Future challenges facing the development of specific active-site-directed synthetic inhibitors of...

Future challenges facing the development of specific active-site-directedsynthetic inhibitors of MMPs

P. Cuniasse a, L. Devel a, A. Makaritis b, F. Beau a, D. Georgiadis b, M. Matziari b,A. Yiotakis b, V. Dive a,*

a CEA, Département d’Ingénierie et d’Etudes des Protéines (DIEP), CE-Saclay, 91191 Gif/Yvette cedex, Franceb Laboratory of Organic Chemistry, Department of Organic Chemistry, University of Athens, Panepistimioplois, Zografou, 15771 Athens Greece

Received 21 July 2004; accepted 23 September 2004

Available online 19 October 2004

Abstract

Despite a deep knowledge on the 3D-structure of several catalytic domains of MMPs, the development of highly specific synthetic active-site-directed inhibitors of MMPs, able to differentiate the different members of this protease family, remains a strong challenge. Due to theflexible nature of MMP active-site, the development of specific MMP inhibitors will need to combine sophisticated theoretical and experi-mental approaches to decipher in each MMP the specific structural and dynamic features that can be exploited to obtain the desired selectivity.© 2004 Elsevier SAS. All rights reserved.

Keywords: MMPs; Inhibitors; Zinc protease; Conformational variability; Inhibitor selectivity

1. Introduction

Over the past 20 years, impressive efforts have been dedi-cated to the development of synthetic compounds interactingpotently and selectively with the active-site of MMPs [1].Despite these efforts, inhibitors able to differentiate fully oneMMP from the others have not been identified so far. In thisreview, possible explanations for this situation are discussed,as well as new strategies to develop more selective inhibitorsof MMPs.

2. Chemical context

MMPs are metalloproteinases characterized by the pres-ence in their active-sites of a catalytically indispensable zincion, a property that has been exploited to design syntheticinhibitors of this enzyme family. Thus, the development of

synthetic inhibitors of zinc metalloproteinases has relied onthe use of a peptide sequence, recognized by the targetedprotease, to which have been grafted different chemical func-tionalities able to interact potently with the zinc ion of theactive-site. This strategy has allowed the identification ofseveral potent pseudo-peptide inhibitors of MMPs. Based onthe chemical structure of the zinc-binding group, four differ-ent classes of zinc metalloprotease inhibitors have been de-veloped, those incorporating a hydroxamate (CONH–O–), acarboxylate (COO–), a thiolate (S–) or a phosphinyl (PO2

–)[1]. Interestingly, while only the last three classes of syn-thetic inhibitors have been successfully applied to clinicalpractice, like those targeting the ACE zinc endopeptidase tocombat cardiovascular diseases [2], most of the MMP inhibi-tors produced by pharmaceutical companies belong to thehydroxamate category. This choice was based on early stud-ies showing that extremely potent inhibitors of MMPs can beobtained by grafting a hydroxamate moiety to a suitablepeptide sequence (see BB-941).Also, as no hydroxamate com-pounds were in clinical use at that time, this left many

Abbreviations: ACE, angiotensin converting enzyme; ADAM, a disinte-grin and metalloproteinase domain containing protein; MMPs, matrix metal-loproteinase; TACE, tumor necrosis factor-a converting enzyme.

* Corresponding author. Tel.: +33-1-69-08-35-85;fax: +33-1-69-08-90-71.

E-mail address: [email protected] (V. Dive).

1 Ki values reported in the text have been determined using synthetic fluo-rogenic substrates. Competitive inhibition for most inhibitors has beendemonstrated.

Biochimie 87 (2005) 393–402

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opportunities for patent applications. Whereas the exploita-tion of hydroxamates has proven very effective in developingpotent inhibitors of MMPs, one drawback of this approachhas been the poor selectivity displayed by this class of inhibi-tors [3–5].

This was illustrated by studies showing that hydroxamateinhibitors developed for MMPs also turned out to be highlyand sometimes more potent inhibitors of TACE, a zinc pro-tease belonging to the ADAMs family (BB-94) [6]. Given thesimilarity between the active-site of TACE and MMPs, thissituation is to be expected. But even unrelated zinc proteaseswere potently inhibited by such compounds. For instance,some hydroxamate MMP inhibitors were observed to blockpotently also the zinc metalloprotease domain of anthraxtoxin, the so-called lethal factor [7]. More unexpected werethe results reported by Saghatelian et al. showing that thehydroxamate MMP inhibitor GM6001 (ilomastat), which iscurrently in clinical evaluation, is also a potent inhibitor ofneprilysin (Ki, 17 nM), leucine aminopeptidase (Ki, 110 nM)and dipeptidylpeptidase III (Ki, 75 nM), three metallopepti-dases displaying no sequence homology with MMPs [8].Altogether, these results suggest that if the hydroxamateavidity for the zinc ion results in the development of highlypotent inhibitors of zinc proteinases, this property may hidethe contribution to the protease binding of the other groups inthe inhibitor structure, an effect reducing the selectivity ofhydroxamates. In contrast to hydroxamate, the phosphinylgroup is a weak zinc-chelating group. As a result, non-optimized phosphinic peptides behave as poor MMP inhibi-tors, as compared to hydroxamates (see 1 and 2).

However, if several rounds of optimization are performed,extremely potent phosphinic inhibitors of MMPs can beprepared from this class of compounds (3) [9]. Interestingly,these kinds of phosphinic inhibitors are weak inhibitors ofTACE. Another interesting difference between hydroxamateand phosphinic peptides relates to their chemical structures.Hydroxamate chemistry only provides access to left or righthand side inhibitors, thus allowing probing of either theprimed or unprimed side of the active-site cleft. In contrast,phosphinic peptide chemistry offers the possibility of devel-oping inhibitors able to interact with both the primed andunprimed side of the active-site cleft, allowing optimization

of inhibitor selectivity by diversification of both P and P′positions of the inhibitors. This possibility was shown toprovide in many cases extremely selective inhibitors ofclosely related zinc metalloproteinases [10–12]. However,the classical chemical approach to synthesizing phosphiniccompounds, which involved tedious preparation of phos-phinic building blocks, limits the full exploitation of thisclass of compounds for development of highly selectiveinhibitors of MMPs. Progress recently achieved in phos-phinic chemistry may, in the near future, allow better exploi-tation of this class of inhibitors [9].

An additional major challenge that chemists have had toface in developing hydroxamate inhibitors concerns the poorin vivo stability of these compounds. Rapid hydrolysis ofhydroxamate compounds was observed in vivo, producing asmetabolic products hydroxylamine and carboxylate deriva-tives [13]. Other concerns were also the rapid and

extensive clearance by biliary excretion reported for the firstgeneration of hydroxamate compounds, as well as their poororal absorption. To overcome these hurdles, medical chem-ists spent considerable time searching for hydroxamate com-pounds with improved in vivo stability and bioavailability,while keeping good potency toward MMPs [1]. RS-113456inhibitor is a nice example of compounds developed to in-crease oral bioavailability and metabolic stability of hydrox-amate derivatives [14]. To limit extensive biliary excretion,chemists have designed inhibitors with no peptide bond,resulting in compounds with no residues at the P2′ position.To compensate for

the loss of several hydrogen bonds formed between a pep-tidic inhibitor like BB-94 and the enzyme active-site resi-dues, a sulfone group (SO2) has been introduced in RS-113456, as a potential hydrogen bond acceptor. Adiphenylether was also placed to interact with the S1′ pocketof MMP active-site. The b position relative to hydroxamatefunction was occupied by a pyran to improve the metabolicprofile of the inhibitor. Several studies have demonstrated

394 P. Cuniasse et al. / Biochimie 87 (2005) 393–402

that the introduction of various substituents in the a or bposition relative to hydroxamate function increases oral bio-availability and metabolic stability of hydroxamate deriva-tives [1]. In these studies, a delicate balance between activeinteractions and the physicochemical properties of the inhibi-tors should be found to keep both high potency towardMMPs and good in vivo efficacy. It might be suggested thatthe chemical efforts to satisfy these requirements were madeat the expense of the selectivity optimization of the inhibitorsfor the different MMPs. But it should be remarked that thereis still no definitive answer about the nature of the MMP thatshould be targeted for a particular disease (see other contri-butions in this issue).

3. Structural context

Potent active-site-directed synthetic inhibitors of MMPshave been developed without knowledge of the 3D-structureof these enzymes. The first structure of an MMP in complexwith a synthetic inhibitor (MMP-1 catalytic domain) wasreported 10 years ago by Glaxo researchers [15]. This struc-ture reveals that the active-site of MMP is a shallow cleft witha flat unprimed side and a narrow primed side centeredaround the S1′ pocket. Since this first report, many otherMMP–inhibitor complexes have been solved (Table 1). Allthe MMP catalytic domains share a marked sequence simi-larity, where the percentage of identical residues ranges froma minimum of 33% between MMP-21 and MMP-23, to amaximum of 86% between MMP-3 and MMP-10 [16]. Asexpected with such sequence similarity, comparison of theavailable 3D-structure shows that the overall topology of theenzyme active-site is highly conserved between the differentMMPs, with two significant features emerging from thesestudies. These concern the depth of the S1′ pocket and thelength and composition of the loop constituting the outsidewall of the S1′ pocket. These features explain why most of thechemical efforts were directed to the development of inhibi-tors able to provide specific interactions with the S1′ pocketof MMPs. The depth of the S1′ pocket was shown to dependon the nature of the residue in position 214 (MMP-1 aminoacid numbering), located in helix B of the catalytic domain(Fig. 1A). When this residue is a leucine, as in most MMPs(exception: MMP-1, MMP-7, MMP-11, MMP-17 andMMP-19), the S1′ pocket corresponds to a very deep channel.In agreement with this observation, compounds harboringvery long side chains in their P1′ position were shown todisplay high potency toward MMPs possessing a deep S1′cavity [1,17]. In MMP-1 and MMP-7, the presence in posi-tion 214 of, respectively, an arginine and a tyrosine wasobserved to reduce the size of the S1′ pocket, as these twovoluminous side chains point inside the S1′ pocket. Thissituation fits with the observation that inhibitors harboringlong side chains in their P1′ position display much loweraffinity toward MMP-1 and MMP-7, as compared to MMPswith leucine in position 214 [17]. However, it should be

noted that exceptions to this rule have been reported. Inter-estingly, subsequent structural studies have explained howMMP-1 and MMP-7 may in some cases accommodate ex-tremely long side chains in their S1′ pocket [17]. Comparisonof the structure of MMP-1 in complex with two inhibitors,one possessing an isobutyl group (Fig. 1A, 1hfc) and thesecond one an SO2(C6H4)O(C6H5) group (Fig. 1B, 966c),illustrates this point [14]. Comparison of these figures high-lights that, depending on the size of the side chain in the P1′position of the inhibitor, two different orientations of thearginine in position 214 of MMP-1 can be observed. The firstone results in a shallow S1′ pocket (closed conformation,Fig. 1A), while the other gives a fully open cavity (openconformation, Fig. 1B) able to accommodate very long sidechains. For MMP-7, the energetic cost of the conformationalshift between the closed and open conformation has beenestimated to be around 3.5 kcal/mol [17]. A similar confor-mational shift may also concern MMP-11. MMP-11 pos-sesses a glutamine in position 214, a property leading someauthors to hypothesize that MMP-11 should possess a shal-low S1′ pocket. However, studies with substrates or inhibi-tors, carrying long side chains in their P1′ positions, were notconsistent with this view [9,18,19]. Whereas the 3D-structure of the free MMP-11 is not available, its structure ininteraction with a phosphinic peptide harboring a phenylpro-pyl side chain in P1′ position reveals that the S1′ pocket ofMMP-11 in this complex corresponds to an open deep chan-nel (1hv5) [20], similar in shape to the one observed inMMPs possessing a leucine in position 214. Currently, thereis still no experimental evidence to support the notion that, inMMP-11, the Gln214 may adopt two different orientations,corresponding to the existence of a closed and open S1′pocket. Binding of inhibitors with long side chains in theirP1′ position to MMP-1 or MMP-7 implies that the intrinsicenergy cost of opening the S1′ cavity is overcome. Thus, inthese inhibitors the P1′ side chain is presumed to form par-ticular tight interactions with residues of the S1′ cavity tocompensate for this energy cost. However, examples in theliterature reveal that this energy barrier may be overcome inanother ways. Strikingly, disubstitution at the a-position ofhydroxamic acid was shown to increase the potency of in-hibitors with long side chains in their P1′ position againstMMP-1, as compared to des-a-analogues (4, Ki MMP-1= 1000 nM; 5 Ki MMP-1 = 2 nM) [17]. Possible contribu-tions of a-substituents in hydroxamic inhibitors to increasingtheir binding of MMP-1 was recently proposed by means ofNMR structural studies [21]. This study suggests that, whilea-substituents contribute to the free energy of bindingthrough enthalpic contribution, due to direct interaction be-tween the inhibitor and the enzyme, many other subtle phe-nomena are involved in the stabilization of the complex (seebelow). Overall, the fact that a-substitution may influencethe accommodation of long side chains in the MMP-1 S1′cavity illustrates how small chemical modifications of theinhibitor may change its selectivity.

395P. Cuniasse et al. / Biochimie 87 (2005) 393–402

As mentioned above, the outside wall of the S1′ subsite isformed by a long loop connecting the Met turn of the enzyme(residue 236, MMP-1 numbering) to the third a-helix of thecatalytic domain. Table 2 reports the S1′ loop sequences in thedifferent MMPs. The length of this loop ranges from 11 resi-dues (MMP-1, MMP-2, MMP-9, MMP-11 and MMP-23) to15 residues (MMP-17 and MMP-25). In addition, the aminoacid composition of the S1′ loop varies significantly. Thisvariation can be quantified by the number of substitutionsfound for each position in the series (Table 2). On average, thevariability is lower at the top of the loop (residue 237–240)than in other parts. In particular, the stretch 241–245 (MMP-1 numbering) displays marked variability in amino acid se-quence. Fig. 2 shows the superimposition of a set of catalyticdomains of MMPs (MMP-1, MMP-3, MMP-7, MMP-8,MMP-11, MMP-12, MMP-13 and MMP-16) in complexwith different inhibitors. For clarity, only the backbone ofMMP-1 is shown in cartoon representation. The overlay ofthese structures was achieved by superimposition of the Cacarbons of the five b-sheets and the three a-helices of thecatalytic domain. When minimizing the root mean squaredeviation on the position of this set of atoms, the deviationsbetween each pair of different structures were lower than0.4 Å. However, as illustrated in Fig. 2, in these 3D-structuresthe S1′ loop adopts various conformations. This propertymight be due, at least in part, to the variation in length andcomposition of this loop between the different MMPs(Table 2). However, this proposal is only partially true as, indifferent crystal structures, the same MMP was observed toadopt different S1′ loop conformations. Fig. 3 shows thesuperimposition of several complexes of MMP-3 with inhibi-tors all possessing an SO2(C6H4)OCH3 side chain in their P1′position. The backbones of the different catalytic domainsare represented by secondary structures and are colored byincreasing b-factor, from blue to yellow. This figure clearlyillustrates that different conformations of the S1′ loop can beobserved in MMP-3 complexes, even when the chemicalgroup in P1′ position of the inhibitor is the same. This vari-ability could be related to crystal packing. However, valuesof the atomic b-factors for the region corresponding to the S1′loop suggest the existence of an intrinsic loop flexibility. Onemay speculate that the presence of longer P1′ side chains willrestrict the flexibility of the S1′ loop and, in some case,impose a particular conformation on the S1′ loop. Thus, thisS1′ loop variability can be exploited by designing inhibitors

with P1′ groups able to establish specific interactions withresidues in the lower part of the S1′ loop as a means to obtainhighly selective inhibitors of MMPs. Yet the inherent flex-ibility of the S1′ loop makes it difficult to predict with highaccuracy the exact binding mode of a particular P1′ group, aswell as the loop conformation in the complex. In this respect,it should be noticed that the structural information availablefrom the different MMP complexes in the PDB databasecannot be used to assess this issue.

To illustrate this point, a subset of the different MMPcomplexes with the inhibitor harboring the longest P1′ sidechains has been selected. This set contains complexes ofMMP-1 (966c [14]), MMP-3 (1c3i [22]; 1ciz [23]; 1g49[24]), MMP-8 (1bzs [25]), MMP-11 (1hv5 [20]). Visualexamination of these complexes (Fig. 4) indicates that the P1′groups only partially fill the S1′ cavity. Another illustration ofthis is depicted in Fig. 5 showing the complex of the catalyticdomain of MMP-3 with an inhibitor containing in the P1′position a diphenylpiperidine side chain, the longest one inthe database. This figure reveals that the bottom part of theS1′ cavity is still partly empty. A simple quantitative estima-tion of the S1′ cavity occupancy in the complexes of thedatabase can be obtained by comparing the distance betweenthe catalytic zinc atom and the last non-hydrogen atom in theP1′ group, as compared to the distance between this zinc atomand the Ca carbon of the last residue of the S1′ loop (residue246 in MMP-1). In these different complexes, the distancesseparating the last non-hydrogen atom of the P1′ group to thezinc atom range from 7.0 to 12.6 Å. The distances separatingthe catalytic zinc from the Ca carbon of the last residue in theS1′ loop range from 16.2 to 18.4 Å. This confirms that most ofthe P1′ side chains of the present set of complexes are still tooshort to interact with residues at the bottom of the S1′ loop.Therefore, these complexes cannot be used to predict how thebinding of long P1′ side chain in the S1′ subsite will modifythe conformation of the lower part of the S1′ loop. However,Fig. 5 suggests that the introduction of various substituents inthe distal phenyl group of this inhibitor will lead to com-pounds able to probe the bottom part of the S1′ cavity.Development of inhibitors containing larger side chains intheir P1′ position raises a number of problematic issues. Infact, it might be speculated that the presence of several rigidgroups in the P1′ side chain of the inhibitor may greatly limitthe exploration of the whole conformational space defined bythe S1′ cavity. In this respect, an examination of the subset of


Table 1Structures of MMP/synthetic inhibitor complexes in the protein data bank

MMP PDBcode Binding mode P1′ group ZBG Resolution (Å)/technique ReferencesMMP-1 966c Primed side SO2(C6H4)O(C6H5) Hydroxamate 1.90 [14]

3AYK/4AYK Primed side SO2(C6H4)OCH3 Hydroxamate RMN (avr/30st) [31]1HFC Primed side CH2CH(CH3)2 Hydroxamate 1.56 [32]1FBL Primed side CH2CH(CH3)2 Hydroxamate 2.50 [33]2TCL Primed side CH2CH(CH3)2 Hydroxamate 2.20 [34]1CGL Primed side CH2CH(CH3)2 Carboxylate 2.40 [15]

MMP-2 1HOV Primed side SO2(C6H4)NHCO(C6H4)(CH2)4CH3 Hydroxamate RMN (11st) [35]MMP-3 1G05 Primed side SO2(C6H4)OCH3 Hydroxamate 2.45 [36]

1G49 Primed side SO2(C6H4)O(CH2)3CH3 Hydroxamate 1.90 [37]1D5J Primed side SO2(C6H4)OCH3 Hydroxamate 2.60 [38]1B3D Primed side POCH3(C6H5) Hydroxamate 2.30 [39]1BQO Primed side SO2(C6H4)OCH3 Hydroxamate 2.30 [40]1D7X Primed side SO2(C6H4)OCH3 Hydroxamate 2.00 [41]1D8F Primed side SO2(C6H4)OCH3 Hydroxamate 2.40 [42]1D8M Primed side SO2(C6H4)OCH3 Hydroxamate 2.44 [36]1BIW Primed side CH2CH(CH3)2 Hydroxamate 2.50 [43]1BM6 Primed side SO2(C6H4)OCH3 Hydroxamate RMN (20st) [44]1UMT/1UMS Primed side CH2CH(CH3)2 Hydroxamate RMN (avr/20st) [45]2SRT Primed side (CH2)2(C6H5) Carboxylate RMN [46]1CIZ Primed side SO2(C6H4)(NC5H4)(C6H5) Carboxylate 1.64 [23]1CAQ Primed side SO2(C6H4)(NC5H4)(C6H5) Carboxylate 1.8 [23]1B8Y Primed side SO2(C6H4)(NC5H4)(C6H5) Carboxylate 2.0 [23]1HFS Primed side (CH2)2(C6H4)(C6H4)F Carboxylate 1.70 [47]1HY7 Primed side SO2(C6H4)(C6H4)OCH3 Carboxylate 1.50 [48]1SLN Primed side (CH2)2(C6H5) Carboxylate 2.27 [49]1C8T Primed side NHCH2(C6H4)(C6H5) Thiolate 2.60 [22]1C3I Primed side NHCH2(C6H4)(C6H5) Thiolate 1.83 [22]1USN Unprimed side None Thiadiazole 1.80 [50]2USN Unprimed side None Thiadiazole 2.20 [50]3USN Unprimed side None Thiadiazole RMN (avr) [51]1G4K Primed side (C6H4)O(C6H5) Pyrimidine trione 2.00 [37]

MMP-7 1MMQ Primed side CH2CH(CH3)2 Hydroxamate 1.90 [52]1MMP Primed side CH2CH(CH3)2 Carboxylate 2.30 [52]1MMR Primed side CH2CH(CH3)2 Sulfodiimine 2.30 [52]

MMP-8 1MNC Primed side CH2CH(CH3)2 Hydroxamate 2.10 [53]1JAQ Primed side CONHCH2CONH2 Hydroxamate 2.25 [54]1JAN. Unprimed side None Hydroxamate 2.50 [55]1JAP Unprimed side None Hydroxamate 1.82 [56]1KBC Primed side CH2CH(CH3)2 Hydroxamate 1.81 [57]1A85 Primed side C0NHCH2(C6H4)NH2 Hydroxamate 2.00 [58]1A86 Primed side C0NHCH2(C6H5) Hydroxamate 2.00 [58]1MMB Primed side CH2CH(CH3)2 Hydroxamate 2.10 [59]1I76 Primed side SO2(C6H4)(C6H5) Carboxylate 1.20 [60]1BZS Primed side SO2(C6H4)(C6H5) Carboxylate 1.701JAO Primed side CH2(C6H5) Thiolate 2.40 [54]1I73 Unprimed side None Phosphonate 1.40 [60]1JH1 Unprimed side None Thiadiazine 2.70 [61]1JJ9 Primed side C6H5 Pyrimidine dione 1.81 [62]

MMP-11 1HV5 Both sides (CH2)3(C6H5) Phosphinyl 2.60 [20]MMP-12 1JIZ Primed side SO2(C6H4)OCH3 Hydroxamate 2.60 [63]

1JK3 Primed side CH2CH(CH3)2 Hydroxamate 1.09 [64]MMP-13 1CXV Primed side SO2(C6H4)O(C6H4)Cl Hydroxamate 2.00 [65]

1EUB Primed side SO2(C6H4)OCH3 Hydroxamate RMN(20st) [66]1FLS/FM1 Primed side SO2(C6H4)OCH3 Hydroxamate RMN (avr/20st) [67]830c Primed side SO2(C6H4)O(C6H4)Cl Hydroxamate 1.60 [14]456C Primed side SO2(C6H4)O(C6H4)Cl Hydroxamate 2.40 [14]

MMP-16 1RM8 Primed side CH2CH(CH3)2 Hydroxamate 1.80 [68]


Fig. 1. Cartoon-type representation of two MMP-1/synthetic inhibitor complexes. a-Helices are shown in red, b-sheet in green, loops in gray with the exceptionof the S1′ loop in yellow. The catalytic zinc atom is shown in magenta. Residue Arg 214 of MMP-1 is shown in blue. A: 1hfc P1′ side chain CH2CH(CH3)2, B:966c, P1′ side chain SO2(C6H4)O(C6H5).

Table 2Amino acid composition of the S1′ loop in the different MMPs. For clarity, the first residue indicated is the Met residue of the “Met turn” (Met 236 inMMP-1 numbering). They are grouped by length and ordered by increasing size. Ni is the number of different residues in each position

236 237 238 239 240 241 242 243 244 245 246 247 248Ni 1 6 3 9 2 9 7 11 9 11 9 6 5 2 6 10 1MMP-1 M Y P S Y T F S G D V Q LMMP-2 M A P I Y T Y T K N F R LMMP-9 M Y P M Y R F T E G P P LMMP-11 M S A F Y T F R Y P L S LMMP-23 M H L N A T L T G W K A LMMP-19 M A P V Y E G Y R P H F K LMMP-20 M Y P T Y K Y K N P Y G F H LMMP-7 M Y P T Y G N G D P Q N F K LMMP-8 M Y P N Y A F R E T S N Y S LMMP-13 M F P I Y T Y T G K S H F M LMMP-14 M A P F Y Q W M D T E N F V LMMP-15 M A P F Y Q W K D V D N F K LMMP-16 M A P F Y Q Y M E T D N F K LMMP-24 M A P F Y Q Y M E T H N F K LMMP-3 M Y P L Y H S L T D L T R F R LMMP-10 M Y P L Y N S F T E L A Q F R LMMP-12 M F P T Y K Y V D I N T F R L LMMP-25 M R P F Y Q G P V G D P D K Y R LMMP-17 M R P Y Y Q G P V G D P L R Y G L

Fig. 2. Cartoon-type representation of several MMP/synthetic inhibitorcomplexes. For clarity only the full backbone of the MMP-1 catalyticdomain is shown in orange (structure 1hfc) and the catalytic zinc atom isshown in sphere representation (magenta). For all complexes, the inhibitorsare omitted. S1′ loops are colored in pink for MMP-2 (1qib), red for MMP-3(1biw), blue for MMP-7 (1mmp), yellow for MMP-8 (1mnc), salmon forMMP-11 (1hv5), cyan for MMP-12 (1jk3), lime for MMP-13 (456c) andgreen for MMP-16 (1rm8).

Fig. 3. Cartoon-type representation of several MMP-3/synthetic inhibitorcomplexes (1d7x, 1g05, 1d5j, 1bq0, 1d8f, 1d8m). The backbones are colo-red according to the b-factors from blue for the lower values to yellow for theupper value. The catalytic zinc atom is shown in sphere representation(magenta). The different inhibitors, all possessing an SO2(C6H4)OCH3 P1′group, are shown in stick representation and colored in red.


the different MMP complexes in Fig. 4, in which the inhibi-tors clearly harbor long P1′ side chains, shows that the posi-tion of the P1′ side chain is remarkably similar in these

complexes, all being aligned along the same axis in the S1′subsite. However, it should be noted that most of the P1′groups found in this set are substituted by a bulky group(phenyl or diphenyl piperidine), which is located at the en-trance of the S1′ subsite. Fig. 6 shows the orientation adoptedby the P1′ group in the complex of MMP-3 with a carboxylateinhibitor (1ciz [23]). In this structure, the plane of the bulkyP1′ group is aligned between residue Val 215 (MMP-1 num-bering), located on the helix II of the catalytic domain, andthe main chain atoms of residues at the top of the S1′ loop(residues 238–240; MMP-1 numbering). These observationssuggest that the steric hindrance imposed on the phenylgroup of these inhibitors by the MMPs residues could re-strain the position of the whole P1′ group. This may preventthe formation of tight specific interactions between the in-hibitor and some enzyme residues, because small adjust-ments in rigid structure are energetically unfavorable. On theother hand, the use of flexible long side chains also involvesextra energy costs, due to unfavorable entropic contributionassociated with the freezing of flexible structure between thefree and the bound state. In addition, the use of flexible unit inthe P1′ side chain of MMP inhibitors and their subsequentstudy in the bound state has revealed some unexpected re-sults. For example, the isobutyl group of the inhibitor incomplex with the MMP-3 do not adopt an extended confor-mation, but a folded one resembling the shape of a cyclo-hexyl ring (1g49, Table 1) [24]. In the same manner, thepropyl group of the inhibitor in complex with MMP-11(1hv5, Table 1) was observed to adopt an unanticipatedconstrained conformation [20]. Thus, a delicate balance be-tween the use of rigid units and groups allowing some degreeof flexibility in the P1′ side chain should be found. Anotherchallenge will be to use groups that both maintain a goodinhibitor solubility and form favorable interactions with theS1′ subsite. Given the high hydrophobicity of the S1′ cavity,this will not be a straightforward issue [26].

4. Future prospects

Structural information as provided by X-ray structures canbe used to improve the selectivity of inhibitors toward aparticular MMP by optimizing the fit and the interactions ofthe potential inhibitor with the target MMP. However, asdiscussed above, the presence of an inherent flexibility in theMMP active-site limits dramatically the accurate modelingof MMP–inhibitor complexes. This situation particularly ap-plies to the modeling of inhibitors in which the size of sidechain is designed to engage in specific interaction with resi-dues located in the segment 241–245 of the S1′ loop. Thissegment exhibits the highest sequence variability betweenthe different MMPs and thus constitutes a hot spot to developselective inhibitors of MMPs. However, this segment ishighly mobile in MMPs. Accurate predictions of bindingenergies in a context of protein mobility is a difficult task, asin most modeling approaches mobility and associated sol-

Fig. 4. Superimposition of several MMPs with synthetic inhibitors posses-sing long P1′ side chains. The inhibitors are shown in stick representation.The catalytic zinc atom is shown in ball representation (magenta). Only theinhibitors and the S1′ loop in cartoon representation are shown for thedifferent complexes. Color code: MMP-1 (966c) in red; MMP-3: 1c3i ingreen, 1ciz in cyan, 1g49 in orange; MMP-8: 1bzs in pink; MMP-11: 1hv5 inslate.

Fig. 5. Top view of the S1′ subsite in the complex of MMP-3 (1ciz). ResidueVal198 is shown in sphere representation and residues Pro221, Leu222 andTyr223 are shown in stick representation. The surface of these residues isshown in mesh type. For clarity, only the diphenylpiperidine group of theinhibitor is displayed.

Fig. 6. Occupation of the S1′ subsite in the complex of MMP-3 with acarboxylate inhibitor possessing a diphenylpiperidine side chain (1ciz). Themolecular surface is shown in mesh type. The inhibitor colored by atom typeis shown in stick representation.


vent effects typically suffer from approximations. In systemsthat feature multiple intermolecular interactions, the strengthof any given intermolecular interaction will depend criticallyon the context in which it is formed. In many cases, the freeenergy contribution that an individual interaction makes tothe stability of a complex as a whole can be significantlylarger than one might expect, based on the properties of thatinteraction studied in isolation. This phenomenon has beentermed positive cooperative effects. Many studies haveshown that experimentally observed cooperative effects canbe extremely difficult to explain, as such effects may dependon both subtle combination of enthalpic and entropic contri-butions [27,28]. In the case of the zinc metalloproteinaseACE, it has been demonstrated that the enthalpically unfa-vorable binding of the inhibitors lisinopril and captoprilresults from a favorable change in entropy related to thehydrophobic effect [29,30]. All these remarks suggest thatpertinent models of MMP–inhibitor complexes should takeexplicitly into account the occurrence of intermolecular mo-tion in the complex. Such modeling approaches should ide-ally be able to determine with good accuracy enthalpic andentropic contributions to the free energy of complexation.These contributions that correspond to a combination ofdifferent processes, involving the target and the ligand in thebound and the free state, but also the solvent, remain difficultto evaluate accurately. It should be kept in mind that aninhibitor selective for one particular MMP, as compared tothe others, by a factor of 1000 will give a binding free energylower by about 4 kcal/mol at 300 K for its preferred target ascompared to the others. Therefore, to be helpful in the devel-opment of selective compounds, modeling methods shouldideally have accuracy on the order of 1 kcal/mol, whichremains below the capabilities of the current computationalmethods used to estimate in silico binding free energy. De-velopment of the modeling approaches must continue toreach this level of accuracy. Furthermore, the reliability ofthese models will have to be checked by experimental stud-ies. Structural information is available for only nine MMPs(Table 1). As discussed above, of the 57 reported structuresalmost 38 correspond to complexes involving either MMP-3 or MMP-8. In many of these complexes, the P1′ position isoccupied by the chain side exhibiting a similar chemicalstructure (23 structures contain substituted sulfone). Ideally,3D-structures of MMPs absent from Table 1, in interactionwith inhibitors bearing longer P1′ side chains than thoselisted in Table 1, will be suitable for the development of newinhibitors. In addition, it should be noted that the databasecontains mainly hydroxamate compounds (34) that bind theMMPs on the primed side. There are only seven inhibitorsthat bind on the unprimed side of the catalytic domain andtherefore very little structural information is available re-garding the unprimed side of the catalytic site. Thermody-namic studies will also be necessary, and to our best knowl-edge no study has yet been estimated the entropic andenthalpic contributions associated with the formation ofMMP–inhibitor complexes. Given the flexible nature of the

MMP active-site, determination of these contributions to thefree energy of binding is essential to improving our under-standing of the factors controlling MMP selectivities. Moresystematic use of NMR analysis of MMP–inhibitor com-plexes in solution will also be required to characterize themotions existing both at the level of the inhibitor and theprotein in the complex. To date, only one NMR study insolution has been dedicated to identifying such motions [21].Interestingly, this study has demonstrated the occurrence oftwo conformations of the complex in solution (MMP-1 ininteraction with an inhibitor bearing a long side chain in theP1′ position), involving both slow and fast exchange betweenthese distinct conformations. In this provocative study, theauthors speculate that motions in this complex through favor-able entropic contributions compensate for the poor fit of thelong side chain in the P1′ position in the S1′ pocket ofMMP-1 and the resulting energetic cost of opening thispocket. Development of highly selective inhibitors for agiven MMP will need to combine sophisticated theoreticaland experimental approaches to decipher in each MMP thespecific structural and dynamic features that can be exploitedto obtain the desired selectivity. In the mean time, efforts inchemistry should be directed toward the sameobjective—inhibitor selectivity. As discussed above, a sig-nificant part of the efforts in chemistry in the MMP field havebeen focused on the oral bioavailability and metabolic stabil-ity of the inhibitors. These options were justified by the factthat most of these studies were performed in private compa-nies. Efforts in chemistry dealing only with the inhibitorselectivity are conceivable in public research institutes. Allresults obtained in this regard would be extremely useful inincreasing our understanding of key molecular interactionscontrolling MMP–inhibitor recognition. Strategies in combi-natorial chemistry can be proposed to check whether coop-erative effects in inhibitor binding can be exploited to iden-tify selective inhibitors. For instance, it would be interestingto see how the P1, P2′ and P3′ preferences of different MMPsevolve when the size of the side chain in the P1′ position issystematically increased. Recent advances in phosphinicpeptide chemistry should allow us to explore these possibili-ties to identify highly selective inhibitors of MMPs.

Acknowledgements

This work was supported by funds from the Commissariatà l’Energie Atomique and from the European Commission(FP5RDT, QLK3-CT02-02136 and FP6RDT, LSHC-CT-2003-503297. All figures were produced with the PyMOLSoftware v0.95 [69].

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