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RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY VIRGINIA COMMONWEALTH UNIVERSITY Date: 22 nd October 2010.

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Presentation on theme: "RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY VIRGINIA COMMONWEALTH UNIVERSITY Date: 22 nd October 2010."— Presentation transcript:

1 RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY VIRGINIA COMMONWEALTH UNIVERSITY Date: 22 nd October 2010

2 THE COMMON PATH 1 Cardiovascular disorders CNS disordersCancer vv Skeletal disordersArthritis The Extracellular Matrix Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3, Matrix metalloproteinase

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

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

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

6 THE TADPOLE ENZYME Gross, J. et. al. Proc. Natl. Acad. Sci. 1965, 54, cleavage site NH 2 COOH Amount of collagen degraded o Area lysed o Degradation of C 14 collagen Lysed collagen gel PDB ID: 1CAG NH 2 collagenase Cleavage of collagen triple helix COOH Microscopic studies

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

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

9 Pro domain Catalytic domain STRUCTURE Murphy, G. Mol. Aspects Med. 2008, 29, 290–308. PDB ID: 1GXD 8 Fibronectin type II domain Hemopexin domain

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

11 MECHANISM OF ACTIVATION Hu, J. Nat. Rev. Drug Discovery, 2007, 6, PDB ID: 1SLM 10 His Cys His Zn 2+ AHEXGHXXGXXH Catalytic site PRCGXPD Cysteine switch peptide Proenzyme catalytic domain

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

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

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

15 Monocyte infiltration MMP -2,-9 Elastin degradation Plaque rupture MMP -2, -9 Smooth muscle cell migration MMP -3,9,12 Foam cell CONDITIONS INVOLVED 14 Atherosclerotic plaque Aneurysms MMP-2 and MMP-9 Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205. Myocardial infarction MMP-2 and MMP-9 Artery

16 GENEDELETIONOVEREXPRESSION MMP-9Reduced LV dilation and inflammation post-MI - MMP-2Reduce LV dilation and rupture rate post MI Reduced LV hypertrophy Severe LV contractile dysfunction Dilated cardiomyopathy MMP-3Defects in cell proliferation and cytokine release Impaired myocardial scar maturation - MT1- MMP Decreased connective tissue malformations - ROLE IN THE CVS Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803, 55–71. 15

17 TARGETING MMPS PDB ID: 1SLM Endogenous inhibitors Synthetic inhibitors 16

18 ENDOGENOUS INHIBITORS Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803, The tissue inhibitors of metalloproteinases Two distinct domains o N-terminal domain o C-terminal domain Four major types o TIMP 1- 4 Broad spectrum inhibitors Bind in a 1:1 stoichiometric ratio N-DomainC-Domain PDB ID: 1BR9

19 S2S2 Cys 1 Cys 70 Ser68 Glu67 Val69 Cys3 Val4 S3S3 S1’S1’ S3’S3’ Zn 2+ Thr2 His Cys Zn 2+ TIMP Active site INHIBITION MECHANISM Brew, K. et. al. Biochim. Biophys. Acta, 2010, 1803, Cys 1 to Val 4Primed subsites Glu 67 to Cys 70Unprimed subsites PDB ID: 1UEA

20 GENEKNOCKOUTOVEREXPRESSION TIMP-1Greater LV dilation and matrix loss post-MI LV systolic dysfunction post- MI Reduced cardiac rupture post MI Improved LV systolic function TIMP-2 Aneurysm developmentInvolved in ECM TIMP-3Greater LV dilation Increased cytokine processing Increased MMP-2 activation in fibroblasts Decreased activation of pro- MMP-2 TIMP-4No phenotypeN.A TIMP: ROLE IN THE CVS Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–

21 TARGETING MMPS PDB ID: 1SLM Endogenous inhibitors Synthetic inhibitors 20

22 21

23 S2S2 S1’S1’ S3’S3’ S2’S2’ S1S1 S3S3 STRUCTURAL BASIS FOR INHIBITION Dorman, G. et. al. Drugs, 2010, 70, PDB ID: 2TCL 22 S3S3 S2S2 S1S1 S1’S1’S2’S2’ S3’S3’ Collagen type peptide inhibitors P3P3 P2P2 P1P1 P1’P1’ P3’P3’P2’P2’ ZBG General inhibitors requirements P1’P1’ P3’P3’P2’P2’ZBG Right side Inhibitors P3P3 P2P2 P1P1 ZBG Left side Inhibitors ZBG : Zinc binding group Zn 2+

24 Methyl group preferred Isobutyl, t-butyl group preferredEssential for activity PEPTIDOMIMETIC INHIBITORS Brown, P. D. Medical Oncology, 1997, 14, I- I0. 23 Based on the structure of natural substrate collagen

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

26 MECHANISM OF ZBG Hu, J. Nat. Rev. Drug Discovery, 2007, 6, Enzyme-hydroxamateActive enzyme

27 BROAD SPECTRUM HYDROXAMATES Skiles, J. W. et. al. Curr. Med. Chem. 2004, 11, The earliest MMP inhibitors Many members of this class entered clinical trials Batimastat Marimastat Ilomastat MMP-1 = 0.4 MMP-2 = 0.39 MMP-3 = 26 MMP-8 = 0.18 MMP-9 = 0.57 MMP-1= 5 MMP-2 = 6 MMP-3 = 200 MMP-7 = 20 MMP-9 = 3 MMP-1=10 MMP-2 = 4 MMP-3 = 20 MMP-8 = 10 MMP-9 = 1 *All IC 50 values are in units of nM

28 27

29 STRUCTURAL BASIS OF INHIBITION Hu, J. Nat. Rev. Drug Discov. 2007, 6, α substituent Improves Pharmacokinetic properties P 2 ’ substituent Wide range of substituent tolerated P 3 ’ substituent Wide range of substituent tolerated P 1 ’ substituent Major determinant of activity Zinc binding group Essential for activity The succinate type backbone was modified

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

31 SULPHONAMIDE BASED INHIBITOR O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, Developed by Parke-Davis showing μ M potency for MMPs EnzymeIC 50 μM MMP-15.4 MMP MMP MMP-771 MMP-926 MMP Sulphonamide typeSuccinic acid type

32 Electron withdrawing group 4’  Increase activity DEVELOPMENT OF PG O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, R MMP (IC 50 μ M) H ’-F ’-Br ’-Cl ’-Br ’-F, 4’-Br ’-NH ’-CF Halogen at position 4’  Increased activityHalogen at position 3’  Decreased activityHalogen at position 2’  Decreased selectivityElectron donating group 4’  Decreased activity

33 DEVELOPING PG O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, MMP IC 50 μ Mt 1/2 (h) H R1R1 Improving the pharmacokinetic profile

34 PG O’Brien, P. M. et. al. J. Med. Chem. 2000, 43, S1’ pocket Schematic representation of crystal structure PG Developed by Parke-Davis EnzymeIC 50 μM MMP-16 MMP MMP-77.2 MMP MMP-97.9 Indicated that it could be used in left ventricular failure

35 SpeciesDoseEffects on MMP Cardiac effects Pigs20mg/kg/dayMMP-2 MMPs LV dilation LV peak wall stress LV load Rats5mg/Kg/dayMMP-2 MMP-9 LV dilation Contractility Thickness of fibrillar collagen Randomized trial were conducted for 90 days Study end points o LV end diastolic index o Ejection fraction Results o No Significant changes o Musculoskeletal syndrome Possible causes STUDIES CONDUCTED Kaludercic, N. et.al. Cardiovascular Therapeutics, 2008, 26, 24– Animal studies Studies in humans Dosing Regimen MMP Selectivity MMP-1

36 Ct Specificity loop Nt S1’S1’ THE S1’ SELECTIVITY POCKET Devel, L. et. al. Biochimie, doi: /j.bioci The depth of the S 1 ’ tunnel is determined by the S 1 ’ specificity loop Pocket differs for different MMP’s Overlap of the active site of major MMP classes MMP- 1 shallow pocketMMP- 13 Deep pocket PDB ID: 456C MMP-1, -7Shallow pocket MMP-2, -9Intermediate pocket MMP-8, -13Deep pocket PDB ID: 2TCL

37 36

38 SulfoneMMP- 1MMP-2MMP-3MMP-9MMP-13BA (%) α β α- Sulfoneβ- Sulfone α-TETRAHYDROPYRANYL SULFONES Becker, D. P. et. al. J. Med. Chem. 2010, 53, RS ( β- Sulfone) Developed by Roche 2 nd Generation sulfone α- Sulphone derivative *All IC 50 values are in units of nM Developed by Pfizer

39 MMP- 1 MMP- 2 MMP- 9 MMP-13BA % α-TETRAHYDROPYRANYL SULFONES Becker, D. P. et. al. J. Med. Chem. 2010, 53, α- Sulphone R All IC 50 values are in units of nM

40 α-PIPERIDINYL SULPHONES Becker, D. P. et. al. J. Med. Chem. 2010, 53, MMP- 1 MMP- 2 MM9- 9 MMP- 13 BA % Piperidine sulphones H R1R1 R2R2 All IC 50 values are in units of nM Selectivity Pharmacokinetics

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

42 ANIMAL STUDIES Becker, D. P. et. al. J. Med. Chem. 2010, 53, Inhibition of post infarction left ventricular dilation investigated in rat model LV volume (mL) LV pressure (mmHg) SHAM-Veh MI-VehMI-10mpkMI-50mpkMI-10mpkMI-50mpk AB α- Tetrahydropyranyl sulfoneα- Piperidine sulfone 50mg/kg VehicleLV vol. (mL) MI- Sham 0.55 MI-Veh0.69 A0.63 B0.62 A B

43 ANIMAL STUDIES Becker, D. P. et. al. J. Med. Chem. 2010, 53, LV volume (mL) LV pressure (mmHg) SHAM-VehMI-VehMI-0.01mpkMI-0.1mpkMI-1mpkMI-10mpk Dose:10mg/kg VehicleLV vol. (mL) MI- Sham 0.49 MI-Veh0.59 C0.51 Pharmacokinetic parameter 10mg/kg SpeciesMouseRatDogMonkey BA (%) C

44 Zn+ 2 His Thiirane inhibitors MECHANISM BASED INHIBITORS Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, Zn+ 2 His Interaction with Zn Covalently bound Zn Enzyme (E) Substrate (S) E:S complex E Product (P) k on k off k on k off E:I complex Inhibitor (I) K cat Rapid Slow

45 Enzymek on M -1.s -1 k off s -1 K i μM MMP-22.1 x x MMP-94.9 x x MMP x x MMP-21.9 x x MMP MMP MMP-21.2 x x MMP MMP THIIRANE TYPE INHIBITORS Ikejiri, M. et. al. J. Biol. Chem. 2005, 280, NO 2 B C A R

46 Gelatin zymography of SMC TETRACYCLINES Franco, C. et. al. Am. J. Pathol. 2006, 168, Doxycycline Weak inhibitors of MMPs Inhibits smooth muscle cell proliferation and migration Inhibits MMP-2 and MMP-9 Untreated SMC Doxycycline treated SMC MMP-9 proenzyme MMP-2 proenzyme MMP-2 active Doxycycline μ M

47 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 SUMMARY 46 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 MMPsInhibitors Future strategies 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 47 Dr. Umesh Desai The Desai group The Department of Medicinal Chemistry at VCU


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