Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of PKD in these processes is severely hampered by the lack of a PKD-specific inhibitor that can be readily applied to cells and in animal models. We now report the discovery of the first potent and selective cell-active small molecule inhibitor for PKD, benzoxoloazepinolone (CID755673). This inhibitor was identified from the National Institutes of Health small molecule repository library of 196,173 compounds using a human PKD1 (PKC)-based fluorescence polarization high throughput screening assay. CID755673 suppressed half of the PKD1 enzyme activity at 182 nM and exhibited selective PKD1 inhibition when compared with AKT, polo-like kinase 1 (PLK1), CDK activating kinase (CAK), CAMKII␣, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cellbased assays, CID755673 blocked phorbol ester-induced endogenous PKD1 activation in LNCaP cells in a concentration-dependent manner. Functionally, CID755673 inhibited the known biological actions of PKD1 including phorbol ester-induced class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis virus glycoprotein transport from the Golgi to the plasma membrane, and the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 inhibited prostate cancer cell proliferation, cell migration, and invasion. In summary, our findings indicate that CID755673 is a potent and selective PKD1 inhibitor with valuable pharmacological and cell biological potential. Protein kinase D (PKD)3 belongs to a subfamily of the Ca 2ϩ / calmodulin-dependent kinases (CAMKs) (1). PKD is a novel target of the second messenger diacylglycerol and phorbol esters, the natural products from plants and potent tumor promoters in mouse skin (2). Three isoforms of PKD (PKD1, -2, and -3) have been identified, which share high sequence homology (3-6). The regulatory domain of PKD contains a C1 domain that binds diacylglycerol/phorbol esters with high affinity and a PH domain that mediates protein-protein interactions. The entire regulatory domain appears to exert a negative effect on catalytic activity, possibly serving as an autoinhibitory domain for PKD (7). The activity of PKD is controlled through a protein kinase C (PKC)-dependent mechanism (8). PKC is the primary target of diacylglycerol/phorbol esters and it activates PKD by directly binding and phosphorylating PKD on two serine residues in the activation loop. In most cellular systems examined, PKD is an effector of selective PKC isoforms, acting in a canonical PKC/PKD pathway that leads to a unique set of biological responses including cell proliferation, survival, protein transport, and immune responses (2, 9).PKD regulates many fundamental cellular functions and has been implicated in the pathogenesis of several diseases. PKD is a key regulator of protein transpo...
Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability.
BackgroundThe parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the γ-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.Methodology/Principal FindingsExploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were ∼20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03≤EC50<3 µM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.Conclusions/SignificanceThe novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.
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