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.

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The nerve growth factor (NGF) receptor was characterized by using a new series of anti-receptor monoclonal antibodies (MAbs). These MAbs (i) showed significantly greater reactivity with a melanoma cell line expressing higher levels of NGF receptor, (ii) inhibited the binding of '251-labeled NGF to its receptor, and (iii) immunoprecipitated both metabolically labeled and '251-labeled NGF affinity-labeled receptor. These experiments defined the receptor as a 75-kDa cell-surface protein. The NGF receptor was visualized by immunoperoxidase staining in tissue sections of human nevi, melanomas, neurofibromas, a pheochromocytoma, and peripheral nerves. Uniform staining of the cytoplasm suggests that, in addition to cell-surface NGF receptors, there is a population of intracellular receptors.Nerve growth factor (NGF) has been the subject of extensive study because of its importance for regulation of development of sympathetic and sensory neurons and possibly other neural crest-derived cell types as well (1). Involvement of NGF in neural crest tumors such as melanoma has been suggested but never directly demonstrated. Efforts to identify oncogenes or oncogene-encoded products in malignant melanoma have been inconclusive (2), but the NGF receptor is a reasonable candidate particularly since, as we demonstrate in this study, it is expressed on melanoma cells in much greater quantities than on normal melanocytes. Progress in determining the relevance of this receptor to the transformed phenotype has been slow because the NGF receptor is not well characterized, nor are there good anti-receptor monoclonal antibodies (MAbs)

A novel dipyridodiazepinone, 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e]- [1,4]diazepin-6-one (BI-RG-587), is a selective noncompetitive inhibitor of HIV-1 reverse transcriptase (RT-1). An azido photoaffinity analogue of BI-RG-587 was synthesized and found to irreversibly inhibit the enzyme upon UV irradiation. BI-RG-587 and close structural analogues competitively protected RT-1 from inactivation by the photoaffinity label. A thiobenzimidazolone (TIBO) derivative, a nonnucleoside inhibitor of RT-1, also protected the enzyme from photoinactivation, which suggests a common binding site for these compounds. Substrates dGTP, template-primer, and tRNA afforded no protection from enzyme inactivation. A tritiated photoaffinity probe was found to stoichiometrically and selectively label p66 such that 1 mol of probe inactivates 1 mol of RT-1.

The accumulation of activated leukocytes in the pulmonary circulation plays an important role in the pathogenesis of lung dysfunction associated with cardiopulmonary bypass. Animal studies have demonstrated that the elimination of leukocytes from the circulation reduces postoperative lung injury and improves postoperative pulmonary function. We conducted a prospective randomized clinical study to evaluate whether postoperative lung function could be improved by use of a leukocyte filter during cardiopulmonary bypass. Elective coronary artery bypass grafting was done with a leukocyte-depleting arterial blood filter incorporated in the extracorporeal circuit (14 patients, leukocyte filter group) or without the filter (18 patients, control group). Blood samples collected at intervals before, during, and after operation were used for analysis of blood cell counts, elastase concentrations, and arterial blood gases. The use of the leukocyte filter caused no significant reduction in leukocyte count (p = 0.86). There were no differences in postoperative lung function between the groups, as assessed through (1) oxygenation index (290 for leukocyte filter group compared with 329 for control group, 95% confidence interval, 286 to 372, p = 0.21), (2) pulmonary vascular resistance (p = 0.10), and (3) intubation time (16.6 hours for leukocyte filter group versus 15.7 hours for control group, 95% confidence interval, 12.1 to 19.1 hours, p = 0.72). The levels of neutrophil elastase were significantly higher at the end of cardiopulmonary bypass in the leukocyte filter group (460 microgram/L in leukocyte filter group versus 230 microgram/L in control group, 95% confidence interval, 101 to 359 microgram/L, p = 0.003). We conclude that the clinical use of the present form of leukocyte-depleting filter did not improve any of the postoperative lung function parameters analyzed in this study.