Synthesis and structure-activity relationship of N-substituted 4-arylsulfonylpiperidine-4-hydroxamic acids as novel, orally active matrix metalloproteinase inhibitors for the treatment of osteoarthritis.

The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. In our preceding paper, we have reported on a series of novel and orally active N-hydroxy-alpha-phenylsulfonylacetamide derivatives. However, these compounds had two drawbacks (moderate selectivity and chirality issues). To circumvent these two problems, a series of novel and orally active N-substituted 4-benzenesulfonylpiperidine-4-carboxylic acid hydroxyamide derivatives have been synthesized. The present paper deals with the synthesis and SAR of these compounds. Among the several compounds synthesized, derivative 55 turned out to be a potent, selective, and an orally active MMP inhibitor in the clinically relevant advanced rabbit osteoarthritis model. Detailed pharmacokinetics and metabolism data are described.

Novel synthesis and structural characterization of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance.

To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing how the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.

Identification of potent and selective MMP-13 inhibitors.

A potent, selective series of MMP-13 inhibitors has been derived from a weak (3.2 microM) inhibitor that did not bear a zinc chelator. Structure-based drug design strategies were employed to append a Zn-chelating group to one end of the molecule and functionality to enhance selectivity to the other. A compound from this series demonstrated rat oral bioavailability and efficacy in a bovine articular cartilage explant model.

Catalytic domain crystal structure of protein kinase C-theta (PKCtheta).

A member of the novel protein kinase C (PKC) subfamily, PKC, is an essential component of the T cell synapse and is required for optimal T cell activation and interleukin-2 production. Selective involvement of PKC in TCR signaling makes this enzyme an attractive therapeutic target in T cell-mediated disease processes. In this report we describe the crystal structure of the catalytic domain of PKC at 2.0-A resolution. Human recombinant PKC kinase domain was expressed in bacteria as catalytically active phosphorylated enzyme and co-crystallized with its subnanomolar, ATP site inhibitor staurosporine. The structure follows the classic bilobal kinase fold and shows the enzyme in its active conformation and phosphorylated state. Inhibitory interactions between conserved features of staurosporine and the ATP-binding cleft are accompanied by closing of the glycine-rich loop, which also maintains an inhibitory arrangement by blocking the phosphate recognition subsite. The two major phosphorylation sites, Thr-538 in the activation loop and Ser-695 in the hydrophobic motif, are both occupied in the structure, playing key roles in stabilizing active conformation of the enzyme and indicative of PKC autocatalytic phosphorylation and activation during bacterial expression. The PKC-staurosporine complex represents the first kinase domain crystal structure of any PKC isotypes to be determined and as such should provide valuable insight into PKC specificity and into rational drug design strategies for PKC selective leads.