Targeting Matrix Metalloproteinases: Exploring the Dynamics of the S1

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Targeting Matrix Metalloproteinases: Exploring the Dynamics of the S1’ Pocket in the Design of Selective, Small Molecule Inhibitors Benjamin Fabre, Ana Ramos, and Beatriz de Pascual-Teresa J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500505f • Publication Date (Web): 29 Sep 2014 Downloaded from http://pubs.acs.org on October 5, 2014

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Journal of Medicinal Chemistry

Targeting Matrix Metalloproteinases: Exploring the dynamics of the S1’ pocket in the design of selective, small molecule inhibitors Benjamin Fabre†, Ana Ramos*, Beatriz de Pascual-Teresa* Departamento de Química y Bioquímica, Facultad de Farmacia, Universidad CEU San Pablo, Urbanización Monteprincipe, 28668 Madrid, Spain. KEYWORDS. Matrix metalloproteinases (MMP), selectivity, inhibition, flexibility, S1’ pocket.

ABSTRACT. Matrix metalloproteinases (MMPs) are important targets for pathological conditions such as arthritis, chronic obstructive pulmonary disease and cancer. The failure of the first broad-spectrum MMP inhibitors in clinical trials has led researchers to address the selectivity as one of their main objectives. The S1' pocket has been widely used to modulate the selectivity of these enzymes because it displays the highest variability in length and shape among MMPs. In this perspective, we encourage medicinal chemists to also consider the dynamics of this pocket as an important parameter to achieve the desired selectivity. To support this proposal, we collect examples from the literature where the flexibility of the S1’ pocket was highlighted as a relevant and significant issue affecting selectivity. We also review the experimental studies on the dynamics of this pocket.

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Introduction In the 90s, inhibition of matrix metalloproteinases (MMPs) aroused great expectations as a possible treatment for diseases such as cancer, emphysema or rheumatoid arthritis.1 However, clinical trials of the first generation of MMP inhibitors were interrupted due to side effects – mainly musculoskeletal syndromes (MSS) – that limited their dosage, and diminished their efficacy.2 The lack of selectivity of these inhibitors was considered the main reason for those side effects. For instance, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-14 have been classified as anti-target for cancer treatment,3 and MMP-1 and MMP-14 inhibition have been linked to the MSS.4, 5 As a consequence, an intensive search for selective inhibitors of MMPs has been pursued since then. As the S1’ pocket is both the most druggable and variable one (in shape and length) of the MMP catalytic center, it received much of the attention.6, 7 Despite more than thirty years of structure-activity relationship (SAR) efforts,8-14 developing selective MMP inhibitors is still a challenge. Few studies proposed that the flexibility of the S1’ pocket is partially responsible for the difficulty of designing selective MMP inhibitors.15-17 Here we proposed that the S1’ pocket flexibility is also an opportunity to reach selectivity. With this purpose, we first review the experimental information available on the dynamics of the S1’ pocket of MMPs, and then discuss the impact of the S1’ pocket flexibility on selectivity, based on examples gathered from the literature. 1. Structure of the catalytic domain of MMPs The vertebrate MMP family regroups 26 proteases, among which 23 are in human, numbered 1-3, 7-17, 19-21, 23-28. MMPs belong to the metzincins clan, which comprises endopeptidases that possess the conserved HExxHxxGxxH/D zinc binding motif and the invariant Met forming 1,4-ß-turn called “Met-turn”.18 The structural similitude to MMP-1, the presence of an N-


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terminus signaling sequence and the cysteine switch motif PRCGxPD are used to assign endopeptidases to the corresponding MMP family.19 The human MMP family is divided into six subfamilies according to their substrate specificity, their localization and their secondary structure; collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9), stromelysins (MMP-3, MMP-10, MMP-11 and MMP-27), membrane type MMPs or MT-MMPs (MMP-14 to 17, MMP-24 and MMP-25), matrilysins (MMP-7 and MMP-26) and others (MMP-12, MMP-19 to 21, MMP-23 and MMP-28). General structure of the catalytic domain: The main structure of the catalytic domain is similar in all MMPs, with a shallow catalytic cleft dividing the catalytic domain into two parts: the N-Term subdomain (NTS) in the upper part, and the C-Term subdomain (CTS) in the lower part (figure 1A). The catalytic domain contains five ß-sheets (ßI-V) and three α-helixes (αΑ−C) represented in figure 1C. The long and highly open loop between αB and αC-helixes, called the Ω-loop, links the NTS and CTS (figures 1B and 1C). Gelatinases (MMP-2 and MMP-9) additionally contains three Fibronectin II-like inserts in their catalytic domains.6 Two conserved zinc ions are present: the catalytic zinc ion that lies in the catalytic cleft and the structural zinc ion trapped in the S-loop. In active MMPs, the three His (His218, His222, His228 in full MMP-3 numbering) and solvent molecule(s) coordinate the catalytic zinc ion. A series of calcium ions (one to five) is also present in the experimental structures of MMPs. Removal of calcium ions from the full MMP-3 results in an inactive protease,20 showing the importance of those structural ions.



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Figure 1A-C. A) Surface of the MMP-2 catalytic domain (Fibronectin II-like insert is missing) in complex with a decapeptide inhibitor (PDB code: 3AYU21). Residues from this inhibitor are


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labeled in orange. The catalytic zinc ion is depicted in CPK representation, in magenta. B) The same complex seen from the back part of the S1’ pocket. Tyr3 from the decapeptide enters into the S1’ pocket. This orientation will be used for the following figures. C) 3D representation of the secondary structure of MMPs (the orientation of figure A is used). The secondary structure elements are labeled. The three His chelating the catalytic zinc ion are highlighted. The Ω-loop that partially delimits the S1’ pocket and the S-loop (between ßIII and ßIV-strand) that contains the structural zinc ion are also labeled. Catalytic cleft and S1’ pocket: The catalytic cleft is delimited by the ßIV-strand, the bulge-edge segment from the S-loop and part of the Ω-loop. The first two elements form its upper-rim, and the Ω-loop forms the bottom part (figure 1A and 1C). The catalytic cleft contains six binding subsites named S3-S3’ pockets, according to the convention for proteases established by Schechter and Berger.22 The S1’ pocket is the most prominent and the less solvent exposed, making it the most attractive to target. Furthermore, due to its variability in length and composition (see figure 2) this pocket has been mainly targeted to achieve selectivity. Three features characterize this pocket: -

The residue at position 214 (the numbering of full MMP-3 is used here for all MMPs): a Leu for the majority of MMPs except for MMP-1, MMP-7, MMP-11 and MMP-20, where bigger Arg, Tyr, Gln and Thr are found, respectively. In these four MMPs, the S1’ pocket is consequently smaller.

-

The “wall-forming-segment” formed by the conserved Pro-X-Tyr residues at positions 238-240.



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-

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The specificity loop extending from residues 241 to 250. The length and the amino-acid composition of this loop are considerably variable, but maintain a hydrophobic site. Showing diversity among MMPs, this loop has received much attention to achieve selective inhibition.

Accordingly, S1’ pockets of MMPs can be classified as small (MMP-1, MMP-7, MMP-11 and MMP-20), medium (MMP-2, MMP-8, MMP-9, MMP-12, MMP-14, MMP-16) and large (MMP3, MMP-10, MMP-13). Small pockets present a bigger amino acid at position 214, the medium size pockets bear a shorter specificity loop (1 to 3 residues shorter than MMP-3) and/or a long residue at position 241 (Arg in MMP-9, Lys in MMP-12) or 244 (Arg in MMP-8 and Met in MMP-14 and MMP-16) that partially closes the S1’ pocket. The sequence alignment of the important residues of the S1’ pocket for the twelve main studied MMPs is indicated in figure 2.

214

238

240

245

250 251

R P S Y T F S G D V Q MMP-1 L P I Y T Y T K N F R MMP-2 L P L Y H S L T D L T R F R MMP-3 Y P T Y G N G D P Q N F K MMP-7 L P N Y A F R E T S N Y S MMP-8 L P M Y R F T E G P P MMP-9 L P L Y N S F T E L A Q F R MMP-10 Q P F Y T F R Y P L S MMP-11 L P T Y K Y V D I N T F R MMP-12 L P I Y T Y T G K S H F M MMP-13 L P F Y Q W M D T E N F V MMP-14 L P F Y Q Y M E T D N F K MMP-16 L P T Y K Y K N P Y G F H MMP-20 Figure 2: Sequence alignment of the residues forming the S1’pocket of the most studied MMPs (numbering of the full MMP-3 is used). Strictly conserved residues are highlighted in red and highly conserved are highlighted in blue. The sequence alignment was taken from the literature.6, 23


L L L L L L L L L L L L L

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2. Protein dynamics and drug binding Protein dynamics: Proteins are dynamic molecules. Conformational dynamics are essential for their activity. For example, the mobility of the catalytic and hemopexin-like domains of the full MMP-1 is crucial to unwrap the triple helix of collagen and to allow its hydrolysis.24 Dynamics of proteins are also essential in molecular recognition events.25 The inhibitory activity of Imatinib - one of the reference examples of rational drug design in medicinal chemistry - lies in its ability to interact with the inactive conformation of Abelson (Abl) Tyrosine Kinase.26 This protein passes from the active to the inactive conformation depending on the conformation of the activation loop.27 The plasticity of this loop, together with the variability of the inactive states of kinases, are mainly responsible for the selectivity of Imatinib.27,28 In other words, the dynamics of Abl tyrosine kinase regulates the recognition of Imatinib. Ideally, the study of protein dynamics would enable us to observe the different existing conformers, to calculate their population and to measure the time scales necessary to pass from one to the other. We are still far from this, but with the progress in nuclear magnetic resonance (NMR), X-ray diffraction, small-angle X-ray scattering (SAXS) and computational techniques, we now possess more accurate and versatile tools to study the different motions - occurring at diverse time scales - in the proteins.29, 30 For instance, a combination of NMR, X-ray and SAXS experiments permitted to characterize a whole range of motions in MMP-12: fast movements of isolated residues, collective motions of residues from the Ω-loop and slower motions between the catalytic and the hemopexin-like domains.31, 32 Figure 3 gives a simplified representation of these motions and links them to the time-scales of protein dynamics: the rotation of a residue



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side chain (expected in the ps to low µs time scale), the collective motion of a loop (ns to µs) and other larger motions such as the reorientation of the catalytic and hemopexin-like domain (µs to low s). In this perspective, we focus on the motion of the S1’ pocket of MMPs. In this pocket, the most flexible part is the long Ω-loop. Accordingly, most of the motions described here correspond to the movement of a single residue or to the collective motion of a few residues composing this loop. The time scale for such motions goes from tens of ps to few µs. Two main experimental techniques allow characterizing such fast dynamics: NMR relaxation and X-ray diffraction. X-ray diffraction gives a “direct” and highly detailed picture (reaching atomic resolution) of the protein structure. Crystals for X-ray diffraction are obtained under nonbiological conditions (temperature, solvation, concentration) and lead to a single frozen structure of low energy. Comparison of two crystal structures of the same protein (e.g. between the apo and holo protein) gives insights into the protein flexibility. The B-factor (which corresponds to the mean square of atoms displacement in the crystal) also presents information on the dynamic of the protein.33 Non-homogenous structure trapped in a single crystal as well as fluctuation occurring in that crystal increase the B-factor. NMR spectroscopy permits the study of the protein in solution at physiological temperatures. However, the determination of the structure is indirect and requires the total assignment of the protein. A set of low-energy conformers is then built using the NMR data (distances and torsion angles in the protein). A major advantage of NMR relaxation is its sensitivity to nuclei motion that makes possible to observe ps to s motions. Fast motions (ps to ns) are characterized in amplitude by the general order parameter S2 (S2 was first introduced by Lipari and Szabo, and represents the degree of spatial restriction of internal motion. S2 varies from 0 to 1 for completely unrestricted and totally rigid motions,


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respectively).34-37 Slow motions (ms to s) are directly observed as distinctive signals for the same nuclei or group of nuclei. Intermediate motions (µs to ms) lead to line broadening of the signal. Molecular dynamic (MD) simulations cover motions up to tenths of µs, with the clear advantage of giving an atomic description of the movement. From this, an estimation of the kinetics (e.g. energy barriers) and thermodynamics quantities (e.g. conformer’s population) can be calculated.

Figure 3: Time scales of the full MMP-12 motions. Residues from the wall-forming segment and the specificity loop (orange) collectively moved as shown by NMR.31 The hinge region (red) that connects the catalytic domain and the hemopexin-like domain is highly flexible and permits



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the slow reorientation of these domains.32 The usual time-scales of protein motions as well as the main techniques used to characterize these motions are indicated.30 Conformational selection and induced-fit: Traditionally, medicinal chemists transcribe the impacts of protein mobility on drug binding through two mechanisms: conformational selection and induced-fit (figure 4A). In conformational selection, the compound (e.g. inhibitor) selects a specific conformation of the apo protein bringing about a change in the population of the preexisting conformers. In the induced-fit pathway, the binding induces the shift of few residues of the protein away from their natural conformations. Rather than opposing, these two models certainly cohabitate in the binding event.25, 38, 39 For instance, binding of Imatinib, which has been proposed to select the inactive conformation from those present in the apo Abl tyrosine kinase,40 may also induce the movement of few residues away from their canonic conformational substrates. Induced-fit motions are generally local and of small amplitude making difficult their use in structure-based drug design (SBDD) (even if they are more and more accurately included in computational techniques such as docking and MD41). In contrast, conformational selection has been used to design inhibitors that interact with (a) specific conformer(s) of a protein. The design of inhibitors of the inactive state of kinases is an example of this strategy.42 Importantly, such strategy allowed the discovery of inhibitors with remarkable selectivity and binding-kinetics. This is the case of inhibitors of the inactive conformation of the p38MAP kinase.43 We will briefly discuss that example as it reveals the relationship between protein conformation, kinetics of the inhibitor binding and selectivity.


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As in Abl tyrosine kinase, the p38MAP kinase (a Ser/thr kinase) presents two conformations: the active and the inactive one (respectively R and R* conformers in figure 4B). In the p38MAP kinase, the active conformation (R conformer in figure 4B) is more stable than the inactive one (R*). Pargellis et al. reported inhibitors of the inactive conformation of p38MAP kinase, which displayed slow binding to the protein, long residence time and high selectivity among kinases.43 They explained the slow binding character of those inhibitors by the conformational change required for the inhibitor to bind to the protein. They proposed that the inactive conformation is less frequently sampled than the active conformation (i.e. R* conformer is of higher energy than R, see figure 4B). This reduces the probability of the inhibitor binding and would explain its low on-rate (kon). The long residence time (the residence time is defined as 1/koff and represent the life time of the protein:inhibitor complex44) could be explained by the high-energy barrier to return from the stable protein/inhibitor complex (R*L) to the active conformer (R). Once the R*L complex is formed, the p38MAP kinase is trapped in the inactive form. Importantly drugs that possess long residence times may show higher in vivo activity and selectivity.44, 45

Figure 4: A) Model for the receptor-ligand binding via conformational selection (left path) and induced-fit (right path). R (receptor) stands for the protein and L (ligand) for the inhibitor. The kinetic rates for each equilibrium are marked. B) Energy landscape for the conformational selection path. In the case of the p38MAP kinase, the apo protein was proposed to pass from the low energy conformer R (the most populated one according to the Boltzmann distribution) to the



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higher energy conformer R*. The ligand then binds to the conformer R* and forms the R*L complex of low energy. The presence of flexibility in the binding site of a compound is often problematic for selectivity: a flexible binding site can accommodate ligands of varied shapes. Thus, if a family of proteins displays a highly flexible binding site, any ligands that bind to one of these proteins might bind to the other members of the family. In this perspective we propose that the mobility of the S1’ pocket of MMPs can be used to achieve partial or complete selectivity among the entire MMP family. In that sense, we think that the dynamics of this pocket deserves to be studied. In the first part we review the most meaningful experimental characterizations of the S1’ pocket mobility. The main evidences come from NMR experiments that allow the study of proteins in solution, although the comparison of various crystal structures from a single MMP gives also additional proofs of the flexibility of this pocket. 3. Experimental evidences for the flexibility of the MMPs S1’ pocket We considered the MMPs for which the flexibility of the S1’ pocket has been experimentally studied. The experimental data are classified by MMP. We decided to start with MMP-3 because a good amount of dynamic studies is available for this protein. MMP-3: Van Doren et al. first observed the flexibility of the MMP-3 Ω-loop. They determined the solution structure (NMR) of the catalytic domain of MMP-3 in complex with a peptidic hydroxamate inhibitor (compound 1, PDB: 1UMS. Refer to figure 10 for the structures of the compounds discussed in this section).46 In those experiments, the Ω-loop and the N-Term loop present the highest root mean square deviations (RMSD) and the most pronounced angular disorder. The Ω-loop adopts an unusual conformation - folded toward the S1’ pocket – and


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displays a different orientation and folding than the NMR structure of MMP-3 previously reported (PDB: 2SRT. In this structure MMP-1 is complexed with an inhibitor).47 Although these results support the flexibility of this loop, they must be taken with caution as the experimental conditions of the two NMR experiments differed in pH and Ca2+ concentration. For instance, computational studies showed that the number of calcium ions present in the MMP-2 catalytic domain influences the folding of the S1’ pocket.48 Gooley et al. showed the flexibility of the Ωloop by solving the structure of the MMP-3:2 complex both through NMR relaxation and X-ray diffraction (structures not register in the protein databank).49 The major differences between the X-ray and NMR structures stand in the long loops (N-Term loop, S-loop and Ω-loop). In particular, residues Tyr240-Arg248 from the Ω-loop displayed high RMSD (> 2 Å) and large Bfactor (22.8 Å2 compared to 11.5 Å2 for the rest of the residues). Latter, Pavlovsky et al. reported an “opening” motion of the S1’ pocket, triggered by residues Tyr240 and His241.50 They solved the crystal structures of MMP-3 in complex with compounds 3-6 (PDB: 1B8Y, 1CAQ and 1CIZ for compound 3 to 5 respectively. No structure for the MMP-3:6 complex has been deposited in the protein databank) and compared them to the crystal structure of the MMP-3:7 complex (PDB: 1SLN).51 Tyr240 and His241 adopt two different orientations depending on the size of the inhibitor. In the complex with 7, a ligand that bears a shorter P1’ fragment, the two residues partially obstruct the entrance to the S1’ pocket. By contrast, the long P1’ fragments of compound 3-6 cause the displacement of these residues towards the outside of the pocket (refer to figure 5 for position of Tyr240). In three other publications a comparison of the catalytic domains of apo MMP-3 and MMP-3 complexed with an inhibitor are reported. Chen et al. solved the crystal structures of the catalytic domain of apo MMP-3 and MMP-3 complexed with compound 8 (PDB: 1CQR and 1B3D



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respectively). Parts of the Ω-loops are strongly shifted between the two structures, which according to the authors accounts for the pronounced induced-fit effect observed for Tyr240. In two subsequent articles, scientists from Pharmacia and Upjohn (now Pfizer) used a unique approach to study MMP-3 without inhibitor in the S1’ pocket. They used P1-P3 inhibitors (inhibitors interacting only with the S1-S3 pockets of the protein).52, 53 First they determined the X-ray structures of the catalytic domain of MMP-3 in complex with the two P1-P3 inhibitors 9 and 10 (PDB: 2USN and 1USN respectively52) and compared them with the X-ray structure of MMP-3 in complex with the P1’ inhibitor 7 (PDB:1SLN).51 The Ω-loop adopts very different folding (figure 5): open with the S1’ inhibitor and packed toward the S1’ pocket with the P1-P3 inhibitors (showing a folding similar to the one described by Chen et al.54 and discussed above). Again, Tyr240 appears as a key residue and presents two conformations: open, when pointing towards the outside of the S1’ pocket, and closed when pointing towards its inside. This movement is accompanied by a shift of up to 7 Å on Leu239-Phe249 backbones. This example highlights the high flexibility of the Ω-loop. Nevertheless, the folding of the free S1’ pocket might be misleading and connected to the crystal packing forces (Pavlovsky et al. showed the sensitivity of this loop to crystal packing, although with much smaller shifts50). Scientists at Pharmacia and Upjohn deepened in this first study and characterized the dynamics of the S1-S3 and S1’-S3’ pockets of MMP-3 in complex with two P1-P3 inhibitors (10 and 11) and the P1’P3’ inhibitor 12.53 Making use of NMR techniques, they demonstrated that the S1-S3 pockets are mainly rigid, with order factors (S2 = 0.88 - 0.89) similar in the three structures. By contrast, residues Tyr240-Ser242 from the Ω-loop behaved differently in the three complexes. The backbone amide resonances of these residues are well defined in the MMP-3:12 complex, in which the inhibitor interacts with the S1’ pocket, but they are not assignable in the two


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complexes with P1-P3 inhibitors. The authors suggested that motions of the prime pockets in the µs to ms range may have led to line broadening of the signals. Interestingly, Sarver et al. previously demonstrated that the binding of inhibitors to non-prime pockets is enthalpy driven, whereas the binding to prime-pockets is both enthalpy and entropy driven.55 The relative rigidity of non-prime pockets and the flexibility of the prime pockets are in agreement with those results. The Ω-loop and, more precisely, the wall-forming segment and the specificity loop, is thus highly flexible in MMP-3. Tyr240 adopts various conformations and partially controls the opening of the S1’ pocket.

Figure 5. Movement of the Ω-loop in MMP-3. Superimposition of the structures of MMP-3 in complex with two P1-P3 inhibitors (9, PDB: 2USN, in grey and 10, PDB: 1USN, in white) and one P1’-P3’ inhibitor (7, PDB: 1SLN, in blue sea). The backbones of residues Pro238-Arg250 are depicted. The chemical structure of 7 is shown. MMP-1: In contrast to MMP-3, only very minor differences were observed by X-ray diffraction between crystal structures of apo and holo MMP-1 (PDB 1CGE and 1CGL respectively).56, 57 However, NMR relaxation experiments suggested the flexibility of the Ω-loop.



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Scientist at Wyeth studied the dynamics of the catalytic domain of apo and inhibited MMP-1. First, they reported the NMR assignment of the catalytic domain of apo MMP-1.58 Part of the specificity loop (Phe242-Gly245) appeared disordered, missing cross peaks in the 2D 1H-15N HSQC spectra. Furthermore, few residues of the Ω-loop (His228, Thr230, Asp231, Ile232, Thr241 and Leu251) experienced slow motion and appeared as doublets. Interestingly, these doublets merged to single peaks upon treatment with an inhibitor (structure not included), indicating the presence of a single conformer of this Ω-loop. Studying the fast dynamics of the protein (via the order parameter S2), the specificity loop appeared as the most flexible part (excluding the highly flexible N-Term part) with S2 values below 0.6 (S2 > 0.8 for the rest of the protein). In contrast to the case of the slower motions, the binding of the inhibitor did not affect the fast dynamics of the protein, as reflected by similar S2 values in both the apo and the holo proteins. The resolution of the solution structure of the MMP-1:13 complex corroborated these results (structure not deposed in the protein databank),59 as the authors reported similar S2 values for the Ω-loop residues.58 Later, scientists at Wyeth reported the solution structure of the apo catalytic domain of MMP-1.60 Since the residues from the specificity loop displayed the highest RMSD and standard deviation of the ψ and φ angles, the specificity loop appeared much more ordered than predicted by the dynamic studies discussed above. Lovejoy et al. effectively demonstrated the flexibility of the S1’ pocket of MMP-1. They determined the X-ray structure of the catalytic domain of MMP-1 in complex with 14 (PDB: 966C), an inhibitor with a long P1’ substituent, which inhibits this protein at unexpected low concentrations (Ki = 23 nM).61 They observed that Arg214 - that closes the S1’ pocket of MMP1 - is displaced to form a longer S1’ pocket and thus to allow the binding of that long inhibitor (see figure 6). Such “open” conformation was not observed for shorter P1’ inhibitors (PDB:


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2TCL).62 It is interesting to note that the movement of Arg214 was observed for both the apo form (PDB: 1AYK)60 and complexes with 13 (PDB: 4AYK).59 This suggests that 14 may select a conformation existing in the apo protein. In summary, the S1’ pocket of MMP-1 appears more rigid than the S1’pocket of MMP-3, with the exception of the movement of Arg214 that significantly changes the length of this pocket. However, such motion observed for compound 14 should be considered as an exception rather than the general rule; and inhibitors bearing long P1’ fragments usually spare MMP-1, which constitutes an advantage since MMP-1 inhibition has been linked to the MSS.

Figure 6. Movement of Arg214 in MMP-1. Superimposition of the X-ray structures of MMP-1 in complex with 15 (sea blue, PDB: 2TCL) and with 14 (tan, PDB: 966C). In the presence of 14, the Arg214 side chain rotates and opens the S1’ pocket. MMP-2: Less experimental data are available for MMP-2. Feng et al. reported the solution structure of MMP-2 in complex with 16 (PDB: 1HOV), an hydroxamate inhibitor with a long and flexible P1’ fragment.63 The residues of the Ω-loop showed moderate mobility apart from Lys245 that was highly disordered. Comparing these solution structures with the two crystal structures available at that time (1CK764 and 1QIB65), the authors reported a shift in the position



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of Phe249 and Arg250. They proposed that Phe249 moves due to the presence of the long npentyl chain of 16 (figure 7).

Figure 7. Movement of Phe249 and Arg250 in MMP-2. Superimposition of the model 7 (11 conformers were submitted in the protein databank, named model 1-11) of the NMR structures of the MMP-2:16 complex (1HOV, sea blue) with the X-ray structure of MMP-2 in complex with 17 (the inhibitor is not shown. PDB: 1QIB). 17 bears a short iPr group in P1’ position. In contrast, 16 contains a long P1’ chain, which induces the displacement of Phe249 and opens up the S1’ pocket. MMP-7: Scientists at AstraZeneca reported the crystal structures of two compounds (18 and 19, obtained from HTS) complexed to MMP-7 (PDB: 2Y6C and 2Y6D for 18 and 19, respectively).66 18 shows partial selectivity and moderate inhibition of MMP-7 (Ki = 10 µM), whereas 19 is a potent broad-spectrum MMP inhibitor (Ki = 79 nM against MMP-7). The potent activity of 19 against MMP-7 is surprising as this compound bears a long P1’ chain that was not predicted to fit into the small S1’ pocket of MMP-7. The crystal structures revealed that, as in MMP-1, the residue at position 214 (Tyr) is mobile and rotates to open the S1’ pocket (figure 8). Furthermore, several residues of the specificity loop gave poor electron densities in the two structures, which suggest their mobility.


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Figure 8. Movement of Tyr214 in MMP-7. Superimposition of the X-ray structures of MMP7:18 (2Y6C, tan) and MMP-7:19 (2Y6D, sea blue) complexes. The long P1’ chain of 19 causes the rotation of Tyr214, opening the S1’ pocket. Several residues of the specificity loop gave unclear electron densities and are not defined in the original structures. MMP-8: MMP-8 presents a medium size S1’ pocket, partially closed by Arg244 (PDB: 1KBC).67 In the crystal structure of the apo catalytic domain of MMP-8 (PDB: 2OY4),68 this residue points toward the S1’ pocket (see figure 9 and 12 for the position adopted by this residue). In the crystal structures of MMP-8:20 (PDB: 1ZS0 and 1ZVX for respectively the S and R enantiomers)69 Arg is displaced towards bulk water to accommodate the long and rigid P1’ chain of 20. In contrast to the X-ray and NMR structures of the apo catalytic domain of MMP-3 (where the Ω-loop tends to collapse; see figure 5), the Ω-loop of the surrogate for apo MMP-8 (MMP-8 complex to a P1-P3 inhibitor. PDB: 1I73) adopts a similar conformation compared to protein complex to a P1’-P3’ oriented inhibitor (PDB: 1I76).70 Interestingly, the original binding mode of the barbiturate inhibitor 21 (IC50 = 1.7 µΜ against MMP-8) to MMP-8 revealed that the wall-forming segment of this protein is flexible.71 Scientists at Roche reported that in MMP-8:21, residues Pro238-Asn239 rotate (~100°) from the position



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observed in other X-ray structures of MMP-8. They explain this movement by an electronic repulsion between one of the carbonyl groups of 21 and the carbonyl group of Pro238 backbone (figure 9).

Figure 9. Mobility of the wall-forming segment in MMP-8. Electronic repulsion between one of the carbonyl groups of 21 and the carbonyl of Pro238 backbone leads to the shift of Pro238Asn239 in MMP-8:21 complex (1JJ9, sea blue). The crystal structure of MMP-8 in complex with 22 (PDB: 1JAQ, tan)72 is superimposed for comparison. The 2-(piperidin-4-yl)ethanol fragment of 21 has been removed for clarity purpose. From one complex to the other, the carbonyl group of Pro238 rotates by ~95°, leading to a shift of ~2.3 Å between the two carbonyl oxygens. In the MMP-8:21 complex, the S1’ pocket is almost unoccupied and Arg244 points towards the inner part of this pocket. MMP-9: No clear evidences of the flexibility of the MMP-9 S1’ pocket is available. Rowsell et al. reported the crystal structure of MMP-9 in complex with a P1' inhibitor (PDB: 1GKC).73 Arg241 displayed poor electron density, suggesting its mobility. Latter Maskos et al. solved the crystal structures of MMP-9 in complex with five inhibitors (all S1’-S3’ oriented and with P1’ fragments of variable lengths. The chemical structures are disclosed in supporting information.


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PDB: 2OVX, 2OVZ, 2OW0, 2OW1, 2OW2).74 The main difference observed in the S1’ pocket was the position of Arg241. This residue is also poorly defined, which prevents direct observation of its side-chain. However, based on the position of the α and β carbons (which are better resolved), the authors concluded that Arg241 is either oriented towards the S1’ pocket (and partially closes it), or displaced towards the outside of the pocket. This “gatekeeper” movement is comparable to the motion of Arg244 in MMP-8. MMP-12: A large amount of experimental data on MMP-12 dynamics comes from the extensive work carried out at the CERM, in Italy. Bertini et al. gave clear evidences of the mobility of the Ω-loop in MMP-12. They solved the crystal structure of the MMP-12:23 complex (PDB: 1Y93).31 Compound 23 lacks P1’ group and therefore the MMP-12:23 complex is a surrogate for the apo MMP-12. They also solved both the crystal and NMR structures of the MMP-12:24 complex (PDB: 1RMZ and 1YCM respectively).31 As in MMP-1 and MMP-8, the binding of the P1’ inhibitor (24) left the Ω-loop mainly unchanged; this loop adopts a similar conformation in the crystal structures of the apo (MMP-12:23) and the inhibited protein. Only residues Pro238 and Asn239 are slightly shifted, describing a motion similar to the one observed in the MMP-3 complex with the barbiturate inhibitor 21 (discussed above). By contrast, when comparing the structure of the MMP-12:23 complex with the crystal structure of the MMP-12:17 complex75 (PDB: 1JK3. The two structures are at atomic resolution; 1.03 Å and 1.09 Å respectively), they observed a different conformation of residues Asp245-Arg250 (specificity loop) with a shift of up to 4 Å on theirs backbones. In parallel, those residues lack long and short range Nuclear Overhauser Effects (NOE) in the NMR spectra of the MMP-12:24 complex, which can be interpreted as a sign of a disordered structure. They further studied the dynamic of this loop in the MMP-12:24 complex, using NMR relaxation. In accordance with the 4 Å shift in



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the backbone position observed in X-ray diffraction, the specificity loop backbone experienced motion in a time scale estimated between ~1 ns to ~10 µs. Such a time scale motion suggests the existence of collective motions in the Ω-loop and leads to the following question: does the 4 Å backbone shift observed by X-ray diffraction cover the whole amplitude of the motion characterized by NMR? The answer remains still unknown, but such a collective motion impacts the binding of the inhibitors and without any doubt should be taken into account in any SBDD approach. MMP-13: MMP-13 has the largest S1’ pocket within MMPs,6, 61 which makes it a suitable target for selective inhibition. Two NMR structures of MMP-13 are available, one in complex with 25 (PDB: 1FM1)76 and other in complex with 13 (PDB: 1EUB).77 In the MMP-13:25 complex, the Ω-loop appears relatively rigid (order parameter S2 in the range of the average value for the entire protein and clear 1H-15N HSQC signals), which differs from the NMR studies of MMP-1, MMP-2, MMP-3 and MMP-12 discussed above. The same year, Zhang et al. reported the solution structure of the MMP-13:13 adduct.77 By contrast, the specificity loop of MMP-13 exhibits flexibility: the order parameter values for residues Lys246-Met250 are similar to those calculated for MMP-1:13 complex (S2 < 0.6)59, and residues Thr244-Phe249 are poorly defined. In particular, the backbones of residues Thr244 and Gly245 could not be assigned due to weak signal intensity, suggesting the mobility of these residues. In this NMR structure, the Ωloop folds towards the S1’ pocket and adopts a conformation close to that observed for MMP-3 in figure 5.


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Comparison of the two NMR experiments involving MMP-13 puts forward the impact that different inhibitors have on the Ω-loop dynamics which, in turn, is expected to affect their binding entropies. MMP-20: Arendt et al. reported the solution structure of MMP-20 in complex with 24 (PDB: 2JSD).23 The Ω-loop presented lower fast motions (order parameter in the 0.8-0.9 range) than in MMP-1, MMP-2, MMP-3, MMP-12 and MMP-13. This comparison is particularly meaningful for MMP-3 and MMP-12, whose complexes with 24 were studied by NMR.78,31 In these two proteins, the order parameters of the Ω-loop residues were below 0.6. All data presented here highlight the fact that the S1’ pocket of MMPs is far from being rigid and cannot be described by a single conformer. These data suggest that particular elements account for the flexibility of the S1’ pocket: 1) the “gate keeper” residues at position 214 (MMP1 and MMP-7), 241 (MMP-9) and 244 (MMP-8); 2) few residues from the specificity loop, as already mentioned by Bertini et al.31 One may wonder if the information covered above is useful for the design of more selective inhibitors. The answer is not trivial. Motions of large amplitude, such as the motions in the activation loops of kinases, can be explicitly introduced in a SBDD program by considering either the activated or the inactivated form of the protein. The movements presented here are of smaller amplitude. Furthermore the case of MMP-13 demonstrated that the flexibility of the S1’ pocket observe experimentally depends on the inhibitor used. Additionally, the amount of data available greatly varies between MMPs, which prevents global conclusions. How to hit a moving target and which motion appears difficult to predict? To help us addressing this question, we will now discuss the most striking examples



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where the flexibility of the S1’ pocket had a negative or positive impact on the discovery of selective MMP inhibitors.

Figure 10. Structures of the compounds discussed in section 3. The coordination modes for the different types of zinc-binding group (ZBG) are indicated.


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4. Impact of the S1’ pocket mobility on the inhibitor selectivity Negative impact Certainly the most straightforward effect of the flexibility of the binding site is that it can accommodate a large variety of ligands with lower energy cost. One of the most striking example in MMPs is the unexpected binding of 14 to MMP-1 (see above).61 Inhibitor 14 contains a long and semi-rigid P1’ chain designed to avoid the inhibition of MMP-1 which is associated with the MSS observed with broad-spectrum MMP inhibitors in clinical trials.2, 79, 80 However, the shift of Arg214 opens the S1’ pocket of MMP-1, making space available for the binding of 14 (figure 6). Similarly, Tyr 214 of MMP-7 moves to open the S1’ pocket and permits the binding of the long P1’ chain of compound 19 (figure 8).66 Scientist at Wyeth faced a similar situation when designing inhibitors selective for MMP-13 over MMP-1.16 Compound 26 (see figure 13 for the structures of the compounds discussed in this section) displayed a high inhibitory activity against MMP-1 (IC50 = 40 nM) despite its long and rigid P1’ fragment. Although the same opening movement of Arg214 is certainly at work, they made an additional observation. They compared the 2D-NOESY spectra of MMP-1:26 and MMP-13:26 complexes. The isopropyl group present in 26 experienced a slow exchange between two conformations (characterized by two peaks in the NMR spectra) in MMP-1, while this was not observed either in MMP-13 or in the inhibitor alone in solution. The butynyl group presented also two orientations, which were quickly inter-converted. They concluded that these movements overcome the energetic cost derived from opening the MMP-1 S1’ pocket, through entropic contributions. This example clearly demonstrates the importance of both protein and



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inhibitor dynamics in the binding event and how this fact can constitute a drawback in the process of finding selective inhibitors. In the three examples above, NMR and X-ray diffraction experiments showed that the flexibility of the S1’ pocket challenges the design of selective inhibitors. Although in many other cases the effect of the flexibility of the S1’ pocket could not be experimentally observed, it is probably involved in the difficulty to achieve selectivity. Positive impact First of all we would like to stress that attempting to attribute the selectivity of one compound to the flexibility of a few residues in the proteins is challenging. In MMPs, the selectivity has been mainly justified by the difference of size and shape of the S1’ pocket. Here we report cases where the selectivity of inhibitors has been reasonably attributed, at least partially, to the flexibility of the Ω-loop. Scientists at Roche proposed that the difference in flexibility of residues Pro238-Asn239 accounts for the MMP-8/MMP-3 selectivity displayed by 21 (IC50 = 30 µM and 1.7 µM against MMP-3 and MMP-8, respectively).71 In the crystal structure of the MMP-8:21 complex (PDB 1JJ9), these residues are shifted from their usual position (the position reported in other X-ray structures). As explained above, the electrostatic repulsion between the carbonyl groups of the inhibitor and the Pro238 backbone provides a reasonable explanation for this movement (figure 9). In MMP-3, H-bond networks between His241 and the backbones of Thr232 and Leu239 rigidify this region. The binding of 21 is thus expected to involve an energy cost that could explain the lower potency of this inhibitor against MMP-3.


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In our research group, we are interested in the development of MMP-2 inhibitors that spare MMP-9.14, 81 This is a difficult task as both proteins present high similarity in their catalytic clefts. We first reported the discovery of compound 27, that showed moderate MMP-2/MMP-9 selectivity (IC50 = 1.7 nM and 43.9 nM against MMP-2 and MMP-9, respectively).82 Due to the difficulty to obtain a crystal structure of this compound in complex with MMP-2, we decided to carry out MD simulations with the aim of understanding the selectivity displayed by this compound. By running MD simulations for the catalytic domains of MMP-2 and MMP-9 both apo and inhibited by compound 27, we observed a major difference of flexibility of the specificity loop in the two proteins: almost rigid in MMP-9 and flexible in MMP-2.83 We proposed that this could be caused by the replacement of the flexible Phe249-Arg250 sequence of MMP-2 (figure 7) by a rigid Pro249-Pro250 sequence in MMP-9. Additionally, we observed better polar interactions between the sulfone group of 27 and the backbone of Leu181 and Ala182 (two conserved residues in MMPs) in the MMP-2:27 trajectory. This interaction is of primary importance and orients the P1’ fragment of the inhibitor toward the S1’ pocket.84 We hypothesized that the higher flexibility of the Ω-loop of MMP-2 allows a better fit of compound 27 into the S1’ pocket, leading to stronger polar interactions at the catalytic cleft. Based on this hypothesis, we developed derivatives of 27 bearing bulkier groups at the specificity loop region. We discovered compound 28, a potent inhibitor of MMP-2 (IC50 = 3.0 nM) with a remarkable selectivity over MMP-9 (IC50 = 346.4 nM). Non zinc-chelating derivatives are the most promising inhibitors of MMPs because they permit exploration of the S1’ pocket and show high selectivity.7 Non zinc-chelating inhibitors have been reported for MMP-8,85 MMP-1286-88 and MMP-1389-99. The enthusiasm for non zinc-chelating inhibitors started with the discovery of compound 29 at Sanofi laboratories.90 This compound



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inhibits MMP-13 at single-digit µM concentrations but does not inhibit the other MMPs tested (MMP-1,-2,-3,-7,-8,-9,-10,-12,-14,-16) at 100 µM. X-ray structure of the MMP-13:29 complex (PDB: 1XUR) revealed the origin of the selectivity: compound 29 opens a non-canonical S1’ pocket (also named S1’*, refer to figure 11) specific to MMP-13.90 The authors proposed that the inhibitor selects a conformation already existing in the apo MMP-13 (conformation selection) and not reachable in the other MMPs. In the MMP-13:29 complex, Gly245 adopts a conformation energetically unfavorable for non-Gly residues. Among MMPs, only MMP-1 presents a Gly at this position (see figure 2) but MMP-1 S1’ pocket is too small to accommodate compound 29. From 29 they developed 30, a highly potent (IC50 = 8 nM) and specific inhibitor of MMP-13,90 demonstrating the potential of such non-zinc chelating derivatives. Two years later, scientists at Pfizer reported the crystal structures of the non-zinc binding derivatives 31 and 32 in complex with MMP-13 (PDB: 2OW9 and 2OZR respectively).91 These potent inhibitors of MMP-13 (IC50 = 30 nM and 0.67 nM for 31 and 32 respectively) also displayed very high selectivity over the other MMPs (IC50 > 30 µM against MMP-1,-2,-3,-7,-8,-9,-12,-14,-17). In these structures, Gly245 adopts the same conformation and the inhibitor fits into the S1’* pocket.

Figure 11. S1’* pocket of MMP-13. In the MMP-13:29 complex (sea green, PDB: 1XUR), compound 29 fits into the S1’* pocket opened thanks to the orientation of Gly245 (blue). In the crystal structure of the full MMP-3100 (tan, PDB:4FVL), Thr244 closes the access to this pocket.


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Pochetti et al. also observed such an S1’* pocket in MMP-8.85 They reported the crystal structures of the non-zinc chelating inhibitors 33 and 34 complexed with MMP-8 (PDB: 3DPE and 3DNG respectively for 33 and (S)-34). These compounds are nM inhibitors of MMP-8 and MMP-13 with very high selectivity over the other MMPs tested (for compound 34, IC50 > 2.5 µM against MMP-1,-2,-3,-7,-9,-12,-14). In the two structures, Tyr248 adopts a different conformation than the one observed in the other experimental structures of MMP-8, and opens an extra binding pocket at the bottom part of the S1’ pocket (figure 12). Consequently, residues Arg244-Tyr248 adopt an orientation previously unobserved. The high selectivity over MMP-3 was not expected, as this protein presents a larger and flexible S1’ pocket. The authors proposed that the conformation of the specificity loop observed in MMP-8 is not reachable in MMP-3 without perturbing the H-bond network observed between the inhibitor and the MMP-8 backbone. Non zinc-chelating inhibitors of MMP-12 were reported but no S1’* pocket was observed.86-88

Figure 12. S1’* pocket of MMP-8. Superimposition of the crystal structures of MMP-8 in complex with a phosphonate inhibitor (tan. The inhibitor is not displayed. PDB: 1ZVX69) and with 33 (the inhibitor is in blue and the protein in sea green. PDB: 3DPE). In MMP-8:33 complex, a new subpocket opens due to the movement of Tyr248.



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These highly selective inhibitors are promising, as they may avoid the MSS observed in clinical trials with broad-spectrum inhibitors.2 32 and 35, two potent and highly selective inhibitors of MMP-13, lacked MSS in rats.91,92

Figure 13. Structures of compounds discussed in sections 4 and 5. 5. Perspectives and future opportunities MMPs are important targets in several pathological conditions such as chronic obstructive pulmonary disease, cardio vascular/congestive heart failure, arthritis and cancer.101 The first MMP inhibitors enrolled in clinical trials failed at showing efficacy, presumably due to their lack of selectivity. Here we defend that the flexibility of that pocket should be considered to rationalize the inhibitors selectivity and to design new drugs.


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How to use the flexibility of the S1’ pocket to design more selective compounds? It is unclear how to use the knowledge accumulated on S1’ pocket flexibility (and compiled in section 3) directly in a drug design program, but clearly the S1’ pocket flexibility impacts the inhibitor selectivity. There is still some way to go before we get a detail understanding on the impact of protein dynamics on the binding event. However, it is essential as it should help us to predict the activity and the selectivity of our compounds in vivo.44 We propose here few general tools to explore the dynamic of the binding pocket and to include its flexibility in the discussion of drugs activity and selectivity. First of all, there is a need to explore the dynamic of the S1’ pocket. Three technics – X-ray diffraction, NMR relaxation and MD simulation – give complementary information about protein dynamics. The most relevant one is NMR relaxation, which permits to directly observe the protein in solution and to record motions at various time scales. However data interpretation is often difficult and requires the assignment of the protein. Furthermore, once the motion of particular residues has been characterized, the structural description of that motion is challenging. In that sense, X-ray diffraction and molecular modeling nicely complements NMR experiments. The work reported by Bertini et al. on MMP-1231 and discussed above, exemplify this complementarity. The motion of the MMP-12 Ω-loop was characterized by NMR, the comparison of two X-ray structures of MMP-12 allows proposing amplitude for that motion and to visualize the displacement of the residues involved. Díaz et al. showed how MD simulations could complement NMR relaxation or X-ray diffraction data.102 During MD simulations, they observed the rearrangement of the catalytic and hemopexin-like domains of MMP-2, consistent with the experimental observation made for MMP-9 (these MMPs with closely related structures). Importantly, MD simulations allow scientists to propose an origin for the motion, to



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draw its free energy landscape (and thus discuss the thermodynamic and kinetics of the motion) and to visualize the motion. The choice of the inhibitor scaffold is also important to explore the S1’ pocket. Non-zinc chelating inhibitors (which show in vitro activity comparable to hydroxamate derivatives!) present a considerable advantage as that they escape from the classical binding mode of zincbinding inhibitors. Additionally ZBGs usually suffers from low selectivity, toxicity (especially hydroxamate) and poor pharmacokinetics properties. The presence of a ZBG and a H-bond acceptor group (sulfone/sulfonamide/carbonyl) that establishes polar contacts with the conserved Leu181 and/or Ala182, orients this type of inhibitors into a particular pose. The removal of these two structural features enables reaching S1’ regions not sufficiently explored, despite the massive amount of structure-activity relationship studies reported. Yet, the non-zinc binding inhibitors reported to date are the result of high-throughput screening (HTS) of compound collections. Exploration of the chemical space might help us to discover new conformations of the S1’ pocket of MMPs. The next step is then to find MMP structures and/or pharmacophores, which permit the design of potent, non-zinc binding inhibitors. This is conceivable for MMP-13, for which various non-zinc binding inhibitors have been reported, as well as few crystal structures of the corresponding complexes. Considering the experimentally and theoretically observed flexibility of the S1’ pocket, we propose that any SBDD project should not be based on a single 3D-structure of the target MMP. For instance, if docking simulations are part of the design process flexible protocols103, 104 or ensemble docking105 should be implemented. Implementation of flexibility in molecular modeling gained much attention and the most significant advancements in that field have been reviewed elsewhere.106-109 MD is the method of choice to consider to flexibility of both the


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ligand and the protein, and it may be the best tool to compare the dynamics of two proteins. In our work, MD simulations of both the apo and holo forms of MMP-2 and MMP-9 gave precious information on the S1’ pocket dynamic of those two proteins. Usually MD simulations are run for a single protein-inhibitor complex to rationalize the in vitro activity of that particular inhibitor. Modeling also the apo protein in solution could help us understanding how the binding of the inhibitor affects the protein motion. Importantly, it should drive us to consider the entropy contribution to binding. Entropy is not directly measurable and is still difficult to predict by computational techniques for such complex systems. However, entropy is expected to play an important role in such flexible proteins, as demonstrated recently for phosphonate inhibitors of MMP-12.110 Medicinal chemists started to shift the way they considered the binding of a molecule to a protein: rather than static it is highly dynamic. The increased interest for the kinetic properties of drugs highlights this change.44, 45 Ki or IC50 values are generally used to characterize the in vitro activity and selectivity of inhibitors. However, those are equilibrium values that lack the kinetics and do not reflect the dynamic nature of the binding event.45 Determination of the association and dissociation rate constants (kon and koff), together with the classical concentration values (Ki or IC50), would be useful to discuss the influence of the receptor and/or the inhibitor flexibility on the binding process. This is particularly true for flexible target such as MMPs. Two classes of MMP inhibitors will help us to demonstrate the potential of studying kinetics. First we saw that the selectivity of non-zinc binding inhibitors stands in the ability of those inhibitor to interact with the non-canonical S1’* pocket of MMP. Two limit mechanisms described the binding of such compound to MMP: the induced-fit effect and conformational selection pathways. Inducedfit effects are usually responsible for the conformational changes promoted by such small



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inhibitors in a protein.45 We could thus hypothesize that induced-fit effects mainly explained the creation of the S1’* pocket. Once the inhibitor (L) binds to the MMP (R) the S1’* opens, which leads to a new conformer R* of the MMP, as shown above: R+L

RL

R*L

R* + L

Once the R*L complex is formed (complex in which the inhibitor interact with the S1’* pocket of the MMP), the inhibitor could be released from the protein either directly from the R*L complex or via the RL complex, after isomerization of the protein (back to its canonical form). Longer residence time could then be expected for the R*L complex compared to MMPs that lack the R* isomer (no S1’* pocket). The occupancy of that MMP (MMP-13) would then be higher than the occupancy of the other MMPs in in vivo experiments (a condition called “temporal target selectivity”45). This explanation is hypothetic but shows us how kinetic parameters could help us understanding the dynamic nature of the binding event. The second example is the case of the thiirane inhibitor SB-3CT (36) that displays partial selectivity for gelatinases (MMP-2 and MMP-9).111 In that compound, the thiirane ring acts as a latent zinc binding group, which opens in the MMP catalytic center and release a highly potent thiolate specie (refer to figure 13).112, 113 This chemical transformation could explain the slow-binding of 36 to gelatinases.113 The origin of the selectivity of that inhibitor toward other MMPs (such as MMP-3, which bears a rather long S1’ pocket) remained unknown. A possible explanation can be found in the dynamic of both the inhibitor and the protein. A particular conformation of the inhibitor into the MMP catalytic cleft is required for the reaction to take place.113-115 In MMPs where such conformation is poorly populated, the opening rate might be too low (compare to the residence time) for the reaction to take place. This may explain the very low apparent on-rate (kon) of 36 in MMP-3 (its kon for MMP-3 is more than 600-times smaller than for MMP-2111). Studying in parallel the kinetic of


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this type of inhibitors, together with the dynamics of their complexes with MMPs (via MD simulations for instance) should help to understand their selectivity. A very important point of studying the kinetics of MMP inhibitors is that an inhibitor that would show longer residence time for a particular MMP, should have better in vivo selectivity. Such inhibitors might constitute very powerful tools for target validation. Conclusion From the examples collected from the literature in this perspective article, we conclude that the S1’ pockets of MMPs are flexible and their dynamic should be considered to justify and improve the selectivity of MMP inhibitors. The recent discovery that the presence of a ZBG is not mandatory to obtain potent MMP inhibitors, together with the differences in flexibility of the S1’ pockets among MMPs, opens up new opportunities for the development of new drugs, based on the inhibition of one particular MMP and minimizing the side effects observed with broadspectrum inhibitors. ASSOCIATED CONTENT Supporting Information. The resolutions of the PDBs discussed here as well as the structures of the inhibitors present in the PDBs 2OVX, 2OVZ, 2OW0, 2OW1 and 2OW2 PDBs are given in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * B. de Pascual-Teresa: phone, (+34) 913724724; fax, (+34) 913510496; e-mail, [email protected]. A. Ramos, email: [email protected]



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Present Addresses † Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic v.v.i. Flemingovo nam. 2, Praha 6166 10, Czech Republic. Funding Sources This work was supported by the Spanish Ministry of Science and Innovation (CTQ2011-24741). ACKNOWLEDGMENT Grant to B. F. from Fundación Universitaria San Pablo CEU is acknowledged. ABBREVIATIONS C-Term, C-terminal; CTS, C-Term subdomain; HSQC, heteronuclear single quantum coherence; HTS, high-throughput screening; MMP, matrix metalloproteinase; NNGH, N-Isobutyl-N-(4methoxyphenylsulfonyl)glycyl hydroxamic acid; NOESY, nuclear Overhauser effect spectroscopy; N-Term, N-terminal; NTS, N-Term subdomain; RMSD, root mean square deviations; SAR, structure-activity relationship; SAXS, small-angle X-ray scattering; SBDD, structure-based drug design; ZBG, zinc binding group. BIOGRAPHIES Benjamin Fabre received his M.Sc. degree in organic chemistry from the École Normale Supérieure de Chimie de Montpellier (2009, France). During his studies he undertook a one-year internship at GlaxoSmithKline’s drug discovery site in Stevenage (U.K.). He completed a Ph.D. in medicinal chemistry – working on matrix metalloproteinase inhibitors - at the Universidad CEU San Pablo (2013, Spain), under the supervision of Ana Ramos and Beatriz de PascualTeresa. This work was awarded the “Premio Ramón Madroñero” given by the Spanish society of


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medicinal and therapeutic chemistry (SEQT) for excellent junior scientists. He is now carrying out a postdoctoral work at the Institute of Organic Chemistry and Biochemistry in Prague (Czech Republic), in the laboratories of Jiři Jiráček. Ana Ramos is Professor of Organic Chemistry at San Pablo CEU University (Madrid). She earned her B.S. (1979) and Ph.D. (Extraordinary Ph.D. Award, 1985) from the Universidad Complutense de Madrid, and conducted postdoctoral studies at University of Dundee (UK) in the area of Organic Photochemistry and visiting Research Fellow at the University of Bath (UK) during a sabbatical period (2004-2005). Since 1996 she conducts a research program that encompasses the design and synthesis of new antitumor agents using different targets, such as DNA (naphthalimide based intercalators), cyclin dependent kinases type 2 (CDK2 inhibitors), PAMP and adrenomedullin (positive and negative modulators), metalloproteinase type 2 (MMP2 inhibitors), Estrogen Receptor (SERMs) and selective CK2 inhibitors. Beatriz de Pascual-Teresa is Professor of Organic Chemistry at San Pablo CEU University (Madrid). She earned her B.S. in Pharmacy (Extraordinary B. S. Award, 1987) and Ph.D. (Extraordinary Ph.D. Award, 1991) from the Universidad de Salamanca, working in the field of natural products. She completed her research training through predoctoral and postdoctoral stays at Louisiana State University (LSU, Baton Rouge, USA), University of California, Los Angeles (UCLA, Los Angeles, USA), Universidad de Alcalá (Alcalá de Henares, Spain) and BASF AG (Ludwigshafen, Germany). She first focused in the stereoselectivity of organic reactions and latter in computer aided drug design. Since 1996 she participates in research projects in the field of medicinal chemistry involving different drug targets, leading the computer aided design of antitumor agents.



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Targeting Matrix Metalloproteinases: Exploring the Dynamics of the S1

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