The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines, such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics, consistent with the requirement for conformational change. Improving interactions in this allosteric pocket, as well as establishing binding interactions in the ATP pocket, enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series, BIRB 796, has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture.

We report on a series of N-pyrazole, N'-aryl ureas and their mode of binding to p38 mitogen activated protein kinase. Importantly, a key binding domain that is distinct from the adenosine 5'-triphoshate (ATP) binding site is exposed when the conserved activation loop, consisting in part of Asp168-Phe169-Gly170, adopts a conformation permitting lipophilic and hydrogen bonding interactions between this class of inhibitors and the protein. We describe the correlation of the structure-activity relationships and crystallographic structures of these inhibitors with p38. In addition, we incorporated another binding pharmacophore that forms a hydrogen bond at the ATP binding site. This modification affords significant improvements in binding, cellular, and in vivo potencies resulting in the selection of 45 (BIRB 796) as a clinical candidate for the treatment of inflammatory diseases.

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We report on the structure-activity relationships (SAR) of 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl]urea (BIRB 796), an inhibitor of p38alpha MAP kinase which has advanced into human clinical trials for the treatment of autoimmune diseases. Thermal denaturation was used to establish molecular binding affinities for this class of p38alpha inhibitors. The tert-butyl group remains a critical binding element by occupying a lipophilic domain in the kinase which is exposed upon rearrangement of the activation loop. An aromatic ring attached to N-2 of the pyrazole nucleus provides important pi-CH(2) interactions with the kinase. The role of groups attached through an ethoxy group to the 4-position of the naphthalene and directed into the ATP-binding domain is elucidated. Pharmacophores with good hydrogen bonding potential, such as morpholine, pyridine, and imidazole, shift the melting temperature of p38alpha by 16-17 degrees C translating into K(d) values of 50-100 pM. Finally, we describe several compounds that potently inhibit TNF-alpha production when dosed orally in mice.

Proliferation of bovine aortic smooth muscle cells (SMCs) induced by thrombin, basic fibroblast growth factor, or serum is inhibited by anionic, nonsulfated aromatic compounds that mimic many of the effects of heparin. Among these compounds are aurintricarboxylic acid (ATA) and a newly synthesized polymer of 4-hydroxyphenoxy acetic acid (compound RG-13577). Iodinated-or 14 C-labeled compound RG-13577 binds to cultured SMCs in a highly specific and saturable manner. Scatchard analysis of the binding data revealed the presence of an estimated lxlO 7 binding sites per cell with an apparent dissociation constant of 3xlO~6 mol/L. Binding of radiolabeled RG-13577 was efficiently competed for by related aromatic anionic compounds and by apolipoprotein E, but not by heparin, heparan sulfate, suramin, or various purified growth factors and extracellular matrix proteins.