No abstract

The high-resolution NMR solution structure of the catalytic fragment of human collagenase-3 (MMP-13) was used as a starting point for structure-based design of selective inhibitors for MMP-13. The major structural difference observed between the MMP structures is the relative size and shape of the S1′ pocket where this pocket is significantly longer for MMP-13, nearly reaching the surface of the protein. On the basis of the extended nature of the MMP-13 S1′ pocket an inhibitor potent and selective for MMP-13 was designed from an initial high throughput screening (HTS) lead. CL-82198 was identified as a weak (10 µM) inhibitor against MMP-13 while demonstrating no activity against MMP-1, MMP-9, or the related enzyme TACE. The drug-like properties of CL-82198 made it an ideal candidate for optimization of enzyme potency and selectivity. On the basis of NMR binding studies, it was shown that inhibitor CL-82198 bound within the entire S1′ pocket of MMP-13 which is the basis of its selectivity against MMP-1, MMP-9, and TACE. A strategy utilizing this information was devised for designing new inhibitors that showed enhanced selectivity toward MMP-13. Our design strategy combined the critical selectivity features of CL-82198 with the known potency features of a nonspecific MMP inhibitor (WAY-152177) to generate a potent and selective MMP-13 inhibitor (WAY-170523). WAY-170523 has an IC50 of 17 nM for MMP-13 and showed >5800-, 56-, and >500-fold selectivity against MMP-1, MMP-9, and TACE, respectively.A structure-based approach to designing potent and selective inhibitors has established itself as an important component of the drug development process (for reviews see refs 1 and 2). This is evident by the extensive structural data available for the matrix metalloproteinase (MMP) family of enzymes and the emergence of unique inhibitors based on this structural information (for reviews see refs 3-7). The MMPs are involved in the degradation of the extracellular matrix that is associated with normal tissue remodeling processes such as pregnancy, wound healing, and angiogenesis. MMP expression and activity is highly controlled because of the degradative nature of these enzymes where the apparent loss in this regulation results in the pathological destruction of connective tissue and the ensuing disease state. Thus, the MMPs are a highly active set of targets for the design of therapeutic agents for the disease areas of arthritis and oncology. The MMP family is composed of a number of enzymes where MMP-13 was recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. Recently, both an NMR and X-ray structure of inhibited MMP-13 have been reported. 8,9 The MMPs are generally categorized based on their substrate specificity, where the collagenase subfamily of MMP-1, MMP-8, and MMP-13 selectively cleaves native interstitial collagens (types I, II, and III). It is likely that only a subset of MMP enzymes will be involved in a particular disease as is evident by the overexpression o...

The development of a kinase structural database, the kinase knowledge base (KKB), is described. It covers all human kinase domain structures that have been deposited in the Protein Data Bank. All structures are renumbered using a common scheme, which enables efficient crosscomparisons and multiple queries of interest to the kinase field. The common numbering scheme is also used to automatically annotate conserved residues and motifs, and conformationally classify the structures based on the DFG-loop and Helix C. Analyses of residue conservation in the ATP binding site using the full human-kinome-sequence alignment lead to the identification of a conserved hydrogen bond between the hinge region backbone and a glycine in the specificity surface. Furthermore, 90% of kinases are found to have at least one stabilizing interaction for the hinge region, which has not been described before.

A protocol is described for rapidly screening small organic molecules for their ability to bind a target protein while obtaining structure-related information as part of a structure-based drug discovery and design program. The methodology takes advantage of and combines the inherent strengths of size exclusion gel chromatography, mass spectrometry, and NMR to identify bound complexes in a relatively universal high-throughput screening approach. Size exclusion gel chromatography in the spin column format provides the high-speed separation of a protein-ligand complex from free ligands. The spin column eluent is then analyzed under denaturing conditions by electrospray ionization mass spectrometry (MS) for the presence of small molecular weight compounds formerly bound to the protein. Hits identified by MS are then individually assayed by chemical shift perturbations in a 2D 1H-15N HSQC NMR spectrum to verify specific interactions of the compound with the protein and identification of the binding site on the protein. The utility of the MS/NMR assay is demonstrated with the use of the catalytic fragment of human fibroblast collagenase (MMP-1) as a target protein and the screening of a library consisting of approximately 32 000 compounds for the identification of molecules that exhibit specific binding to the RGS4 protein.

In earlier work from our laboratory, we have described the use of the ring system and ring scaffold as descriptors. We showed that these descriptors could be used for fast compound clustering, novelty determination, compound acquisition, and combinatorial library design. Here we extend the concept to a whole family of structural descriptors with the ring system as the centerpiece. We show how this simple idea can be used to build powerful search tools for mining chemical databases in useful ways. We have also built recursive partition trees using these fragments as descriptors. We will discuss how these trees can help in analyzing complex structure-activity data.