Inhibition of MMP-1 and MMP-13 with phosphinic acids that exploit binding in the S2 pocket

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.     

Novel matrix metalloproteinase inhibitors: generation of lead compounds by the in silico fragment-based approach

Generation of structurally new matrix metalloproteinase inhibitors was successfully carried out using an in silico technique. In order to identify the small fragment interacting with residues in the S1' pocket of MMP-1 through hydrogen bonds, we performed in silico screening using the LUDI program. As a result, acetyl-L-alanyl-(N-methyl)amide (Ac-L-Ala-NHMe) was selected to link with another fragment, hydroxamic acid that interacted with catalytic zinc. By this approach, the L-glutamic acid derivative 2b was discovered to be a new type of matrix metalloproteinase inhibitor. Further transformation to reduce its peptidic nature and improve activity yielded nonpeptidic lead compounds as inhibitors of MMP-1, -2, -3, and -9.     

Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: design,synthesis and structure-activity relationship

The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1' hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1' directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1' directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1' directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.     

Substrate recognition and selectivity of peptide deformylase. Similarities and differences with metzincins and thermolysin.

The substrate specificity of Escherichia coli peptide deformylase was investigated by measuring the efficiency of the enzyme to cleave formyl- peptides of the general formula Fo-Xaa-Yaa-NH2, where Xaa represents a set of 27 natural and unusual amino acids and Yaa corresponds to a set of 19 natural amino acids. Substrates with bulky hydrophobic side-chains at the P1' position were the most efficiently cleaved, with catalytic efficiencies greater by two to five orders of magnitude than those associated with polar or charged amino acid side-chains. Among hydrophobic side-chains, linear alkyl groups were preferred at the P1' position, as compared to aryl-alkyl side-chains. Interestingly, in the linear alkyl substituent series, with the exception of norleucine, deformylase exhibits a preference for the substrate containing Met in the P1' position. Next, the influence in catalysis of the second side-chain was studied after synthesis of 20 compounds of the formula Fo-Nle-Yaa-NH2. Their deformylation rates varied within a range of only one order of magnitude. A 3D model of the interaction of PDF with an inhibitor was then constructed and revealed indeed the occurrence of a deep and hydrophobic S1' pocket as well as the absence of a true S2' pocket. These analyses pointed out a set of possible interactions between deformylase and its substrates, which could be the ground driving substrate specificity. The validity of this enzyme:substrate docking was further probed with the help of a set of site-directed variants of the enzyme. From this, the importance of residues at the bottom of the S1' pocket (Ile128 and Leu125) as well as the hydrogen bond network that the main chain of the substrate makes with the enzyme were revealed. Based on the numerous homologies that deformylase displays with thermolysin and metzincins, a mechanism of enzyme:substrate recognition and hydrolysis could finally be proposed. Specific features of PDF with respect to other members of the enzymes with motif HEXXH are discussed.     

Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger polypeptide which plays a key role regulating cell growth and differentiation in the urogenital system. Using the whole-genome PCR approach, we searched murine genomic DNA for high-affinity WT1 binding sites and identified a 10-bp motif 5'GCGTGGGAGT3' which we term WTE). The WTE motif is similar to the consensus binding sequence 5'GCG(G/T)GGGCG3' recognized by EGR-1 and is also suggested to function as a binding site for WT1, setting up a competitive regulatory loop. To evaluate the underlying biochemical basis for such competition, we compared the binding affinities of WT1 and EGR1 for both sequences. WT1 shows a 20- to 30-fold-higher affinity for the WTE sequence compared with that of the EGR-1 binding motif. Mutational analysis of the WTE motif revealed a significant contribution to binding affinity by the adenine nucleotide at the eighth position (5'GCGTGGGAGT3') as well as by the 3'-most thymine (5'GCGTGGGAGT3'), whereas mutations in either flanking nucleotides or other nucleotides in the core sequence did not significantly affect the specific binding affinity. Mutations within WT1 zinc fingers II to IV abolished the sequence-specific binding of WT1 to WTE, whereas alterations within the first WT1 zinc finger reduced the binding affinity approximately 10-fold but did not abolish sequence recognition. We have thus identified a WT1 target, which, although similar in sequence to the EGR-1 motif, shows a 20- to 30-fold-higher affinity for WT1. These results suggest that physiological action of WT1 is mediated by binding sites of significantly higher affinity than the 9-bp EGR-1 binding motif. The role of the thymine base in contributing to binding affinity is discussed in the context of recent structural analysis.     

Crystal structures of MMP-1 and -13 reveal the structural basis for selectivity of collagenase inhibitors

The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether. In contrast, the collagenase-1 S1' pocket must undergo a conformational change to accommodate comparable P1' groups. The selectivity of the diphenylether series of inhibitors for collagenase-3 is largely determined by their affinity for the preformed S1' pocket of collagenase-3, as compared to the induced fit in collagenase-1.     

Molecular modeling of binding between amidinobenzisothiazoles, with antidegenerative activity on cartilage, and matrix metalloproteinase-3

The aim of the work was to investigate the mechanism of binding between human metalloproteinase-3 (MMP-3) and new compounds belonging to the benzisothiazolylamidines class. In vitro tests suggest that these molecules, endowed with antinflammatory and cartilage antidegenerative activity, could act as ligands toward MMP-3. In lack of experimental structural informations, we performed molecular docking simulations to probe the interactions of benzisothiazolylamidines with matrix metalloproteinase-3, using the docking package GOLD and the software HINT as a post-process scoring function. Both GOLD and HINT predicted a binding mode for the compounds under analysis within the hydrophobic S1' pocket of MMP-3, without interaction with the catalytic Zn(2+) ion. The scores assigned by the programs to the interaction between the tested benzisothiazolylamidines and human MMP-3 were consistent with a potential direct enzyme inhibitory activity. The highest affinity was predicted for the N-(benzo[d]isothiazol-3-yl)-4-chlorobenzamidine (2), emerged as the most active derivative also in the in vitro tests.     

Binding affinities for sulfonamide inhibitors with matrix metalloproteinase-2 using a linear response method

Due to their involvement in many pathological conditions, matrix metalloproteinases (MMPs), are very attractive therapeutic targets. Our study focuses on one of them, MMP-2, which is involved in tumor progression and metastasis. Recently, the solution structure of the catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020) was published by Feng et al. Using the Hanessian group published binding affinity data and the structure published by Feng as a basis, we have built a binding affinity model by targeting the S(2)' pocket of the enzyme with a set of nine alpha-N-sulfonylamino hydroxamic acid derivatives. Two binding geometries of each ligand have been generated corresponding to two binding modes denoted A and B, respectively, of which the first one is targeting the S(2)' pocket and the second one the S(1) pocket. For the binding affinity model developed for mode A the computed activities show a rmsd of 0.583 kcal/mol as compared with the experimental data, and a correlation coefficient r(2) of 0.779, while in the case of the binding mode B a rmsd of 0.834 kcal/mol and correlation coefficient r(2) of 0.500, respectively, were obtained. In conclusion, our data suggest a higher probability for the Phe(76) gated S(2)' open form pocket to accommodate the substituent alpha versus the wide solvent exposed S(1) subsite, probability which some research groups could have overlooked due to extensive use in their calculations of non revealing S(2)' pocket open state crystallographic structures instead of NMR ones.     

Crystal structures of MMP-9 complexes with five inhibitors: contribution of the flexible Arg424 side-chain to selectivity

Human matrix metalloproteinase 9 (MMP-9), also called gelatinase B, is particularly involved in inflammatory processes, bone remodelling and wound healing, but is also implicated in pathological processes such as rheumatoid arthritis, atherosclerosis, tumour growth, and metastasis. We have prepared the inactive E402Q mutant of the truncated catalytic domain of human MMP-9 and co-crystallized it with active site-directed synthetic inhibitors of different binding types. Here, we present the X-ray structures of five MMP-9 complexes with gelatinase-specific, tight binding inhibitors: a phosphinic acid (AM-409), a pyrimidine-2,4,6-trione (RO-206-0222), two carboxylate (An-1 and MJ-24), and a trifluoromethyl hydroxamic acid inhibitor (MS-560). These compounds bind by making a compromise between optimal coordination of the catalytic zinc, favourable hydrogen bond formation in the active-site cleft, and accommodation of their large hydrophobic P1' groups in the slightly flexible S1' cavity, which exhibits distinct rotational conformations of the Pro421 carbonyl group in each complex. In all these structures, the side-chain of Arg424 located at the bottom of the S1' cavity is not defined in the electron density beyond C(gamma), indicating its mobility. However, we suggest that the mobile Arg424 side-chain partially blocks the S1' cavity, which might explain the weaker binding of most inhibitors with a long P1' side-chain for MMP-9 compared with the closely related MMP-2 (gelatinase A), which exhibits a short threonine side-chain at the equivalent position. These novel structural details should facilitate the design of more selective MMP-9 inhibitors.     

The intermediate S1' pocket of the endometase/matrilysin-2 active site revealed by enzyme inhibition kinetic studies, protein sequence analyses, and homology modeling

Human matrix metalloproteinase-26 (MMP-26/endometase/matrilysin-2) is a newly identified MMP and its structure has not been reported. The enzyme active site S1' pocket in MMPs is a well defined substrate P1' amino acid residue-binding site with variable depth. To explore MMP-26 active site structure-activity, a series of new potent mercaptosulfide MMP inhibitors (MMPIs) with Leu or homophenylalanine (Homophe) side chains at the P1' site were selected. The Homephe side chain is designed to probe deep S1' pocket MMPs. These inhibitors were tested against MMP-26 and several MMPs with known x-ray crystal structures to distinguish shallow, intermediate, and deep S1' pocket characteristics. MMP-26 has an inhibition profile most similar to those of MMPs with intermediate S1' pockets. Investigations with hydroxamate MMPIs, including those designed for deep pocket MMPs, also indicated the presence of an intermediate pocket. Protein sequence analysis and homology modeling further verified that MMP-26 has an intermediate S1' pocket formed by Leu-204, His-208, and Tyr-230. Moreover, residue 233 may influence the depth of an MMP S1' pocket. The residue at the equivalent position of MMP-26 residue 233 is hydrophilic in intermediate-pocket MMPs (e.g. MMP-2, -8, and -9) and hydrophobic in deep-pocket MMPs (e.g. MMP-3, -12, and -14). MMP-26 contains a His-233 that renders the S1' pocket to an intermediate size. This study suggests that MMPIs, protein sequence analyses, and molecular modeling are useful tools to understand structure-activity relationships and provides new insight for rational inhibitor design that may distinguish MMPs with deep versus intermediate S1' pockets.     

Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.     

Insight into the stereochemistry in the inhibition of carboxypeptidase A with N-(hydroxyaminocarbonyl)phenylalanine: binding modes of an enantiomeric pair of the inhibitor to carboxypeptidase A

Both D- and L-isomers of N-(hydroxyaminocarbonyl)phenylalanine () were shown to have strong binding affinity towards carboxypeptidase A (CPA) with D- being more potent than its enantiomer by 3-fold (Chung, S. J.; Kim, D. H. Bioorg. Med. Chem. 2001, 9, 185.). In order to understand the reversed stereochemical preference shown in the CPA inhibition, we have solved the crystal structures of CPA complexed with each enantiometer of up to 1.75 A resolution. Inhibitor L- whose stereochemistry belongs to the stereochemical series of substrate binds CPA like substrate does with its carbonyl oxygen coordinating to the active site zinc ion. Its hydroxyl is engaged in hydrogen bonding with the carboxylate of Glu-270. On the other hand, in binding of D- to CPA, its terminal hydroxyl group is involved in interactions with the active site zinc ion and the carboxylate of Glu-270. In both CPA small middle dot complexes, the phenyl ring in is fitted in the substrate recognition pocket at the S(1)' subsite, and the carboxylate of the inhibitors forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and a hydrogen bond with the guanidinium of Arg-127. In the complex of CPA small middle dotD-, the carboxylate of the inhibitor is engaged in hydrogen bonding with the phenolic hydroxyl of the down-positioned Tyr-248. While the L- binding induces a concerted movement of the backbone amino acid residues at the active site, only the downward movement of Tyr-248 was noted when D- binds to CPA.     

Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating arylsulfonylureido and 5-dibenzo-suberenyl/suberyl moieties

Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.     

Virtual screening identification and chemical optimization of substituted 2-arylbenzimidazoles as new non-zinc-binding MMP-2 inhibitors

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases known to exert multiple regulatory roles in tumor progression and invasiveness. This encouraged over the years the approach of MMP, and particularly MMP-2, targeting for anticancer treatment. Early generations of MMP inhibitors, based on aspecific zinc binding groups (ZBGs) assembled on (pseudo)peptide scaffolds, have been discontinued due to the clinical emergence of toxicity and further drawbacks, giving the way to inhibitors with alternative zinc-chelator moieties or not binding the catalytic zinc ion.In the present paper, we continue the search for new non-zinc binding MMP-2 inhibitors: exploiting previously identified compounds, a virtual screening (VS) campaign was carried out and led to the identification of a new class of ligands. The structure-activity relationship (SAR) of the benzimidazole scaffold was explored by synthesis of several analogues whose inhibition activity was tested with enzyme inhibition assays. By performing the molecular simplification approach, we disclosed different sets of single-digit micromolar inhibitors of MMP-2, with up to a ten-fold increase in inhibitory activity and ameliorated selectivity towards off-target MMP-8, compared to selected lead compound. Molecular dynamics calculations conducted on complexes of MMP-2 with docked privileged structures confirmed that analyzed inhibitors avoid targeting the zinc ion and dip inside the S1′ pocket. Present results provide a further enrichment of our insights for the design of novel MMP-2 selective inhibitors.     

Insights into the function of the zinc hydroxide-Thr199-Glu106 hydrogen bonding network in carbonic anhydrases

The exact functional role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network in the carbonic anhydrases is unknown. However, from the results of molecular dynamics simulations (MD) we are able to better define its function. From computer graphics analysis and MD simulations on the zinc hydroxide form of human carbonic anhydrase II we find that this interaction forces the hydroxide hydrogen atom to be in a "down" position relative to the deep water-binding pocket. From previous work we have found that this pocket is a high-affinity binding site for CO2. We also note that during the timescale of our simulation (126 ps) the hydrogen bonds between the hydroxide hydrogen atom and Thr199 and the one between Thr199 and Glu106 are not fluxional. We propose that the role of the zinc hydroxide (water)-Thr199-Glu106 hydrogen bond network is to lock the hydrogen atom in the down position in order to expose the CO2 molecule bound in the deep water pocket to a lone pair of the hydroxide oxygen atom. This would allow for the rapid reaction of the CO2 molecule around the zinc ion. Furthermore, if the hydroxide hydrogen atom were not locked in the down position the binding of CO2 to the deep water pocket could be interfered with by the unrestrained hydroxide hydrogen atom (e.g. the N-Zn-O-H torsion could undergo rotational transitions that would partially block the deep water pocket). In summary, the roles we ascribe to this hydrogen bonding network are (1) to allow for facile access of CO2 to the deep water pocket and (2) to allow for maximal exposure of a hydroxide oxygen lone pair to the CO2 carbon atom.     

Design, synthesis and molecular modeling study of iminodiacetyl monohydroxamic acid derivatives as MMP inhibitors

As the matrix metalloproteinases (MMPs) can be massively up-regulated in degenerative tissues and degrade the extracellular matrix, these key enzymes are promising targets for the therapy of cancer and other degenerative diseases. Here, we are presenting a series of new non-peptidic hydroxamate-based matrix metalloproteinase inhibitors, MMPIs, incorporating the iminodiacetic (IDA) hydroxamic acid scaffold, as mimics of truncated peptidic MMPIs. A series of alkylaryl and sulfonylaryl groups, on the IDA basic scaffold, was investigated with the aim of improving potency and selectivity against MMPs involved in degenerative diseases. The sulfonamide based IDA derivatives studied (compounds B1-B3) showed to be potent (nM range) against deep S1' pocket MMPs enzymes (i.e., MMP-2).     

Discovery of novel hydroxamates as highly potent tumor necrosis factor-alpha converting enzyme inhibitors. Part II: optimization of the S3' pocket

A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1'-S3' pocket.     

Substrate hydrolysis by matrix metalloproteinase-9

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.     

Pivotal molecular determinants of peptidic and collagen triple helicase activities reside in the S3' subsite of matrix metalloproteinase 8 (MMP-8): the role of hydrogen bonding potential of ASN188 and TYR189 and the connecting cis bond

The mechanism of triple helical collagen unwinding and cleavage by collagenases in the matrix metalloproteinase (MMP) family is complex and remains enigmatic. Recent reports show that triple helicase activity is initiated by the hemopexin C domain of membrane type 1-MMP, whereas catalytically inactive full-length interstitial collagenase (MMP-1) exhibits full triple helicase functionality pointing to active site determinants that are needed to complete the triple helicase mechanism. In MMP-8, the neutrophil collagenase, a conserved Gly at the S(3)' substrate specificity subsite is replaced by Asn(188) that forms a highly unusual cis bond with Tyr(189), a conserved active site residue in the collagenases. Only in MMP-1 is the S(3)' Gly also replaced, and there too a cis configured Glu-Tyr occurs. Thus, this high energy peptide bond coupled to the canonical Tyr may be important in the collagenolytic process. In a systematic mutagenesis investigation of the MMP-8 S(3)' subsite we found that introducing an S(3)' Gly(188) into MMP-8 reduced collagenolytic efficiency by approximately 30% with a corresponding reduction in cleavage of a synthetic peptide fluorescence resonance energy transfer substrate analogue of the alpha2(I) collagen chain cleavage site. The substitution of Asn(188) to Leu, a hydrophobic residue of similar size to the highly polar Asn and designed to retain the cis bond, revealed the importance of hydrogen bonding to bound substrate with both collagenolytic and peptidic activities reduced approximately 3-fold. In contrast, the specificity for type I collagen of the mutant Y189F dropped 3-fold without any significant alteration in general peptidase activity. Therefore, S(3)' and in particular the hydrogen bonding potential of Tyr(189) is a specific molecular determinant for MMP-8 triple helicase activity. The cis bond connection to Asn(188) juxtaposes these two side chains for closely spaced hydrogen bonding with substrate that improves collagenolytic and general catalytic efficiency that could be exploited for new collagenase-specific inhibitor drugs.