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MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM

MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM. RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY VIRGINIA COMMONWEALTH UNIVERSITY EMAIL: boothellors@vcu.edu. Date: 22 nd October 2010. THE COMMON PATH. vv. Cardiovascular disorders. Arthritis.

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MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM

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  1. MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM • RIO BOOTHELLO • DEPARTMENT OF MEDICINAL CHEMISTRY • VIRGINIA COMMONWEALTH UNIVERSITY • EMAIL: boothellors@vcu.edu. • Date: 22nd October 2010

  2. THE COMMON PATH vv Cardiovascular disorders Arthritis Skeletal disorders The Extracellular Matrix Matrix metalloproteinase CNS disorders Cancer Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3, 207-214.

  3. EXTRACELLULAR MATRIX Elastin Laminin Collagen Integrins Plasma membrane • Rozario, T. Dev. Biol. 2010, 341, 126–140.

  4. FUNCTIONS • Enzymes involved in ECM remodelling Provides structure Tracks migratory cells Presents growth factors to receptors Senses/transduces mechanical signals • Bone morphogenetic protein 1 • ADAMS • Serine proteases • Matrix metalloproteinases • Rozario, T. Dev. Biol. 2010, 341, 126–140.

  5. THE TADPOLE ENZYME 1962: Discovered by Jerome Gross and Charles Lapiere • Anuran tadpole explants • Placed on collagen gel • Collagen degraded • Gross, J. et. al. Proc. Natl. Acad. Sci.1962, 48, 1014-1022.

  6. THE TADPOLE ENZYME • Amount of collagen degraded • Area lysed • Degradation of C14 collagen Lysed collagen gel • Microscopic studies NH2 COOH NH2 collagenase cleavage site COOH Cleavage of collagen triple helix PDB ID: 1CAG Gross, J. et. al. Proc. Natl. Acad. Sci. 1965, 54, 1197-1204.

  7. HISTORY • 1970 • Purification of human collagenase • 1979 • Purification of TIMP-1 • 1984 • Development of genomic clones • 1992 • Batimastat Phase I trial • 1993 • First crystal structure solved • Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3, 207-214.

  8. MATRIX METALLOPROTEINASES • Belong to the metzincin group of proteases • Synthesized as inactive precursors • Degrade the extracellular matrix in a concerted manner PDB ID: 1CK7 • McCaw, A. et. al. Nat. Rev. Mol. Cell Biol., 2007, 8, 221-233.

  9. STRUCTURE Fibronectin type II domain Catalytic domain Pro domain Hemopexin domain • Murphy, G. Mol. Aspects Med. 2008, 29, 290–308. PDB ID: 1GXD

  10. CLASSIFICATION Zn2+ MMP -1, -8, -13 Collagenase N C Zn2+ C MMP -7, -26 N Matrilysins Zn2+ Membrane type C N MT-MMP 1-8 N C Zn2+ Gelatinase MMP -2, -9 Furin domain Fibronectin repeat N Cysteine switch Propeptide Signal sequence Hinge region Transmembrane domain Catalytic domain Hemopexin domain Cytosolic domain Tail • Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

  11. MECHANISM OF ACTIVATION PRCGXPD Cysteine switch peptide AHEXGHXXGXXH Catalytic site His His Cys His Zn2+ Proenzyme catalytic domain • Hu, J. Nat. Rev. Drug Discovery, 2007, 6, 480-498. PDB ID: 1SLM

  12. ACTIVATION OF THE PROENZYME His Zn+2 His His • Stepwise activation SH Pro His • Activation by MT-MMP His Zn2+ His Active form • Chemical activation His Zn2+ His His Intermediate Proenzyme SH SH Pro Pro • Ra, H. J.; Parks, W. C. Matrix Biol. 2007, 26, 587–596.

  13. ROLE PLAYED IN ECM • Path clearing through the ECM • ECM proteolysis generates signaling molecules • Degradation of basement membrane • Activation of latent signal MMP MMP Epithelial cells MMP Mesenchymal cells Degradation of basement membrane Proliferation MMP Cell death Cell motility Mesenchymal cell McCaw, A. et. al. Nat. Rev. Mol. Cell Biol. 2007, 8, 221-233.

  14. THE CELLULAR MILIEU IN CVS Myocytes Collagen IV Collagen VI Laminin Proteoglycans Endothelial Cells Collagen IV Laminin Fibronectin Vascular Smooth Muscle cells Collagen I Collagen III Collagen IV Laminin Fibronectin Fibroblasts Collagen I Collagen III Periostin Fibronectin MMPs Mast cells/Leukocytes/ Macrophages Cytokines Growth factors MMPs • Bowers, S. L. K. et.al. J. Mol. Cell. Cardiol. 2010, 48, 474-482.

  15. CONDITIONS INVOLVED MMP -3,9,12 Artery Smooth muscle cell migration Elastin degradation MMP -2,-9 Foam cell Monocyte infiltration Plaque rupture MMP -2, -9 Aneurysms Myocardial infarction MMP-2 and MMP-9 MMP-2 and MMP-9 Atherosclerotic plaque Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

  16. ROLE IN THE CVS Brew, K. et. al.Biochim. Biophys. Acta2010, 1803, 55–71.

  17. TARGETINGMMPS • Endogenous inhibitors • Synthetic inhibitors • PDB ID: 1SLM

  18. ENDOGENOUS INHIBITORS • The tissue inhibitors of metalloproteinases • Two distinct domains • N-terminal domain • C-terminal domain • Four major types • TIMP 1- 4 • Broad spectrum inhibitors • Bind in a 1:1 stoichiometric ratio N-Domain C-Domain • PDB ID: 1BR9 • Brew, K. et. al.Biochim. Biophys. Acta2010, 1803, 55-71.

  19. INHIBITION MECHANISM Glu67 Cys 70 His His Zn2+ Active site TIMP Ser68 Zn2+ S2 Cys Cys3 Cys 1 Val4 His Val69 Thr2 S3’ S3 S1’ PDB ID: 1UEA Brew, K. et. al.Biochim. Biophys. Acta, 2010, 1803, 55-71.

  20. TIMP: ROLE IN THE CVS Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

  21. TARGETINGMMPS • Endogenous inhibitors • Synthetic inhibitors • PDB ID: 1SLM

  22. THE FIRST GENERATION

  23. STRUCTURAL BASIS FOR INHIBITION Zn2+ S3 S2 S1 S3’ S1’ S2’ S2’ S3 S1 Collagen type peptide inhibitors P2’ P3’ P1’ P2 P3 P1 ZBG S3’ ZBG P2’ P3’ P1’ P2 P1 S2 P3 ZBG S1’ General inhibitors requirements Right side Inhibitors ZBG : Zinc binding group Left side Inhibitors • Dorman, G. et. al. Drugs, 2010, 70, 949-964. PDB ID: 2TCL

  24. PEPTIDOMIMETIC INHIBITORS Based on the structure of natural substrate collagen Isobutyl, t-butyl group preferred Methyl group preferred Essential for activity • Brown, P. D. Medical Oncology, 1997, 14, I- I0.

  25. THE ZINC BINDING GROUP • Hydroxamates • Thiol • Phosphinates • Carboxylates Hu, J. Nat. Rev. Drug Discov.2007, 6, 480-498.

  26. MECHANISM OF ZBG Active enzyme Enzyme-hydroxamate Hu, J. Nat. Rev. Drug Discovery, 2007, 6, 480-498.

  27. BROAD SPECTRUM HYDROXAMATES • The earliest MMP inhibitors • Many members of this class entered clinical trials Ilomastat Batimastat MMP-1= 5 MMP-2 = 6 MMP-3 = 200 MMP-7 = 20 MMP-9 = 3 MMP-1 = 0.4 MMP-2 = 0.39 MMP-3 = 26 MMP-8 = 0.18 MMP-9 = 0.57 MMP-1=10 MMP-2 = 4 MMP-3 = 20 MMP-8 = 10 MMP-9 = 1 Marimastat *All IC50 values are in units of nM • Skiles, J. W. et. al. Curr. Med. Chem. 2004, 11, 2911-2977.

  28. THE SECOND GENERATION

  29. STRUCTURAL BASIS OF INHIBITION P2’ substituent Wide range of substituent tolerated The succinate type backbone was modified P3’ substituent Wide range of substituent tolerated P1’ substituent Major determinant of activity α substituent Improves Pharmacokinetic properties Zinc binding group Essential for activity • Hu, J. Nat. Rev. Drug Discov.2007, 6, 480-498.

  30. MODIFICATIONS OF THE BACKBONE • Malonic acid type • Glutaric acid type • Sulphonamide type • Sulphone type Hu, J. Nat. Rev. Drug Discov.2007, 6, 480-498.

  31. SULPHONAMIDE BASED INHIBITOR Sulphonamide type Succinic acid type Developed by Parke-Davis showing μM potency for MMPs • O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.

  32. DEVELOPMENT OF PG-116800 Electron withdrawing group 4’  Increase activity Halogen at position 4’  Increased activity Halogen at position 3’  Decreased activity Halogen at position 2’  Decreased selectivity Electron donating group 4’  Decreased activity O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.

  33. DEVELOPING PG-116800 R1 H Improving the pharmacokinetic profile O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.

  34. PG-116800 PG-116800 Developed by Parke-Davis S1’ pocket Schematic representation of crystal structure Indicated that it could be used in left ventricular failure O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, 156-166.

  35. STUDIES CONDUCTED Studies in humans Animal studies • Randomized trial were conducted for 90 days • Study end points • LV end diastolic index • Ejection fraction • Results • No Significant changes • Musculoskeletal syndrome • Possible causes Kaludercic, N. et.al. Cardiovascular Therapeutics, 2008, 26, 24–37.

  36. THE S1’ SELECTIVITY POCKET • The depth of the S1’ tunnel is determined by the S1’ specificity loop • Pocket differs for different MMP’s S1’ Ct Specificity loop MMP- 1 shallow pocket MMP- 13 Deep pocket Nt PDB ID: 2TCL PDB ID: 456C Overlap of the active site of major MMP classes Devel, L. et. al. Biochimie, 2010. doi:10.1016/j.bioci.2010.07.017.

  37. THE THIRD GENERATION

  38. α-TETRAHYDROPYRANYL SULFONES RS-130830 (β-Sulfone) Developed by Roche 2nd Generation sulfone α- Sulfone β- Sulfone Developed by Pfizer α- Sulphone derivative *All IC50 values are in units of nM Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  39. α-TETRAHYDROPYRANYL SULFONES R α- Sulphone All IC50 values are in units of nM Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  40. α-PIPERIDINYL SULPHONES R1 R2 Selectivity H Pharmacokinetics Piperidine sulphones All IC50 values are in units of nM • Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  41. STRUCTURAL SELECTIVITY Arg214 Leu218 Crystal structure of α-Piperidine sulfone MMP-13, MMP-1 S1’overlap Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  42. ANIMAL STUDIES • Inhibition of post infarction left ventricular dilation investigated in rat model A B LV pressure (mmHg) α- Tetrahydropyranyl sulfone α- Piperidine sulfone LV volume (mL) SHAM-Veh MI-Veh MI-10mpk MI-50mpk MI-10mpk MI-50mpk A B Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  43. ANIMAL STUDIES C LV pressure (mmHg) LV volume (mL) SHAM-Veh MI-Veh MI-0.01mpk MI-0.1mpk MI-1mpk MI-10mpk Becker, D. P. et. al. J. Med. Chem. 2010, 53, 6653-6680.

  44. MECHANISM BASED INHIBITORS His kon Rapid His His + koff Slow E:I complex Zn+2 Zn+2 Enzyme (E) Inhibitor (I) + Substrate (S) Interaction with Zn His kon koff E:S complex Thiirane inhibitors His His Kcat + Product (P) E Covalently bound Zn • Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, 33992-34002.

  45. THIIRANE TYPE INHIBITORS R 1 A NO2 2 B 3 C • Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, 33992-34002.

  46. TETRACYCLINES • Weak inhibitors of MMPs • Inhibits smooth muscle cell proliferation and migration • Inhibits MMP-2 and MMP-9 Doxycycline MMP-9 proenzyme MMP-2 proenzyme 0 42 104 208 416 MMP-2 active Doxycycline μM Untreated SMC Doxycycline treated SMC Gelatin zymography of SMC • Franco, C. et. al. Am. J. Pathol. 2006, 168, 1697-1709.

  47. SUMMARY • MMPs • Inhibitors • Future strategies • Physiological and pathological functions of MMP are broad • Role of individual MMP in disease progression is not known • Modulators of MMP activity needs to be recognized • Inhibitors of MMPs have evolved from potent broad spectrum to selective cardiovascular agents • Development of selective inhibitors holds the key to the progress of these agents • Understanding the variation in binding site of MMPs may be essential • Understanding the role of extracellular matrix metalloproteinase inducer and other modulators of MMP • Developing newer strategies to first understand the role and then target MMPs

  48. ACKNOWLEDGEMENTS • Dr. Umesh Desai • The Desai group • The Department of Medicinal Chemistry at VCU

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