Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-abl, a 210-kDa fusion protein with deregulated tyrosine kinase activity. Encouraged by the clinical validation of Bcr-abl as the target for the treatment of CML by imatinib, we sought to identify pharmacological agents that could target this kinase by a distinct mechanism. We report the discovery of a new class of Bcr-abl inhibitors using an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library. Compounds in this class (exemplified by GNF-2) show exclusive antiproliferative activity toward Bcr-abl-transformed cells, with potencies similar to imatinib, while showing no inhibition of the kinase activity of full-length or catalytic domain of c-abl. We propose that this new class of compounds inhibits Bcr-abl kinase activity through an allosteric non-ATP competitive mechanism.

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.C urrent hypotheses (1, 2) identify dysfunction of the endothelial cell (EC) layer as a cardinal event in the pathophysiology of multiple medical conditions, including sepsis. The mortality associated with sepsis alone is similar (3) to that of acute myocardial infarction (MI). In contrast to MI, available therapies are limited for the treatment of sepsis and its associated tissue injuries (3, 4). Mechanical ventilation is often required for the support of patients with sepsis but is itself associated with additional, iatrogenic pulmonary injury that also appears to involve EC barrier dysfunction (5). Recent progress in the use of controlled ventilator strategies, such as positive end-expiratory pressure (PEEP), lessens the potential for ventilator-induced lung injury (VILI), but a need still exists for therapies that would prevent this clinical complication (5). Most recently, in vitro studies have linked a variety of EC signal transduction pathways to the physiological mechanisms of EC barrier function and identified several endothelial protein kinases as potential drug discovery targets (1). However, the integration of the knowledge regarding in vitro signal transduction pathways with in vivo pathophysiology related to compromised EC barrier function and the validation of potential EC therapeutic targets have not occurred.Homeostasis and resistance to tissue injury are maintained by a balance between intracellular EC cytoskeletal contractionrelaxation cycles and modulation of EC extracellular adhesion properties, whi...

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Excessive glial activation, with overproduction of cytokines and oxidative stress products, is detrimental and a hallmark of neurodegenerative disease pathology. Suppression of glial activation is a potential therapeutic approach, and protein kinases are targets of some antiinflammatory drugs. To address an unmet need for selective inhibitors of glial activation, we developed a novel 3-amino-6-phenylpyridazine derivative that selectively blocks increased IL-1 beta, iNOS, and NO production by activated glia, without inhibition of potentially beneficial glial functions.

Barrier dysfunction, involving the endothelium or epithelium, is implicated in the pathophysiology of many disease states, including acute and ventilator-associated lung injury. Evidence from cell culture, in vivo and clinical studies, has identified myosin light chain kinase as a drug discovery target for such diseases. Here, we measured disease-relevant end points to test the hypothesis that inhibition of myosin light chain kinase is a potential therapeutic target for treatment of barrier dysfunction resulting from acute lung injury. We used a combined gene knockout and chemical biology approach with an in vivo intact lung injury model. We showed that inhibition of myosin light chain kinase protects lung function, preserves oxygenation, prevents acidosis, and enhances survival after endotoxin exposure with subsequent mechanical ventilation. This protective effect provided by the small molecule inhibitor of myosin light chain kinase is present when the inhibitor is administered during a clinically relevant injury paradigm after endotoxin exposure. Treatment with inhibitor confers additional protection against acute lung injury to that provided by a standard protective mode of ventilation. These results support the hypothesis that myosin light chain kinase is a potential therapeutic target for acute lung injury and provide clinical end points of arterial blood gases and pulmonary compliance that facilitate the direct extrapolation of these studies to measures used in critical care medicine.