NMR-based approaches for lead discovery

Current Opinion in Drug Discovery and Development

Volumn 5, Issue 4, 2002, Pages 630-647

  Wyss, Daniel F ^a   McCoy, Mark A ^a   Senior, Mary M ^a  

^a MERCK RESEARCH LABORATORIES   (United States)

Author keywords

Chemical shift perturbations; Lead identification; Ligand screening; NMR screening; NMR screening libraries; NMR SOLVE; SAR by NMR; SHAPES; STD NMR; WaterLOGSY

Indexed keywords

LEAD;

ANALYTIC METHOD; BIOAVAILABILITY; LIGAND BINDING; NUCLEAR MAGNETIC RESONANCE; NUCLEAR MAGNETIC RESONANCE IMAGING; NUCLEAR OVERHAUSER EFFECT; PROCESS TECHNOLOGY; RELIABILITY; REVIEW; SCREENING; TECHNIQUE; ANIMAL; DRUG DESIGN; HUMAN; METHODOLOGY; PHARMACEUTICS;

ANIMALS; DRUG DESIGN; HUMANS; NUCLEAR MAGNETIC RESONANCE, BIOMOLECULAR; TECHNOLOGY, PHARMACEUTICAL;

EID: 0036653390     PISSN: 13676733     EISSN: None     Source Type: Journal    
DOI: None     Document Type: Review

References (100)

1. Sem DS, Pellecchia M: NMR in the acceleration of drug discovery. Curr Opin Drug Discovery Dev (2001) 4:479-492. An overview of the role of NMR in the drug discovery and development process that argues that NMR is primed to revolutionize the high-throughput structural characterization of protein-ligand interactions.
2. Pochapsky SS, Pochapsky TC: Nuclear magnetic resonance as a tool in drug discovery, metabolism and disposition. Curr Top Med Chem (2001) 1:427-441.
3. Pellecchia M, Sem DS, Wüthrich K: NMR in drug discovery. Nat Rev Drug Disc (2002) 1:211-219. Reviews the principles that enable NMR to characterize molecular interactions and discusses three NMR-based drug discovery strategies.
4. van Dongen M, Weigelt J, Uppenberg J, Schultz J, Wikström M: Structure-based screening and design in drug discovery. Drug Disc Today (2002) 7:471-478. Discusses the principles of a structure-based screening and design approach for lead discovery and shows its application to the discovery of a selective lead inhibitor of FABP-4 using site-selective NMR screening [38].
5. Peng JW, Lepre CA, Fejzo J, Abdul-Manan N, Moore JM: Nuclear magnetic resonance-based approaches for lead generation in drug discovery. Methods Enzymol (2001) 338:202-230. Provides details on theoretical, experimental and practical aspects of NMR screening approaches, with particular focus on ligand-detected methods.
6. Diercks T, Coles M, Kessler H: Applications of NMR in drug discovery. Curr Opin Chem Biol (2001) 5:285-291.
7. Roberts GCK: Applications of NMR in drug discovery. Drug Disc Today (2000) 5:230-240.
8. Hajduk PJ, Meadows RP, Fesik SW: NMR-based screening in drug discovery. Q Rev Biophys (1999) 32:211-240. Discusses fragment-based NMR strategies for discovering high-affinity ligands for proteins, and reviews the early examples of SAR by NMR and variations thereof.
9. Nicholson JK, Connelly J, Lindon JC, Holmes E: Metabonomics: A platform for studying drug toxicity and gene function. Nat Rev Drug Disc (2002) 1:153-161. Illustrates the role of NMR in metabonomics.
10. Rudin M, Beckmann N, Porszasz R, Reese T, Bochelen D, Sauter A: In vivo magnetic resonance methods in pharmaceutical research: Current status and perspectives. NMR Biomed (1999) 12:69-97. Reviews in vivo NMR methods in clinical and preclinical studies.
11. Karet G: Weak binders aid discovery. Drug Disc Dev (2002) 5:32-38.
12. Hajduk PJ, Meadows RP, Fesik SW: Discovering high-affinity ligands for proteins. Science (1997) 278:497-499.
13. Nienaber VL, Richardson PL, Klighofer V, Bouska JJ, Giranda VL, Greer J: Discovering novel ligands for macromolecules using X-ray crystallographic screening. Nat Biotechnol (2000) 18:1105-1108.
14. Carr R, Jhoti H: Structure-based screening of low-affinity compounds. Drug Disc Today (2002) 7:522-527.
15. Zuiderweg ERP: Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry (2002) 41:1-7.
16. 13C-labeled methyl groups. J Am Chem Soc (2000) 122:7898-7904.
17. Eichmüller C, Tollinger M, Kräutler B, Konrat R: Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy. J Biomol NMR (2001) 20:195-202.
18. McCoy MA, Wyss DF: Alignment of weakly interacting molecules to protein surfaces using simulations of chemical shift perturbations. J Biomol NMR (2000) 18:189-198. A method is described that enables the rapid determination of the binding site location and orientation of protein-bound aromatic ring-containing small molecules by minimizing the difference between experimental and simulated chemical shift perturbations. Such structural information is essential for the optimization of NMR screening hits into potent leads by focused chemistry approaches.
19. McCoy MA, Wyss DF: Spatial localization of ligand binding sites from electron current density surfaces calculated from NMR chemical shift perturbations. J Am Chem Soc (2002) manuscript submitted. Describes a rapid approach in which NMR chemical shift perturbations of the protein are used to construct an electron current density surface in coordinate space, which localizes the most probable position of the electron current density source from the small molecule ligand. This permits the spatial location of the ligand, bearing some resemblance to difference maps of electron density used in X-ray crystallography.
20. Medek A, Hajduk PJ, Mack J, Fesik SW: The use of differential chemical shifts for determining the binding site location and orientation of protein-bound ligands. J Am Chem Soc (2000) 122:1241-1242. Illustrates the utility of differential chemical shift perturbations of a series of closely related ligands to rapidly determine the precise location of the ligand binding site and the orientation of the ligand in the binding pocket, which provides critical structural information for lead development and optimization.
21. Pellecchia M, Meininger D, Dong Q, Chang E, Jack R, Sem DS: NMR-based structural characterization of large protein-ligand interactions. J Biomol NMR (2002) 22:165-173. Describes an NMR methodology that, with a suitable suite of NMR experiments and selective isotope-labeling schemes, rapidly obtains structural information of how a molecule binds to a protein, even in the absence of assignments or a complete 3D structure for the protein. The information is useful in the design of bi-ligand combinatorial libraries [78,106].
22. Hajduk PJ, Boyd S, Nettesheim D, Nienaber V, Severin J, Smith R, Davidson D, Rockway T, Fesik SW: Identification of novel inhibitors of urokinase via NMR-based screening. J Med Chem (2000) 43:3862-3866. Describes a cost-effective approach for the large-scale production of urokinase from mammalian cells, extending the applicability of target-detected NMR screening to proteins derived from mammalian systems. NMR screening, together with X-ray crystallography, identified 2-aminobenzimidazoles as a novel class of urokinase inhibitors, which serve as a starting point for the structure-based design of more potent leads.
23. Hajduk PJ, Gerfin T, Boehlen J-M, Häberli M, Marek D, Fesik SW: High-throughput nuclear magnetic resonance-based screening. J Med Chem (1999) 42:2315-2317.
24. Ross A, Schlotterbeck G, Klaus W, Senn H: Automation of NMR measurements and data evaluation for systematically screening interactions of small molecules with target proteins. J Biomol NMR (2000) 16:139-146.
25. Hann M, Hudson B, Lewell X, Lifely R, Miller L, Ramsden N: Strategic pooling of compounds for high-throughput screening. J Chem Inf Comput Sci (1999) 39:897-902.
26. Günther UL, Schaffhausen B: NMRKIN: Simulating line shapes from two-dimensional spectra of proteins upon ligand binding. J Biomol NMR (2002) 22:201-209.
27. Sattler M, Fesik SW: Use of deuterium labeling in NMR: Overcoming a sizeable problem. Structure (1996) 4:1245-1249.
28. Kay LE, Gardner KH: Solution NMR spectroscopy beyond 25 kDa. Curr Opin Struct Biol (1997) 7:722-731.
29. 2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA (1997) 94:12366-12371. An introduction to the TROSY method. TROSY enables the study of very high molecular weight proteins by NMR, because it produces a significant narrowing of NMR resonance line-widths and, thus, expands the scope of NMR-based lead/drug discovery approaches that rely on the detection of NMR signals from the target.
30. Riek R, Pervushin K, Wüthrich K: TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution. Trends Biochem Sci (2000) 25:462-468.
31. Wüthrich K: The second decade - into the third millenium. Nat Struct Biol (1998) 5(Suppl):492-495.
32. Goto NK, Kay LE: New developments in isotope labeling strategies for protein solution NMR spectroscopy. Curr Opin Struct Biol (2000) 10:585-592. Reviews novel isotope labeling strategies for proteins that have facilitated the study of structures of large proteins and protein dynamics. Such developments will also be essential for the application of target-detected NMR screening to larger proteins.
33. 13C for assignment and interpretation of nuclear magnetic resonance spectra of proteins. Q Rev Biophys (1996) 23:1-38.
34. 15N-amino acids. J Biomol NMR (1996) 8:184-192.
35. Yamazaki T, Otomo T, Oda N, Kyogoku Y, Uegaki K, Ito N, Ishino Y, Nakamura H: Segmental isotope labeling for protein NMR using peptide splicing. J Am Chem Soc (1998) 120:5591-5592.
36. Otomo T, Teruya K, Uegaki K, Yamazaki T, Kyogoku Y: Improved segmental isotope labeling of proteins and application to a larger protein. J Biomol NMR (1999) 14:105-114.
37. Xu R, Ayers B, Cowbum D, Muir TW: Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies. Proc Natl Acad Sci USA (1999) 96:388-393.
38. Weigelt J, van Dongen M, Uppenberg J, Schultz J, Wikström M: Site-selective screening by NMR spectroscopy with labeled amino acid pairs. J Am Chem Soc (2002) 124:2446-2447.
39. Pellecchia M, Meininger D, Shen AL, Jack R, Kasper CB, Sem DS: SEA-TROSY (solvent exposed amides with TROSY): A method to resolve the problem of spectral overlap in very large proteins. J Am Chem Soc (2001) 123:4633-4634.
40. Lin M, Shapiro MJ, Wareing JR: Diffusion-edited NMR-affinity NMR for direct observation of molecular interactions. J Am Chem Soc (1997) 119:5249-5250.
41. Hajduk PJ, Olejniczak ET, Fesik SW: One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules. J Am Chem Soc (1997) 119:12257-12261.
42. Chen A, Shapiro MJ: Affinity NMR. Anal Chem (1999) 71:669A-675A.
43. Ni F: Recent developments in transferred NOE methods. Prog NMR Spectros (1994) 26:517-606.
44. Fejzo J, Lepre CA, Peng JW, Bemis GW, Ajay, Murcko MA, Moore JM: The SHAPES strategy: An NMR-based approach for lead generation in drug discovery. Chem Biol (1999) 6:755-769. A lead discovery strategy based on the use of a library of low molecular weight fragments collected from the statistical analysis of established drugs. Protein targets are screened against weakly binding, drug-like molecular fragments by ligand-detected NMR methods, and hits are used to construct drug candidates computationally or in the laboratory.
45. Mayer M, Meyer B: Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy. J Med Chem (2000) 43:2093-2099.
46. Meyer B, Weimar T, Peters T: Screening mixtures for biological activity by NMR. Eur J Biochem (1997) 246:705-709.
47. Henrichsen D, Emst B, Magnani JL, Wang, W-T, Meyer B, Peters T: Bioaffinity NMR spectroscopy: Identification of an E-selectin antagonist in a substance mixture by transfer NOE. Angew Chem Int Ed (1999) 38:98-102.
48. Vogtherr M, Peters T: Application of NMR based binding assays to identify key hydroxy groups for intermolecular recognition. J Am Chem Soc (2000) 122:6093-6099.
49. Herfurth L, Weimar T, Peters T: Application of 3D-TOCSY-tr-NOESY for the assignment of bioactive ligands from mixtures. Angew Chem Int Ed (2000) 39:2097-2099.
50. Chen A, Shapiro MJ: NOE pumping: A novel NMR technique for identification of compounds with binding affinity to macromolecules. J Am Chem Soc (1998) 120:10258-10259.
51. Chen A, Shapiro MJ: NOE pumping. 2. A high-throughput method to determine compounds with binding affinity to macromolecules by NMR. J Am Chem Soc (2000) 122:414-415.
52. Mayer M, Meyer B: Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed (1999) 38:1784-1788. Introduces STD NMR as a very sensitive ligand-detected NMR screening method.
53. Mayer M, Meyer B: Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc (2001) 123:6108-6117. Describes a protocol for 'group epitope mapping' by STD NMR, which can be used to identify groups of a weakly binding ligand that are in direct contact with a target.
54. Dalvit C, Pevarello P, Tatò M, Veronesi M, Vulpetti A, Sundström M: Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J Biomol NMR (2000) 18:65-68.
55. Dalvit C, Fogliatto G, Stewart A, Veronesi M, Stockman B: WaterLOGSY as a method for primary NMR screening: Practical aspects and range of applicability. J Biomol NMR (2001) 21:349-359. Discusses the practical aspects and range of applicability of this very sensitive and recently introduced ligand-detected NMR screening method, which relies on the direct transfer of polarization from protein-bound water to the protein-bound ligand.
56. Klein J, Meinecke R, Mayer M, Meyer B: Detecting binding affinity to immobilized receptor proteins in compound libraries by HR-MAS STD NMR. J Am Chem Soc (1999) 121:5336-5337.
57. 3. J Med Chem (2001) 44:3059-3065. Describes the application of STD NMR to characterize the binding specificity of RGD peptide ligands to the heterodimeric integrin αIIbβ3 when embedded into the lipid bilayer of a liposome.
58. Jayalakshmi V, Krishna NR: Complete relaxation and conformational exchange matrix (CORCEMA) analysis of intermolecular saturation transfer effects in reversibly forming ligand-receptor complexes. J Magnetic Resonance (2002) 155:106-118.
59. Takahashi H, Nakanishi T, Kami K, Arata Y, Shimada I: A novel NMR method for determining the interfaces of large protein-protein complexes. Nat Struct Biol (2000) 7:220-223.
60. Ramos A, Kelly G, Hollingworth D, Pastore A, Frenkiel T: Mapping the interfaces of protein-nucleic acid complexes using cross-saturation. J Am Chem Soc (2000) 122:11311-11314.
61. Jahnke W, Rüdisser S, Zurini M: Spin label enhanced NMR screening. J Am Chem Soc (2001) 123:3149-3150.
62. 19F relaxation measurements for the study of fluorinated ligand-receptor interactions. J Magnetic Resonance (2001) 153:32-47.
63. 13C-NMR. Chem Pharm Bull (1998) 46:718-720.
64. 31P nuclear magnetic resonance spectroscopy. Anal Biochem (2001) 299:31-36. Describes the development and application of an NMR-based bioassay for the activity of the response regulator Che Y, illustrating the potential of NMR as both a binding assay and a bioassay that can reliably characterize low affinity leads.
65. Shuker SB, Hajduk PJ, Meadows RP, Fesik SW: Discovering high-affinity ligands for proteins: SAR by NMR. Science (1996) 274:1531-1534. Introduction of an NMR-based drug discovery strategy that develops high-affinity ligands by linking together fragments, identified and optimized independently by NMR, based on structural information of the ternary complex.
66. Hajduk PJ, Sheppard G, Nettesheim DG, Olejniczak ET, Shuker SB, Meadows RP, Steinman DH, Carrera GM Jr, Marcotte PA, Severin J, Walter K, Smith H, Gubbins E, Simmer R, Holzman TF, Morgan DW, Davidsen SK, Summers JB, Fesik SW: Discovery of potent nonpeptide inhibitors of stromelysin using SAR by NMR. J Am Chem Soc (1997) 119:5818-5827.
67. Hajduk PJ, Dinges J, Miknis GF, Merlock M, Middleton T, Kempf DJ, Egan DA, Walter KA, Robins TS, Shuker SB, Holzman TF, Fesik SW: NMR-based discovery of lead inhibitors that block DNA binding of the human papillomavirus E2 protein. J Med Chem (1997) 40:3144-3150.
68. Hajduk PJ, Dinges J, Schkeryantz JM, Janowick D, Kaminski M, Tufano M, Augeri DJ, Petros A, Nienaber V, Zhong P, Hammond R, Coen M, Beutel B, Katz L, Fesik SW: Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis. J Med Chem (1999) 42:3852-3859.
69. Hajduk PJ, Zhou M-M, Fesik SW: NMR-based discovery of phosphotyrosine mimetics that bind to the Lck SH2 domain. Bioorg Med Chem Lett (1999) 9:2403-2406.
70. Hajduk PJ, Gomtsyan A, Didomenico S, Cowart M, Bayburt EK, Solomon L, Severin J, Smith R, Walter K, Holzman TF, Stewart A, McGaraughty S, Jarvis MF, Kowaluk EA, Fesik SW: Design of adenosine kinase inhibitors from the NMR-based screening of fragments. J Med Chem (2000) 43:4781-4786.
71. Liu G, Huth JR, Olejniczak ET, Mendoza R, DeVries P, Leitza S, Reilly EB, Okasinski GF, Fesik SW, von Geldem TW: Novel p-arylthio cinnamides as antagonists of leukocyte function-associated antigen-1/intracellular adhesion molecule-1 interaction. 2. Mechanism of inhibition and structure-based improvement of pharmaceutical properties. J Med Chem (2001) 44:1202-1210. Demonstrates the power of the NMR-based fragment optimization approach for rapidly modifying high-affinity ligands to improve their pharmaceutical properties.
72. Ross A, Senn H: Automation of measurements and data evaluation in biomolecular NMR screening. Drug Disc Today (2001) 6:583-593. Reviews automation of industrial-scale NMR-based screening, presenting a fully integrated hardware set-up for 'just-in-time' sample preparation and automated analysis of target-detected NMR screening data.
73. Jahnke W, Perez LB, Paris CG, Strauss A, Fendrich G, Nalin CM: Second-site NMR screening with a spin-labeled first ligand. J Am Chem Soc (2000) 122:7394-7395.
74. Jahnke W: Spin labels as a tool to identify and characterize protein-ligand interactions by NMR spectroscopy. ChemBioChem (2002) 3:167-173.
75. Bemis GW, Murcko MA: The properties of known drugs. 1. Molecular frameworks. J Med Chem (1996) 39:2887-2893.
76. Bemis GW, Murcko MA: Properties of known drugs. 2. Side chains. J Med Chem (1999) 42:5095-5099.
77. Walters WP, Stahl MT, Murcko MA: Virtual screening - an overview. Drug Disc Today (1998) 3:160-178.
78. Jack RM, Smith JS, Villar HO, Sem DS, Coutts SM: Triad therapeutics: Integration of NMR structural determinations and smart chemistry to speed drug discovery. Drug Disc Today (2002) 7:S35-S38.
79. Wyss DF: NMR-based approaches for lead discovery. Cambridge Healthtech Institute's 2nd Annual Structure-Based Drug Design, Cambridge, MA, USA (2002).
80. Lugovskoy AA, Degterev AI, Fahmy AF, Zhou P, Gross JD, Yuan J, Wagner G: A novel approach for characterizing protein ligand complexes: Molecular basis for specificity of small-molecule Bcl-2 inhibitors. J Am Chem Soc (2002) 124:1234-1240.
81. McCoy MA, Wyss DF: Structures of protein - protein complexes are docked using only NMR restraints from residual dipolar coupling and chemical shift perturbations. J Am Chem Soc (2002) 124:2104-2105.
82. Fahmy A, Wagner G: TreeDock: A tool for protein docking based on minimizing van der Waals energies. J Am Chem Soc (2002) 124:1241-1250.
83. Moy FJ, Haraki K, Mobilio D, Walker G, Powers R, Tabei K, Tong H, Siegel MM: MS/NMR: A structure-based approach for discovering protein ligands and for drug design by coupling size exclusion chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Anal Chem (2001) 73:571-581. Comprehensive description of a relatively universal high-throughput screening approach combining the inherent strengths of size exclusion gel chromatography, MS and NMR.
84. Stockman BJ, Farley KA, Angwin DT: Screening of compound libraries for protein binding using flow-injection nuclear magnetic resonance spectroscopy. Methods Enzymol (2001) 338:230-246.
85. Lepre CA: Library design for NMR-based screening. Drug Disc Today (2001) 6:133-140. Reviews the current state-of-the-art methodologies for designing primary and follow-up libraries for NMR-based screening.
86. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev (2001) 46:3-26. Characterization of some current drugs in terms of properties, such as number of hydrogen bond donors and acceptors, hydrophobicity (ClogP) and molecular weight, to derive rules for defining compounds as 'drug-like'.
87. Walters WP, Murcko MA: Prediction of 'drug-likeness'. Adv Drug Deliv Rev (2002) 54:255-271. Reviews various computational techniques for identifying drug-like molecules, and provides some insights into challenges facing the field.
88. Maly DJ, Choong IC, Ellman JA: Combinatorial target-guided ligand assembly: Identification of potent subtype-selective c-Src inhibitors. Proc Natl Acad Sci USA (2000) 97:2419-2424.
89. Oprea TI, Davis AM, Teague SJ, Leeson PD: Is there a difference between leads and drugs? A historical perspective. J Chem Inf Comput Sci (2001) 41:1308-1315. Describes interesting differences between the properties of leads and drugs, providing information for the design of libraries aimed at lead discovery.
90. Hajduk PJ, Bures M, Praestgaard J, Fesik SW: Privileged molecules for protein binding identified from NMR-based screening. J Med Chem (2000) 43:3443-3447.
91. Muegge I, Martin YC, Hajduk PJ, Fesik SW: Evaluation of PMF scoring in docking weak ligands to the FK506 binding protein. J Med Chem (1999) 42:2498-2503.
92. Kim JL, Morgenstem KA, Lin C, Fox T, Dwyer MD, Landro JA, Chambers SP, Markland W, Lepre CA, O'Malley ET, Harbeson SL, Rice CM, Murcko MA, Caron PR, Thomson JA: Crystal structure of the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell (1996) 87:343-355.
93. McCoy MA, Senior MM, Gesell JJ, Ramanathan L, Wyss DF: Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex. J Mol Biol (2001) 305:1099-1110.
94. Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, Weber PC: Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol (1997) 4:463-467.
95. SPECTROSPIN AG (Busse-Grawitz ME, Roeck W): NMR probe head with cryogenically cooled preamplifiers. US-05814992 (1998).
96. BRUKER AG (Pervushin K, Wider G, Riek R, Wüthrich K): Transverse relaxation-optimized spectroscopy. US-06133736 (2000).
97. ABBOTT LABORATORIES (Fesik SW, Hajduk PJ): Use of nuclear magnetic resonance to identify ligands to target biomolecules. US-05804390 (1998).
98. ABBOTT LABORATORIES (Fesik SW, Hajduk PJ, Olejniczak ET): Use of nuclear magnetic resonance to design ligands to target biomolecules. US-05989827 (1999).
99. TRIAD THERAPEUTICS INC (Sem DS, Pellecchia M, Tempczyk-Russell A): NMR-SOLVE method for rapid identification of bi-ligand drug candidates. WO-00075364 (2000).
100. TRIAD BIOTECHNOLOGY INC (Sem D): Multi-partite ligands and methods of identifying and using same. WO-09960404 (1999).