ABSTRACT
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
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
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
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
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.
