2. FRAGMENT- BASED DRUG DESIGN Yemane Mengistu Michigan State University January 30, 2008

3. Annual Research and Development Expense Expense ($ Billions) 40 80 120 160 Annual NME Approvals R & D Investment NME ( New Medical Entities ) Source : Pharma, FDA, Lehman :

4. Drug Discovery Process Target molecule eg. Enzymes High-throughput Screening (HTS) Combinatorial Chemistry Natural products Lead & Drug optimization Toxicity Clinical trials

5. Creating a Library Thymidinyl nucleoside A Natural Product Sun, D., Lee, R.E. Tetrahedron Lett. 2005, 46, 8497-8501

6. Thymidinyl nucleoside library Creating a Library Using Ugi Chemistry

7. Walters, W.P., Stahl, M.T., Murcko, M.A. Drug Discovery Today 1998, 3,160-178

8. Creating a Thymidinyl Ugi Library Sun, D., Lee, R.E. Tetrahedron Lett. 2005, 46, 8497-8501

9. Thymidinyl Ugi Library Sun, D.; Lee, R.E. Tetrahedron Lett. 2005, 46, 8497-8501

10. High-throughput Screening (HTS) A process of assaying a large number of compounds against biological targets. Up to 100,000 compounds can be analyzed in a day. Robots can usually prepare and analyze many plates simultaneously. http://www.metprog.org.uk/images/manufacturing_icon.jpg

11. What types of compounds become leads from an HTS?

12. HTS Drug like (Rule of 5) Lead-likeness Molecular weight500 # Hydrogen Bond acceptors10 Sum of N and O # Hydrogen Bond Donors 5 Sum of NH and OH ClogP<5 Molecular weight ~300 Fewer Hydrogen Bond Acceptors Sum of N and O ClogP<3 Low to high affinity for the target receptor Lead like behavior Drug like behavior ≤ ≤ ≤ Lipophilicity Congreve, M. et al. Drug Disco. Today.2003,8, 876-877 Lipinski, C.A. et al. Adv.Drug Deli.Rev.1997,23,3-25. Lipinski’s Rules (Pfizer)

13. 0 100 200 300 400 500 600 Potency Relative Molecular Mass 1µM 10 nM 1mM HTS hits Drugs Lead optimization Drug Candidates HTS Library

14. A Typical Drug Discovery Cascade Increased risk of failure HTS HTS Hits Hits actives Lead series Drug candidates Drug 1,000,000 2000 1200 50-200 10 1 Opera,T.I. J Comput. Aided Mol Des 2002, 16, 323-334

15. HTS 1996199920032004 Compounds screened100,000430,000615,0001,050,000 Average lead potency3,000 nM400 nM10 nM Screen success20%50%58%65% Leads per target1.01.71.92.0 GlaxoSmithKline’s HTS Scoreboard Chemical engineering news, 2004, 82,23-32

16. Fragnomics: Fragment Based Drug Design An approach that uses small and relatively simple molecules to make lead compounds Fragments Lead Target Merge and Expand Potential Medicinal compounds Look for affinity ~Enzymes, etc

17. MW of Average HTS Hits and Fragments 0 100 200 300 400 500 600 Potency Relative Molecular Mass 1µM 10 nM 1mM Fragments HTS hits Drugs Lead optimization Drug Candidates Rees D.C, Congreve M, Murray C.W, Carr R.Nat. Rev. Drug Discov. 2004, 3:660

18. Conventional HTS approach Fragment based drug design Fragnomics: Fragment Based Drug Design Erlason D.A, McDowell RS, O’Brien T. J Med Chem. 2004, 47:3463-82 Lewis, W.G.et al Angew. Chem. Int. Ed. Engl. 2002, 41,1053-1057 K d >100µM

19. Fragnomics: Fragment Based Drug Design Swayze, E.E, et al.J.Med.Chem. 2003, 46, 4232-4235 Conventional HTS approach Fragment based drug design

20. What Qualifies Compounds to be Fragments? Congreve, M. et al. Drug Discov.Today 2003,8, 876-877 Molecular Weight Mr ~300 Da H-bond donors (HBD) <3 H-bond acceptors (HBA) <3

21. What Qualifies Compounds to be Fragments? M r =200 HBD= 2 HBA=3 IC 50 = 1.3mM IC 50 = 65 nM Fragment Lead for protein kinase inhibitor Congreve, M. et al. Drug Discov.Today 2003,8, 876-877 M r =456 Molecular Weight Mr ~300 Da H-bond donors (HBD) <3 H-bond acceptors (HBA) <3

22. What Qualifies Compounds to be Fragments? Clog P=1.92 PSA=48.14 Clog P=3.07 PSA= 77.6 Congreve, M. et al. Drug Discov.Today 2003,8, 876-877 Ertl, P.et. al. J.Med.Chem. 2000, 43,3714-3717 Clog P <3  A measure of Lipophilicity of a compound Polar Surface Area (PSA) <60  A measure of permeability through the cell membrane. Fragment Lead for protein kinase inhibitor

23. Some Common Drug-Based Fragments Hartshorn, M.J., Murray, C.W.et.al. J. Med. Chem. 2005, 48, 403-413 Ring system from drug Heterocyclic system Side chains

24. Conventional HTS vs. Fragonomics Based on Central Scaffold A library with 1 million compounds 100 X R 1 100 X R 2, and 100 X R 3 yields 100 Variations yield a library of only 300 compounds Carr, R, and Hann, M. Modern Drug Discov. 2002, 45-48

25. Conventional (HTS) Drug Design Erlanson, D.A, Hansen, K.S. Curr Opin Chem Biol. 2004, 8,399-406.

26. Conventional (HTS) and Fragment Based Drug Design Erlanson, D.A, Hansen, K.S. Curr Opin Chem Biol. 2004, 8,399-406.

27. 1. Prepare set of potential binding elements with a common chemical linkage group Maly, D.J., Choong, D.J., and Ellman, J.A. Proc.Natl.Acad.Sci.USA. 2000,97,2419-2424 Fragment Based Drug Design

28. 1. Prepare set of potential binding elements with a common chemical linkage group 2.Screen Potential binding elements Maly, D.J., Choong, D.J., and Ellman, J.A. Proc.Natl.Acad.Sci.USA. 2000,97,2419-2424 Fragment Based Drug Design

29. 1. Prepare set of potential binding elements with a common chemical linkage group 2.Screen Potential binding elements 3. Prepare library of all possible combinations of linked binding elements. Maly, D.J., Choong, D.J., and Ellman, J.A. Proc.Natl.Acad.Sci.USA. 2000,97,2419-2424

30. Fragment Based Drug Design 1. Prepare set of potential binding elements with a common chemical linkage group 2.Screen Potential binding elements 3. Prepare library of all possible combinations of linked binding elements. 4.Screen library of linked binding elements Maly, D.J., Choong, D.J., and Ellman, J.A. Proc.Natl.Acad.Sci.USA. 2000,97,2419-2424

31. Application of Fragment based drug design Maly, D.J., Choong, D.J., and Ellman, J.A. Proc.Natl.Acad.Sci.USA. 2000,97,2419-2424 1. Protein kinase inhibitors  Tyrosine kinase (Src) activate numerous signaling pathways within cells, leading to cell proliferation, differentiation, migration and metabolic changes  Src kinases have been implicated in the pathology of tumors, osteoclast-mediated Bone resorption and disorders associated with T-cell proliferation Scapin,G. Drug Discovery today. 2002, 7,2002

32. Library for the Protein kinase inhibitor D.J.Maly, I.C.Choong and J.A, Ellman,Proc.Natl.Acad.Sci.USA,2000,97,2419-2424

33. Library for the Protein kinase inhibitor D.J.Maly, I.C.Choong and J.A, Ellman,Proc.Natl.Acad.Sci.USA,2000,97,2419-2424 K i =41µM

34. Library of protein kinase inhibitor D.J.Maly, I.C.Choong and J.A, Ellman,Proc.Natl.Acad.Sci.USA,2000,97,2419-2424

35. Fragment-Based Design : Protein Kinase Inhibition D.J.Maly, I.C.Choong and J.A, Ellman,Proc.Natl.Acad.Sci.USA,2000,97,2419-2424 CompoundIC 50 µM C-SrcFynLynLck [7]41± 5>1000 [16]40± 1664± 50400± 170>500 [7,16]0.064± 0.0385.0± 2.413 ± 2.4>250 716

36. Correlation of linker structure with IC 50 values for c-Src Inhibition

37. Application of Fragment Based Drug Design 2. Matrix Metalloproteinase inhibitors Matrix Metalloproteinases is a family of zinc-dependent endopeptidases. Implicated in a variety of diseases including arthritis and tumor metastasis. Conventional high-throughput screening failed to get non-peptide inhibitor. Haiduk, P.J. et al. JACS. 1997,119, 5818-5827

38. Application of Fragment Based Drug Design 2. Matrix Metalloproteinase Inhibitors K d =17 mM K d =0.2 mM IC 50 =57 nM Haiduk, P.J. et al. JACS. 1997,119, 5818-5827 Puerta, D.T, Lewis J.A. JACS. 2004, 126, 8389

39. Application of Fragment Based Drug Design 2 Matrix Metalloproteinase Inhibitors IC 50 = 0.5 nM Wada, C.K, et al. J.Med.Chem. 2002, 45, 219-232 ABT-518, a drug candidate in clinical trial by Abbot Pharmaceutical Company

40. Application of Fragment Based Drug Design 3. Thymidylate synthase (TS) Is the sole source for production of thymidine monophosphate (dTMP). dTMP plays a central role in DNA synthesis. It has been a target for dividing cancer cells. Banerjee D, Mayer-Kuckuk P, Capiaux G, et al. Biochim. Biophys. Acta, 2002, 1587,:164-73.

41. Application of Fragment based Drug Design Screen against library of Disulfide-containing small Molecules Erlanson, D.A, Braisted, A.C.Proc.Natl.Acad.Sci.USA. 2000, 97,9367–9372 TS Site Directed Ligand Discovery for TS

42. Preparation of Disulfide-Containing Library Members Parlow, J.J. & Normansell, J.E.Mol.Diversity 1995,1, 266-269

43. Synthesis of Sulfonyl Libraries Erlanson, D.A, Braisted, A.C.Proc.Natl.Acad.Sci.USA. 2000, 97,9367–9372

44. Thymidylate Synthase Inhibitor Erlanson, D.A, Braisted, A.C.Proc.Natl.Acad.Sci.USA. 2000, 97,9367–9372 Selected Non selected

45. Erlanson, D.A, Braisted, A.C.Proc.Natl.Acad.Sci.USA. 2000, 97, 9367–9372

46. Thymidylate Synthase Inhibitor Erlanson, D.A, Braisted, A.C.Proc.Natl.Acad.Sci.USA. 2000, 97,9367–9372

47. Application of Fragment Based Drug Design 4. Cysteine Aspartyl Protease-3 ( Caspase-3) Mediator of apoptosis ( programmed cell death). They are responsible for the cleavage of the key cellular proteins such as cytoskeleton proteins. Reducing the apoptotic response in diseases with dysregulated apoptosis such as myocardial infarction, stroke, traumatic brain, Alzheimer’s disease, and Parkinson diseases could benefit. Hotchkiss, R.S. et al. Nat. Immunol. 2000, 1, 496-501

48. Tethering with Extenders-dynamically Assembling Fragments Caspase-3 using extender A Caspase-3 using extender B Erlanson, D.A, Hansen, K.S. Curr Opin Chem Biol. 2004, 8,399-406.

49. Assembly of the Extender with Enzyme and with Fragment Library

50. Erlanson, D.A, Lam, J.W, Wesmann,C. Nat. Biotechnol. 2003, 21, 308-314 A B c

51. Assembling the inhibitor

52. Erlanson, D.A, Lam, J.W, Wesmann,C. Nat. Biotechnol. 2003, 21, 308-314 Assembling the inhibitor

53. Caspase-3 Superimposition of Inhibitor 1(Gray) and compound 4 (salmon) with Capsase 3 Erlanson, D.A, Lam, J.W, Wesmann,C. Nat.Biotechnol.2003, 21,308-314

54. Summary The use of fragment based drug design accompanied by different means of detection could increase the chance of finding new medical entities. Site directed ligand discovery and fragment based lead discovery are still in their infancy, but the success of these emerging approaches could success. No single technology will suffice, and the combination of HTS, site directed, and fragment-based lead discovery will likely become increasingly important.

55. Acknowledgements Prof. Kevin D. Walker Prof. Babak Borhan Prof. Bill Wulff Prof. Bob Hausinger Dr. Philip J. Hajduk, Abbott Laboratories Lab members:,Mark, Irosha, Washington, Danielle, Behnaz Friends: Khassay, Mercy, Rahman, Anil, Munmun, Luis