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RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY

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1 MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM
RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY VIRGINIA COMMONWEALTH UNIVERSITY Date: 22nd October 2010

2 The Extracellular Matrix
THE COMMON PATH Cancer vv Skeletal disorders Cardiovascular disorders The Extracellular Matrix Arthritis CNS disorders If we have a look at a list of some of the major diseases. Certain diseases like cancer, arthritis , skeletal disorders, CVS and CNS disorders are sure to be to be mentioned. All these conditions are known to cause immense suffering and in some cases mortality in humans However, one similarity between these that could be used to our advantage is their relation to the ECM and the plethora of enzymes which are involved in the functioning and remodelling of the ECM. One such group of enzymes the MMP has been long known to have a role in each of these disease but recently its role especially in the field of CVS disorders in garnering a lot of interest. It is this role of the MMPs in the CVS and its implication that I’ll be talking about in detail. However to understand the role of MMPs in the CVS it is quite essential to understand the ECM and its functioning. Matrix metalloproteinase Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3,

3 EXTRACELLULAR MATRIX Collagen Laminin Integrins Elastin
Plasma membrane Laminin Integrins The extracellular matrix (ECM) is a complex structural entity surrounding and supporting cells Collagen :Insoluble, high tensile strength Laminin: Adhesion Integrins: receptors which establish cell- extra cellular environment contact Elastin: elastic properties in vasculature and lung Each of these are present in varying proportion which depend on the tissue they surround however in general as a group they perform a number of functions which include Elastin Rozario, T. Dev. Biol , 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 The most important function is its ability to maintain the characteristic structure of the tissue No longer considered static and the dynamic property seen through properties such as its action on migratory cell The ability to presents growth factor aswell as sensing and acting on the mechanical stimuli it is subjected to. A no. of enzymes are involved which include bone morphogenetic protein-1, adams, serine protease and MMPs However the MMPs are the focus of my seminar today Matrix metalloproteinases Rozario, T. Dev. Biol , 341, 126–140.

5 THE TADPOLE ENZYME 1962: Discovered by Jerome Gross and Charles Lapiere Anuran tadpole explants Placed on collagen gel The discovery of these enzyme was done other quite unconventional circumstances and hence I’ll just briefly describe the story MMPs were discovered in 1962 by Jerome gross and charles Lapiere Placed tadpole explants from the metamorphising organs of the gills, fins and tail on collagen gel and realised that a substance relaeased from these cleaved collagen . The called the substance the tadpole enzyme in their early papers Collagen degraded Gross, J. et. al. Proc. Natl. Acad. Sci. 1962, 48,

6 THE TADPOLE ENZYME Amount of collagen degraded Area lysed
Degradation of C14 collagen Microscopic studies Lysed collagen gel NH2 collagenase Cleavage of collagen triple helix COOH cleavage site NH2 COOH On further studying it, they checked the amount cleaved usually by measuring the amount of lysis and the C14 present aswellas checked the type of cleavage the enzyme was making on the triple helix structure of collagen which was normally cut at a point 1/3 rd from the C terminal PDB ID: 1CAG Gross, J. et. al. Proc. Natl. Acad. Sci. 1965, 54,

7 HISTORY 1970 1979 1984 1992 1993 Purification of human collagenase
Purification of TIMP-1 1984 Development of genomic clones 1992 Batimastat Phase I trial 1993 First crystal structure solved Following this important discovery the field of MMP has made a lot of progress Purified in 1970 and in 1979 an endogenous inhibitor was found which was purified and named the tissue inhibitor of MMP In 1984 development of genomic clones allowed further studies of the role of these enzymes and the therefore led to development of many inhibitors leading the path was Batimastat in1992 which moved into phase I trials in 1992 for cancer 1993 when the first crystal structure was solved further paved the path for MMP research So what are MMPs in general Brinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3,

8 MATRIX METALLOPROTEINASES
Belong to the metzincin group of proteases Synthesized as inactive precursors Degrade the extracellular matrix in a concerted manner which are named after the zinc ion and the conserved Met pdb id 1CK7 residue at the active site PDB ID: 1CK7 McCaw, A. et. al. Nat. Rev. Mol. Cell Biol., 2007, 8,

9 STRUCTURE Fibronectin type II domain Catalytic domain Pro domain
Hemopexin domain Pro domain MMPs consists of four major domains and these include Prodomain which holds the MMP in the inactive form Catalytic domain which is responsible for the activity of the enzyme And two ancilliary domains the fibronectin type II domain and the hemopexin domain Murphy, G. Mol. Aspects Med. 2008, 29, 290–308. PDB ID: 1GXD

10 CLASSIFICATION MMP -1, -8, -13 C N Collagenase MMP -7, -26 Matrilysins
Zn2+ Collagenase MMP -7, -26 Matrilysins C N Zn2+ Membrane type MT-MMP 1-8 N C Zn2+ Gelatinase MMP -2, -9 N C Zn2+ 1st collagenase General arrangement of the MMP include the Ndomain signal sequence the propeptide region , the cysteine switch region followed by the cataytic domain which also contains the zinc ion , then the hinge region, the hemopexin domain and the C terminal Other classes have the same arrangement the only minor changes with certain domains being included and certain being excluded 2nd matrilysin same arrangement, but lack the hinge and hemopexin domain 3rd Membrane type attached to the membrane as the name suggests, same arrangement with extr feature to aid attachment to the membrane TM domain and the cytosolic domain no. from 1 to 8 4th gelatinase same with a extra fibronectin domain MMP-2 and MMP-9 Catalytic domain Cysteine switch Propeptide Hinge region Hemopexin domain Tail Signal sequence N Transmembrane domain Cytosolic domain Furin domain Fibronectin repeat Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

11 MECHANISM OF ACTIVATION
PRCGXPD Cysteine switch peptide AHEXGHXXGXXH Catalytic site His Cys Zn2+ The catalytic site basically contains of preserved 3 histidine moiety which co-ordinates with the zinc ion these are a part of almost every MMP The cysteine switch peptide is part of the propeptide domain of the MMPs and is responsible for forming the fourth co-ordination bond with the zinc atom This is is basically seen in the proform Proenzyme catalytic domain Hu, J. Nat. Rev. Drug Discovery, 2007, 6, PDB ID: 1SLM

12 ACTIVATION OF THE PROENZYME
Active form His Zn2+ SH Pro SH Pro His Zn2+ His Zn+2 SH Pro Stepwise activation Activation by MT-MMP 1st step cleavage of the Zn Cysteine coordination bond 2nd step involves formation of an intermediate 3rd step involves replacement by water molecule which forms a H bond with the nearby glutamate residue activates a zincbound H2O molecule providing the nucleophile that cleaves peptide bonds. The cysteine-thiol and zinc ion interaction keeps proMMPs in a latent state. by three mechanisms: 1)direct cleavage of the pro-domain by another proteinase; 1) reduction of the free thiol by oxidants or by nonphysiologic reagents such as alkylating agents, heavy metal ions, and disulfides and 3) allosteric perturbation of zymogen Thiol reduction and allosteric controls would lead to Inter or intramolecular autolytic cleavage of the prodomain Once activated these perform a no. of function which include……… Chemical activation Proenzyme Intermediate 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 Mesenchymal cells MMP Epithelial cells MMP Degradation of basement membrane Proliferation Cell death Cell motility Mesenchymal cell MMP Clears the path helping the migration of the cells during ECM remodelling And doing so these can also activate other factor which could have an effect in the ECM remodelling process Also involved in degradation of the basement membrane And the has an effect on the fate of the cell through the activation of latent signals which may either cause cell proliferation, motility or cell death Similarly also affects active signals which eventually have an effect on the fate of the cells All these properties and functions that are performed by the MMP made it probable that these set of enzymes could play a important role in vital systems like the cardiovascular system by shaping or remodeling its ECM. McCaw, A. et. al. Nat. Rev. Mol. Cell Biol. 2007, 8,

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 Validating the role of MMPs in the cardiovascular system On checking the different cells of the CVS and their ECM content It was found that of the 7 different types of cells the the fibroblasts and the Mast cells were responsible for direct MMP production which could inturn have a effect on the ECM of each of they other cell types To further understand its role in the cardiovascular system a no. of common CVS pathologies were studied, these included…….. This is based on histopathological findings such as the activation of foamy macrophages, the local production of cytokines and chemokines, and the involvement of MMPs The use of animal models with genetically altered mice(both transgenic and knockout mice) has only strengthened the view that MMPs are key players in vascular pathologies Mast cells/Leukocytes/ Macrophages Cytokines Growth factors MMPs Bowers, S. L. K. et.al. J. Mol. Cell. Cardiol. 2010, 48,

15 CONDITIONS INVOLVED Aneurysms Myocardial infarction MMP-2 and MMP-9
Monocyte infiltration MMP -2,-9 Elastin degradation Plaque rupture MMP -2, -9 Smooth muscle cell migration MMP -3,9,12 Foam cell Myocardial infarction MMP-2 and MMP-9 Artery Aneurysms Aneurysm Now aneurysm are basically a dilation of the aorta due to weakening of the walls of the blood vessel And checking the MMPs involved in these cases, MMPs 2 and 9 were found to be present in higher amounts suggesting that these may be involved Another condition that was checked was the atherosclerotic plaques where the MMPs were found to be involved in various steps of the plaque progression such as……. Similarly myocardial infarction was also checked and it was seen that especially post myocardial infarction ie the time when most of the left ventricular remodelling takes place the content of MMP-2 and 9 increases drastically indicating a role played by these MMPs in the remodelling However , though each of these conditions showed the presence of a particular group of MMPs it was necessary to check which MMPs where actually having a negative effect and the ones having a positive one Therefore studies were conducted first in knockout mice………. MMP-2 and MMP-9 Atherosclerotic plaque Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

16 ROLE IN THE CVS GENE DELETION OVEREXPRESSION MMP-9
Reduced LV dilation and inflammation post-MI - MMP-2 Reduce LV dilation and rupture rate post MI Reduced LV hypertrophy Severe LV contractile dysfunction Dilated cardiomyopathy MMP-3 Defects in cell proliferation and cytokine release Impaired myocardial scar maturation MT1-MMP Decreased connective tissue malformations Which showed that deletion of MMP-9 and -2 decreased LV dilation and inflammation post-MI As well as reduced LV hypertrophy in MMP-2 knockout mice. MMP-3 deletion indicated defects in cell proliferation and impaired myocardial scar maturation Whereas MT-MMP1 was involved in connnective tissue malformation To further confirm these finding overexpression studies were conducted which indicated that overexpression MMP-2 produced totally opposite effects of severe LV contractile dysfunction and cardiomyopathy However, in general MMP-2 and MMP-9 were deemed essential in the CVS This also implicated the need for inhibitors of these enzymes which could therefore modulate the remodelling process controlled by these enzymes. And therefore the need for MMP inhibitors Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803, 55–71.

17 TARGETING MMPS Endogenous inhibitors Synthetic inhibitors PDB ID: 1SLM
Now endogenous inhibitors served as a basis for why the MMP inhibitor therapy in CVS could be a sucessful one however before I get to that I will just briefly decribe these endogenous inihibitors 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 These were basically called the Tissue inhibitor of MMP In general these have two distinct domains The N-terminal and the C-terminal domain out of which the N-terminal domain is important for MMP inhibition These are grouped in a class of 4 enzymes And are known to be broad spectrum inhibitors of various MMPs and bind in a 1:1 stoichiometric ratio So coming to the mode of binding and the peptides involved making important interactions with the enzymes N-Domain C-Domain PDB ID: 1BR9 Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803,

19 INHIBITION MECHANISM TIMP Active site Cys 1 to Val 4 Primed subsites
Ser68 Glu67 Val69 Cys3 Val4 S3 S1’ S3’ Zn2+ Thr2 His Cys Zn2+ TIMP Active site Cys 1 to Val 4 Primed subsites Glu 67 to Cys 70 Unprimed subsites The most important contact is the replacement of the Cys of the prodomain by a Cys belonging to the TIMP Other important contacts involve the ones made by two set of peptides the Cys1 to Pro-5 and the Glu-67 to Cys-70 which essentially interact with the various subsites of MMPs And now coming back to why inhibitors could be important in CVS ……... PDB ID: 1UEA Brew, K. et. al. Biochim. Biophys. Acta, 2010, 1803,

20 TIMP: ROLE IN THE CVS GENE KNOCKOUT OVEREXPRESSION TIMP-1
Greater LV dilation and matrix loss post-MI LV systolic dysfunction post-MI Reduced cardiac rupture post MI Improved LV systolic function TIMP-2 Aneurysm development Involved in ECM TIMP-3 Greater LV dilation Increased cytokine processing Increased MMP-2 activation in fibroblasts Decreased activation of pro-MMP-2 TIMP-4 No phenotype N.A Knockout studies in mice revealed that TIMP-1 deletion Increased LV dilation and dysfunction following myocardial infarction TIMP-2 deletion enhanced susceptibility to aortic aneurysm Whereas TIMP-3 deletion produced cardiac Myocardial LV dysfunction and increased MMP-2 activation TIMP-4/ Unknown Overexpression studies produced the opposite effects like……reduced cardiac rupture Therefore confirming that the inhibitor therapy could have a beneficial role in the CVS and……….. Chow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.

21 TARGETING MMPS Endogenous inhibitors Synthetic inhibitors PDB ID: 1SLM
Hence were created, the synthetic inhibitors Now the MMPs had a long way to go before they were actually used as inhibitors for cardiovascular system and hence I’ll talking about the development of this class over the years starting with the first generation agents PDB ID: 1SLM

22 THE FIRST GENERATION

23 STRUCTURAL BASIS FOR INHIBITION ZBG : Zinc binding group
Zn2+ S2 S1’ S3’ S2’ S1 S3 Collagen type peptide inhibitors P3 P2 P1 P1’ P3’ P2’ ZBG General inhibitors requirements P3 P2 P1 ZBG Left side Inhibitors These were basically design to mimic the collagen substrate and therefore checking the interaction made by the substrate and according to the Schechter and Berger nomenclature of proteases the subsites on the left of the scissile amide of the substrate are known as the primed subsites and those on the right the unprimed subsites. Therefore general requirements would be the presence of a group to replace the scissile amide bond and substituents to bind to the primed and the unprimed subsites However, binding to all the subsites is not essential for activity and therefore the concept of left side inhibitors which bind to the unprimed sites and the right sided inhibitors which bind to the primed subsites Therefore looking it to the early inhibitors, P1’ P3’ P2’ ZBG Right side Inhibitors ZBG : Zinc binding group Dorman, G. et. al. Drugs, 2010, 70, 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 It could easily be summarized that the isobutyl group was essential at positions P1’ and the P2’ position The methyl group at P3’ position however the most important was the ZBG which was going to replace the scissile amide bound a no. these ZBG groups were tried at this position and these included Brown, P. D. Medical Oncology, 1997, 14, I- I0.

25 THE ZINC BINDING GROUP Hydroxamates Thiol Phosphinates Carboxylates
The Hydroxamates, thiol , phoshinates, carboxylates so how do these function taking the example of the hydroxamtes out of which the hydroxamates where the most potent as ZBG Now the basic mechanism by which these acted involves Carboxylates Hu, J. Nat. Rev. Drug Discov. 2007, 6,

26 MECHANISM OF ZBG Active enzyme Enzyme-hydroxamate
…….. a activated enzyme in which the cys residue is replaced by a water molecule which forms a H bond with a Glutamate residue which makes the OH behave as a nucleophile which could cleave a substrate However in the presence of a ZBG for example Hydroxamate would break such an interaction and therefore inhibit the protein cleaving property of the MMP And hence based on these basic requirements …………… Active enzyme Enzyme-hydroxamate Hu, J. Nat. Rev. Drug Discovery, 2007, 6,

27 BROAD SPECTRUM HYDROXAMATES
The earliest MMP inhibitors Many members of this class entered clinical trials Ilomastat Batimastat MMP-1 = 0.4 MMP-2 = 0.39 MMP-3 = 26 MMP-8 = 0.18 MMP-9 = 0.57 Marimastat MMP-1= 5 MMP-2 = 6 MMP-3 = 200 MMP-7 = 20 MMP-9 = 3 ……A no of inhibitors especially the ones containing hydroxamate were found to be quite active as anticancer agents these included ilomastat and batimastat which were the earliest agents but the lack of pharmacokinetic profile prompted the need of agents like marimastat which showed good broad spectrum activity but nevertheless none of these progressed in clinical trials basically due to the development of a no. of side effects which thought to be due to inhibition of other closely related agents like the ADAMS This made researchers to look in to the need for selectivity in such agents and therefore was born the concept of the second generation of MMP inhibitors MMP-1=10 MMP-2 = 4 MMP-3 = 20 MMP-8 = 10 MMP-9 = 1 *All IC50 values are in units of nM Skiles, J. W. et. al. Curr. Med. Chem. 2004, 11,

28 THE SECOND GENERATION The second generation of MMP inhibitors were basically the result of extensive SAR studies which tried to establish the necessities of a good inhibitor and hence to summarize the sar study

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 To summarize the SAR study the following effects were seen on changing the substituents at each of the positions The zBG was of prime importance The alpha substituent was considered essential interms of maintaining the PK profile of the drug The P2 did not have a large effect on potency similarly the P3 position was also not essential for activity The P1 position was the most essential for selectivity towards certain groups and Hence due to the established importance of only the P1 and the alpha substituent only the succinate backbone was considered essential for activity This ring was modified to reduce the size of the inhibitors and hence the basic protein backbone was modified to a number of other systems which included….. α substituent Improves Pharmacokinetic properties Zinc binding group Essential for activity Hu, J. Nat. Rev. Drug Discov. 2007, 6,

30 MODIFICATIONS OF THE BACKBONE
Malonic acid type Glutaric acid type Sulphonamide type Malonic , glutaric, sulfonamide and the sulfone type……however 2 backbones the sulfonamide and the sulfones were successful in producing good potency However , I’ll be talking about one such sulfonamide in detail which was developed by Parke-Davis…….. Sulphone type Hu, J. Nat. Rev. Drug Discov. 2007, 6,

31 SULPHONAMIDE BASED INHIBITOR
Sulphonamide type Succinic acid type Developed by Parke-Davis showing μM potency for MMPs Enzyme IC50 μM MMP-1 5.4 MMP-2 0.040 MMP-3 0.038 MMP-7 71 MMP-9 26 MMP-13 0.062 It all started with them finding out that in their group of sulfonamide containing moiety the sulfonamide portion of their library of MMP inhibitors had good uM potency and similarly in the Succinic acid type groups the one with the biphenyl group had shown uM potency and therefore combining these two moieties they developed a biphenyl sulfonamide type moiety which showed nanomolar selectivity for selective MMPs……..therefore to further develop more potent agents a no. of modification were made and included….. O’Brien, P. M. et. al. J. Med. Chem. 2000, 43,

32 DEVELOPMENT OF PG Halogen at position 2’  Decreased selectivity Electron donating group 4’  Decreased activity Halogen at position 3’  Decreased activity Halogen at position 4’  Increased activity Electron withdrawing group 4’  Increase activity R MMP (IC50 μM) 1 2 3 7 9 13 H 5.4 0.04 0.038 71 26 0.062 4’-F 4.2 0.039 0.010 4.8 64 0.043 4’-Br 6.0 0.004 0.007 7.2 7.9 0.008 4’-Cl 6.5 0.011 0.009 7.5 16 0.048 3’-Br 100 0.535 0.290 0.710 2’-F, 4’-Br 3.6 0.005 0.016 2.1 4.9 4’-NH2 0.036 31 20 0.105 4’-CF3 0.013 0.023 First modifying the 4’ position of the biphenyl ring 4-X group in general increased the potency of these inhibitors 3-X group however was found to decrease activity for all MMPs 2-X and 4-x increased the potency to all MMPs but was found to decrease the selectivity EDG at the 4- position in general decreased activity, whereas ewg at 4-position increased activity However among all the substitution the one containg the Br was selected for further advancement due to it having a good potency and especially a good profile for MMP-2 and MMP-9 Next they tried to improve the pharmacokinetics of the compound O’Brien, P. M. et. al. J. Med. Chem. 2000, 43,

33 Improving the pharmacokinetic profile
DEVELOPING PG R1 H Improving the pharmacokinetic profile MMP IC50 μM t1/2 (h) 1 2 3 7 9 13 50 0.004 0.010 77 6.7 0.026 15.6 0.012 11 9.5 25.1 6 0.007 7.2 7.9 0.008 43.6 0.009 3.1 4.9 0.005 41.8 27 0.002 15 2.3 3.88 Which they started By first placing a methyl group which increased the t1/2 of the drug increased to 25.1 h as compared to the unsubstituted one Next isopropyl group also further increased the t ½ Similarly the iso butyl moiety However the ethyl sulfonyl methyl benzene group was found to decrease t 1/2 Hence the isobutyl group was selected since it had the best potency and pk profiles and hence was developed……… O’Brien, P. M. et. al. J. Med. Chem. 2000, 43,

34 PG-116800 Developed by Parke-Davis S1’ pocket PG-116800 Enzyme IC50 μM
MMP-1 6 MMP-3 0.007 MMP-7 7.2 MMP-2 0.004 MMP-9 7.9 PG-11680 Looking at its crystal structure indicated that the biphenyl group was basically interacting with the S1’ pocket whereas the sulfonamide group developed interaction with ………….. AA and the Zinc binding group interacted with the zinc A look at the inhibitory profile indicated good activity for MMP-2 and MMP-9 prompting further animal studies for this compound in LV failure and hence commensed the animal studies ….. 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,

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 Species Dose Effects on MMP Cardiac effects Pigs 20mg/kg/day MMP-2 MMPs LV dilation LV peak wall stress LV load Rats 5mg/Kg/day MMP-9 Contractility Thickness of fibrillar collagen Which indicated beneficial effects like decrease LV dilation etc And this therefore validated human trials Human trial were conducted for 90days as a randomised trial with LV end diastolic index and the ejection fraction acting as the study endpoints However results indicated that the drug had no significant effect and that it produce side effect which was basically thought to be due to two major reason Improper dosing regimen and lack of MMP-1 selectivity The MMP selectivity was a structural problems and hence this got the researchers thinking about ways to improve the selectivity among MMPs especially to produce MMP-1 sparing effect ….. Dosing Regimen MMP Selectivity MMP-1 Kaludercic, N. et.al. Cardiovascular Therapeutics, 2008, 26, 24–37.

36 THE S1’ SELECTIVITY POCKET
Specificity loop Nt S1’ The depth of the S1’ tunnel is determined by the S1’ specificity loop Pocket differs for different MMP’s MMP-1, -7 Shallow pocket MMP-2, -9 Intermediate pocket MMP-8, -13 Deep pocket Around this time the crystal structures of a no of MMPs were known and hence on overlapping the major classes it was found that the only region that different among the MMP was the S1’ site which was named the selectivity pocket Further analysing the S1’ selectivity pocket of various MMP it was seen that S1 loop determined the depth of the pocket and the MMP s could be grouped in to 3 categories The Narrow 1 and 7 Intermediate 2 and 9 Deep 13 and 8 As an eg this structure indicates the difference between the shallow pocket of MMP-1 and the deep pocket of MMP-13 This therefore laid the foundation for the 3rd generation of agents MMP- 1 shallow pocket MMP- 13 Deep pocket PDB ID: 2TCL PDB ID: 456C Overlap of the active site of major MMP classes Devel, L. et. al. Biochimie, doi: /j.bioci

37 THE THIRD GENERATION which were designed with MMP-1 sparing activity in mind…one such agent was recently developed ……

38 α-TETRAHYDROPYRANYL SULFONES
2nd Generation sulfone RS (β-Sulfone) Developed by Roche α- Sulphone derivative Sulfone MMP- 1 MMP-2 MMP-3 MMP-9 MMP-13 BA (%) α 435 0.1 18.1 0.3 0.015 45.8 β 800 0.4 17.5 1 0.6 21.2 Based on the 2nd generation sulfone backbone which were potent broad spectrum agents and taking advantage of the S1’ selectivity pocket Roche developed a no of beta sulfones initially as anti athritic agents with MMP-1 sparing activity , however these compound lacked good Pk properties Using the same basic principle recently Pfizer developed an alpha sulfone which in addition to increased potency to MMP-2 and MMP-9 also showed excellent MMP-1 sparing properties as seen by this comparision With increased potency this change also conferred increased BA Therefore with a view to improve the MMP-1 sparing property even further, a no. of modification were made at the R position and these included Developed by Pfizer *All IC50 values are in units of nM Becker, D. P. et. al. J. Med. Chem. 2010, 53,

39 α-TETRAHYDROPYRANYL SULFONES
α- Sulphone R MMP-1 MMP-2 MMP-9 MMP-13 BA % 268 0.1 0.4 5.8 1800 0.3 2.9 0.45 - 435 0.15 45.8 400 0.2 1140 35.9 8000 1.2 0.8 33 5000 0.6 1 42.3 A group of alpha sulfones were prepared based on the principle of varying the R group thought to be interacting with the P1’ specificity pocket , A no. of groups were tried which included The unsubstituted phenyl group which showed good potency but low potency and BA Substituting the 3 or the meta position with a Cl group decreased potency A Cl group at the 4-position increased both activity and BA but the group was interested in achieving a 10,000 fold selectivity over MMP-1. Placing a OH grp at 4- position again produced concerns of the decreased selectivity Next a –OCF3 group was place which achieved both activity and selectivity aswellas had a good BA profile Replacing with a –CF3 group also improved potency with an even better selectivity profile Looking at both the OCF3 and CF3 substitution showed good potency but the improved selectivity led to the –CF3 substituted compound to be selected for further clinical trials similarly the piperidine were also developed All IC50 values are in units of nM Becker, D. P. et. al. J. Med. Chem. 2010, 53,

40 α-PIPERIDINYL SULPHONES
Selectivity Pharmacokinetics Piperidine sulphones MMP-1 MMP-2 MM9-9 MMP-13 BA % 10000 1.7 4 1.2 12.4 0.3 1.9 5.5 48.3 0.1 2.3 0.2 36.3 6000 0.5 16.6 4000 0.15 23 0.18 67.9 Similarly the alpha piperidines were also prepared these were substituted at two site The R2 position was the one which was going to interact with the S1 selectivity site and was therefore going to decide the selectivity of the molecule The R1 position was a part of the piperidine N and was basically placed to confer pk properties to the molecules Following the same successful R2 substituent as the previous tetahydropyranyl group ie the phenyl with a 4-CF3 First the P1’ substituent was replaced by p-CF3, whereas the R1 was left unsubstituted This produced good selectivity , pK needed improvement hence the next few involve substitution at the R1 position Keeping the R2 unchanged, first a isopropyl group was placed which improved Pk properties drastically indicating this position indeed has a effect on the pk properties but attenuated the activity for MMP-13 Next a cyclopropane ring was placed to improve activity which retained high potency and had a respectable BA Now keeping the cyclopropane ring unchanged a no of substitution were made at the R2 position , The 4-OCF3 position was found to increase selectivity against MMP-1 by about 10-fold and improved selectivity for MMP-9 aswell Replacing the cyclopropane ring by a methoxy ethyl ring not only increased activity but also improved the pK properties and hence was selected for further animal studies along with compound containing the cyclopropane ring as a back up All IC50 values are in units of nM Becker, D. P. et. al. J. Med. Chem. 2010, 53,

41 STRUCTURAL SELECTIVITY
Arg214 Leu218 A look at their crystal structures indicated that the substituted phenylphenoxy ring was interacted with the s1’ pocket and overlaying the structure of MMP-2 and MMP-13 and MMP-1 indicated that the MMP-1 had ARG at a position corresponding to the leu in MMP-13 and the conformation of this Arg was the reason for the sparing and lack of activity of MMP-1 since it ltd the size of the MMp-1 pocket to a shallow one Hence preventing MMP-1 inhibitory activity……. Further animal studies were therefore conducted on three of their compound so far and this indicated………. Crystal structure of α-Piperidine sulfone MMP-13, MMP-1 S1’overlap Becker, D. P. et. al. J. Med. Chem. 2010, 53,

42 ANIMAL STUDIES Inhibition of post infarction left ventricular dilation investigated in rat model LV volume (mL) LV pressure (mmHg) SHAM-Veh MI-Veh MI-10mpk MI-50mpk A B 50mg/kg Vehicle LV vol. (mL) MI-Sham 0.55 MI-Veh 0.69 A 0.63 B 0.62 A B α- Tetrahydropyranyl sulfone α- Piperidine sulfone For A which is THp and B which is piperidinyl Inhibition of post MI LV dilation in a rat model indicated significant decrease in LV volume for both A and B as compared to control Similar test conducted for C……. Becker, D. P. et. al. J. Med. Chem. 2010, 53,

43 Pharmacokinetic parameter 10mg/kg
ANIMAL STUDIES Pharmacokinetic parameter 10mg/kg Species Mouse Rat Dog Monkey BA (%) 51.7 66.6 64 53.8 C Dose:10mg/kg Vehicle LV vol. (mL) MI-Sham 0.49 MI-Veh 0.59 C 0.51 LV volume (mL) LV pressure (mmHg) SHAM-Veh MI-Veh MI-0.01mpk MI-0.1mpk MI-1mpk MI-10mpk Which is a piperidinyl showed significant changes compared to control as well as this agent showed a good PK profile in a no of species tested…… Another agent that has been recently developed as selective MMP-2 and MMP-9 inhibitors are………… Becker, D. P. et. al. J. Med. Chem. 2010, 53,

44 MECHANISM BASED INHIBITORS
kon Rapid + koff Slow E:I complex Enzyme (E) Inhibitor (I) Zn+2 His + Substrate (S) Interaction with Zn Covalently bound Zn Thiirane inhibitors E:S complex Zn+2 His The mechanism based inihibitors….Normal enzyme reaction involve reaction of a enzyme with a substrate which result in the formation of an enzyme substrate complex followed by the formation of the product and the release of the enzyme In the presence of an inhibitor the enzyme forms a tight complex with the inhibitor which prevents the formation of a product or the release of the enzyme MBI involve inhibitors which from a strong covalent bonds thus having a rapid Kon time and a slower koff time there by inhibiting the enzyme for longer period of time. The sulphur containing thirane group is a similar type of inhibitor which covalently binds to the Zn atom and there produces a faster kon rate however koff rate drastically decreases resulting in longer inihibition of the enzymes On taking a closer look at the profile invoved…… kon koff Kcat + E Product (P) Ikejiri, M. et. al. J. Biol. Chem. 2005, 280,

45 THIIRANE TYPE INHIBITORS
Enzyme kon M-1.s-1 koff s-1 Ki μM MMP-2 2.1 x 104 3.5 x 10-4 0.016 MMP-9 4.9 x 103 9.0 x 10-4 0.18 MMP-14 6.9 x 102 6.4 x 10-4 0.9 1.9 x 103 1.3 x 10-3 0.7 - 1.0 4.9 1.2 x 104 0.11 0.13 0.68 1 2 3 A NO2 B The parent compound showed a rapid kon rates for MMP-2 , 9 and 14 but very slow koff rates for MMP-2 -9 and 14 Modification at the R position resulted loss of this effect on MMP9 and 14 but this effect continued for MMP-2 Similarly substitution with a N-methyl amide had the same effect These agents could therefore have some potential for CVS disorders Another agents which is could be called a quite atypical agents is………… C Ikejiri, M. et. al. J. Biol. Chem. 2005, 280,

46 TETRACYCLINES Weak inhibitors of MMPs
Inhibits smooth muscle cell proliferation and migration Inhibits MMP-2 and MMP-9 Doxycycline Doxycycline treated SMC Untreated SMC 42 416 104 208 MMP-9 proenzyme MMP-2 proenzyme MMP-2 active Doxycycline μM Tetracylines are a quite exceptional case of MMP inhibitors . Whose MMP inihibiting potential was first found from their use in periodontal disease Where they are known to inhibit MMP at subantimicrobial doses with out any of the side effects seen with other MMP inhibitors and therefore could be used Recent studies also indicate that these inhibit smc proliferation and migration And inhibit MMP-2 and MMP-9 present in smc in a dose dependent manner however the MOA is not Known and should be looked in to Gelatin zymography of SMC Franco, C. et. al. Am. J. Pathol. 2006, 168,

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 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 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 Physiological and pathological roles of MMP are vast since it is spread out throughout the body’s ECM and therefore each of these may have diverse roles in different tissues Hence this makes it necessary to understand the role of each of these play in both normal physiological processes and in pathological conditions Understanding the modulators involved could also give us an insight at understanding these roles A look at the inhibitors indicate the development of MMPs from broad spectrum inhibitors to selective inhibitors of the MMP involved in the CVS And through the development of inhibitors the importance of selectivity and approaches with which these could be achieved were also realised The most important of which included understanding the various intricacies of the binding site In addition to this other approaches such as targeting a modulator such emmprin could also be seen as strategy to attain selectivity

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


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