Presentation on theme: "RIO BOOTHELLO DEPARTMENT OF MEDICINAL CHEMISTRY"— Presentation transcript:
1 MATRIX METALLOPROTEINASES: ITS IMPLICATIONS IN THE CARDIOVASCULAR SYSTEM RIO BOOTHELLODEPARTMENT OF MEDICINAL CHEMISTRYVIRGINIA COMMONWEALTH UNIVERSITYDate: 22nd October 2010
2 The Extracellular Matrix THE COMMON PATHCancervvSkeletal disordersCardiovascular disordersThe Extracellular MatrixArthritisCNS disordersIf 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 diseasebut 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 metalloproteinaseBrinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3,
3 EXTRACELLULAR MATRIX Collagen Laminin Integrins Elastin Plasma membraneLamininIntegrinsThe extracellular matrix (ECM) is a complex structural entity surrounding and supporting cellsCollagen :Insoluble, high tensile strengthLaminin: AdhesionIntegrins: receptors which establish cell- extra cellular environment contactElastin: elastic properties in vasculature and lungEach 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 includeElastinRozario, T. Dev. Biol , 341, 126–140.
4 FUNCTIONS Enzymes involved in ECM remodelling Provides structure Tracks migratory cellsPresents growth factors to receptorsSenses/transduces mechanical signalsBone morphogenetic protein 1ADAMSSerine proteasesThe most important function is its ability to maintain the characteristic structure of the tissueNo longer considered static and the dynamic property seen through properties such as its action on migratory cellThe 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 MMPsHowever the MMPs are the focus of my seminar todayMatrix metalloproteinasesRozario, T. Dev. Biol , 341, 126–140.
5 THE TADPOLE ENZYME1962: Discovered by Jerome Gross and Charles LapiereAnuran tadpole explantsPlaced on collagen gelThe discovery of these enzyme was done other quite unconventional circumstances and hence I’ll just briefly describe the storyMMPs were discovered in 1962 by Jerome gross and charles LapierePlaced 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 papersCollagen degradedGross, J. et. al. Proc. Natl. Acad. Sci. 1962, 48,
6 THE TADPOLE ENZYME Amount of collagen degraded Area lysed Degradation of C14 collagenMicroscopic studiesLysed collagen gelNH2collagenaseCleavage of collagen triple helixCOOHcleavage siteNH2COOHOn 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 terminalPDB ID: 1CAGGross, J. et. al. Proc. Natl. Acad. Sci. 1965, 54,
7 HISTORY 1970 1979 1984 1992 1993 Purification of human collagenase Purification of TIMP-11984Development of genomic clones1992Batimastat Phase I trial1993First crystal structure solvedFollowing this important discovery the field of MMP has made a lot of progressPurified in 1970 and in 1979 an endogenous inhibitor was found which was purified and named the tissue inhibitor of MMPIn 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 cancer1993 when the first crystal structure was solved further paved the path for MMP researchSo what are MMPs in generalBrinckerhoff, C. E. et. al. Nat. Rev. Mol. Cell Biol. 2002, 3,
8 MATRIX METALLOPROTEINASES Belong to the metzincin group of proteasesSynthesized as inactive precursorsDegrade the extracellular matrix in a concerted mannerwhich are named after the zinc ion and the conserved Met pdb id 1CK7residue at the active sitePDB ID: 1CK7McCaw, A. et. al. Nat. Rev. Mol. Cell Biol., 2007, 8,
9 STRUCTURE Fibronectin type II domain Catalytic domain Pro domain Hemopexin domainPro domainMMPs consists of four major domains and these includeProdomain which holds the MMP in the inactive formCatalytic domain which is responsible for the activity of the enzymeAnd two ancilliary domains the fibronectin type II domain and the hemopexin domainMurphy, 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+CollagenaseMMP -7, -26MatrilysinsCNZn2+Membrane typeMT-MMP 1-8NCZn2+GelatinaseMMP -2, -9NCZn2+1st collagenaseGeneral 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 terminalOther classes have the same arrangement the only minor changes with certain domains being included and certain being excluded2nd matrilysin same arrangement, but lack the hinge and hemopexin domain3rd 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 84th gelatinase same with a extra fibronectin domain MMP-2 and MMP-9Catalytic domainCysteine switchPropeptideHinge regionHemopexin domainTailSignal sequenceNTransmembrane domainCytosolic domainFurin domainFibronectin repeatChow, A. K. et. al. Brit. J. Pharmacol. 2007, 152, 189–205.
11 MECHANISM OF ACTIVATION PRCGXPDCysteine switch peptideAHEXGHXXGXXHCatalytic siteHisCysZn2+The catalytic site basically contains of preserved 3 histidine moiety which co-ordinates with the zinc ion these are a part of almost every MMPThe 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 atomThis is is basically seen in the proformProenzyme catalytic domainHu, J. Nat. Rev. Drug Discovery, 2007, 6, PDB ID: 1SLM
12 ACTIVATION OF THE PROENZYME Active formHisZn2+SHProSHProHisZn2+HisZn+2SHProStepwise activationActivation by MT-MMP1st step cleavage of the Zn Cysteine coordination bond2nd step involves formation of an intermediate3rd step involves replacement by water molecule which forms a H bond with the nearby glutamate residue activates a zincboundH2O 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 nonphysiologicreagents 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 prodomainOnce activated these perform a no. of function which include………Chemical activationProenzymeIntermediateRa, 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 moleculesDegradation of basement membraneActivation of latent signalMMPMesenchymal cellsMMPEpithelial cellsMMPDegradation of basement membraneProliferationCell deathCell motilityMesenchymal cellMMPClears the path helping the migration of the cells during ECM remodellingAnd doing so these can also activate other factor which could have an effect in the ECM remodelling processAlso involved in degradation of the basement membraneAnd 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 deathSimilarly also affects active signals which eventually have an effect on the fate of the cellsAll 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 MyocytesCollagen IVCollagen VILamininProteoglycansEndothelial CellsCollagen IVLamininFibronectinVascular SmoothMuscle cellsCollagen ICollagen IIICollagen IVLamininFibronectinFibroblastsCollagen ICollagen IIIPeriostinFibronectinMMPsValidating the role of MMPs in the cardiovascular systemOn checking the different cells of the CVS and their ECM contentIt 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 typesTo 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 onlystrengthened the view that MMPs are key players in vascular pathologiesMast cells/Leukocytes/MacrophagesCytokinesGrowth factorsMMPsBowers, S. L. K. et.al. J. Mol. Cell. Cardiol. 2010, 48,
15 CONDITIONS INVOLVED Aneurysms Myocardial infarction MMP-2 and MMP-9 Monocyte infiltrationMMP -2,-9Elastin degradationPlaque ruptureMMP -2, -9Smooth muscle cell migrationMMP -3,9,12Foam cellMyocardial infarctionMMP-2 and MMP-9ArteryAneurysmsAneurysmNow aneurysm are basically a dilation of the aorta due to weakening of the walls of the blood vesselAnd checking the MMPs involved in these cases, MMPs 2 and 9 were found to be present in higher amounts suggesting that these may be involvedAnother 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 remodellingHowever , 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 oneTherefore studies were conducted first in knockout mice……….MMP-2 and MMP-9Atherosclerotic plaqueChow, 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-2Reduce LV dilation and rupture rate post MIReduced LV hypertrophySevere LV contractile dysfunctionDilated cardiomyopathyMMP-3Defects in cell proliferation and cytokine releaseImpaired myocardial scar maturationMT1-MMPDecreased connective tissue malformationsWhich showed that deletion of MMP-9 and -2 decreased LV dilation and inflammation post-MIAs well as reduced LV hypertrophy in MMP-2 knockout mice.MMP-3 deletion indicated defects in cell proliferation and impaired myocardial scar maturationWhereas MT-MMP1 was involved in connnective tissue malformationTo further confirm these finding overexpression studies were conducted which indicated that overexpression MMP-2 produced totally opposite effects of severe LV contractile dysfunction and cardiomyopathyHowever, in general MMP-2 and MMP-9 were deemed essential in the CVSThis 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 inhibitorsBrew, 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 inihibitorsPDB ID: 1SLM
18 ENDOGENOUS INHIBITORS The tissue inhibitors of metalloproteinasesTwo distinct domainsN-terminal domainC-terminal domainFour major typesTIMP 1- 4Broad spectrum inhibitorsBind in a 1:1 stoichiometric ratioThese were basically called the Tissue inhibitor of MMPIn general these have two distinct domainsThe N-terminal and the C-terminal domain out of which the N-terminal domain is important for MMP inhibitionThese are grouped in a class of 4 enzymesAnd are known to be broad spectrum inhibitors of various MMPs and bind in a 1:1 stoichiometric ratioSo coming to the mode of binding and the peptides involved making important interactions with the enzymesN-DomainC-DomainPDB ID: 1BR9Brew, K. et. al. Biochim. Biophys. Acta 2010, 1803,
19 INHIBITION MECHANISM TIMP Active site Cys 1 to Val 4 Primed subsites Ser68Glu67Val69Cys3Val4S3S1’S3’Zn2+Thr2HisCysZn2+TIMPActive siteCys 1 to Val 4Primed subsitesGlu 67 to Cys 70Unprimed subsitesThe most important contact is the replacement of the Cys of the prodomain by a Cys belonging to the TIMPOther 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 MMPsAnd now coming back to why inhibitors could be important in CVS ……...PDB ID: 1UEABrew, 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-MILV systolic dysfunction post-MIReduced cardiac rupture post MIImproved LV systolic functionTIMP-2Aneurysm developmentInvolved in ECMTIMP-3Greater LV dilationIncreased cytokine processingIncreased MMP-2 activation in fibroblastsDecreased activation of pro-MMP-2TIMP-4No phenotypeN.AKnockout studies in mice revealed thatTIMP-1 deletionIncreased LV dilation and dysfunction following myocardial infarctionTIMP-2 deletion enhanced susceptibility to aortic aneurysmWhereas TIMP-3 deletion produced cardiac Myocardial LV dysfunction and increased MMP-2 activationTIMP-4/ UnknownOverexpression studies produced the opposite effects like……reduced cardiac ruptureTherefore 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 inhibitorsNow 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 agentsPDB ID: 1SLM
23 STRUCTURAL BASIS FOR INHIBITION ZBG : Zinc binding group Zn2+S2S1’S3’S2’S1S3Collagen type peptide inhibitorsP3P2P1P1’P3’P2’ZBGGeneral inhibitors requirementsP3P2P1ZBGLeft side InhibitorsThese were basically design to mimic the collagen substrate and therefore checking the interaction made by the substrateand 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 subsitesHowever, 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 subsitesTherefore looking it to the early inhibitors,P1’P3’P2’ZBGRight side InhibitorsZBG : Zinc binding groupDorman, G. et. al. Drugs, 2010, 70, PDB ID: 2TCL
24 PEPTIDOMIMETIC INHIBITORS Based on the structure of natural substrate collagenIsobutyl, t-butyl group preferredMethyl group preferredEssential for activityIt could easily be summarized that the isobutyl group was essential at positions P1’ and the P2’ positionThe methyl group at P3’ position however the most important was the ZBG which was going to replace the scissile amide bounda no. these ZBG groups were tried at this position and these includedBrown, 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 ZBGNow the basic mechanism by which these acted involvesCarboxylatesHu, 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 substrateHowever in the presence of a ZBG for example Hydroxamate would break such an interaction and therefore inhibit the protein cleaving property of the MMPAnd hence based on these basic requirements ……………Active enzymeEnzyme-hydroxamateHu, J. Nat. Rev. Drug Discovery, 2007, 6,
27 BROAD SPECTRUM HYDROXAMATES The earliest MMP inhibitorsMany members of this class entered clinical trialsIlomastatBatimastatMMP-1 = 0.4MMP-2 = 0.39MMP-3 = 26MMP-8 = 0.18MMP-9 = 0.57MarimastatMMP-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 profileprompted 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 ADAMSThis 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 inhibitorsMMP-1=10 MMP-2 = 4 MMP-3 = 20 MMP-8 = 10 MMP-9 = 1*All IC50 values are in units of nMSkiles, J. W. et. al. Curr. Med. Chem. 2004, 11,
28 THE SECOND GENERATIONThe 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’ substituentWide range of substituent toleratedThe succinate type backbone was modifiedP3’ substituentWide range of substituent toleratedP1’ substituentMajor determinant of activityTo summarize the SAR study the following effects were seen on changing the substituents at each of the positionsThe zBG was of prime importanceThe alpha substituent was considered essential interms of maintaining the PK profile of the drugThe P2 did not have a large effect on potency similarly the P3 position was also not essential for activityThe P1 position was the most essential for selectivity towards certain groups andHence due to the established importance of only the P1 and the alpha substituent only the succinate backbone was considered essential for activityThis 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 substituentImproves Pharmacokinetic propertiesZinc binding groupEssential for activityHu, J. Nat. Rev. Drug Discov. 2007, 6,
30 MODIFICATIONS OF THE BACKBONE Malonic acid typeGlutaric acid typeSulphonamide typeMalonic , glutaric, sulfonamide and the sulfone type……however 2 backbones the sulfonamide and the sulfones were successful in producing good potencyHowever , I’ll be talking about one such sulfonamide in detail which was developed by Parke-Davis……..Sulphone typeHu, J. Nat. Rev. Drug Discov. 2007, 6,
31 SULPHONAMIDE BASED INHIBITOR Sulphonamide typeSuccinic acid typeDeveloped by Parke-Davis showing μM potency for MMPsEnzymeIC50 μMMMP-15.4MMP-20.040MMP-30.038MMP-771MMP-926MMP-130.062It 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 potencyand 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 PGHalogen at position 2’ Decreased selectivityElectron donating group 4’ Decreased activityHalogen at position 3’ Decreased activityHalogen at position 4’ Increased activityElectron withdrawing group 4’ Increase activityRMMP (IC50 μM)1237913H5.40.040.03871260.0624’-F4.20.0390.0104.8640.0434’-Br6.00.0040.0077.27.90.0084’-Cl6.50.0110.0097.5160.0483’-Br1000.5350.2900.7102’-F, 4’-Br3.60.0050.0162.14.94’-NH20.03631200.1054’-CF30.0130.023First modifying the 4’ position of the biphenyl ring4-X group in general increased the potency of these inhibitors3-X group however was found to decrease activity for all MMPs2-X and 4-x increased the potency to all MMPs but was found to decrease the selectivityEDG at the 4- position in general decreased activity, whereas ewg at 4-position increased activityHowever 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-9Next they tried to improve the pharmacokinetics of the compoundO’Brien, P. M. et. al. J. Med. Chem. 2000, 43,
33 Improving the pharmacokinetic profile DEVELOPING PGR1HImproving the pharmacokinetic profileMMP IC50 μMt1/2(h)1237913500.0040.010776.70.02615.60.012119.525.160.0077.27.90.00843.60.0093.14.90.00541.8270.002152.33.88Which they startedBy first placing a methyl group which increased the t1/2 of the drug increased to 25.1 h as compared to the unsubstituted oneNext isopropyl group also further increased the t ½Similarly the iso butyl moietyHowever the ethyl sulfonyl methyl benzene group was found to decrease t 1/2Hence 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-16MMP-30.007MMP-77.2MMP-20.004MMP-97.9PG-11680Looking 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 zincA 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 structureIndicated that it could be used in left ventricular failureO’Brien, P. M. et. al. J. Med. Chem. 2000, 43,
35 STUDIES CONDUCTED Studies in humans Animal studies Randomized trial were conducted for 90 daysStudy end pointsLV end diastolic indexEjection fractionResultsNo Significant changesMusculoskeletal syndromePossible causesSpeciesDoseEffects on MMPCardiac effectsPigs20mg/kg/dayMMP-2MMPsLV dilationLV peak wall stressLV loadRats5mg/Kg/dayMMP-9ContractilityThickness of fibrillar collagenWhich indicated beneficial effects like decrease LV dilation etcAnd this therefore validated human trialsHuman trial were conducted for 90days as a randomised trial with LV end diastolic index and the ejection fraction acting as the study endpointsHowever 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 reasonImproper dosing regimen and lack of MMP-1 selectivityThe 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 RegimenMMP SelectivityMMP-1Kaludercic, N. et.al. Cardiovascular Therapeutics, 2008, 26, 24–37.
36 THE S1’ SELECTIVITY POCKET Specificity loopNtS1’The depth of the S1’ tunnel is determined by the S1’ specificity loopPocket differs for different MMP’sMMP-1, -7Shallow pocketMMP-2, -9Intermediate pocketMMP-8, -13Deep pocketAround 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 pocketFurther 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 categoriesThe Narrow 1 and 7Intermediate 2 and 9Deep 13 and 8As an eg this structure indicates the difference between the shallow pocket of MMP-1 and the deep pocket of MMP-13This therefore laid the foundation for the 3rd generation of agentsMMP- 1 shallow pocketMMP- 13 Deep pocketPDB ID: 2TCLPDB ID: 456COverlap of the active site of major MMP classesDevel, L. et. al. Biochimie, doi: /j.bioci
37 THE THIRD GENERATIONwhich were designed with MMP-1 sparing activity in mind…one such agent was recently developed ……
38 α-TETRAHYDROPYRANYL SULFONES 2nd Generation sulfoneRS (β-Sulfone)Developed by Rocheα- Sulphone derivativeSulfoneMMP- 1MMP-2MMP-3MMP-9MMP-13BA (%)α43220.127.116.11.01545.8β8000.417.510.621.2Based 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 propertiesUsing 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 comparisionWith increased potency this change also conferred increased BATherefore with a view to improve the MMP-1 sparing property even further, a no. of modification were made at the R position and these includedDeveloped by Pfizer*All IC50 values are in units of nMBecker, D. P. et. al. J. Med. Chem. 2010, 53,
39 α-TETRAHYDROPYRANYL SULFONES α- SulphoneRMMP-1MMP-2MMP-9MMP-13BA %2618.104.22.1688000.32.90.45-4350.1545.84000.2114035.980001.20.83350000.6142.3A 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 includedThe unsubstituted phenyl group which showed good potency but low potency and BASubstituting the 3 or the meta position with a Cl group decreased potencyA 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 selectivityNext a –OCF3 group was place which achieved both activity and selectivity aswellas had a good BA profileReplacing with a –CF3 group also improved potency with an even better selectivity profileLooking 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 developedAll IC50 values are in units of nMBecker, D. P. et. al. J. Med. Chem. 2010, 53,
40 α-PIPERIDINYL SULPHONES SelectivityPharmacokineticsPiperidine sulphonesMMP-1MMP-2MM9-9MMP-13BA%100001.722.214.171.124.95.5126.96.36.199.236.360000.516.640000.15230.1867.9Similarly the alpha piperidines were also prepared these were substituted at two siteThe 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 moleculeThe R1 position was a part of the piperidine N and was basically placed to confer pk properties to the moleculesFollowing the same successful R2 substituent as the previous tetahydropyranyl group ie the phenyl with a 4-CF3First the P1’ substituent was replaced by p-CF3, whereas the R1 was left unsubstitutedThis produced good selectivity , pK needed improvement hence the next few involve substitution at the R1 positionKeeping 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-13Next a cyclopropane ring was placed to improve activity which retained high potency and had a respectable BANow 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 aswellReplacing 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 upAll IC50 values are in units of nMBecker, D. P. et. al. J. Med. Chem. 2010, 53,
41 STRUCTURAL SELECTIVITY Arg214Leu218A 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 oneHence 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 sulfoneMMP-13, MMP-1 S1’overlapBecker, D. P. et. al. J. Med. Chem. 2010, 53,
42 ANIMAL STUDIESInhibition of post infarction left ventricular dilation investigated in rat modelLV volume (mL)LV pressure (mmHg)SHAM-VehMI-VehMI-10mpkMI-50mpkAB50mg/kgVehicleLV vol. (mL)MI-Sham0.55MI-Veh0.69A0.63B0.62ABα- Tetrahydropyranyl sulfoneα- Piperidine sulfoneFor A which is THp and B which is piperidinylInhibition of post MI LV dilation in a rat model indicated significant decrease in LV volume for both A and B as compared to controlSimilar test conducted for C…….Becker, D. P. et. al. J. Med. Chem. 2010, 53,
43 Pharmacokinetic parameter 10mg/kg ANIMAL STUDIESPharmacokinetic parameter 10mg/kgSpeciesMouseRatDogMonkeyBA (%)51.766.66453.8CDose:10mg/kgVehicleLV vol. (mL)MI-Sham0.49MI-Veh0.59C0.51LV volume (mL)LV pressure (mmHg)SHAM-VehMI-VehMI-0.01mpkMI-0.1mpkMI-1mpkMI-10mpkWhich 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 konRapid+koffSlowE:I complexEnzyme (E)Inhibitor (I)Zn+2His+Substrate (S)Interaction with ZnCovalently bound ZnThiirane inhibitorsE:S complexZn+2HisThe 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 enzymeIn 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 enzymeMBI 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 enzymesOn taking a closer look at the profile invoved……konkoffKcat+EProduct (P)Ikejiri, M. et. al. J. Biol. Chem. 2005, 280,
45 THIIRANE TYPE INHIBITORS Enzymekon M-1.s-1koff s-1Ki μMMMP-22.1 x 1043.5 x 10-40.016MMP-94.9 x 1039.0 x 10-40.18MMP-146.9 x 1026.4 x 10-40.91.9 x 1031.3 x 10-30.7-1.04.91.2 x 1040.110.130.68123ANO2BThe parent compound showed a rapid kon rates for MMP-2 , 9 and 14 but very slow koff rates for MMP-2 -9 and 14Modification at the R position resulted loss of this effect on MMP9 and 14 but this effect continued for MMP-2Similarly substitution with a N-methyl amide had the same effectThese agents could therefore have some potential for CVS disordersAnother agents which is could be called a quite atypical agents is…………CIkejiri, M. et. al. J. Biol. Chem. 2005, 280,
46 TETRACYCLINES Weak inhibitors of MMPs Inhibits smooth muscle cell proliferation and migrationInhibits MMP-2 and MMP-9DoxycyclineDoxycycline treated SMCUntreated SMC42416104208MMP-9 proenzymeMMP-2 proenzymeMMP-2 activeDoxycycline μMTetracylines are a quite exceptional case of MMP inhibitors . Whose MMP inihibiting potential was first found from their use in periodontal diseaseWhere 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 usedRecent studies also indicate that these inhibit smc proliferation and migrationAnd 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 toGelatin zymography of SMCFranco, C. et. al. Am. J. Pathol. 2006, 168,
47 SUMMARY MMPs Inhibitors Future strategies Physiological and pathological functions of MMP are broadRole of individual MMP in disease progression is not knownModulators of MMP activity needs to be recognizedUnderstanding the role of extracellular matrix metalloproteinase inducer and other modulators of MMPDeveloping newer strategies to first understand the role and then target MMPsInhibitors of MMPs have evolved from potent broad spectrum to selective cardiovascular agentsDevelopment of selective inhibitors holds the key to the progress of these agentsUnderstanding the variation in binding site of MMPs may be essentialPhysiological 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 tissuesHence this makes it necessary to understand the role of each of these play in both normal physiological processes and in pathological conditionsUnderstanding the modulators involved could also give us an insight at understanding these rolesA look at the inhibitors indicate the development of MMPs from broad spectrum inhibitors to selective inhibitors of the MMP involved in the CVSAnd through the development of inhibitors the importance of selectivity and approaches with which these could be achieved were also realisedThe most important of which included understanding the various intricacies of the binding siteIn 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