Insights from Selective Non-phosphinic Inhibitors of MMP-12 Tailored to Fit with an S1′ Loop Canonical Conformation
2010; Elsevier BV; Volume: 285; Issue: 46 Linguagem: Inglês
10.1074/jbc.m110.139634
ISSN1083-351X
AutoresLaurent Devel, Sandra Garcia, Bertrand Czarny, Fabrice Beau, E. Lajeunesse, Laura Vera, Dimitris Georgiadis, E.A. Stura, Vincent Dive,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAfter the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S1′ cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S1′ specificity cavity, maximizing surface/volume ratios, without perturbing the S1′ loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S1′ loop conformation. The search for such compounds that fit precisely to preponderant S1′ loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs. After the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S1′ cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S1′ specificity cavity, maximizing surface/volume ratios, without perturbing the S1′ loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S1′ loop conformation. The search for such compounds that fit precisely to preponderant S1′ loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs. IntroductionThe association of matrix metalloproteinases (MMPs) 2The abbreviations used are: MMPmatrix metalloproteinaseACEangiotensin-converting enzymeNEPneprilysinAHAacetohydroxamic acidRMSDroot mean square deviation. with a variety of pathological states has stimulated impressive efforts over the past 20 years to develop synthetic compounds able to block efficiently (1Babine R.E. Bender S.L. Chem. Rev. 1997; 97: 1359-1472Crossref PubMed Scopus (907) Google Scholar, 2Whittaker M. Floyd C.D. Brown P. Gearing A.J. Chem. Rev. 1999; 99: 2735-2776Crossref PubMed Scopus (934) Google Scholar, 3Cuniasse P. Devel L. Makaritis A. Beau F. Georgiadis D. Matziari M. Yiotakis A. Dive V. Biochimie. 2005; 87: 393-402Crossref PubMed Scopus (106) Google Scholar, 4Fisher J.F. Mobashery S. Cancer Metastasis Rev. 2006; 25: 115-136Crossref PubMed Scopus (218) Google Scholar, 5Nuti E. Tuccinardi T. Rossello A. Curr. Pharm. Des. 2007; 13: 2087-2100Crossref PubMed Scopus (99) Google Scholar, 6Tu G. Xu W. Huang H. Li S. Curr. Med. Chem. 2008; 15: 1388-1395Crossref PubMed Scopus (100) Google Scholar, 7Yiotakis A. Dive V. Mol. Aspects Med. 2008; 29: 329-338Crossref PubMed Scopus (38) Google Scholar) and selectively the uncontrolled activity of these enzymes (8Fingleton B. Curr. Pharm. Des. 2007; 13: 333-346Crossref PubMed Scopus (217) Google Scholar). Extremely potent inhibitors of MMPs have been developed, but in most cases these compounds act as broad spectrum inhibitors of MMPs (9Brown S. Meroueh S.O. Fridman R. Mobashery S. Curr. Top. Med. Chem. 2004; 4: 1227-1238Crossref PubMed Scopus (63) Google Scholar). The arguments that have been proposed to explain the difficulties in identifying inhibitors able to differentiate one MMP from another include: (a) marked sequence similarities between the catalytic domains of MMPs, (b) a well conserved enzyme active site topology (backbone RMSD between the MMP catalytic domains is 0.7–0.8 Å), and (c) the mobility of residues in the so-called S1′ specificity loop (10Moy F.J. Chanda P.K. Chen J. Cosmi S. Edris W. Levin J.I. Rush T.S. Wilhelm J. Powers R. J. Am. Chem. Soc. 2002; 124: 12658-12659Crossref PubMed Scopus (58) Google Scholar, 11Bertini I. Calderone V. Cosenza M. Fragai M. Lee Y.M. Luchinat C. Mangani S. Terni B. Turano P. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5334-5339Crossref PubMed Scopus (137) Google Scholar).MMPs form a group of 23 proteins in humans, all of which contain a catalytic domain belonging to the zinc metalloproteinase family (12Bode W. Maskos K. Biol. Chem. 2003; 384: 863-872Crossref PubMed Scopus (142) Google Scholar, 13Tallant C. Marrero A. Gomis-Rüth F.X. Biochim. Biophys. Acta. 2010; 1803: 20-28Crossref PubMed Scopus (293) Google Scholar). Retrospective analysis suggests that the incorporation of strong zinc-binding groups, such as hydroxamate functions, potentiates MMP inhibition but unfortunately in an indiscriminate manner affecting most members of the MMP family (7Yiotakis A. Dive V. Mol. Aspects Med. 2008; 29: 329-338Crossref PubMed Scopus (38) Google Scholar), as well as other unrelated zinc-proteinases (14Saghatelian A. Jessani N. Joseph A. Humphrey M. Cravatt B.F. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10000-10005Crossref PubMed Scopus (367) Google Scholar). The use of a less avid zinc-binding group, like the phosphoryl group present in phosphinic peptide transition state analogs, has led to a second generation of more selective MMP inhibitors, like the selective inhibitors reported for MMP-12 (macrophage-metalloelastase) (15Devel L. Rogakos V. David A. Makaritis A. Beau F. Cuniasse P. Yiotakis A. Dive V. J. Biol. Chem. 2006; 281: 11152-11160Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The third generation MMP inhibitors possess no zinc-binding group and exploit mainly the depth of the S1′ cavity present in most MMPs (16Engel C.K. Pirard B. Schimanski S. Kirsch R. Habermann J. Klingler O. Schlotte V. Weithmann K.U. Wendt K.U. Chem. Biol. 2005; 12: 181-189Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 17Johnson A.R. Pavlovsky A.G. Ortwine D.F. Prior F. Man C.F. Bornemeier D.A. Banotai C.A. Mueller W.T. McConnell P. Yan C. Baragi V. Lesch C. Roark W.H. Wilson M. Datta K. Guzman R. Han H.K. Dyer R.D. J. Biol. Chem. 2007; 282: 27781-27791Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This strategy has led to the discovery of the first extremely selective MMP-13 inhibitors, such as compound 1 (Scheme 1). The x-ray structure of MMP-13 in complex with compound 1 confirms that this compound enters deeply into the S1′ cavity of MMP-13 and involves an unusual S1′ loop conformation characterized by the presence of an additional S1′ side pocket, a feature absent from other MMP-13·inhibitor complex x-ray structures. Part of the S1′ side pocket of MMP-13 involves residues of the so-called S1′ loop, whose size and sequence varies among MMPs. The selectivity of compound 1 toward MMP-13 has been explained by the presence of Gly248, a unique feature of the S1′ loop of MMP-13 that allows it to adopt a main chain conformation that is energetically disfavored for other MMPs (16Engel C.K. Pirard B. Schimanski S. Kirsch R. Habermann J. Klingler O. Schlotte V. Weithmann K.U. Wendt K.U. Chem. Biol. 2005; 12: 181-189Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Another selective inhibitor without zinc-binding group has also been recently reported (compound 2; Scheme 1) for the MMP-8, MMP-13 pair (18Pochetti G. Montanari R. Gege C. Chevrier C. Taveras A.G. Mazza F. J. Med. Chem. 2009; 52: 1040-1049Crossref PubMed Scopus (76) Google Scholar). Interestingly, the x-ray structure of 2 bound to MMP-8 again reveals the presence of an S1′ side pocket, with the distal part of 2 protruding into this pocket, in a manner similar to that observed in the MMP-13·1 complex. This suggests that the presence of an S1′ side pocket is not restricted to MMP-13 and might also occur in other MMPs through a displacement of the S1′ loop relative to the protein body. To explore this possibility, a series of compounds with no phosphinic zinc-binding group have been developed and screened against various MMPs, with the objective to identify highly selective MMP-12 inhibitors. Such attempts previously achieved only the identification of compounds with low potency toward MMP-12 and poor selectivity profile (like 3 in Scheme 1) (19Morales R. Perrier S. Florent J.M. Beltra J. Dufour S. De Mendez I. Manceau P. Tertre A. Moreau F. Compere D. Dublanchet A.C. O'Gara M. J. Mol. Biol. 2004; 341: 1063-1076Crossref PubMed Scopus (73) Google Scholar).The drive toward the development of highly selective MMP-12 inhibitors is justified by recent studies showing the overexpression of MMP-12 in several human pathologies, such as emphysema (20Lagente V. Le Quement C. Boichot E. Expert Opin. Ther. Targets. 2009; 13: 287-295Crossref PubMed Scopus (79) Google Scholar), osteoarthritis (21Liu M. Sun H. Wang X. Koike T. Mishima H. Ikeda K. Watanabe T. Ochiai N. Fan J. Arthritis Rheum. 2004; 50: 3112-3117Crossref PubMed Scopus (56) Google Scholar), atherosclerosis (22Halpert I. Sires U.I. Roby J.D. Potter-Perigo S. Wight T.N. Shapiro S.D. Welgus H.G. Wickline S.A. Parks W.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 9748-9753Crossref PubMed Scopus (320) Google Scholar), aneurisms (23Curci J.A. Liao S. Huffman M.D. Shapiro S.D. Thompson R.W. J. Clin. Invest. 1998; 102: 1900-1910Crossref PubMed Scopus (403) Google Scholar), giant cell arteritis (24Rodríguez-Pla A. Martínez-Murillo F. Savino P.J. Eagle Jr., R.C. Seo P. Soloski M.J. Rheumatology. 2009; 48: 1460-1461Crossref PubMed Scopus (12) Google Scholar), and chronic obstructive pulmonary disease (25Demedts I.K. Morel-Montero A. Lebecque S. Pacheco Y. Cataldo D. Joos G.F. Pauwels R.A. Brusselle G.G. Thorax. 2006; 61: 196-201Crossref PubMed Scopus (182) Google Scholar). Recently, based on a large clinical study, testing for an association between both asthma and chronic obstructive pulmonary disease and single-nucleotide polymorphisms in the gene encoding MMP-12, a detrimental role was attributed to the overexpression of MMP-12 (26Hunninghake G.M. Cho M.H. Tesfaigzi Y. Soto-Quiros M.E. Avila L. Lasky-Su J. Stidley C. Melén E. Söderhäll C. Hallberg J. Kull I. Kere J. Svartengren M. Pershagen G. Wickman M. Lange C. Demeo D.L. Hersh C.P. Klanderman B.J. Raby B.A. Sparrow D. Shapiro S.D. Silverman E.K. Litonjua A.A. Weiss S.T. Celedón J.C. N. Engl. J. Med. 2009; 361: 2599-2608Crossref PubMed Scopus (263) Google Scholar). In animal models, mice deficient in MMP-12 were shown to be less susceptible to emphysema (27Hautamaki R.D. Kobayashi D.K. Senior R.M. Shapiro S.D. Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1233) Google Scholar, 28Morris D.G. Huang X. Kaminski N. Wang Y. Shapiro S.D. Dolganov G. Glick A. Sheppard D. Nature. 2003; 422: 169-173Crossref PubMed Scopus (427) Google Scholar, 29Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. Mizuno-Horikawa Y. Nakano M. Asahi M. Takahashi M. Uozumi N. Ihara S. Lee S.H. Ikeda Y. Yamaguchi Y. Aze Y. Tomiyama Y. Fujii J. Suzuki K. Kondo A. Shapiro S.D. Lopez-Otin C. Kuwaki T. Okabe M. Honke K. Taniguchi N. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15791-15796Crossref PubMed Scopus (344) Google Scholar), atherosclerosis (30Johnson J.L. George S.J. Newby A.C. Jackson C.L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15575-15580Crossref PubMed Scopus (287) Google Scholar), and aneurisms (21Liu M. Sun H. Wang X. Koike T. Mishima H. Ikeda K. Watanabe T. Ochiai N. Fan J. Arthritis Rheum. 2004; 50: 3112-3117Crossref PubMed Scopus (56) Google Scholar). Furthermore, the need for MMP inhibitors with better selectivity profile is also justified by the poor outcomes observed in preclinical studies using broad spectrum inhibitors (31Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2349) Google Scholar, 32Overall C.M. Kleifeld O. Br. J. Cancer. 2006; 94: 941-946Crossref PubMed Scopus (290) Google Scholar) and the opposing roles that MMPs play in pathologies like cancer (33López-Otín C. Palavalli L.H. Samuels Y. Cell Cycle. 2009; 8: 3657-3662Crossref PubMed Scopus (102) Google Scholar) and atherosclerosis progression (30Johnson J.L. George S.J. Newby A.C. Jackson C.L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15575-15580Crossref PubMed Scopus (287) Google Scholar). These studies have clearly indicated the absolute need to specifically target the MMPs involved in pathology progression and not those counteracting it. Ultimately, highly selective inhibitors are essential tools to obtain proof of principle for the efficacy of a chemical intervention in animal models, as compared with gene invalidation.DISCUSSIONThe development of most MMP inhibitors for the last 15 years has relied on the use of a strong zinc-binding group, such as hydroxamate, and by targeting the entrance of the S1′ cavity. This strategy was selected to obtain potent inhibition through tight hydroxamate/zinc ion interaction and maintaining the inhibitor in a low molecular weight range by limiting the size of the P1′ side chain. Unfortunately, although providing extremely potent MMP inhibitors, all of the compounds obtained through this strategy displayed poor selectivity toward MMPs and also targeted other unrelated zinc-metalloproteinases like NEP (14Saghatelian A. Jessani N. Joseph A. Humphrey M. Cravatt B.F. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10000-10005Crossref PubMed Scopus (367) Google Scholar). The use of a weaker zinc-binding group (carboxylate or phosphoryl group) led to more selective inhibitors, in particular for MMP-12 (15Devel L. Rogakos V. David A. Makaritis A. Beau F. Cuniasse P. Yiotakis A. Dive V. J. Biol. Chem. 2006; 281: 11152-11160Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 48Li W. Li J. Wu Y. Rancati F. Vallese S. Raveglia L. Wu J. Hotchandani R. Fuller N. Cunningham K. Morgan P. Fish S. Krykbaev R. Xu X. Tam S. Goldman S.J. Abraham W. Williams C. Sypek J. Mansour T.S. J. Med. Chem. 2009; 52: 5408-5419Crossref PubMed Scopus (32) Google Scholar, 49Li W. Li J. Wu Y. Wu J. Hotchandani R. Cunningham K. McFadyen I. Bard J. Morgan P. Schlerman F. Xu X. Tam S. Goldman S.J. Williams C. Sypek J. Mansour T.S. J. Med. Chem. 2009; 52: 1799-1802Crossref PubMed Scopus (66) Google Scholar). First, the present study demonstrates that pseudo-peptides with no phosphinic zinc-binding group in their structures can behave as potent MMP-12 inhibitors. Second, this study shows that exploring the bottom part of the S1′ cavity of MMP-12 by using long and bent P1′ side chain resulted in inhibitors showing even better selectivity profile than the best selective MMP-12 reported so far (compound 4) (15Devel L. Rogakos V. David A. Makaritis A. Beau F. Cuniasse P. Yiotakis A. Dive V. J. Biol. Chem. 2006; 281: 11152-11160Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Third, this study indicates that the inhibitor P1′ side chain accommodation into the S1′ cavity occurs without major conformational shift of the S1′ loop, in contrast to what was observed with MMP-13 selective inhibitors (16Engel C.K. Pirard B. Schimanski S. Kirsch R. Habermann J. Klingler O. Schlotte V. Weithmann K.U. Wendt K.U. Chem. Biol. 2005; 12: 181-189Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) and mixed MMP13/MMP-8 inhibitors (18Pochetti G. Montanari R. Gege C. Chevrier C. Taveras A.G. Mazza F. J. Med. Chem. 2009; 52: 1040-1049Crossref PubMed Scopus (76) Google Scholar). Selective MMP-13 inhibitors have been proposed to select a particular/minor conformation of MMP-13 from a conformational ensemble. Even if this concept seems to apply also to MMP-8, noncanonical S1′ loop conformation in MMPs is poorly documented; thus translation of this approach for developing selective inhibitor toward other MMPs approach remains challenging. In contrast a lot of three-dimensional structures (x-ray or NMR) of MMPs are available in which a preferred S1′ loop conformation can be potentially targeted by inhibitors probing the bottom part of the S1′ cavity for developing more selective MMP inhibitors. Although this strategy is appealing, the present study shows that subtle variations in the inhibitor structure entail profound selectivity and potency variations. Potency and selectivity are in fact a function of several linked and complex parameters. Thus, the exact positioning of the P1′ side chain inside the S1′ cavity seems to be critical. Such positioning depends on the nature of the ring (isoxazole versus phenyl) in the inhibitor P1′ side chain at the entrance of S1′ cavity. The shape of the P1′ side chain is also important. A long P1′ linear side chain like in 23 strongly increases the inhibitor potency but yields a poor selectivity profile. Only with a bent side chain have the best results in terms of selectivity been achieved. Compounds 5 and 36 highlight how very subtle structural differences act in synchrony to yield a different selectivity profile. The small adjustments of the residues lining the S1′ cavity in response to inhibitor binding are difficult to predict, yet such knowledge could potentiate the use of structure-based design approach to develop highly selective inhibitors. These limitations explain why the development of selective MMP inhibitors has been so disappointing in the past 20 years.In summary, the present study reports how the systematic modification of the P1′ side chain has resulted in the identification of selective inhibitors. The absence of a zinc-binding group has facilitated the discovery process by strengthening the role played by the P1′ side chain and by removing the positional constraints associated with the zinc interaction. Considering the previous successes obtained with MMP-13, it appears extremely likely that more non-zinc-binding inhibitors of MMPs with high selectivity profile will be reported in the future. Whether such compounds will select highly or less populated S1′ loop conformations of MMPs is more difficult to foresee. IntroductionThe association of matrix metalloproteinases (MMPs) 2The abbreviations used are: MMPmatrix metalloproteinaseACEangiotensin-converting enzymeNEPneprilysinAHAacetohydroxamic acidRMSDroot mean square deviation. with a variety of pathological states has stimulated impressive efforts over the past 20 years to develop synthetic compounds able to block efficiently (1Babine R.E. Bender S.L. Chem. Rev. 1997; 97: 1359-1472Crossref PubMed Scopus (907) Google Scholar, 2Whittaker M. Floyd C.D. Brown P. Gearing A.J. Chem. Rev. 1999; 99: 2735-2776Crossref PubMed Scopus (934) Google Scholar, 3Cuniasse P. Devel L. Makaritis A. Beau F. Georgiadis D. Matziari M. Yiotakis A. Dive V. Biochimie. 2005; 87: 393-402Crossref PubMed Scopus (106) Google Scholar, 4Fisher J.F. Mobashery S. Cancer Metastasis Rev. 2006; 25: 115-136Crossref PubMed Scopus (218) Google Scholar, 5Nuti E. Tuccinardi T. Rossello A. Curr. Pharm. Des. 2007; 13: 2087-2100Crossref PubMed Scopus (99) Google Scholar, 6Tu G. Xu W. Huang H. Li S. Curr. Med. Chem. 2008; 15: 1388-1395Crossref PubMed Scopus (100) Google Scholar, 7Yiotakis A. Dive V. Mol. Aspects Med. 2008; 29: 329-338Crossref PubMed Scopus (38) Google Scholar) and selectively the uncontrolled activity of these enzymes (8Fingleton B. Curr. Pharm. Des. 2007; 13: 333-346Crossref PubMed Scopus (217) Google Scholar). Extremely potent inhibitors of MMPs have been developed, but in most cases these compounds act as broad spectrum inhibitors of MMPs (9Brown S. Meroueh S.O. Fridman R. Mobashery S. Curr. Top. Med. Chem. 2004; 4: 1227-1238Crossref PubMed Scopus (63) Google Scholar). The arguments that have been proposed to explain the difficulties in identifying inhibitors able to differentiate one MMP from another include: (a) marked sequence similarities between the catalytic domains of MMPs, (b) a well conserved enzyme active site topology (backbone RMSD between the MMP catalytic domains is 0.7–0.8 Å), and (c) the mobility of residues in the so-called S1′ specificity loop (10Moy F.J. Chanda P.K. Chen J. Cosmi S. Edris W. Levin J.I. Rush T.S. Wilhelm J. Powers R. J. Am. Chem. Soc. 2002; 124: 12658-12659Crossref PubMed Scopus (58) Google Scholar, 11Bertini I. Calderone V. Cosenza M. Fragai M. Lee Y.M. Luchinat C. Mangani S. Terni B. Turano P. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5334-5339Crossref PubMed Scopus (137) Google Scholar).MMPs form a group of 23 proteins in humans, all of which contain a catalytic domain belonging to the zinc metalloproteinase family (12Bode W. Maskos K. Biol. Chem. 2003; 384: 863-872Crossref PubMed Scopus (142) Google Scholar, 13Tallant C. Marrero A. Gomis-Rüth F.X. Biochim. Biophys. Acta. 2010; 1803: 20-28Crossref PubMed Scopus (293) Google Scholar). Retrospective analysis suggests that the incorporation of strong zinc-binding groups, such as hydroxamate functions, potentiates MMP inhibition but unfortunately in an indiscriminate manner affecting most members of the MMP family (7Yiotakis A. Dive V. Mol. Aspects Med. 2008; 29: 329-338Crossref PubMed Scopus (38) Google Scholar), as well as other unrelated zinc-proteinases (14Saghatelian A. Jessani N. Joseph A. Humphrey M. Cravatt B.F. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10000-10005Crossref PubMed Scopus (367) Google Scholar). The use of a less avid zinc-binding group, like the phosphoryl group present in phosphinic peptide transition state analogs, has led to a second generation of more selective MMP inhibitors, like the selective inhibitors reported for MMP-12 (macrophage-metalloelastase) (15Devel L. Rogakos V. David A. Makaritis A. Beau F. Cuniasse P. Yiotakis A. Dive V. J. Biol. Chem. 2006; 281: 11152-11160Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The third generation MMP inhibitors possess no zinc-binding group and exploit mainly the depth of the S1′ cavity present in most MMPs (16Engel C.K. Pirard B. Schimanski S. Kirsch R. Habermann J. Klingler O. Schlotte V. Weithmann K.U. Wendt K.U. Chem. Biol. 2005; 12: 181-189Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 17Johnson A.R. Pavlovsky A.G. Ortwine D.F. Prior F. Man C.F. Bornemeier D.A. Banotai C.A. Mueller W.T. McConnell P. Yan C. Baragi V. Lesch C. Roark W.H. Wilson M. Datta K. Guzman R. Han H.K. Dyer R.D. J. Biol. Chem. 2007; 282: 27781-27791Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). This strategy has led to the discovery of the first extremely selective MMP-13 inhibitors, such as compound 1 (Scheme 1). The x-ray structure of MMP-13 in complex with compound 1 confirms that this compound enters deeply into the S1′ cavity of MMP-13 and involves an unusual S1′ loop conformation characterized by the presence of an additional S1′ side pocket, a feature absent from other MMP-13·inhibitor complex x-ray structures. Part of the S1′ side pocket of MMP-13 involves residues of the so-called S1′ loop, whose size and sequence varies among MMPs. The selectivity of compound 1 toward MMP-13 has been explained by the presence of Gly248, a unique feature of the S1′ loop of MMP-13 that allows it to adopt a main chain conformation that is energetically disfavored for other MMPs (16Engel C.K. Pirard B. Schimanski S. Kirsch R. Habermann J. Klingler O. Schlotte V. Weithmann K.U. Wendt K.U. Chem. Biol. 2005; 12: 181-189Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Another selective inhibitor without zinc-binding group has also been recently reported (compound 2; Scheme 1) for the MMP-8, MMP-13 pair (18Pochetti G. Montanari R. Gege C. Chevrier C. Taveras A.G. Mazza F. J. Med. Chem. 2009; 52: 1040-1049Crossref PubMed Scopus (76) Google Scholar). Interestingly, the x-ray structure of 2 bound to MMP-8 again reveals the presence of an S1′ side pocket, with the distal part of 2 protruding into this pocket, in a manner similar to that observed in the MMP-13·1 complex. This suggests that the presence of an S1′ side pocket is not restricted to MMP-13 and might also occur in other MMPs through a displacement of the S1′ loop relative to the protein body. To explore this possibility, a series of compounds with no phosphinic zinc-binding group have been developed and screened against various MMPs, with the objective to identify highly selective MMP-12 inhibitors. Such attempts previously achieved only the identification of compounds with low potency toward MMP-12 and poor selectivity profile (like 3 in Scheme 1) (19Morales R. Perrier S. Florent J.M. Beltra J. Dufour S. De Mendez I. Manceau P. Tertre A. Moreau F. Compere D. Dublanchet A.C. O'Gara M. J. Mol. Biol. 2004; 341: 1063-1076Crossref PubMed Scopus (73) Google Scholar).The drive toward the development of highly selective MMP-12 inhibitors is justified by recent studies showing the overexpression of MMP-12 in several human pathologies, such as emphysema (20Lagente V. Le Quement C. Boichot E. Expert Opin. Ther. Targets. 2009; 13: 287-295Crossref PubMed Scopus (79) Google Scholar), osteoarthritis (21Liu M. Sun H. Wang X. Koike T. Mishima H. Ikeda K. Watanabe T. Ochiai N. Fan J. Arthritis Rheum. 2004; 50: 3112-3117Crossref PubMed Scopus (56) Google Scholar), atherosclerosis (22Halpert I. Sires U.I. Roby J.D. Potter-Perigo S. Wight T.N. Shapiro S.D. Welgus H.G. Wickline S.A. Parks W.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 9748-9753Crossref PubMed Scopus (320) Google Scholar), aneurisms (23Curci J.A. Liao S. Huffman M.D. Shapiro S.D. Thompson R.W. J. Clin. Invest. 1998; 102: 1900-1910Crossref PubMed Scopus (403) Google Scholar), giant cell arteritis (24Rodríguez-Pla A. Martínez-Murillo F. Savino P.J. Eagle Jr., R.C. Seo P. Soloski M.J. Rheumatology. 2009; 48: 1460-1461Crossref PubMed Scopus (12) Google Scholar), and chronic obstructive pulmonary disease (25Demedts I.K. Morel-Montero A. Lebecque S. Pacheco Y. Cataldo D. Joos G.F. Pauwels R.A. Brusselle G.G. Thorax. 2006; 61: 196-201Crossref PubMed Scopus (182) Google Scholar). Recently, based on a large clinical study, testing for an association between both asthma and chronic obstructive pulmonary disease and single-nucleotide polymorphisms in the gene encoding MMP-12, a detrimental role was attributed to the overexpression of MMP-12 (26Hunninghake G.M. Cho M.H. Tesfaigzi Y. Soto-Quiros M.E. Avila L. Lasky-Su J. Stidley C. Melén E. Söderhäll C. Hallberg J. Kull I. Kere J. Svartengren M. Pershagen G. Wickman M. Lange C. Demeo D.L. Hersh C.P. Klanderman B.J. Raby B.A. Sparrow D. Shapiro S.D. Silverman E.K. Litonjua A.A. Weiss S.T. Celedón J.C. N. Engl. J. Med. 2009; 361: 2599-2608Crossref PubMed Scopus (263) Google Scholar). In animal models, mice deficient in MMP-12 were shown to be less susceptible to emphysema (27Hautamaki R.D. Kobayashi D.K. Senior R.M. Shapiro S.D. Science. 1997; 277: 2002-2004Crossref PubMed Scopus (1233) Google Scholar, 28Morris D.G. Huang X. Kaminski N. Wang Y. Shapiro S.D. Dolganov G. Glick A. Sheppard D. 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