDP) as a 
heterotrimeric AP is presented. The G ail p lY2 - -GDP complex was 

30 identified as an AP for the m2 mAChR because its presence 
enhanced the affinity of the m2 mAChR for an antagonist, N- 
methylscapolamine (NMS) by 5 -fold as demonstrated by the 
following method. 3 H-NMS binding assays performed by the 
method of Rinken and Haga (Rinken, A. and Haga, T. (1993) 

35 Arch. Biocehm. Biophys . 301:158-164) showed that the 

59 



wo oiMmi 



TOT501/500M 



apparent Kd of ^ ^OR f or » 

Scribed in ^" PLs «« prepared as 

5 a 7 rd »3 to an y of the ^.^T ** ^ 

«* «M or modified AU S pretnt ed h """"^ 

* 50 PM OOP to the MnaC b Blti ^ addi «°* 

As a second ^.^1 

'« >*terodimeri 2e d * and Tl, - 

anu o subtypes of 
receptor is presented «, , he hUman °P*oid 

identified as an » for the h * ° Pi ° id was 
Presence caused the ^t ^ 
M opioid receptor to chanle T " h »» • 

* ^strated b, tha ^d ofTord^ * « 

T° prepare the 6/k onioin ' 
- -eenin,, the foil ^ ZTT ^ 
"ceptor i s purlf . J ^ " thod " »<>ed. Human K -opioid 
» concentration of 50 ^ *~ at a 

and dialed lnto 0£>1 ° ld "captor is purified 

of these t„o preparatZ entratiOT ° f 50 ^ E ^ 
"em to dimerise. ^"tions „ e re combined to allow 

* ~ — «- then 

utilise affinity selection ^ ^ ° f th * ""hods 
Presented herein. ttd MIS ° r modified AI|IS 

mME ' lE METHODS FOR THE ». 

to add ■"^'s.r- 

Cn^;^ ^ detect- 
exchanged f or „„i u ■ , . er9enfc nacelle solubili za t ion • 

ror solubilization -in j ""mzation is 

meetino the foll 0 „ lng orlCeria . » Proteoliposomes 

a - ID the Upid composition of 



WO 02/057792 



PCT/US01/50088 



the PL is defined and characterized as having no more than 
six discrete lipid entities making up 90% of the lipid 
content of the PL; (2) 90% of the HP- PL preparation has a 
defined, unimodal size distribution of particles spanning no 
5 more than three orders of magnitude and wherein mean 
particle size is in the range of 5-10000 nm; (c) the PL 
preparation yields >50 nM HP. 

To prepare HP- PL bearing m2 mAChR protein, the 
following procedure is used. Synthetic lipids (Avanti Polar 

10 Lipids, Alabaster, AL) D-ribo-phytospingosine-1 -phosphate 
and ceramide-C18 : 0 each in chloroform are combined in a 
volume of 1 mL each at 5 mM in a glass test tube, and the 
chloroform is evaporated at room temperature under a stream 
of argon for 24 hrs. The lipid residue is then wetted with 

15 0.40 mL of TBS, sonicated for 30 minutes at 28°C / and the 
mixture is transferred to a 1.5 mL polypropylene tube. To 
this lipid mixture is added 0,2 mL 50 (1M m2 mAChR prepared 
as described in Peterson, G.L. et al. (Peterson, G.L. et al. 
(1995) J. Biol, Chem. 270: 17808), This yields a crude 

20 mixture of lipid and protein. 

The crude protein-lipid mixture is then incubated for 3 
hours at 8°C with 5 minute, 22°C bath sonication at 30 minute 
intervals (6 times) . This mixture is then subjected to 11 
passages through a 100 nm polycarbonate membranes in a small 

25 volume extruder according to the manufacturer's protocol 
(Avanti Polar Lipids, Alabaster, AL) at 2 0°C. This yields a 
crude PL preparation of 0.6 mL volume, 16 |iM m2 mAChR, and 
excess amphiphile (DbM and lipid) . To remove excess lipid 
and detergent, the crude PL preparation is passed through a 

30 10.0 mL desalting (G-50 sephadex column) previously 
equilibrated with TBS at 4°C. After application of the 
crude PL preparation to the desalting column, TBS at 4°C is 
used to elute the material from the column. This desalting 
procedure is conducted with a flow rate of 0.5 mL/minute. 

35 The protein concentration is estimated by combining a 10 |XL 

61 



PCT/US01/50088 



WOOJ/05T792 



10 



15 



, of 0 2% Triton X-100 and using that 

sample with 10 I* <* °- 2% assay (Bio-Pad 

mlx ture in a Bio-Rad colori M tri pr ^ fcy 
Inc ., Hercules, CM. The concentration 

dilution with TBS to 10 |M. a Coulter N4 

- d T r »tCer follow the 

submioron particle analyzer attribution 
pro tocols. » independent asses-** of ^ ^ by 

ffid me an particle site is with . running 

meeting 20 ,b onto an «^ » ^ ^ , , 300 
TBS at a flow » J 1^0 ^ ^ aetector monltoring 
5 |» ToSo-Haas) , fitted ^ lt 

«t 2 S0 and chilled to 12 ^ ^ ^ opcR . pL , s 

S. 23 minutes identified the reten ^ Bi2e 

which was compared to Btan the mea n apparent 

m ar*er elution times ^^f^, dalto „ protein, 
size was comparable to that d wlth hps complexed 

Pr oteoliposcmes may also be prepa 

this procedure, _. , 



20 



25 



with » this ^7 o ^ iU2atlon in defined 

sol obilisation is ™f h ;t u B o ;Lg criteria: (1) the 
proteoliposcmes meeting ^ ^ charaoterize d as 

lipid composition of the p entities making up 

having no more than six discrete I P ^ ^ ^ ^ 
90% o£ the upid cogent of *e , ^ di3trlbutlon of 
preparation has a defined, rf 
particles spanning no more than thre ^ ^ ^ 
and wherein mean particle size (<) ^ pL 

nm, O. the PL -^^f^^ protein designated 
preparation included at lea ^ ^ supports 

t he accessory protein ^h ^ ^ target 

the maintenance of a tavo 

HP. . „•> „&ChR, the following 

To prepare HP-PL bearing - polar Uplds , 

procedure was used. Sy^etic -^-phosphate and 
, ^ClS^n chloroform are combined in a volume 



WO 02/057792 



PCT/US01/50088 



of 1 mL each at 5 inM in a glass test tube and the chloroform 
is evaporated at room temperature under a stream of argon 
for 24 hrs. The lipid residue is then wetted with 0.40 mL 
of TBS plus 2 mM MgCl 2 (TBSM) , sonicated for 3 0 minutes at 
5 28°C / and mixture transferred to a 1.5 mL polypropylene 
tube. Separately, 50 [iM G ail p iy2 was prepared by combining 
equal volumes of 100 \M G au with 100 \iM Gp lY2 purified as 
described (ref) and dialyzed into TBSM plus 800 mM DbM 
(TBSM- AG) . To the lipid mixture is added 0.2 mL 50 [AM m2 
10 mAChR prepared as described in Peterson, G.L. et al. 
(Peterson, G.L. et al. (1995) J. Biol. Chem. 270: 17808) and 
0.2 mL 50 (lM G ail p lY2 . To this mixture is added 10 ^L of 50 mM 
GDP. This yields a crude mixture of lipid, HP, and AP. 

The crude HP/AP-lipid mixture is then incubated for 3 
15 hours at 8°C with 5 minute, 22°C bath sonication at 30 minute 
intervals (6 times) . This mixture is then subjected to 11 
passages through a 100 nm polycarbonate membranes in a small 
volume extruder according to the manufacturer's protocol 
(Avanti Polar Lipids, Alabaster, AL) at 20°C. This yields a 
20 crude PL preparation of 0.8 mL volume, 12 jo,M mAChR, 12 [AM 
G «iiPiY 2 ' and excess amphiphile (DbM and lipid) . To remove 
excess lipid and detergent, the crude PL preparation is 
passed through a 10.0 mL desalting (G-50 sephadex column) 
previously equilibrated with TBS at 4°C. After application 
25 of the crude PL preparation to the desalting column, TBS at 
4°C is used to elute the material from the column. This 
desalting procedure is conducted with a flow rate of 0.5 
mL/minute. The protein concentration is estimated by 
combining a 10 |aL sample with 10 |iL of 0.2% Triton X-100 and 
30 using that mixture in a Bio-Rad colorimetric protein assay 
following the manufacture's instructions. The concentration 
is then adjusted by dilution with TBS to 20 \M. 

To define size distribution, a Coulter N4 submicron 
particle analyzer is used according to the manufacturers 

63 



WO 02/057792 



PCT/USOl/50088 



protocol. An independent assessment of size distribution 
and mean particle size is accomplished by analytical SEC by 
injecting 20 uL onto an analytical SEC column with a running 
buffer TBS at a flow rate of 1.0 mL/minute (02000^, 5 x 300 

5 mm, 5 ^m; ToSo-Haas) , fitted with a UV detector monitoring 
at 280 nm, and chilled to 12°C. A single peak eluting at 
8.23 minutes post -inject ion identified the retention time of 
the HP/AP-PL's which is compared to standard curve of 
molecular size marker elution times. The resultant mean 

10 apparent size is comparable to that of a 940,000 dalton 
protein. 

Control AP-PLs for use as screening controls are made 
to meet the following criteria: (1) the lipid composition of 
the PL was defined and characterized as having no more than 
15 six discrete lipid entities making up 90% of the lipid 
content of the PL; (2) 90% of the HP-PL preparation has a 
defined, unimodal size distribution of particles spanning no 
more than three orders of magnitude and wherein mean 
particle size is in the range of 5-10000 nm; (3) the PL 
20 preparation is designed to exactly mimic the analogous 
HP/AP-PL except no target HP is present. 

To prepare AP-PL bearing m2 mAChR, the following 
procedure is used. Synthetic lipids (Avanti Polar Lipids 
Alabaster, AL) D-ribo-phytospingosine-l-phosphate and 
25 ceramide-C18:0 each in chloroform are combined in a volume 
of 1 mL each at 5 mM in a glass test tube and the chloroform 
is evaporated at room temperature under a stream of argon 
for 24 hours. The lipid residue is then wetted with 0.40 mL 
of TBS plus 2 mM MgCl 2 (TBSM), sonicated for 30 minutes at 
30 28°C, and mixture transferred to a 1.5 mL polypropylene 
tube. Separately, 50 |AM G ailPlY2 was prepared by combining 
equal volumes of 100 uM G 0il with 100 uM G PlY2 purified as 
described Hou, Y. et al., J. Biol. Chem. (2000) 275:38961-6 
and dialyzed into TBSM plus 800 mM DbM (TBSM-AG) . To the 

35 lipid mixture is added 0.2 mL TBS -AG and 0.2 mL 50 G^,- 

64 



WO 02/057792 



PCT/US01/50088 



To this mixture is added 10 |IL of 50 mM GDP. This yields a 
crude mixture of lipid and AP. The protein concentration is 
estimated by combining a 10 |1L sample with 10 (XL of 0.2% 
Triton X-100 and using that mixture in a Bio-Rad 
5 colorimetric protein assay following the instructions of the 
manufacturer. The concentration is then adjusted by 
dilution with TBS to 10 |iM. 

This crude AP-lipid mixture is then incubated for 3 
hours at 8°C with 5 minute, 22°C bath sonication at 30 minute 

10 intervals (6 times) . This mixture is then subjected to 11 
passages through a 100 nm polycarbonate membranes in a small 
volume extruder according to the manufacturer's protocol 
(Avanti Polar Lipids, Alabaster, AL) at 20°C. This yields a 
crude PL preparation of 0.8 mL volume, 12 JIM mAChR, 12 |J,M 

15 G ailPlY2 , and excess amphiphile (DbM and lipid) . To remove 
excess lipid and detergent, the crude PL preparation is 
passed through a 10.0 mL desalting (G-50 sephadex column) 
previously equilibrated with TBS at 4°C. After application 
of the crude PL preparation to the desalting column, TBS at 

20 4°C is used to elute the material from the column. This 
desalting procedure is conducted with a flow rate of 0.5 
mL/minute . 

To define size distribution, a Coulter N4 submicron 
particle analyzer is used according to the manufacturers 

25 protocol. An independent assessment of size distribution 
and mean particle size is accomplished by analytical SEC by 
injecting 20 \xh onto an analytical SEC column with a running 
buffer TBS at a flow rate of 1.0 mL/minute (02000^, 5 x 300 
mm, 5 \im; ToSo-Haas) , fitted with a UV detector monitoring 

30 at 280 nm, and chilled to 12°C. A single peak eluting at 
8.23 minutes identified the retention time of the AP-PL's 
which is compared to standard curve of molecular size marker 
elution times. The resultant mean apparent size is 
comparable to that of a 940,000 dalton protein. 

65 



WO 02/057792 



PCT/US01/50088 



The proteoliposomes described herein are then screened 
affinity selection and ALIS or moani 



herein. 



12 . DUAL EPWOPI AFFINITY PHRIFICATION OF HPS 

^construction of nucieic acid seguence, , enco ingj 
tagg ed HP proteins of the invention is wel = £*ai 
of those in the art, utilizing routing procedures 

1 Tald^'r^lUtTn-nag Tag-Human ml mAChR-RB- 

tag protean or JeUitin-Flag Ta g -Hum*n Beta 2 Adrenergic 
epC-BB-tag protein, are produced from a . recombin n 
baculovirus following the methodology prov a ded by the 

5 manufacturer of the viral expression system (Pha-ingen, San 

Die3 °;r!efiy, recombinant virus is selected based on its 

Brier xy, recombinant 

anility to direct the astern blot 

protein<s). Protein expression «* * q£ the 

» analysis of ^ bodies «- 

::::: js. - - — - r - 

;:^n itself, the -stern blot reveals whether or no and 
lo what degree, the HP prota^s essed^ ^ 

25 a functional assay is performed to 

expressed protein is functional. For example for the 
^litin-Flag Tag-Human ml m.Chl.-BH-tag Jrote n, a ceU 
hased •H-H-methyls=apolamina binding analyses is perfo 
by the method of Rinhen and Haga (Rinhen, A. «a,J- 

30 a,93) Arch. Biocehm. Biopsy*. 3 01 = 158-164) confirm^ that 

" h virus-directed protein expression was func icnal, 
indicating a Bmax of 0.5x10' receptors per cell and a Kd of 
Tl o l This baculovirus was then amplified by to a high 
ItL ot a. 0X10- p«u/mb by conventional methods (Pharmmgen, 

35 San Diego, CA) . 



66 



WO 02/057792 



PCT/US01/50088 



Next, large-scale insect cell cultures are obtained for 
the isolation of the proteins. Briefly, once high titer 
virus stocks are generated the constructs of interest, ten 
10 liter production runs were executed, growing Sf21 insect 
cells to a density of 2xl0 6 cells/mL in a bioreactor with 
wave agitation (Wave Biotechnology, Bedminster, NJ) . For 
each protein produced, these cells were inoculated with high 
titer virus stock at an multiplicity of infection (MOI) of 5 
and two days post-infection the cells were harvested. 

' The HP proteins are then solubilzed. For example, 
mAChR protein expressing cells are collected by 
centrifugation for 30 minutes at 10,000 x g. The cell 
pellets from all ten bioreactor production runs are 
combined, and the cell pellets are suspended in 500 mL of 
ice cold TBS buffer plus 1 mM EDTA, 10 |ig/ml pepstatin and 1 
p.g/ml pmethylsulfonyl fluoride, and 20 mM dodecyl-P-D- 
maltoside (DbM) . This cell slurry is subjected to 50 
strokes in a pestle A dounce homogenizer at 4°C to break 
open the cells and solubilize the membrane proteins. To 
clarify this suspension, the material is centrifuged for 60 
minutes at 40000xg at 4°C to remove insoluble material. The 
supernatant is then collected and used as a source of 
soluble Mellitin-Flag Tag-Human ml mAChR- EE protein. (Note: 
The honey bee mellitin signal sequence is cleaved off by 
cellular proteases in the process of expression and 
trafficking to the plasma membrane of the insect cell. From 
this point on the solubilized protein is referred to as Flag 
Tag -Human ml mAChR -EE.) 

Each solubilized recombinant HP is then purified with 
two rounds of affinity purification over an antibody 
affinity column. Both the anti-EE-tag ■ and anti-Flag-tag 
columns are prepared similarly. Briefly, 35 mg of purified 
anti- epitope antibody is dialyzed into coupling buffer and 
coupled to 10 mL CNBr-activated sepharose beads according 
the manufacturers protocol (Amersham Pharmacia, Piscataway, 

67 



10 



pcT/csni/swra* 

WO 02/057792 

i 9 x 15 era 
af erred to a * 
NJ) . ^ resin is then a£Jinity colons «e 

Polypropylene c ° lm °°' ► a-c 

CeriH^tedwithTB^at^C n ^ ag . 

Hext, BO m* of solubalazed » " a££lnity column 

Human ml mAChR-EE, is applied to uas then 

« a flow rate of 0.2 » ^ at the same flow 

.ashed with 5 column volumes of ^ £ron the 

rate . The spe=ifieaUy-boun^ HP (HH2-EEEEYMPME- 

cclumn with a bolus of excess Ui- ln . 

CCOH; Sigma-Genosys, St Lours, ^ ^ applia d 

o£ 1S mh in TBS-M at 10 mM. ™ column , similarly 

5 This material ^ o c> 

£ol d volume of TBS-AG ™^ £ugation is then used to 
sucrose gradient ^^^tly, the material as 
temove misfolded PoWspt^ q£ 5% . 25% suor ose 

applied to a discontinuous step « ^ ^ t 

» ln TBS-AC and centrafuged m y con£ormed „P 

25 TBS-AG overnight at 4°C. ^ ffl soluti on re 

The protein pr otein assay (Bio-Rad 

determined by adjusted as necessary, 

^stories. Inc., Hercules. OA) ^filtration 
by dilution or concentrataon ^ ^ ^ ^ 



30 



by dilution " 60 jaM. The 

oell (Millipore, Bedford M. ^ , Hlggins 
concentration is also detained ^ ^ ^ ^ ^ 

c . 18 column whereupon 50 uh meter for analysis 

el uted into a nigh-resolutaon^ % £ormlc aold 

using a gradient of * ac° ^ ^ 2(> „. 
35 counterion) in water (w/ 0.1% 

68 



WO 02/057792 



PCT/USO 1/50088 



The Rt of the DbM is determined by a separate control 
experiments under identical conditions. To quantitate the 
amount of detergent, the area under the DbM peak from the 
protein sample is compared to a standard curve generated 
5 from multiple RPC runs with identical conditions assaying 
the DbM peak heighth for DbM samples of known concentration. 
If the detergent concentration is estimated to be below 0.48 
mM (4x cmc) , it is adjusted with the addition of a small 
amount of concentrated DbM in TBS. 

10 

EXAMPLE 13: EPITOPE AFFINITY AND METAL CHELATE AFFINITY 
CHROMATOGRAPHY (MCAC) OF HPS 

HPs are also constructed with an epitope affinity tag 

and a metal chelate affinity tag, as represented, for 

15 example, by Hemagglutinin SS-Rat m3 mAChR-HSV-OctaHis. 
Briefly, an isolated nucleic acid molecule encoding 
Hemagglutinin SS-Rat m3 mAChR-HSV-OctaHis is used in the 
production of a recombinant baculovirus following methods 
provided by the manufacturer (Pharmingen, San Diego, CA) . 

20 Virus production, the large-scale insect cell culture and 
expression of protein, the preparation of epitope affinity 
columns, the preparation of solubilized HP, and epitope 
affinity purification were all performed as previosly 
described herein except that anti-HSV antibody replaces the 

25 anti-FLAG antibody in the preparation of the HSV epitope 
affinity column and the HSV peptide (NH2 -QPELAPEDPED-COOH; 
Sigma-Genosys) is used to elute the Hemagglutinin SS-Rat m3 
mAChR-HSV-OctaHis protein from the column instead of the 
FLAG peptide. Also, since there is no EE epitope on the 

30 Hemagglutinin SS-Rat m3 mAChR-HSV-OctaHis protein, no 
epitope affinity purification using the EE epitope is used* 
(Note: The Hemagglutinin SS signal sequence may be cleaved 
off in some cell lines by cellular proteases in the process 
of expression and trafficking to the plasma membrane of the 



69 



PCT/CS01/5008R 

WO 02/057792 

. , „„ the solubili^d protein is 
. nl From this point on the s 
^ Tto'as ^ *3 .nAChR-HSV-OetaH^ ^ ^ 

referl ; r he Unal a ££ inity ^ * * '^es, to 
HSV .OctaHis is appUe^ at a ^ ^ ^ 

a MC *C ooi-n PteP« e d W colun* 

— 3 1 eo^ainin, B * JT—O 

** imidazole xn co ntainxng actxve ^ 

mL fractions. ^rac binding assay, el 
assessed by a known radx°l g ^ a volume o£ urn 

% „ Q le concentrations of 190 24 exchang es xnto a 

15 ^ lUal U then dialed wxth three ^ 
This materxal ove rnight at 4 C to g 

-°;°r Serial is of 

-rr - adjusted as 

and detergent are 

TO oonfi- the .f r :^ sam pie is suhjected to «» » 
25 „«., Ptodnoed ere^a ^ - ^ 

analysis on 5-12* rfurer . s Instruotions . Ten v* 

aeeordin, to the *»- ^ ^ les ar 

sample are loaded per gel lane ^ ^ , 99 ^ 

, ■ ori bv silver staxnxng v activities of the 

visualxzed by Bi ^ spe cxfxc acci 

• , rhem 270: 17808). ^ e s * methods are 

30 J. Bxol. Chem. * acc ording to these 

^ChR proteins prepay ^ peterson , G ^L. . 

determined accordxng the ^ 270: 17 B08) 

r , e t al. ( 199b ' --aiiv the specxfxc 

(Peterson, ^ typica lly 

For the methods 



70 



WO 02/057792 



PCT/US01/S0088 



activity of the proteins is determined to be in the range of 
11-16 nmol of specific ligand binding per mg mAChR protein. 

Ligand affinity chromatography is another measure by 
which the HPs of the invention may be evaluated for specific 
activity. For example, the mAChR purified by the methods 
described herein may be subjected to known ligand affinity 
chromatography over a column of immobilized mAChR ligand 3- 
(2 ' -aminobenzhydryloxy) - tropane (ABT) . 



71 



WO 02/057792 



PCT/USO 1/50088 



EQUIVALENTS 

Those skilled in the art will recognize, or be able to 
ascertain, using no more than routine experimentation, many 
equivalents to the specific embodiments of the invention 
described herein. Such equivalents are intended to be 
encompassed by the following claims. 



72 



WO 02/057792 



PCT/USO 1/50088 



What is claimed is: 

1. A method for identifying a ligand for a hydrophobic 
protein, the method comprising 
5 (a) selecting a ligand molecule by affinity selection 

by exposing a hydrophobic target protein bound by 
an amphiphile to a multiplicity of molecules to 
promote the formation of at least one complex 
between the hydrophobic target protein and the 
10 ligand molecule, 

(b) separating the complex from the unbound molecules, 
and 

(c) identifying the ligand molecule. 

15 2. The method of claim 1, wherein exposure of the 
hydrophobic target protein to a multiplicity of 
molecules occurs under homogeneous solution phase 
conditions. 

20 3. The method of claim 1, wherein exposure of the 
hydrophobic target protein to a multiplicity of 
molecules occurs under heterogeneous solution phase 
conditions. 

25 4. The method of claim 1, wherein selection of the ligand 
molecule is done using multi -dimensional 
chromatography . 

5. The method of claim 1, wherein the hydrophobic target 
30 protein is selected from the group consisting of: 

(a) of a membrane protein, 

(b) an integral membrane protein, 

(c) a transmembrane protein, 

(d) a monotopic membrane protein, 
35 (e) a polytopic membrane protein, 

(f) a pump protein, 

(g) a channel protein, 

73 



PCT/US01/5008R 



WO (12/057792 



( \. . associated enzyme, and 

a membrane-associai- 

a transporter protein 



(j 
00 



• , wherein the multiplicity of 
Th e method of cl- | l£ rf moleCuleS . 

molecules is a mass co 

wein the multiplicity of 
7 . Th e method of claim 1, * ^ ^ ^ ^ mags . 

0 molecules is a library 

coded . 

, f claim 1. w^xein the amphiphile « 
™e method o£ clarm^ ing of : 

selected from the gro f 

15 ( ^ 3 ^"hiohlu; macromolecular polymer, 

(b) an amphophilic m and 

(c) a surfactant or detergent, 

(d) an amphiphilic polypeptide. 

, „wein ligand molecule 

^convoluted by mass spectral an 

^ method of claim 1. "^1,^1^ « ith 
complex from the unbo und molecule 
eolid Phase chromatography medra. 
^method accords to claim 1, herein, the 

hydrophobic target P" tel "° in seq uence, 

w at least one transmembrane ^ ^ 



20 9- 
10. 



25 

11. 



30 12. 



35 



at xeast one t***-" ^ 

(b) at least two tag sequences 
selection, and seque nce. 

(c) a hydrophobic protein (HP) 



74 



WO 02/057792 



PCT/US01/50088 



13. The method according to claim 12, wherein the 

hydrophobic protein sequence is selected from the group 
consisting of 





ot a meniDrane protein, 


/K\ 
\0) 


an integral membrane protein, 


(c) 


a transmembrane protein, 


(d) 


a monotopic membrane protein, 


(e) 


a polytopic membrane protein, 


(f) 


a pump protein, 


(g) 


a channel protein, 


(h) 


a receptor kinase protein, 


(i) 


a G protein- coupled receptor protein, 


(j) 


a membrane-associated enzyme, and 


(k) 


a transporter protein. 



14 . The method according to claim 12 , wherein the tag 
sequences comprise epitope tag sequences selected from 
the group consisting of 

(a) a FLAG tag ( NH2 - DYKDDDDK- COOH ) (SEQ ID NO: 29) , 

(b) an EE tag (NH2 - EEEEYMPME - COOH ) (SEQ ID N0:30), 

(c) a hemagglutinin tag (NH2 - YPYDVPDYA- COOH ) (SEQ ID 
N0:31) , 

(d) a myc tag (NH2 - KHKLEQLRNSGA- COOH ) (SEQ ID NO: 32), 
and 

(e) an HSV tag (NH2 -QPELAPEDPED-COOH) (SEQ ID NO:33). 

15. The method according to claim 12, wherein the 
hydrophobic target protein comprises a sequence with an 
amino terminus to carboxy terminus order selected from 
the group consisting of 

(a) Tagl-Tag2-HP, 

(b) Tagl-HP-Tag2, and 

(c) HP-Tagl-Tag2. 



75 



WO 02/057792 



PCT/USOl/50088 



16. The method according to claim 15, wherein the 

hydrophobic target protein is selected from the group 
consisting of * 

(a) Myc tag-EE tag-Human m2 mAChR (SEQ id NO-7) 

(b) Flag tag-Human Beta 2 Adrenergic Receptor-EE tag 
(SEQ ID NO: 8) , 

Human Neurokinin 3 Receptor-HSV tag-Myc tag (SEQ 

Flag tag-Human ml mAChR -EE tag (SEQ ID N0:10) , and 
Rat m3 mAChR- HSV tag-OctaHis tag (SEQ ID N0:ll) . 



17. 



18. 



(c) 

(d) 
(e) 



The method according to claim 15, wherein the 
hydrophobic target protein further comprises a 
heterologous signal sequence (SS) at the amino 
terminus . 

The method according to claim 17, wherein the 
heterologous signal sequence is selected from the group 
consisting of K 

(a) the Mellitin signal sequence of NH - 
KFLVNVALVFMWYISYIYA- COOH (SEQ ID NO: 12), 

(b) the GP signal sequence of NH 2 -VRTAVLILLLVRFSEP- 
COOH (SEQ ID NO: 13) , 

(c) the Hemagglutinin signal sequence of NH - 
KTIIALSYIPCLVFA-COOH (SEQ ID N0:14), ' 

(d) the rhodopsin tag 1 si gnal sequenced NH - 

MNGTEGPNPYVPFSNKTGWRSPFEAPQYYLAEP-COOH (SEQ ID 
NO: 15), and 

(e) the rhodopsin tag ID4 signal sequence of NH - 
GKNPLGVRKTETSQVAPA - COOH (SEQ ID N0:16) . 

19. The method according to claim 18, wherein the tag 
sequences further comprise a hexahistidine sequence 
SEQ ID NO:17) and a decahistidine sequence (SEQ ID 
NO: 18) . 



76 



WO 02/057792 



PCT/US0 1/50088 



20. The method according to claim 19, wherein the 

hydrophobic target protein is selected from the group 
consisting of 

(a) GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ ID 
5 NO: 19), 

(b) Mellitin SS-Flag tag-Human Beta 2 Adrenergic 
Receptor-EE tag(SEQ ID NO:20), 

(c) Hemagglutinin SS -Human Neurokinin 3 Receptor-HSV 
tag-Myc tag (SEQ ID NO: 21) , 

10 (d) Mellitin SS-Flag tag-Human ml mAChR-EE tag (SEQ ID 

NO:22) , and 

(e) Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHis tag 
(SEQ ID NO: 23) . 

15 21. A method of isolating a hydrophobic protein, the method 
comprising 

(a) purifying the hydrophobic protein by sucrose 
gradient ultracentrifugation, 

(b) purifying the hydrophobic protein by antibody 
20 affinity purification, and 

(c) purifying the hydrophobic protein by immobilized 
metal affinity chromatography. 

22. The method of claim 21, wherein the hydrophobic protein 
25 comprises 

(a) at least one transmembrane domain sequence, 

(b) at least two tag sequences useful for affinity 
selection, and 

(c) a hydrophobic protein (HP) sequence. 

30 

23. The method according to claim 22, wherein the 
hydrophobic protein sequence is selected from the group 
consisting of 

(a) a membrane protein, 
35 (b) an integral membrane protein, 

(c) a transmembrane protein, 

77 



WO 02/057792 



PCT/US01/50088 



(d) 



(h) 

(i) 

(j) 
(k) 



10 24. 



lQ , a tnonotopic membrane protein, 

(e) a polytopic membrane protein, 

(f) a pump protein, 

(g) a channel protein, 
a receptor kinase protein, 
a G protein-coupled receptor protexn, 
a membrane-associated enzyme, and 
a transporter protein. 

T he method accord to claim 22, from 
seq uences comprise epitope tag sequences selecte 

^ T^C'S— K-COOH, (SEQ IB .0,23, , 
b an^ tag (NH2-EEEEYMPME-C00H) (SEQ ID N0:30) , 
<W ^agglutinin tag <NH2-™oVPB*A-C0OH> (SEQ IB 

Tlyotao (NH2 -KHKLEQLRMSGA- COOH ) (SEQ IB NO : 32) , 

,., an d HSV tag (NH2-QPELAPEDPEB-COOH) (SEQ IB NO : 33>. 

» 25 . method according to claim 22 wherein the 

hydrophobic protein composes a segnenc w 
terminus to carboxy terminus order selected 
group consisting of 

25 (a) Tagl-Tag2-HP, 

(b) Tagl-HP-Tag2, and 

(c) HP-Tagl-Tag2 . 



15 (O 
(d) 



26. 

30 



The method according to claim 22 wherein the 
hydrophobic protein is selected from the group 

consisting of wo -7} 

«yc tag-EE tag-Human m2 mAChR (SEQ IB NO 
(b) nag tag-Human Beta 2 Adrenergic Receptor tag 
(SEQ ID NO: 8) , 

Human Neurokinin 3 R eceptor-HSV tag-«yc tag (SEQ 



35 (c) 

ID NO: 9) , 



78 



WO 02/057792 



PCT/US01/50088 



(d) Flag tag-Human ml mAChR-EE tag (SEQ ID NO:10), and 

(e) Rat m3 mAChR-HSV tag-OctaHis tag (SEQ ID NO: 11). 

27. The method according to claim 22, wherein the 
hydrophobic protein further comprises a heterologous 
signal sequence (SS) at the amino terminus. 

28. The method according to claim 27, wherein the 
•heterologous signal sequence is selected from the group 
consisting of 

(a) the Mellitin signal sequence of NH 2 - 
KFLWVALVFMWYISYIYA-COOH (SEQ 10 1*0:12), 

(b) the GP signal sequence of NH 2 - VRTAVLI LLLVRFSEP - 
COOH (SEQ ID NO: 13) , 

(c) the Hemagglutinin signal sequence of NH 2 - 
KTIIALSyiFCLVFA-COOH (SEQ ID NO: 14), 

(d) the rhodopsin tag 1 signal sequence of NH 2 - 
MNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEP-COOH (SEQ ID 
NO: 15), and 

(e) the rhodopsin tag ID4 signal sequence of NH2- 
GKNPLGVRKTETSQVAPA-COOH (SEQ ID NO: 16). - 

29. The method according to claim 14, wherein the tag 
sequences further comprise a hexahistidine sequence 
(SEQ ID NO: 17) and a decahistidine sequence (SEQ ID 
N0:18) . 

30. The method according to claim 29, wherein the 
hydrophobic target protein is selected from the group 
consisting of 

(a) GP67 SS-Myc tag-EE tag-Human m2 mAChR (SEQ ID 
N0:19), 

(b) Mellitin SS-Flag tag-Human Beta 2 Adrenergic 
Receptor-EE tag(SEQ ID NO:20) , 

(c) Hemagglutinin SS-Human Neurokinin 3 Receptor-HSV 
tag-Myc tag (SEQ ID N0:21) , 



79 



WO 02/057792 



PCT7US01/50088 



31 



15 



(d) Mellitin SS-Flag tag-Human ml mAChR-EE tag (SEQ ID 
NO:22) , and 

(.) Hemagglutinin SS-Rat m3 mAChR-HSV tag-OctaHxs tag 
(SEQ ID NO:23) . 

An isolated nucleic acid molecule suitable for 
hydrophobic protein expression, comprising 
(a) a vector polynucleotide sequence for protein 
expression in a eukaryotic cell, and 
10 (b) a polynucleotide sequence encoding an engineered 

hydrophobic protein comprising the following 
elements 

(i) an N-terminal methionine residue, 

(ii) a heterologous signal sequence (SS) , 

(iii) at least one transmembrane domain sequence, 

(iv) at least two tag sequences useful for 
affinity selection, and 

(v) a hydrophobic protein (HP) sequence. 

The isolated nucleic acid molecule of claim 32, wherein 
the N-terminal methionine sequence and the heterologous 
signal sequence are selected from the group consisting 
of 

(a) MKFLVNVALVFMWYISYIYA (SEQ ID NO:24) , 
25 (b) MVRTAVLILLIiVRFSEP (SEQ ID NO: 25) , 

( C ) MKTIIALSYIFCLVFA (SEQ ID NO: 26) 

(d) MMNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEP-COOH (SEQ ID 
NO: 27) and 

(e) MGKNPLGVRKTETSQVAPA-COOH (SEQ ID NO:28) . 

The isolated nucleic acid molecule of claim 33, wherein 
the tag sequences comprise epitope tag sequences 
selected from the group consisting of 
(a) a FLAG tag (NH2 - DYKDDDDK- COOH) (SEQ ID NO:l) , 
35 (b) an EE tag (NH2-EEEEYMPME-CO0H) (SEQ ID NO: 2), 



20 32. 



30 



33. 



80 



WO 02/057792 



PCT/US01/50088 



(c) a hemagglutinin tag (NH2 - YPYDVPDYA- COOH) (SEQ ID 
NO:3), 

(d) a myc tag (NH2 - KHKLEQLRNSGA- COOH ) (SEQ ID NO:4) , 
and 

5 (e) an HSV tag (NH2-QPELAPEDPED-COOH) (SEQ ID NO: 5). 

34. The isolated nucleic acid molecule of claim 33, wherein 
the elements of the engineered hydrophobic protein are 
arrayed from an amino to carboxy terminus order 
10 selected from the group consisting of 

( a ) SS - Tag 1 - Tag2 - HP , 

(b) SS-Tagl-HP-Tag2, and 

(c) SS-HP-Tagl-Tag2 . 

15 35. The isolated nucleic acid molecule of claim 34, wherein 
the tag sequences further comprise a hexahistidine 
sequence (SEQ ID NO: 17) and a decahistidine sequence 
(SEQ ID NO: 18) . 

20 36. The isolated nucleic acid molecule of claim 35, wherein 
the engineered hydrophobic protein is selected from the 
group consisting of 

(a) GP 67- Myc - EE - Human m2 mAChR (SEQ ID NO:19), 

(b) Mellitin-Flag Tag-Human ml mAChR- EE (SEQ ID 
25 NO: 20), and 

37. A method for identifying a ligand for a hydrophobic 
protein, the method comprising 

(a) selecting a hydrophobic target protein from the 
30 group consisting of 

(i) of a membrane protein, 

(ii) an integral membrane protein, 

(iii) a transmembrane protein, 

(iv) a monotopic membrane protein, 
35 (v) a poly topic membrane protein, 

(vi) a pump protein, 

(vii) a channel protein, 

81 



PCT/CS01/50088 



WO 02/057792 



10 



15 



20 



25 



(b) 



« * "^C^oPc protein * 
(xii) wherein the nyu 

by amphiphile; hydrophobic 

(iv) an^xph^^ ultl _ 
, electing a Ugand moleeul Belectlon 

diB ensional ^"^"^l^ solution pha se 

by exposing under homoganoue a ^ ^ 

an a^iphiXe to a the £oM ation oi 

a »ass-coded U*« » ^ ^ hydroph ooic 
at least one complex moleou le; 

-^CS-p---— * olecules: 

separating tne o 
identifying tne i y 



(d) 
(e) 



30 



35 



analysis- 

.f ,na a ligand for a hydrophobic 
<-W for identify^ a ng 
38 . a method fo met hod comprising tbe 
protein, the m ic targe t protein 

(a) selecting a hydro? 
croup consisting oi 
* ° of a membrane protein, 
^ an integral membrane protein, 

v a monotopic membrane P 
^ a polytopic membrane protein, 
(vi) a pump Protein, 



82 



WO 02/057792 



PCT/US01/50088 



(vii) a channel protein, 

(viii) a receptor kinase protein, 

(ix) a G protein- coupled receptor protein, 

(x) a membrane -associated enzyme, and 

(xi) a transporter protein, 

(xii) wherein the hydrophobic protein is bound 
by amphiphile; - 

(b) selecting an amphiphile to bind' the hydrophobic 
protein from the group consisting of: 

(i) a polar lipid, 

(ii) an amphiphilic macromolecular polymer, 

(iii) a surfactant or detergent, and 

(iv) an amphiphilic polypeptide; 

(c) selecting a ligand molecule using multi- 
dimensional chromatography by affinity selection 
by exposing under heterogeneous solution phase 
conditions a hydrophobic target protein bound by 
an amphiphile to a multiplicity of molecules from 
a library that is not mass-coded to promote the 
formation of at least one complex between the 
hydrophobic target protein and the ligand 
molecule; 

(d) separating the complex from the unbound 
molecules, ;and 

(e) identifying the ligand molecule by mass spectral 
analysis. 

39. A method of isolating a hydrophobic protein, the method 
comprising 

(a) selecting a hydrophobic protein comprising 

(i) at least one transmembrane domain 
sequence, 

(ii) at least two tag sequences useful for 
affinity selection, selected from the group 
consisting of: 



83 



WO 02/057792 



PCT/US01/50088 



(1) a FLAG tag (NH2-DYKDDDDK-COOH) (SEQ 
ID NO:29) , 

(2) an EE tag ( NH2 - EEEE YMPME - COOH ) (SEQ 
ID NO: 30) , 

5 (3) a hemagglutinin tag (NH2-YPYDVPDYA- 

COOH) (SEQ ID NO: 31) , 

(4) a myc tag (NH2 -KHKLEQLRNSGA-COOH) 

(SEQ ID NO: 32) , and 

(5) an HSV tag ( NH2 - QPELAPEDPED - COOH ) 
10 (SEQ ID NO: 33) ; 

(iii) a hydrophobic protein (HP) sequence 

selected from the group consisting of: 

(1) a membrane protein, 

(2) an integral membrane protein, 
15 (3) a transmembrane protein, 

(4) a monotopic membrane protein, 

(5) a polytopic membrane protein, 

(6) a pump protein, 

(7) a channel protein, 

20 (8) a receptor kinase protein, 

(9) a G protein- coupled receptor 
protein, 

(10) a membrane -associated enzyme, and 

(11) a transporter protein; 

25 (b) purifying the hydrophobic protein by sucrose 

gradient ultracentrifugation; 

(c) purifying the hydrophobic protein by antibody 
affinity purification; and 

(d) purifying the hydrophobic protein by immobilized 
30 metal affinity chromatography. 

40. An isolated nucleic acid molecule suitable for 
hydrophobic protein expression, comprising 
(a) a vector polynucleotide sequence for protein 
35 expression in a eukaryotic cell; and 

84 



WO 02/057792 



PCT/US01/50088 



(b) a polynucleotide sequence encoding an engineered 
hydrophobic protein comprising the following 
elements 

(i) an N- terminal methionine residue, 

(ii) a heterologous signal sequence (SS) , 
wherein the N- terminal methionine sequence 
and the heterologous signal sequence are 
selected from the group consisting of 

(1) MKFLVNVALVFMWYISYIYA (SEQ ID 
NO:24), 

(2) MVRTAVLILLLVRFSEP (SEQ ID NO: 25), 

(3) MKTIIALSYIPCLVFA (SEQ ID NO: 26) 

( 4 ) MMNGTEGPNFYVPFSNKTGWRSPFEAPQYYLAEP - 
COOH (SEQ ID NO: 27) and 

(5) MGKNPLGVRKTETSQVAPA- COOH (SEQ ID 
NO:28) , 

(iii) at least one transmembrane domain 
sequence ; 

(iv) at least two tag sequences useful for 
affinity selection selected from the group 
consisting of 

(1) a FLAG tag (NH2 -DYKDDDDK- COOH) (SEQ 
ID NO:l) , 

(2) an EE tag ( NH2 - EEEE YMPME - COOH ) (SEQ 
ID NO:2) , 

(3) a hemagglutinin tag (NH2 - YPYDVPDYA- 
COOH) (SEQ ID NO: 3) , 

(4) a myc tag (NH2 - KHKLEQLRNSGA- COOH) 

(SEQ ID N0:4) , and 

(5) an HSV tag ( NH2 - QPELAPEDPED - COOH ) 

(SEQ ID NO: 5) , and 

(v) a hydrophobic protein (HP) sequence 
selected from the group consisting of: 

(1) a membrane protein, 

(2) an integral membrane protein, 



85 



WO 02/057792 



PCTAJS01/50088 



5 



10 



(3) 


a 


f vanampmhranp T)T*Ofc 6 in » 

UiailSlUvillU-'J- CHIC? ^f-*- \s »- w.*.** , 




ct 


monotopic membrane protein, 


(5) 


a 


polytopic membrane protein, 


(6) 


a 


pump protein, 


(7) 


a 


channel protein, 


(8) 


a 


receptor kinase protein, 


(9) 


a 


G protein- coupled receptor 




protein, 


(10) 


a 


membrane-associated enzyme, and 


(ID 


a 


transporter protein. 



86 



WO 02/057792 



PCT/US01/50088 



1/10 





> 




CO 


CO 


O 


u 






Eh 


> 




a 


CO 


fa 




>* 




3: 


CO 


2 


CO 


< 


H 


fa 


>J 


< 


< 


CO 


O 


u 


• • 


CO 




< 


fa 


CO 


> 


fa 


o 


H 


^ 


< 


CO 


fa 


> 


Eh 


2 


»4 


J 


H 


H 


^ 


fa 


2 






2 


2 


fa 


fa 


> 


H 


Q 




2 


2 


s 


fa 


Eh 




H 


CO 


CO 


O 


JH 


o 


Eh 


> 




2 


fa 


*C 




a 


2 


2 


a 


2 


> 




> 


> 


Eh 


> 




CO 


o 




H 


u 


fa 


2 


CO 


CO 


M 


H 


2 


2 


Eh 








M 


H 


H 


fa 


CJ 






Eh 






M 


H 


CO 


< 


fa 


CO 


Q 




> 


> 


Q 










> 


fa 


fa 


CO 


H 




5s 




fa 




a 




H 


O 


2 


< 


>* 




fa 


fa 


H 


H 


fa 


3 




fa 


H 




fa 


2 


2 


CO 




< 


id 




< 






fa 


fa 


< 


2 


H 






2 








a: 


O 




fa 


>H 


fa 


Eh 


< 


tu 


M 


H 


U 


w 


>« 


> 


Eh 


j 


OS 




2 


w 


2 


CJ 


O 


o 


M 






u 




fa 


fa 


Q 


U 


H 




w 






Eh 


Q 


H 


H 


fa 






a: 


> 


CO 


Q 


fa 




< 


a 


Q 




CO 




H 


fa 


< 




fa 


< 


fa 


CO 


> 




o 




CO 


2 


2 


2 






CO 


OS 


H 


CO 


2 


fa 








2 


H 


fa 


2 


Q 


fa 


fa 




> 


►J 


fa 


2 




fa 


Eh 


•J 






a 


2 


CO 


CO 


< 


o 


M 


2 


H 


fa 




fa 


2 


w 


CO 




>< 




o 


fa 


U 




> 




U 


> 




fa 






2 


CO 


u 


H 


CO 




< 




> 


< 


o 


fa 


Eh 




>* 




J 


2 


Q 


o 


CO 


hi 


U 


fa 


M 


CO 


fa 


a 


> 


< 


< 


w 


2 


> 


> 


> 


Eh 


fa 


fa 


CO 


O 


> 






2 


a 


2 


fa 


H 




fa 


co 


fa 




H 


Di 


M 


Q 


> 


Oi 


Q 


H 


H 


> 


H 






CO 


a 


2 


CO 


•J 


> 


< 


> 


> 


E^ 




2 








H 


fa 


M 


> 


M 




CO 




fa 


Q 


fa 


H 




M 




M 


o 


O 


Q 




U 




CO 


D 




2 


Q 


Di 


J 


> 


o 


CJ 


fa 


1 


fa 




5 


< 




> 






2 


£■ 








> 


H 


fa 


2 


H 


O 


a: 




fa 


< 


fa 


CO 




M 


> 


> 


O 


fa 






s 


Eh 




H 




< 








S 




fa 


fa 




Q 


< 


w 


> 








J 




CO 


Q 


Eh 






>• 


H 


id 


> 




2 




w 


o 


fa 


M 


CO 




Cb 




fa 


H 


CO 


fa 


h 







WO (12/057792 



PCT/US01/50088 



2/10 



> 

w 

Q 
Ol 
O 
O 

> 
Q 
DC 
Q 

< 

as 
oi 
a 
z 

a 

fa 

o 
z 
o 

fa 

a 
o 

« 
Q 
Q 
Q 



§ 

H 

01 
H 



2 
fa 

s 

> 
fa 

2 



5 

M 

3 

< 
O 

fa 
> 

O 



0) 

> 



2 
J 

H 
H 
> 
Ol 
< 



o) 

H 
fa 

Z 

a 

01 

w 
fa 

s 

H 

H 
> 

> 

fa 
> 

H 

> 
H 

01 

2 
> 

H 
O 

2 
O 



J, 
01 

a 

i*: 
fa 
fa 

01 
H 

H 
fa 

Q 

H 
> 

u 

H 
fa 

H 

01 
< 

> 
U 

> 

a 

H 
01 
H 

fa 

w 
a 

fa 
z 
o 
fa 

s 



w 
a 
fa 

> 
fa 

01 

X 
> 

fa 
> 

2 

M 
> 

fa 
> 
>* 
fa 
03 

> 
H 
03 
03 
< 

< 

a 

E-« 
fa 
fa 
Q 
U 
U 
H 
fa 



H 
fa 
fa 
fa 

S 

u 
J 

H 
fa 
H 
O 
2 

M 
M 
O 
J 



U 

z 

H 

< 
fa 

a 

s 

ol 
>* 

fa 

2 

o 

H 
fa 

fa 

. 01 
H 
J 

O 



fa 

j 
u 
fa 

01 
01 

fa 

01 

•J 
o 

fa 

o 

H 
fa 
O 
D 

a 
fa 
> 
a 

01 

j 
z 
a 
> 

fa 



01 
01 
>i 

a 
z 
o 

a 

a 

01 
03 
01 

s 

u 

J 
fa 
o 
fa 

H 

Ol 
fa 
Q 
fa 
01 
Ol 

u 

H 
fa 

z 
fa 
o 

03 



2 
fa 
2 
>< 
fa 
fa 
fa 
fa 
•J 

oi 

Q 

Z 

H 

oi 

U 

Z 

Ol 

O 

O 

01 

P 

M 

z 

Q 
01 
fa 



fa 
Ol 

O 
fa 

01 
Q 

O 

a 

ol 



>* 
o 

M 

z 

> 
fa 
^ 
01 
H 
J 

z 

Q 
O 
w 
> 
X 
> 



o 
a 
a 
o 
> 
fa 

Q 
fa 
H 
O 
fa 

Q 
fa 
U 
>J 
►J 

z 
fa 

fa 
a 
fa 
> 
a: 
>* 
o 

01 

a 
fa 

o 

E-« 
Z 
O 
Z 



CM 

6 



o 

CM 

• • 

o 
z 

Q 

H 

O 
fa 
01 



fa 



WO 02/057792 



PCT/US01/50088 



3/10 



•J 

3f 
8 

< 

1 

CO 

< 

•J 



Q 

I 

o 

C5 
O 
O 
Q 
H 

W 
2 

fa 
> 

CJ 

fa 

H 

co 
< 

H 
H 

2 



s 

> 
M 

J 

3 

1 



s 

cn 

M 

a: 

CO 

fa 
a 
> 
fa 
a 
s 

fa 
O 

CO 

fa 

< 
fa 

CO 

fa 
> 
fa 

o 

< 

CO 

fa 

CO 
CO 
CO 

•J 

< 
o 

Q 

►4 





w 


> 


fa 


fa 




HI 


fa 


HI 




Q 
i—i 




wj 




M 




rv* 


1—4 




rv 




W 




HI 
1 1 


fa 


> 










•> 


Cu 




ry 
Um 






2 


r i 




uu 


5 

uu 






MH 








rn 


LJ 


tr 1 






►u 




f\t 
mm 


LX« 






rV 




f? . 

M-l 






i—4 


UJ 




ft. 






r % 


r a 
UJ 




•yr 




MM 


r 1 
W 


rV 
U4 








M- 1 




r . 
f 










1— 1 




1 




C-t 




HI 






CJ 


5 












rn 






0 


H-l 












MM 




P-H 


1— 1 




Qj 




CO 


rj 


CJ 




U- 1 




Cu 


ry 




00 


[u 




* 






rn 


*> 




m^ 


Cu 












< 


04 






Cd 


00 




M 






CU 






&3 






M-* 




Cu 


O 


Cu 


Cd 


M f 


< 


< 






» 














MS 


VH 


M 


J 




Hi 


M^ 


Cu 


M* 






> 


w 




H 


H 


> 


Hi 


M** 


> 


CO 




ry 


r 


ry 




u 

v*/ 








00 


L 


H 


H 


HI 


00 






*> 


Fh 

L* 


Si 




H 
m 


E 

M«* 


H 


bH 


1^ 


rv 


L— 1 






u* 












H 






rn 




S 


s 


z 


fa 


H 








1— 1 


fa 












fa 


Qj 
MM 


Q 


CO 




Of 


fa 


00 


GO 


J 








00 


Cu 


D£ 


0< 


M 




M 


< 


04 


Cu 






Cu 






CJ 




J 


00 










W 


00 


$ 


04 


> 




J 


Eh 




Q 


J 


fa 


fa 


< 


Cu 


H 


H 


in 


Q 


CO 


>< 


M 


M 


w 








< 


> 
H 


fa 


SS 


S 






H 


fa 




co 


h 


fa 


DC 


J 






fa 


Q 


>i 


S 


H 


Di 


s 


> 




O 


fa 


Oi 




< 


Cu 


H 


fa 


CO 










U 


CJ 




< 




fa 








H 


s 


H 


fa 


§ 


•j 


2 






fa 


fa 


H 


CO 


0 


> 


< 


CO 



CO 

O 

LL 



(M 

• • 

o 



WO 02/057792 



PCT/CS01/50088 



4/10 



H 
H 

O 
M 

> 
o 

o 
< 
> 

H 

2 

co 
> 
< 

0« 
O. 

01 
2 



Q 
Q 
Q 



8 

Q 

H 
>< 
CO 
H 
X 



2 
> 

> 



Eh 

o 

< 

a: 
o 

:* 

Eh 
H 
>< 

£ 
CO 
En 
En 
O 



Q 
U 

j 

co 

>< 
2 
2 
> 

oi 



O* 

2 



O* 

CO 
H 

> 
J 

2 
o 

> 
Eh 
<C 

CO 

o 

Eh 



H 
< 

> 

CO 
O 

H 

2 

•< 

OS 

os 

8 

os 

CO 

a 

04 

oS 

Eh 

> 

co 
u* 
>* 

OS 

Q 

04 

CO 
M 

>4 
3 



o 

h3 



01 
OS 



2 
H 
Eh 
01 

os 

H 

OS 

3 

H 
U 
2 
> 
Eh 
> 

0< 



CO 
CO 
Eh 
J 
CO 

w 
2 
co 

o 
oi 

Q 
01 
U 
Id 

w 
w 

CO 

a 

a 
►a 

05 

04 



CO 

a 
o 

OS 
O* 

i*S 

a 

s 

'S 

OS 

a 

OS 

s 

*s 

H 

04 



CO 

a: 

2 

CO 

< 
> 

Q 

i 

Q 
U 
< 



< 

Eh 

O 
fa 
Eh 
H 
H 

04 

a 

CO 

A 

04 

O 

H 
>• 
U 

o 
> 

Eh 

os 

01 

o 

3 

a 
s 

04 



u 
u 

OS 

u 
u 

OS 

o 

Ot 

04 

Eh 

oi 

04 

CO 

o 

01 

o 

04 

a 

CO 
OS 
01 
CO 
CO 
CO 
CO 
CO 

0 

a 

s 

o 

04 

Eh 
01 
CO 

o 



> 

H 
2 

04 

CO 
CO 

os 

04 
04 

§ 

Eh 

04 

3 

01 

04 

Q 

> 

2 

04 

2 
H 

> 
01 
CO 

o 

04 

01 

' 01 

u 

01 



u 
< 
u 

2 

04 
2 

M 
Eh 
CO 
2 
> 

U 
J 

s 
>H 
o 
J 

01 

Eh 
03 

04 

> 

u 

Q 
CJ 

04 

H 
CO 
> 
tJ 
> 
2 

H 

04 

Eh 
Eh 

»4 

H 

04 

< 

H 



CN 
CM 

• • 

o 

2 
Q 

H 

a 

01 
CO 



oi 
2 

2 
* 
01 
01 
01 
01 

u 
a 

OS 
CO 

04 

Eh 
OS 

X 

> 

CO 

o 

04 



< 

CO 

J 

H 
OS 

01 

3 



04 

M 
*S 

OS 

OS 
OS 

xs 

D 
OS 

u 
J 

OS 

04 

Eh 
Q 
OS 

04 

2 



55 

IL. 



WO 02/057792 



PCT/TS01/50088 



5/10 



Of 




F-i 


b 


C/l 


CO 






H 


J 












H 


r 








CO 




1 1 






*z 


r/i 

V/J 


fO 


r— 1 




Ct. 

M-4 


Cli 


ry 


•> 




C— c 

tr* 








|_4 




5 


U4 


C_i 

lT 1 




J 


Hi 


04 


O 


C 


rn 


w 


|J 




h-i 


(/) 


ft 




> 












CD 








< 


b 




f— 1 


b 






b 


CO 


> 


> 


O 


b 




CO 


b 


CO 


> 




O 


x 




2 


b 


> 


M 


CO 


b 




2 


< 


> 


CO 


o 


> 




CO 


H 






CO 


H 


Q 




M 




M 




2 


> 


CO 


> 


ft 


•J 


a 


b 


b 


< 


£ 


>* 








O 


b 


b 


Q 


£ 


CO 


o 


U 


b 


H 


Eh 


< 




Eh 






M 


CO 


b 


2 




2 




0 


b 


CO 


H 




< 


as 


b 




£ 


j 


> 


£ 






> 


a: 


> 


2 


O 


2 


b 


< 






CO 


b 


H 


H 


H 


> 


h 


H 


> 










U 


o 






b 


o 


Eh 


J 


H 




b 


O 




b 


►J 


H 


CO 


Q 


2 




►J 


CO 


2 


> 




CO 


CO 




H 


E-» 


H 


b 


H 


Q 


> 




2 


O 






CO 


H 


Eh 




CO 


H 


Eh 




b 










Q 


s 




O 


< 


< 






u 


b 




b 


< 





b 

CO 
CO 



> 
O 

a 
o 
a 

CO 
CO 

o 

CO 

b 

CO 

co 
O 

Eh 
Oi 

X 
> 
b 
2 

b 

b 

Eh 
CD 
CO 

a 

a 

b 

Eh 



b 
H 
b 

H 

b 

M 
Eh 
2 
H 
Eh 
> 
b 



> 

H 
CO 

in 

H 



b 
CO 
O 



Q 
b 
b 
Q 
CO 
CO 

< 

CO 
b 
CO 

< 
< 

Q 
2 
53 
2 
£ 
CO 
Q 
CO 
CO 

co 
b 

Q 
O 
Q 
S 

o 
b 

CO 

b 

CO 

H 
Eh 
b 
£ 
b 
b 
U 

b 
O 

b 



co 
b 

O 

H 
b 

Eh 
Cl, 



r— 1 Ob 



a 
u 

Q 
O 
Q 
O 
2 
CO 

a 
< 
o 
j 

s 

b 
b 
> 
Q 
> 

Eh 

o 
•J 
a 
b 
2 

CO 

> 
a 

Q 
CO 
CO 
b 

CO 

z 

M 

CO 
CO 
X 

o 

Pu 



b 

H 
J 

co 

2 

b 
b 

Eh 

a 

CO 

b 

•J 
< 
b 



S 

b 

b 
CO 

J 

b 
►J 

S 
H 

a 
< 

CO 
CO 
2 

Eh 

a 

CO 

o 

Eh 

a 



a 
u 
a 
a 

►j 

b 
Eh 

u 

b 
Eh 

2 
O 

u 
> 

ft 
2 
> 
Eh 
CO 

2 

M 

O 
•J 

£ 

O 
J 
2 
£ 

b 

H 
U 
CO 
Q 
U 
b 



•J 
> 
2 

H 

b 

£ 

Eh 
H 



CN 

• • 

o 
3 



a 
b 

CO 



X 
X 
X 
X 
X 
X 
X 
X 



a 
b 
b 

Q 

b 
b 

b 
b 
a 

s 

b 

b 
> 



X 
b 

H 

> 
CO 

a 

b 
a 
a 

a 
a 
* 
b 

b 

&d 

b 



LO 

cri 



WO 02/057792 



PCT/US01/50088 



6/10 



D 

3 
z 

E 
+ 

T- 

8^ 
3 CO N 
< C CO 
COc> - 
O *- io 

Z5V 
O cJj 

z ?z 



O 

z 
E 

10 
CM 



oo 

Q.CL 

g 20 



g 

< 

CO 

z 
E 

CM 
+ 

« CD 
GO Q- 
O) p 



CD ^ 6 

sis 



2 o 

^ CO L 



co 



S5 



„ o <b 
(3 t- 
^ 2 V 

UJ o CD 



z 

_l 

Q 

< 

CO 

z 
E 

+ 

i 

O 

1- CL 

O p 

§•8 

CO O - 

UJ o CD 
z ? z 



3 

z 



LO 
CM 



o 

a 
o £q 

^ co CD 
CO § co 
CD ° 

SI* 

sis 



a 

3 



E 
io 

CM 
+ 

O 

o 

2 2 a 

CO § N 
CD T ~ t 

Sis 



CM 
© 

o 
CO 



1— 

> 
O 



□ H 




< 

DC 
< 
111 
-J 

o 



v 



o lo o mo io o to o in o in 
ooooooo N N (O co io m ^ 

T^dd do ddddddd 

30Nvayosav 



o to o 

co co 

odd 



WO 02/057792 



PCT/US01/50088 



7/10 



CO 

.2 
2 
□ 

CO 

x: 
CO 



X 

o 
o 



o 
9 

CD 

T— 

6 

LU 

3 

111 

S 

Q 

CO 
Q 
O 
X 
I- 
UJ 



* 
o 
o 

LU 
> 

o 
o 

LU 

DC 

>< 
Q 

I 

CD 

Q 

< 
CO 
Z 




m 

CM 



8 

CL 
LU 

DC 
CL 

LO 



LO 
CVJ 



X 

o 
o 

CL 
LU 

DC 
CL 

00 



XL 
O 



LO 
CVJ 

+ 

O 

o 

CQ 
Q 

CO 
CO 
LU 
— I 
00 



O 



LO 
CM 

+ 
■ 

§ 

o 

ID 

o 

DC 
X 



oo 



3 



z CO 



=*-CL LO ^CL 
U)0 CM U .ZTCS 
CM i LO ^ 



o 
o 

CL 
LU 
DC 
CL 

00 



o 
o 

CL 
LU 
DC 
CL 

OO 



CM 
+ 



CL 

LU 
DC 
CL 

OO 



XL 

LO 
CM 

4- 

1 

CL 
LU 
DC 
CL 
CO 



3 



< 

CO 



1 1 



o ~ 



XL 
O 



o 



■S: XL 

=*- O 
O i- 



xL 
O 



XL 
O 



LO 

CM 



X 

8 

CL 
LU 
DC 
CL 

00 



XL 
O 



LO 
CM 



X 

o 
o 

CL 
LU 
DC 
CL 

CO 



XL 

LO 
CVJ 



X 

8 
& 

DC 
CL 

LU 
LU 



0O 



XL 
O 



02/057792 



8/10 



PCT/CS01/50088 




WO 02/057792 



PCT/US01/50088 



LU 

e e ^ 

F + E lu 

qC QUI £ 

zq. zo- H 

< ^ < ^ o 

gogo a 

J O — l o ^ 

Q E3 □ 



o 
o 

I 

CO 



p 

z 

LU 

«5 LU 

it 
Si 

X z 

LU LU 

Z u- 

is 

b o 

Q+l 
— ' m 

is 



9/10 



d 

LL 



o 

CO 



ll 



LU 

II 



CO 

1 



So" 

CM 



o 



'in 



O) CO 
CM in n< ~ 



o 

d 
o 
co 



o 

d 
o 



o 
d 



o 
o 



LU 
CO 

z 
O 

CL 
CO 



CM 
CO 



co in 
DC 
CO 



o 

d 
o 



o 
o 



cp cm t 



t i t i i r 

CO CO t- r- CM CO ^- CVJ 



o 
d 



0> CD CD t— ■ i— i— CD CD 
^ LO LQ LQ CO CO CO CO CO 



£? I? t t ? w 4 

I — N N I s - N o5 Q) 



CM 



3 



WO 02/057792 



PCT/US01/50088 



10/10 



CD 
(0 

c 
o 
a 

CO 
0) 

a: 

CO 




(5 

LL