Development of Selective Inhibitors and Substrate of Matrix Metalloproteinase-12

Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO2-CH2)-Xaa′-Yaa′-Zaa′-NH2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with Ki values of 0.19 and 4.4 nm toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa′-Zaa′ positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr239 and Lys177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm. Four phosphinic peptide libraries with compounds having the general formula p-Br-Ph-(PO2-CH2)-Xaa′-Yaa′-Zaa′-NH2 have been prepared and screened against 10 matrix metalloproteinases (MMPs). We identified two phosphinic peptides with Ki values of 0.19 and 4.4 nm toward MMP-12 (macrophage elastase) that are more than 2-3 orders of magnitude less potent toward the other MMPs tested. These highly selective MMP-12 inhibitors contain a Glu-Glu motif in their Yaa′-Zaa′ positions. Incorporation of this Glu-Glu motif into the sequence of a nonspecific fluorogenic peptide cleaved by MMPs provides a highly selective substrate for MMP-12. A model of one of these inhibitors interacting with MMP-12 suggests that the selectivity observed might be due, in part, to the presence of two unique polar residues in MMP-12, Thr239 and Lys177. These MMP-12-selective inhibitors may have important therapeutic applications to diseases in which MMP-12 has been suggested to play a key role, such as in emphysema, atherosclerosis, and aortic abdominal aneurysm. The matrix metalloproteinases (MMPs) 2The abbreviations used are: MMP, matrix metalloproteinase; Mca, (7-methoxycoumarin-4-yl)acetyl; Dpa, N3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl; DMF, N,N-dimethylformamide; DIC, N,N′-diisopropylcarbodiimide; HOBt, N-hydroxybenzotriazole; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography. form a group of structurally related extracellular zinc endoproteases able to degrade at least one protein component of the extracellular matrix (1Bode W. Biochem. Soc. Symp. 2003; : 1-14PubMed Google Scholar). Based on this property, MMPs are considered to be critical mediators of both normal and pathological tissue remodeling processes (2Brinckerhoff C.E. Matrisian L.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 207-214Crossref PubMed Scopus (974) Google Scholar, 3Mott J.D. Werb Z. Curr. Opin. Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (887) Google Scholar). Their overexpression is observed in and associated with a variety of diseases, including cancer (4Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5219) Google Scholar, 5Folgueras A.R. Pendas A.M. Sanchez L.M. Lopez-Otin C. Int. J. Dev. Biol. 2004; 48: 411-424Crossref PubMed Scopus (490) Google Scholar), arthritis (6Burrage P.S. Mix K.S. Brinckerhoff C.E. Front. Biosci. 2006; 11: 529-543Crossref PubMed Scopus (1023) Google Scholar), multiple sclerosis (7Yong V.W. Power C. Forsyth P. Edwards D.R. Nat. Rev. Neurosci. 2001; 2: 502-511Crossref PubMed Scopus (899) Google Scholar, 8Rosenberg G.A. Lancet. 2005; 365: 1291-1293Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), and atherosclerosis (9Dollery C.M. Libby P. Cardiovasc. Res. 2006; 69: 625-635Crossref PubMed Scopus (211) Google Scholar, 10Newby A.C. Physiol. Rev. 2005; 85: 1-31Crossref PubMed Scopus (714) Google Scholar). Therefore, there is substantial interest in developing MMP synthetic inhibitors for a variety of therapeutic indications (11Whelan C.J. Curr. Opin. Investig. Drugs. 2004; 5: 511-516PubMed Google Scholar, 12Daheshia M. Curr. Med. Res. Opin. 2005; 21: 587-594Crossref PubMed Scopus (27) Google Scholar, 13Mannello F. Tonti G. Papa S. Curr. Cancer Drug Targets. 2005; 5: 285-298Crossref PubMed Scopus (138) Google Scholar, 14Vihinen P. Ala-aho R. Kahari V.M. Curr. Cancer Drug Targets. 2005; 5: 203-220Crossref PubMed Scopus (256) Google Scholar, 15Tayebjee M.H. Lip G.Y. MacFadyen R.J. Curr. Med. Chem. 2005; 12: 917-925Crossref PubMed Scopus (72) Google Scholar). Results of the first clinical trials with broad spectrum MMP inhibitors in cancer therapy were disappointing, highlighting the need for better understanding of the exact role of each MMP during the different stages of tumor progression (16Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Crossref PubMed Scopus (2408) Google Scholar). Recent research in this field has focused on the development of inhibitors that fully differentiate one MMP from another (17Brown S. Meroueh S.O. Fridman R. Mobashery S. Curr. Top. Med. Chem. 2004; 4: 1227-1238Crossref PubMed Scopus (64) Google Scholar). This is a particularly difficult task, since the topology and nature of the residues in the enzyme's active site are highly conserved among the different MMPs (18Cuniasse P. Devel L. Makaritis A. Beau F. Georgiadis D. Matziari M. Yiotakis A. Dive V. Biochimie (Paris). 2005; 87: 393-402Crossref PubMed Scopus (108) Google Scholar). Moreover, parts of the MMP catalytic domain, which play a critical role in enzyme specificity, seem to be highly flexible (19Moy 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 (60) Google Scholar, 20Bertini 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 (138) Google Scholar). This situation may explain why most previously reported MMP synthetic inhibitors preferentially inhibit some MMPs but are not exclusive inhibitors of a single MMP. A recent breakthrough in this field was achieved by identifying the first highly selective synthetic inhibitor of MMP-13 (21Engel 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 (203) Google Scholar). Selective inhibitors for MMP-2 and MMP-9 have also been reported recently, but their degree of selectivity toward MMPs is less than that achieved for the MMP-13-selective inhibitor (22Ikejiri M. Bernardo M.M. Meroueh S.O. Brown S. Chang M. Fridman R. Mobashery S. J. Org. Chem. 2005; 70: 5709-5712Crossref PubMed Scopus (43) Google Scholar). To identify highly selective MMP inhibitors, libraries of phosphinic peptides were prepared. Phosphinic peptides are good transition state mimics and have been shown to behave as highly potent inhibitors of different zinc metalloproteinases (23Dive V. Georgiadis D. Matziari M. Makaritis A. Beau F. Cuniasse P. Yiotakis A. Cell Mol. Life Sci. 2004; 61: 2010-2019Crossref PubMed Scopus (79) Google Scholar). To probe the S1′ cavity of MMPs, a chemical strategy that makes it possible to prepare phosphinic peptides harboring various substituents in their P1′ position was used (see Scheme 1). This strategy relies on the use of a common precursor, which can be modified in one step, to prepare phosphinic peptides displaying substituted isoxazole side chains in their P1′ position (24Makaritis A. Georgiadis D. Dive V. Yiotakis A. Chemistry. 2003; 9: 2079-2094Crossref PubMed Scopus (64) Google Scholar). In such phosphinic peptides, the isoxazole ring is used as a rigid scaffold to project in the right orientation various chemical groups able to interact with the S1′ subsite of MMPs, which corresponds to a deep cavity. Based on this strategy, four libraries of phosphinic peptides, containing four different isoxazole side chains in their P1′ position, were prepared by introducing additional chemical diversity in the P2′ and P3′ positions of the inhibitors (see Scheme 1). Screening of these libraries against 10 different MMPs allowed us to identify highly selective inhibitors of MMP-12. Based on the unique structure of these inhibitors, a highly selective substrate of MMP-12 has been developed, and determinants of MMP-12 selectivity have been tentatively mapped by developing a model of interaction of one of these selective inhibitors with MMP-12. Commercial reagents were used as received without additional purification. Solvents were of the reagent grade available from commercial sources and used without further purification. SynPhase lanterns®, colored cogs, and spindles were from Mimotopes. Fmoc-amino acids were from Novabiochem. N,N′-diisopropylcarbodiimide (DIC), N-hydroxybenzotriazole (HOBt), trifluoroacetic acid, and triisopropylsilane were from Aldrich. N 2-Fmoc-N 3-2,4-dinitrophenyl-l-2,3-diaminopropionic acid and (7-methoxycoumarin-4-yl)acetyl (Mca) were from Bachem. Anhydrous N,N-dimethyl formamide (DMF) was from Fluka. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 was from Novabiochem. Catalytic domains of mouse MMP-11, human MMP-7, and MMP-14 expressed in Escherichia coli BL21 DE3 were and as R. M. I. M. A. Dive V. P. 16: PubMed Scopus Google Scholar). of the other human MMPs were from and were on a and and with a of and with a at peptide and was by on a phosphinic acid J. Am. Chem. Soc. 2001; Scopus Google and Scopus Google Scholar), was prepared by of A. S. J. Dive V. J. Org. Chem. 61: PubMed Scopus Google Scholar). Four were prepared from and previously (24Makaritis A. Georgiadis D. Dive V. Yiotakis A. Chemistry. 2003; 9: 2079-2094Crossref PubMed Scopus (64) Google Scholar, A. A A.R. Text of Chemistry. Scholar). These were for which was prepared to A. M. Med. Chem. PubMed Scopus Google The and to four phosphinic (24Makaritis A. Georgiadis D. Dive V. Yiotakis A. Chemistry. 2003; 9: 2079-2094Crossref PubMed Scopus (64) Google Scholar). of the of the by the group and of the by to the four and (see Scheme 1). and were by on by and by of phosphinic peptides of the general formula (see Scheme were prepared by were on SynPhase by a of compounds was prepared as a of containing compounds in each in a of two from the and of the P1′ (see Scheme 1). of the was with in 10 were with and and used for The position was from of Fmoc-amino containing the acid and by of the position was 10 of the and and in the step, were and for at of each acid was by acid and the were of colored spindles and were associated with a and into to of acids and in for at These containing were used to the each a single was to prepare a of different each containing a acid in the position and a of acids in the and the were in the presence of phosphinic and in at a was the each was for with a of trifluoroacetic and with additional each was from a to a of with a acid at the and were by and In each were were the of the of each was by acid of compounds were to the of compounds and (see were by purification. The and of the P1′ position in these compounds were as previously (24Makaritis A. Georgiadis D. Dive V. Yiotakis A. Chemistry. 2003; 9: 2079-2094Crossref PubMed Scopus (64) Google Scholar). the form first in to be the most potent toward values of compounds and MMPs is in Scheme in a of substrate was prepared on SynPhase by a N 2-Fmoc-N 3-2,4-dinitrophenyl-l-2,3-diaminopropionic acid was first into the of each acid, was into the peptide sequence from the the was and from a This substrate was by HPLC, and were used to the and of the were in 10 at as S. A. Cuniasse P. Georgiadis D. Beau F. R. G. V. P. Yiotakis A. Dive V. J. Med. Chem. PubMed Scopus Google Scholar). the of MMP-11, of the were in were in to of substrate were by the in at nm by the of the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 fluorogenic substrate by were a with a and a The of a were of nm MMP, and a of the In the of MMP-11, were in with by a a Life was used as as to the of A. Cuniasse P. R. Beau F. Yiotakis A. P. Dive V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). at various were with the MMPs the of The of inhibitor was by acid each inhibitor the was in at were in to a of values were the by and A. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This into the of the and inhibitor and to the situation of both and values were from with the by and is substrate at and is enzyme MMP were by a phosphinic peptide inhibitor displaying Ki values than nm toward site for the of by MMP-12, and was by to These that these MMPs cleaved the peptide the sequence of this The interaction of with the catalytic of was a on with the S. M. J. Chem. 4: Scopus Google Scholar). field was used D. M. J.D. S. J. S. D. L. C. S. B. B. M. R. J. M. J. D. M. J. Chem. B. 102: PubMed Scopus Google Scholar). The structure of MMP-12 was that of the catalytic of this enzyme in with acid inhibitor by 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 (138) Google Scholar). The position of was by the catalytic of with the phosphinic inhibitor M. R. Cuniasse P. Yiotakis A. Dive V. P. D. J. Mol. Biol. 2001; PubMed Scopus Google on that of the catalytic of MMP-12. The structure of was by of the structure of the of and for the phosphinic inhibitor were from with the S. O. J. V. M. R. B. C. C. S. J. G.A. P. J. G. A. P. R. R. A. M. B. Chen W. C. M. A. Scholar). These were at the of a The for the phosphinic inhibitor were to and for the and In to the structure of the protein during the of the were to the positions of of The were to and for the and and the residues these the and the side were to the residues were at a than from the In a of the phosphinic inhibitor and the catalytic MMP-12 was used in to the of the the of the inhibitor and the the the were a and a with a of The was to The first of the of of of the were The was to The was by to These were by of The structure was with the Four with the general formula were by were by four different isoxazole side chains in the position of the inhibitor to probe the S1′ cavity of MMPs 1). The of the side in the position from to In libraries and the side in the position is of to on the group to the isoxazole the group a different a different of the cavity. was prepared as a of each containing a single acid in the position, different acids in were in the The of the by each of the of MMPs for the position to the P2′ position of the in each of the P1′ in at toward and for these two MMPs are not shown in and This is related to the presence of a side in the inhibitor P1′ In to most MMPs, these two MMPs are to a S1′ cavity (1Bode W. Biochem. Soc. Symp. 2003; : 1-14PubMed Google Scholar). reported in and that the presence of a side in the position in the of inhibitor high toward most of the MMPs tested. on the of the side and the MMP for the for the are reported in this MMP-12, inhibitors harboring the side the the was observed for inhibitors containing the and side chains are by MMP-12, with the more selective inhibitors for MMP-12. The of MMP-12 for the group was also observed for MMP-2 and but for these two MMPs, the inhibitors a as with MMP-12 1). and form a group in which the side provides the most potent In this inhibitors with toward and with MMP-14 a unique with a for and 1). Based on this of inhibitors harboring this group good selectivity toward the observed for the this was not that the presence of the and side chains in the inhibitor position compounds that some selectivity for MMP-12. The can be by reported in of the position on the of MMPs by phosphinic peptide of the general formula The first to libraries and as in Scheme and the The a Ki of of the P2′ in most MMPs, the presence of in the P2′ position of the inhibitor that selectivity in the P2′ position of the nature of the group in the P1′ to the presence of the side selectivity for MMP-12 of the that the nature of the in the P2′ position the selectivity of toward MMP-12. The selectivity for MMP-12 was observed the P2′ position was by a containing a related such as and in the P2′ position less In the presence of a in the P2′ position a toward most MMPs, selectivity toward MMP-12. The containing was less potent toward MMP-12, selectivity was as with the with a In the presence of a in the P2′ position of the inhibitor also in a selectivity toward MMP-12, but in this the also good toward MMP-13 shown in MMP-13 the side in the P1′ position of the the good selectivity toward MMP-12 by the of compounds in in the P2′ position is by a phosphinic peptides of the general formula were with the position by the isoxazole side and the position by different peptides with the position by the side were also to identify selective MMP-12 of the in the was in the position, the reported in that the presence of a in the position inhibitors displaying high selectivity toward MMP-12, in the position is by and the inhibitor the selectivity toward MMP-12 is the position is by with the side in the position, the presence of and in the position the selectivity of the inhibitors toward MMP-12 to a MMP-12 and MMP-13 and and toward to the presence of at the P1′ position of the of the phosphinic peptides reported in this were prepared as a of two to of the inhibitor in two inhibitors were and and by to these for the the in were reported in since this the selectivity for MMP-12, both for compounds and A has that for this of isoxazole the first in the in the P1′ position (24Makaritis A. Georgiadis D. Dive V. Yiotakis A. Chemistry. 2003; 9: 2079-2094Crossref PubMed Scopus (64) Google Scholar). with the group as a highly potent inhibitor of MMP-12 and selectivity from to orders of magnitude with other is less potent toward MMP-12 but a of has selectivity on the one might be more than the These that in to the of the isoxazole side in the P1′ position, also the presence of a Glu-Glu motif selectivity for MMP-12. Moreover, compounds and behave as inhibitors of and Selective of further a Glu-Glu sequence be a motif to a by MMP-12, this motif was into the sequence of a fluorogenic The of this substrate was on the structure of a fluorogenic peptide used to MMP F. G. PubMed Scopus Google MMP-12 was observed to the Glu-Glu substrate with high and the sequence of this substrate (see this substrate is cleaved by MMP-12, the Glu-Glu motif with the and of MMP-12. In with the of MMP-9 and the peptide to be a substrate of other MMPs, a that the Glu-Glu motif may also the selectivity of synthetic toward MMP-12 Based on the values of the catalytic reported in the Glu-Glu substrate is cleaved and more by MMP-12, as with MMP-13 and of with model of interacting with MMP-12 was to the residues of MMP-12 that are in to the Glu-Glu side chains of the inhibitor This model that the in the P2′ position is to with the of and MMPs residues in of Thr239 in MMP-12. interaction of with and is to the presence of a side in the P1′ position of this the presence of a polar side in these two MMPs, in a position to Thr239 in MMP-12, the of the P1′ in this This model also that the in the P3′ position of is in with Lys177. the of MMP-2 and most other MMPs not have a at the MMP-12 position but a in MMP-2 and the is by in MMP-12 a was observed the of the of MMP-2 and that the in MMP-2 and MMP-9 toward the P3′ of the selective on this it is to that a interaction the of and of MMP-12 be by the presence of the in MMP-2 and the of toward MMP-2 and of the S1′ that the P1′ side of is in with residues of this two positions by and may since a in acid the different MMPs is observed for these two positions in acid of two positions in the S1′ of MMPs in a This that the of a of phosphinic peptide the of highly potent and selective inhibitors of MMP-12. In to some for MMP the inhibitors reported in this behave as inhibitors of and A. A. M. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). The of the P1′ both to the selectivity and of this of compounds toward in the of MMP-12, this that the inhibitor in to the P1′ also on the nature of the side in the P2′ and P3′ positions. of the side from to the toward MMPs 1). This suggests that some the MMP S1′ cavity may with the of the since further of the group from to toward MMPs and the by the inhibitors in other than of the S1′ cavity for most MMPs screened in this not the presence of residues the the with toward the of the S1′ cavity This may explain the of for inhibitors harboring the group in this to the side MMPs can be in two different one a for the side and the other for 1). The model of in with MMP-12 that among the residues of the S1′ that are in to the P1′ of two may play a role in the of the P1′ These two colored in in are of interest are not conserved MMPs The diversity of residues in these two positions of the S1′ may the to MMPs of inhibitors harboring P1′ be these S1′ residues and the reported in and in the the at the of the S1′ in most MMPs makes it difficult to of the that may P1′ group and the different residues of the S1′ that toward the S1′ cavity. This by recent (19Moy 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 (60) Google Scholar, 20Bertini 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 (138) Google Scholar), the use of MMP to fully explain the to inhibitor and be in the role of these residues in inhibitor with the role of active site residues of MMPs have been reported A.R. C.M. M. PubMed Scopus Google Scholar, C.M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and to the of there are the S1′ Results from such may the of the P1′ side chains that be into the inhibitor structure to The selective MMP-12 inhibitors reported in this contain a Glu-Glu motif in their The side chains of these two are in to the side chains of Thr239 and to model of in interaction with MMP-12 The model suggests that the selectivity of might be by a interaction the and side chains that a in position as observed in MMPs, for the side The that and in P2′ also selective in of the The can be used to explain the of MMP-12 toward inhibitors a in the P3′ of the MMP-12 structure in with with be to the role of Thr239 and in MMP-12 The catalytic by MMP-12 in the fluorogenic substrate the Glu-Glu motif is another that the role by this motif in the of a by MMP-12. of the sequence of this peptide to cleaved by MMP-12. To the of compounds and are the first of highly potent and selective MMP-12 inhibitors V. S. Med. Chem. 2004; PubMed Scopus Google Scholar, R. S. J. S. I. P. A. F. D. A.C. M. J. Mol. Biol. 2004; PubMed Scopus Google Scholar, A.C. P. C. M. R. D. A. S. P. J. F. A. Med. Chem. 2005; PubMed Scopus Google Scholar). have the role of MMP-12 in different diseases, Based on these MMP-12 was to play a key role in the development of Science. PubMed Scopus Google Scholar, G. A. D. 2003; PubMed Scopus Google Scholar, S. J. W. M. M. M. S. Lee J. A. Lopez-Otin C. M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar), atherosclerosis A. A. D. P. L. 2004; PubMed Scopus Google Scholar, A.R. B. P. S. 2004; PubMed Scopus Google Scholar, A.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar), and abdominal aortic S. J. 102: PubMed Scopus Google Scholar, W. S. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The role of MMP-12 in cancer progression is more M. J. Google Scholar, G. G. C. A. S. Cancer Res. 2005; 11: Google Scholar, R. U. 2001; PubMed Scopus Google Scholar). the of MMP-12 in different it be of interest to the development of the diseases in can be with compounds and Screening of a phosphinic libraries has allowed the of highly selective MMP-12 with the these compounds not other such as and These that selective MMP inhibitors can be by phosphinic peptide