ABSTRACT
Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.
Subject(s)
Matrix Metalloproteinase 13/drug effects , Matrix Metalloproteinase Inhibitors/chemical synthesis , Matrix Metalloproteinase Inhibitors/pharmacology , Osteoarthritis/drug therapy , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Tetrazoles/chemical synthesis , Tetrazoles/pharmacology , Animals , Cartilage, Articular/drug effects , Cartilage, Articular/pathology , Collagenases/drug effects , Dogs , Drug Design , Humans , Kidney/metabolism , Macaca fascicularis , Male , Matrix Metalloproteinase Inhibitors/toxicity , Models, Molecular , Organic Anion Transporters, Sodium-Independent/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded ß-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.
Subject(s)
ADAM Proteins/chemistry , Catalytic Domain , Membrane Proteins/chemistry , Phenylalanine/analogs & derivatives , Protease Inhibitors/chemistry , Thiophenes/chemistry , ADAM Proteins/metabolism , Humans , Ligands , Membrane Proteins/metabolism , Models, Molecular , Phenylalanine/chemistry , Phenylalanine/metabolism , Protease Inhibitors/metabolism , Protein Binding , Protein Unfolding , Thiophenes/metabolismABSTRACT
PH-797804 ((aS)-3-{3-bromo-4-[(2,4-difluorobenzyl)oxy]-6-methyl-2-oxopyridin-1(2H)-yl}-N,4-dimethylbenzamde) is a diarylpyridinone inhibitor of p38 mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. Due to steric constraints imposed by the pyridinone carbonyl group and the 6- and 6'-methyl substituents of PH-797804, rotation around the connecting bond of the pyridinone and the N-phenyl ring is restricted. Density functional theory predicts a remarkably high rotational energy barrier of >30â kcal mol(-1), corresponding to a half-life of more than one hundred years at room temperature. This gives rise to discrete conformational spaces for the N-phenylpyridinone group, and as a result, two atropic isomers that do not interconvert under ambient conditions. Molecular modeling studies predict that the two isomers should differ in their binding affinity for p38α kinase; whereas the atropic S (aS) isomer binds favorably, the opposite aR isomer incurs significant steric interference with p38α kinase. The two isomers were subsequently identified and separated by chiral chromatography. IC50 values from p38α kinase assays confirm that one atropisomer is >100-fold more potent than the other. It was ultimately confirmed by small-molecule X-ray diffraction that the more potent atropisomer, PH-797804, is the aS isomer of the racemic pair. Extensive pharmacological characterization supports that PH-797804 carries most activity both inâ vitro and inâ vivo, and it has a stability profile compatible with oral formulation and delivery options.
Subject(s)
Benzamides/chemistry , Protein Kinase Inhibitors/chemistry , Pyridones/chemistry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Arthritis/drug therapy , Benzamides/pharmacology , Benzamides/therapeutic use , Computer Simulation , Crystallography, X-Ray , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Female , Lipopolysaccharides/toxicity , Male , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary , Pyridones/pharmacology , Pyridones/therapeutic use , Quantum Theory , Rats , Receptors, Cell Surface , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.
Subject(s)
Drug Design , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyridones/chemical synthesis , Pyridones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Disease Models, Animal , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Male , Microsomes, Liver/enzymology , Molecular Structure , Pyridazines/chemistry , Pyridazines/pharmacology , Pyridones/chemistry , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
The synthesis and SAR studies of a novel N-aryl pyridinone class of p38 kinase inhibitors are described. Systematic structural modifications to the HTS lead, 5, led to the identification of (-)-4a as a clinical candidate for the treatment of inflammatory diseases. Additionally, the chiral synthesis and properties of (-)-4a are described.
Subject(s)
Benzamides/chemical synthesis , Benzamides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyrones/chemical synthesis , Pyrones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Benzamides/chemistry , Disease Models, Animal , Dogs , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Humans , Inhibitory Concentration 50 , Macaca fascicularis , Male , Molecular Structure , Pyridones , Pyrones/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/chemistry , p38 Mitogen-Activated Protein Kinases/pharmacologyABSTRACT
A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-α in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.
Subject(s)
Protein Kinase Inhibitors/therapeutic use , Pyrimidinones/therapeutic use , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Administration, Oral , Animals , Arthritis/drug therapy , Arthritis, Experimental , Caco-2 Cells , Crystallography, X-Ray , Humans , Inhibitory Concentration 50 , Male , Models, Molecular , Protein Kinase Inhibitors/chemistry , Pyrimidinones/chemistry , Rats , Rats, Inbred Lew , Structure-Activity RelationshipABSTRACT
A ((1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl) succinamide derivative (here referred to as Compound 12) shows significant activity toward many matrix metalloproteinases (MMPs), including MMP-2, MMP-8, MMP-9, and MMP-13. Modeling studies had predicted that this compound would not bind to ADAMTS-5 (a disintegrin and metalloproteinase with thrombospondin motifs-5) due to its shallow S1' pocket. However, inhibition analysis revealed it to be a nanomolar inhibitor of both ADAMTS-4 and -5. The observed inconsistency was explained by analysis of crystallographic structures, which showed that Compound 12 in complex with the catalytic domain of ADAMTS-5 (cataTS5) exhibits an unusual conformation in the S1' pocket of the protein. This first demonstration that cataTS5 can undergo an induced conformational change in its active site pocket by a molecule like Compound 12 should enable the design of new aggrecanase inhibitors with better potency and selectivity profiles.
Subject(s)
ADAM Proteins/chemistry , Amides/chemistry , Protein Conformation , ADAMTS5 Protein , Animals , Catalytic Domain , Cattle , Drug Design , Humans , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , SuccinatesABSTRACT
Starting from an initial HTS screening lead, a novel series of C(5)-substituted diaryl pyrazoles were developed that showed potent inhibition of p38alpha kinase. Key to this outcome was the switch from a pyridyl to pyrimidine at the C(4)-position leading to analogs that were potent in human whole blood based cell assay as well as in a number of animal efficacy models for rheumatoid arthritis. Ultimately, we identified a clinical candidate from this substrate; SD-0006.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Catalytic Domain , Humans , Models, Molecular , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The structure based drug design, synthesis and structure-activity relationship of a series of C6 sulfur linked triazolopyridine based p38 inhibitors are described. The metabolic deficiencies of this series were overcome through changes in the C6 linker from sulfur to methylene, which was predicted by molecular modeling to be bioisosteric. X-ray of the ethylene linked compound 61 confirmed the predicted binding orientation of the scaffold in the p38 enzyme.
Subject(s)
Benzamides/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Triazoles/chemistry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Benzamides/chemical synthesis , Benzamides/pharmacology , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Crystallography, X-Ray , Drug Design , Humans , Microsomes, Liver/metabolism , Models, Chemical , Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacology , Rats , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.
Subject(s)
Matrix Metalloproteinase Inhibitors , Osteoarthritis/drug therapy , Picolinic Acids/chemistry , Protease Inhibitors/chemistry , Tetrazoles/chemistry , Administration, Oral , Animals , Binding Sites , Cartilage/drug effects , Cartilage/metabolism , Catalytic Domain , Crystallography, X-Ray , Disease Models, Animal , Drug Discovery , Matrix Metalloproteinase 13/metabolism , Picolinic Acids/chemical synthesis , Picolinic Acids/pharmacology , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Rats , Tetrazoles/chemical synthesis , Tetrazoles/pharmacology , Zinc/chemistryABSTRACT
The identification and evolution of a series of potent and selective p38 inhibitors is described. p38 inhibitors based on a N-benzyl pyridinone high-throughput screening hit were prepared and their SAR explored. Their design was guided by ligand bound co-crystals of p38alpha. These efforts resulted in the identification of 12r and 19 as orally active inhibitors of p38 with significant efficacy in both acute and chronic models of inflammation.
Subject(s)
Anti-Inflammatory Agents/chemistry , Protein Kinase Inhibitors/chemistry , Pyridones/chemistry , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Administration, Oral , Animals , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/pharmacokinetics , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Drug Discovery , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Male , Microsomes, Liver/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Pyridones/chemical synthesis , Pyridones/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
SD0006 is a diarylpyrazole that was prepared as an inhibitor of p38 kinase-alpha (p38alpha). In vitro, SD0006 was selective for p38alpha kinase over 50 other kinases screened (including p38gamma and p38delta with modest selectivity over p38beta). Crystal structures with p38alpha show binding at the ATP site with additional residue interactions outside the ATP pocket unique to p38alpha that can confer advantages over other ATP competitive inhibitors. Direct correlation between inhibition of p38alpha activity and that of lipopolysaccharide-stimulated TNFalpha release was established in cellular models and in vivo, including a phase 1 clinical trial. Potency (IC(50)) for inhibiting tumor necrosis factor-alpha (TNFalpha) release, in vitro and in vivo, was <200 nmol/l. In vivo, SD0006 was effective in the rat streptococcal-cell-wall-induced arthritis model, with dramatic protective effects on paw joint integrity and bone density as shown by radiographic analysis. In the murine collagen-induced arthritis model, equivalence was demonstrated to anti-TNFalpha treatment. SD0006 also demonstrated good oral anti-inflammatory efficacy with excellent cross-species correlation between the rat, cynomolgus monkey, and human. SD0006 suppressed expression of multiple proinflammatory proteins at both the transcriptional and translational levels. These properties suggest SD0006 could provide broader therapeutic efficacy than cytokine-targeted monotherapeutics.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arthritis, Experimental/drug therapy , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Administration, Oral , Animals , Bone Density/drug effects , Cell Line , Endotoxemia/drug therapy , Endotoxemia/metabolism , Female , Humans , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/physiopathology , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , Macaca fascicularis , Male , Mice , Mice, Inbred DBA , Models, Molecular , Pain/drug therapy , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
The disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of metalloproteases consists of 19 members. These enzymes play an important role in the turnover of extracellular matrix proteins in various tissues and their altered regulation has been implicated in diseases such as cancer, arthritis and atherosclerosis. Unlike other metalloproteinases, ADAMTS members demonstrate a narrow substrate specificity due to the various exosites located in the C-terminal regions of the enzymes, which influence protein recognition and matrix localization. The tight substrate specificity exhibited by ADAMTS enzymes makes them potentially safe pharmaceutical targets, as selective inhibitors designed for each member will result in the inhibition or cleavage of only a limited number of proteins. With the recent elucidation of crystal structures for ADAMTS-1, -4 and -5, the design of potent and selective small molecule inhibitors is underway and will lead to drug candidates for evaluation in clinical trials in the next 5-10 years.
Subject(s)
ADAM Proteins/drug effects , ADAM Proteins/chemistry , ADAM Proteins/classification , ADAM Proteins/metabolism , Amino Acid Sequence , Enzyme Activation , Humans , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Several inhibitors of a series of cis-1(S)2(R)-amino-2-indanol-based compounds were reported to be selective for the aggrecanases, ADAMTS-4 and -5 over other metalloproteases. To understand the nature of this selectivity for aggrecanases, the inhibitors, along with the broad spectrum metalloprotease inhibitor marimastat, were independently bound to the catalytic domain of ADAMTS-5, and the corresponding crystal structures were determined. By comparing the structures, it was determined that the specificity of the relative inhibitors for ADAMTS-5 was not driven by a specific interaction, such as zinc chelation, hydrogen bonding, or charge interactions, but rather by subtle and indirect factors, such as water bridging, ring rigidity, pocket size, and shape, as well as protein conformation flexibility.
Subject(s)
Endopeptidases/chemistry , Enzyme Inhibitors/chemistry , ADAM Proteins/chemistry , ADAMTS4 Protein , ADAMTS5 Protein , Animals , Cattle , Humans , Hydrogen Bonding , Procollagen N-Endopeptidase/chemistry , Protein Structure, Tertiary , Structural Homology, Protein , Zinc/chemistryABSTRACT
PH-797804 is a diarylpyridinone inhibitor of p38alpha mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38alpha inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38alpha kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180 degrees rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38alpha kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38alpha kinase inhibitors.
Subject(s)
Benzamides/pharmacology , Computational Biology , Protein Kinase Inhibitors/pharmacology , Pyrones/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Benzamides/chemistry , Crystallography, X-Ray , Humans , Hydrogen Bonding , Models, Molecular , Molecular Structure , Phosphorylation , Protein Kinase Inhibitors/chemistry , Pyridones , Pyrones/chemistry , Substrate Specificity , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to 1.4A resolution in the presence of an inhibitor. A high resolution structure of an ADAMTS/aggrecanase protein provides an opportunity for the development of therapeutics to treat osteoarthritis.
Subject(s)
ADAM Proteins/chemistry , Catalytic Domain , ADAM Proteins/antagonists & inhibitors , ADAM Proteins/isolation & purification , ADAM Proteins/metabolism , ADAMTS5 Protein , Amino Acid Sequence , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Enzyme Stability/drug effects , Humans , Models, Molecular , Molecular Sequence Data , Protein Folding , Protein Structure, Secondary , Temperature , Tissue Inhibitor of Metalloproteinase-3/metabolismABSTRACT
A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.
Subject(s)
Antirheumatic Agents/chemical synthesis , Piperazines/chemical synthesis , Pyrazoles/chemical synthesis , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Antirheumatic Agents/chemistry , Antirheumatic Agents/pharmacology , Arthritis, Experimental/chemically induced , Arthritis, Experimental/drug therapy , Collagen , Crystallography, X-Ray , Male , Mice , Mice, Inbred DBA , Models, Molecular , Piperazines/chemistry , Piperazines/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Rats , Rats, Inbred Lew , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/chemistryABSTRACT
5(S)-Fluoro-N6-(iminoethyl)-l-lysine (14), an analogue of the potent, selective induced nitric oxide synthase (iNOS) inhibitor iminoethyl-l-lysine (1), was synthesized and found to be a selective iNOS inhibitor.
Subject(s)
Lysine/chemical synthesis , Nitric Oxide Synthase/antagonists & inhibitors , Crystallography, X-Ray , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Lysine/analogs & derivatives , Lysine/chemistry , Models, Molecular , Nitric Oxide Synthase/chemistry , Nitric Oxide Synthase Type IIABSTRACT
In the literature, the introduction of fluorine into bioactive molecules has been known to enhance the biological activity relative to the parent molecule. Described in this article is the synthesis of 4R-fluoro-L-NIL (12) and 4,4-difluoro-L-NIL (23) as part of our iNOS program. Both 12 and 23 were found to be selective iNOS inhibitors as shown in Table 2 below. Secondarily, methodology to synthesize orthogonally protected 4-fluoro-L-lysine and 4,4-difluoro-L-lysine has been developed.
Subject(s)
Enzyme Inhibitors/chemistry , Fluorine/chemistry , Lysine/analogs & derivatives , Nitric Oxide Synthase/antagonists & inhibitors , Animals , Crystallography, X-Ray , Humans , Lysine/chemistry , Mice , Models, Biological , Models, Chemical , Molecular Structure , Nitric Oxide Synthase Type IIABSTRACT
MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.