Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

Protein kinases are a growing drug target class in disorders in peripheral tissues, but the development of kinase-targeted therapies for central nervous system (CNS) diseases remains a challenge, largely owing to issues associated specifically with CNS drug discovery. However, several candidate therapeutics that target CNS protein kinases are now in various stages of preclinical and clinical development. We review candidate compounds and discuss selected CNS protein kinases that are emerging as important therapeutic targets. In addition, we analyse trends in small-molecule properties that correlate with key challenges in CNS drug discovery, such as blood-brain barrier penetrance and cytochrome P450-mediated metabolism, and discuss the potential of future approaches that will integrate molecular-fragment expansion with pharmacoinformatics to address these challenges.Protein kinases regulate diverse cellular functions through the orchestrated propagation and amplification of cellular stimuli into distinct biological responses through coordinated signal transduction cascades. With several hundred kinases encoded in the human genome, almost every signal transduction process is influenced by interconnected phosphorylation events. Deregulation of kinase activity has been implicated in various diseases, ranging from vascular disorders and inflammatory diseases to neurological disorders and cancer 1,2 . This has generated intense interest in the pursuit of protein kinases as drug targets.However, most kinase-targeted drugs and potential kinase targets that have been investigated are for non-central nervous system (CNS) disorders. CNS disease indications for kinasetargeted drugs seem to be lagging behind those for other disease areas, such as cancer (FIG. 1), and ~25% of publications related to CNS disorders are for CNS cancers. This pattern is also seen in pharmaceutical industry pipelines that are publicly disclosed. For example, Novartis, the manufacturer of the kinase inhibitor cancer therapeutics imatinib (Gleevec) and nilotinib (Tasigna), has various oncology candidates and CNS-targeted therapies in clinical development. Half of the oncology pipeline are kinase inhibitors, but none of the disclosed CNS candidates seems to target protein kinases. Nevertheless, there is increasing interest in the development of kinase-targeted therapeutics for CNS indications [3][4][5][6][7][8][9][10][11] , and the established success in other disease indications provides encouragement for the development of smallmolecule kinase modulators for CNS disorders. NIH Public Access Author ManuscriptNat Rev Drug Discov. Author manuscript; available in PMC 2010 February 19. Published in final edited form as:Nat Rev Drug Discov. 2009 November ; 8(11): 892-909. doi:10.1038/nrd2999. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThis Review focuses on the special challenge of targeting protein kinases for CNS disease indications. A recent review 2 provides an up-to-date overview of targeting protein kinases i...

BackgroundOverproduction of proinflammatory cytokines from activated microglia has been implicated as an important contributor to pathophysiology progression in both acute and chronic neurodegenerative diseases. Therefore, it is critical to elucidate intracellular signaling pathways that are significant contributors to cytokine overproduction in microglia exposed to specific stressors, especially pathways amenable to drug interventions. The serine/threonine protein kinase p38α MAPK is a key enzyme in the parallel and convergent intracellular signaling pathways involved in stressor-induced production of IL-1β and TNFα in peripheral tissues, and is a drug development target for peripheral inflammatory diseases. However, much less is known about the quantitative importance of microglial p38α MAPK in stressor-induced cytokine overproduction, or the potential of microglial p38α MAPK to be a druggable target for CNS disorders. Therefore, we examined the contribution of microglial p38αMAPK to cytokine up-regulation, with a focus on the potential to suppress the cytokine increase by inhibition of the kinase with pharmacological or genetic approaches.MethodsThe microglial cytokine response to TLR ligands 2/3/4/7/8/9 or to Aβ1-42 was tested in the presence of a CNS-penetrant p38α MAPK inhibitor, MW01-2-069A-SRM. Primary microglia from mice genetically deficient in p38α MAPK were used to further establish a linkage between microglia p38α MAPK and cytokine overproduction. The in vivo significance was determined by p38α MAPK inhibitor treatment in a LPS-induced model of acute neuroinflammation.ResultsIncreased IL-1β and TNFα production by the BV-2 microglial cell line and by primary microglia cultures was inhibited in a concentration-dependent manner by the p38α MAPK-targeted inhibitor. Cellular target engagement was demonstrated by the accompanying decrease in the phosphorylation state of two p38α MAPK protein substrates, MK2 and MSK1. Consistent with the pharmacological findings, microglia from p38α-deficient mice showed a diminished cytokine response to LPS. Further, oral administration of the inhibitor blocked the increase of IL-1β in the cerebral cortex of mice stressed by intraperitoneal injection of LPS.ConclusionThe p38α MAPK pathway is an important contributor to the increased microglial production of proinflammatory cytokines induced by diverse stressors. The results also indicate the feasibility of targeting p38α MAPK to modulate CNS proinflammatory cytokine overproduction.

Background: An accumulating body of evidence is consistent with the hypothesis that excessive or prolonged increases in proinflammatory cytokine production by activated glia is a contributor to the progression of pathophysiology that is causally linked to synaptic dysfunction and hippocampal behavior deficits in neurodegenerative diseases such as Alzheimer's disease (AD). This raises the opportunity for the development of new classes of potentially disease-modifying therapeutics. A logical candidate CNS target is p38 MAPK, a well-established drug discovery molecular target for altering proinflammatory cytokine cascades in peripheral tissue disorders. Activated p38 MAPK is seen in human AD brain tissue and in AD-relevant animal models, and cell culture studies strongly implicate p38 MAPK in the increased production of proinflammatory cytokines by glia activated with human amyloid-beta (A ) and other disease-relevant stressors. However, the vast majority of small molecule drugs do not have sufficient penetrance of the bloodbrain barrier to allow their use as in vivo research tools or as therapeutics for neurodegenerative disorders. The goal of this study was to test the hypothesis that brain p38 MAPK is a potential in vivo target for orally bioavailable, small molecules capable of suppressing excessive cytokine production by activated glia back towards homeostasis, allowing an improvement in neurologic outcomes.

Serine-threonine protein kinases are critical to CNS function, yet there is a dearth of highly selective, CNS-active kinase inhibitors for in vivo investigations. Further, prevailing assumptions raise concerns about whether single kinase inhibitors can show in vivo efficacy for CNS pathologies, and debates over viable approaches to the development of safe and efficacious kinase inhibitors are unsettled. It is critical, therefore, that these scientific challenges be addressed in order to test hypotheses about protein kinases in neuropathology progression and the potential for in vivo modulation of their catalytic activity. Identification of molecular targets whose in vivo modulation can attenuate synaptic dysfunction would provide a foundation for future disease-modifying therapeutic development as well as insight into cellular mechanisms. Clinical and preclinical studies suggest a critical link between synaptic dysfunction in neurodegenerative disorders and the activation of p38αMAPK mediated signaling cascades. Activation in both neurons and glia also offers the unusual potential to generate enhanced responses through targeting a single kinase in two distinct cell types involved in pathology progression. However, target validation has been limited by lack of highly selective inhibitors amenable to in vivo use in the CNS. Therefore, we employed high-resolution co-crystallography and pharmacoinformatics to design and develop a novel synthetic, active site targeted, CNS-active, p38αMAPK inhibitor (MW108). Selectivity was demonstrated by large-scale kinome screens, functional GPCR agonist and antagonist analyses of off-target potential, and evaluation of cellular target engagement. In vitro and in vivo assays demonstrated that MW108 ameliorates beta-amyloid induced synaptic and cognitive dysfunction. A serendipitous discovery during co-crystallographic analyses revised prevailing models about active site targeting of inhibitors, providing insights that will facilitate future kinase inhibitor design. Overall, our studies deliver highly selective in vivo probes appropriate for CNS investigations and demonstrate that modulation of p38αMAPK activity can attenuate synaptic dysfunction.

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...

The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (= MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK:MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior.

Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS) associated with superoxide dismutase 1 (SOD1) mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT) deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.