Potent Mechanism-based Inhibitors for Matrix Metalloproteinases

Abstract

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (kon of 102 to 104 M-1 s-1 and the reversal of the process is slow (koff of 10-3 to 10-4 s-1). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases. Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (kon of 102 to 104 M-1 s-1 and the reversal of the process is slow (koff of 10-3 to 10-4 s-1). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases. Extracellular proteolysis is an essential aspect of both physiological and pathological processes. Several enzyme families have been implicated in extracellular proteolysis, of which the matrix metalloproteinases (MMPs) 3The abbreviations used are: MMP, matrix metalloproteinase; mCPBA, 3-chloroperoxybenzoic acid; DMEM, Dulbecco's modified Eagle's medium; TIMP, tissue inhibitor of metalloproteinases, Dnp, 2,4-dinitrophenyl. constitute an important group. The MMPs are zinc-dependent endopeptidases that play key roles in embryonic development, neurological processes, wound healing, angiogenesis, arthritis, cardiovascular diseases, and cancer, just to mention a few examples. In cancer, for instance, MMPs are implicated at all stages of tumor progression, including tumor growth, angiogenesis, and metastasis (1Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5169) Google Scholar). Two MMPs, gelatinases A and B (MMP-2 and MMP-9, respectively), are highly expressed in human cancer, and a direct relationship between cancer progression and gelatinase expression and activity has been well established in many studies (2McCawley L.J. Matrisian L.M. Mol. Med. Today. 2000; 6: 149-156Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). As tumors manifest high levels of gelatinase activity, inhibitors specific for the gelatinases are highly sought. In the past 8 years, there have been numerous approaches aimed at targeting MMP activities in tumors, and several clinical trials were carried out to test the efficacy of various inhibitors. Unfortunately, the results of these trials were disappointing due to the lack of an objective clinical response and undesired side effects. Many reasons have been postulated for these effects, but at the core of the problem remains the issue of inhibitor selectivity (3Pavlaki M. Zucker S. Cancer Metastasis Rev. 2003; 22: 177-203Crossref PubMed Scopus (194) Google Scholar, 4Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2390) Google Scholar). Indeed, virtually all MMP inhibitors tested so far have been broad-spectrum inhibitors, designed around chelation of the active site zinc ion (5Skiles J.W. Gonnella N.C. Jeng A.Y. Curr. Med. Chem. 2004; 11: 2911-2977Crossref PubMed Scopus (191) Google Scholar), and their spectrum of inhibition includes, in addition to MMPs, other metalloenzymes. Because targeting gelatinases remains of great promise in cancer therapy (6Matrisian L.M. Sledge Jr., G.W. Mohla S. Cancer Res. 2003; 63: 6105-6109PubMed Google Scholar), efforts aimed at developing better and selective gelatinase inhibitors continue. A mere handful of selective inhibitors for MMPs have been reported in the literature (for a review, see Ref. 7Brown S. Meroueh S.O. Fridman R. Mobashery S. Curr. Top. Med. Chem. 2004; 4: 1227-1238Crossref PubMed Scopus (63) Google Scholar). We previously described the design and properties of inhibitor 1 (Fig. 1), which is a selective mechanism-based inhibitor for gelatinases. This compound binds to the active sites of MMP-2 and MMP-9, with the thiirane moiety coordinating with the zinc ion. This coordination to the active site metal ion activates the thiirane ring for opening by the nucleophilic attack of the active site glutamate in these enzymes (Fig. 2). A unique property of this inhibitor is that on binding to the active site zinc ion, a pattern of slow binding for inhibition sets in, leading to a rapid process for the onset of inhibition with an attendant slow process for recovery from slow binding at the noncovalent stage of inhibition. This noncovalent inhibited species leads to covalent inhibition by modification of the glutamate.FIGURE 2Mechanism of action of inhibitor 1. A, coordination of the thiirane with the zinc ion is a prerequisite for the inhibition process. B, stereoview of the computational model for the noncovalent binding of inhibitor 1 in the active site of MMP-2. The active site of the enzyme is depicted as a Connolly surface in green. The active site zinc ion is depicted as an orange sphere, with the three coordinating histidine residues depicted in capped sticks. Inhibitor 1 (in capped sticks and colored according to atom types) is shown coordinated to the active site zinc ion via the thiirane sulfur. The loop that constitutes the S1′ subsite of the enzyme is drawn as a tube in purple, so the terminal phenyl group of the inhibitor is visible. The site of structure elaboration in arriving at molecules 2-7 is indicated by the white arrow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Whereas inhibitor 1, the prototype of this type of novel mechanism-based inhibitor for gelatinases, is showing promise in mouse models for diseases involving gelatinases (8Gu Z. Cui J. Brown S. Fridman R. Mobashery S. Strongin A.Y. Lipton S.A. J. Neurosci. 2005; 25: 6401-6408Crossref PubMed Scopus (373) Google Scholar, 9Kruger A. Arlt M.J. Gerg M. Kopitz C. Bernardo M.M. Chang M. Mobashery S. Fridman R. Cancer Res. 2005; 65: 3523-3526Crossref PubMed Scopus (115) Google Scholar), the poor solubility of this inhibitor in aqueous medium is a limitation of the molecule, which we have attempted to remedy. Furthermore, we have been interested in exploiting the concept behind the inhibitor design in targeting other MMPs. A computational model of the inhibitor bound in the active site of MMP-2 within the constraints of the data from x-ray absorption spectroscopy has been generated (10Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) (Fig. 2B). This model for inhibition of inhibitor 1 led the way in exploration of the next generation of this type of MMP inhibitor. The possibility for specific electrostatic interactions near the terminal phenyl group in inhibitor 1 bound to the active site of MMP-2 was anticipated for judiciously designed chemical functionalities into the molecular template of compound 1. We have introduced three functional groups, the methylsulfonamide (compounds 2 and 3), the nitro (compounds 4 and 5), and the acetamide (compounds 6 and 7), at the terminal phenyl ring system to exploit these electrostatic interactions. It was expected that these molecules would improve the solubility in aqueous solutions while exhibiting high selectivity in inhibition toward gelatinases and the membrane-anchored MMP, MT1-MMP (MMP-14), which all share a S1′ binding As described these have been these inhibitors in studies of the of MMPs in processes. Furthermore, we have the of these molecules (compounds and The that the are poor inhibitors no properties toward MMPs the of the thiirane group for this inhibitor and were on a a are reported in from on the were on and high was was with other were from a of in were and at and the was for to at the addition of 1 aqueous the was with The was with and over and reduced The was by to to as a white The and and spectrum were to the reported C. Scopus Google Scholar). a of in were and at and the was at the for 2 with the was into The was with and over and reduced to as a for a of in were and zinc at and the was for with the was The was with and over and reduced to a as an orange which was in the next a of in were and at the was to and the was for an 2 to the addition of the was with The was with 1 aqueous and over and reduced The was by to from as a for a of which was from in the as described for compound in were and at and the was at the for 1 to the addition of the was with The was with 1 aqueous and over and reduced The was by to from as a white for a of in was at and the was at for the was with and and the was with The was with and over and reduced The was by to 2 as a white for was in the as described for with the that was used in of The was by to 4 as a for was in the as described for with the that was used in of The was by to 6 as a white for a of 2 in 2 was at and the was at the reduced the was into The was with and over and reduced The was by to 3 as a white for was in the as described for 3, with the that 4 was used in of The was by to 5 as a for was in the as described for 3, with the that 6 was used in of The was by to to as a white for of Inhibitor of the solutions of the thiirane 1, 3, and in (e.g. and were to of 5 at was for to the of activity was with from The activities of MMP-9, and MMP-14 were with at and of and in R. was the used to at and in R. was with at and in a of 5 was PubMed Scopus Google Scholar). was a with and and The and were 1 and 3 of 4 was used for data The were carried out at and the was at the from and were binding enzyme inhibition was for by the enzyme to a of the and of the inhibitor 2 in with The were to 1 O. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the is the is the the formation of the enzyme-inhibitor and is the the and (kon and were from the and of of the the inhibitor according to a 1), is the and the is the of slow binding inhibition. The expression for the for the formation of The used for the of MMP-9, and MMP-14 with the were Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), The inhibition was by were by the inhibitor according to 3) analysis of inhibition, the enzyme and of the in 1 were for h at in The activity was with the for The for the of the enzyme with the were by analysis of the These were to 4 Scholar), and the in the presence and of the of enzyme in the presence and of inhibitor were at for The enzyme activity was with the as described of the was in in a 1 no at for to of to for over a The of the inhibition was on a at over the of Both the and solutions were tested for MMP activity the activity was expressed as a to that in the of inhibitor. were from the and in in an with and human were from and in Dulbecco's modified Eagle's medium with and for pro-MMP-2 MT1-MMP and were by as previously described B. Mol. Biol. PubMed Scopus Google Scholar). and pro-MMP-2 and and were expressed in with the and to from the as previously described Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and were by with 1 for h at as previously described Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was out in 5 and active and were from and and the of human and MMP-14 were from enzyme was by active site with solutions of with The MMP inhibitor was in the Mobashery and GM6001 was from solutions of GM6001, and 2-7 were in in the on in were with and for in medium with and at as described S. M. Bernardo M.M. M. Gervasi D.C. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The medium was and the were with with and of the MMP inhibitors. The were with and for 6 h with pro-MMP-2 The were with and with inhibitors of inhibitor from of The were to to pro-MMP-2 activation and to analysis to MT1-MMP expression and and was as previously described M. Gervasi D.C. Fridman R. Cancer Res. Google Scholar). The for analysis were to by to MT1-MMP was with to MT1-MMP from and migration were in in medium to the migration the were with medium in the presence and of A to pro-MMP-2 activation D.C. A. M. M. M. Fridman R. Res. PubMed Scopus Google Scholar). were in the a with to of inhibitor 3 were and the were at for various were an model to a at at the indicated The of wound in the presence of inhibitor was by the of the wound with a an cell invasion was carried out in with of in and various of inhibitor 3 were on the The was with an at in a with the surface of the in was with a to all of the The that to the side of the were and with and a in three was in of the to the inhibitors was by 104 were in in the medium was with and and with inhibitor 3 medium was with an was and the in at and was a were the The of medium but no was was in and of MMP computational model for binding of inhibitor 1 to the active site of MMP-2 is shown in The site for at the of the terminal phenyl ring of the inhibitor is indicated by an 3 the for and and 3, and of C. Scopus Google Scholar) in which was with in the presence of to the in The nitro group of was reduced over and the was with to the and in high of and to their and 6 an of and due to the of the the of the were the of and 6 to the 3, and was by the with in 3 and had solubility in aqueous with the inhibitor 1. was in 5 which was the that we used for all of the kinetic The solubility for the was as 1, 3, of action of this of MMP inhibitors that the thiirane would with the active site zinc ion (Fig. 2). with this the three of this (compounds 3, and are inhibitors of gelatinases of MT1-MMP in the of inhibitor 3) the (compounds and are poor inhibitors at all toward all of the tested MMPs for MMP inhibition by 3, and The enzymes were to a of the and of the inhibitor in R. was for to The kinetic were as described to in a new for MMP inhibition by and 6 The enzymes were with of inhibitor in R. The activity was with the The kinetic for inhibition were as described to to to to to to to to to in a new In slow binding inhibition, binding of the inhibitor to the the a that is for the reversal of the inhibition PubMed Scopus Google Scholar, Mol. Biol. Google Scholar, PubMed Scopus Google Scholar). The slow binding inhibitor a unique profile for the onset of inhibition that is by We see slow binding for inhibitor 3 MMP-9, and for inhibitor 5 with and for inhibitor with (Fig. binding MMP inhibition by inhibitors. were by the of the in solutions of MMP-2 and and inhibitor 3, as described of activity by and the conditions. The of the data to 1, the of the with inhibitor to a Large Image Figure ViewerDownload Hi-res image Download (PPT) The for the onset of slow binding inhibition are rapid to 104 and the for the reversal of the process from the noncovalent enzyme-inhibitor are slow to 10-3 the for reversal for inhibitor 3 with MMP-2 and are and The for the noncovalent (Ki) that from slow binding inhibition are from the of 3, and are selective for gelatinases, with 3 showing the slow binding with MMP-14 as The of the slow binding for inhibition by 3 and for MMP-9, and respectively), 5 for and for are in It is that the inhibition for inhibitors 1, 3, and as mechanism-based inhibitors are from the template for the inhibitor 1 both MMP-2 and inhibitor 3 the gelatinases and inhibitors 5 and are mechanism-based inhibitors only for MMP-2. Furthermore, these are nanomolar inhibitors for their enzymes and for the and for the slow binding of their of thiirane of MMP inhibitors was designed to covalent enzyme inhibitors. formation of the noncovalent enzyme-inhibitor the active site of MMPs for for were expected to modified by the inhibitor with the thiirane ring opening (Fig. 2). The of inhibition a noncovalent stage and a stage that to the covalent modification of the active site as The covalent of inhibition results in modification of the glutamate as an on side The x-ray absorption spectroscopy analysis with inhibitor 1 (10Kleifeld O. Kotra L.P. Gervasi D.C. Brown S. Bernardo M.M. Fridman R. Mobashery S. Sagi I. J. Biol. Chem. 2001; 276: 17125-17131Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) had for the covalent in that the onset of inhibition, the the formation of a from the thiirane of the inhibitor coordinated to the active site zinc ion. Whereas a slow binding a prerequisite for covalent both the mechanism-based process leading to covalent enzyme modification and the slow binding for the of activity seen with these inhibitors (Fig. with inhibitor 1 had shown that slow binding led to covalent with a for the inhibited species the that would have been anticipated from 4 the for recovery of activity from the slow binding of inhibition (in other leading to an anticipated of activity due to the noncovalent This is the with inhibitors 3, and as The for recovery of activity from the noncovalent slow binding species for the best inhibition 3 with is a mere of activity recovery was seen for MMP-2 inhibited by inhibitor 3 48 h of dialysis. for recovery from inhibition anticipated with this inhibitor and MMP-2 in just h were the slow binding that for MMP-2 inhibition. This is the and the inhibited enzyme is the which is The results of for inhibitors 3, and are in that mere slow binding for the inhibition that we we to recovery of activity seen we with covalent The is that the of covalent is are the covalent in aqueous Science. PubMed Scopus Google Scholar). This would in recovery of The process there is a of the to that the is on an at the and was attempted on MMP-2 in the presence and of inhibitor 3 to a in molecular with a of active MMP-2 with the inhibitor. However, several with we to a addition in molecular to the The is that at this high of the are and the of the due to the of the inhibitor was within the of the of on by has been as the physiological of pro-MMP-2 A.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This is at and is by by binding active as a for pro-MMP-2 on the cell surface A.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A.Y. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of pro-MMP-2 to the the pro-MMP-2 by a MT1-MMP A.Y. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). activation a B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), leading to full We have previously shown that broad spectrum MMP inhibitors (e.g. enhance pro-MMP-2 activation by MT1-MMP in the presence of M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), a process that to of MT1-MMP at the cell surface by the MMP inhibitor. This effect on pro-MMP-2 activation was the were to inhibitor 1 M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which toward a of selectivity for inhibition of gelatinases. we that targeting of MT1-MMP by broad spectrum MMP inhibitors a effect by the activity of the A M.M. Brown S. Fridman R. Mobashery S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Because inhibitor 3 is selective for the gelatinases, we postulated that like inhibitor 1 in a system of pro-MMP-2 activation by MT1-MMP in the presence of this which levels of were to MT1-MMP and with pro-MMP-2 in the presence of GM6001, a broad spectrum MMP inhibitor 3, as described M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). activation was by As shown in of the to as little as GM6001 pro-MMP-2 as by the of the active inhibitor pro-MMP-2 these conditions. this effect of broad spectrum MMP inhibitors as GM6001 the as we have previously shown M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). GM6001 a of active MT1-MMP (Fig. In the were with inhibitor 3 to 4 pro-MMP-2 activation was the of active MT1-MMP was with inhibitor 3, with reduced for this with MMP-2 inhibitor 3 is a mechanism-based inhibitor for to MMP-2 is to this inhibitor pro-MMP-2 activation these conditions. It is that covalent inhibition of as to a inhibition, the of the active site of MT1-MMP for a prerequisite for pro-MMP-2 activation M. Bernardo M.M. Gervasi D.C. Z. Brown S. Mobashery S. Fridman R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). studies are these results that the concept behind inhibitor 3 is a promising from which to and selective MT1-MMP inhibitors, a key in tumor cell these studies the selectivity of inhibitor 3 in a system and to the that key to the of MMP inhibitors. Inhibitor 3 and is well established that tumor cell migration and invasion on gelatinase we to the effect of inhibitor 3, which is selective for the gelatinases, on the migration and invasion of as described migration was of pro-MMP-2 which was by A and inhibition of cell As shown in A and B, of to various of inhibitor 3 inhibited at 2 their migration in a wound with the of to was reduced by inhibitor 3, and as little as of inhibitor inhibition of cell invasion (Fig. These of inhibitor 3 to since no of cell was were to inhibitor 3 to of as the the high selectivity by this compound toward MMP-2 to other MMPs the migration in the presence of inhibitor 3, a to other MMPs, including that the effect was due to MMP-2 inhibition. These results the of inhibitor 3 to as a selective gelatinase inhibitor in and to processes. The new of inhibitor 3 and high selectivity this inhibitor an for in in relevant human models in The thiirane of mechanism-based inhibitors was and by for the in of selectivity in inhibition of MMPs of to several processes. We have in the that inhibitor 3 targets and inhibitors 5 and are only toward MMP-2. The activities for these new inhibitors a unique in of the roles of these MMPs in various processes.