Malaria caused by Plasmodium falciparum is a catastrophic disease worldwide (880,000 deaths yearly). Vaccine development has proved difficult and resistance has emerged for most antimalarials. In order to discover new antimalarial chemotypes, we have employed a phenotypic forward chemical genetic approach to assay 309,474 chemicals. Here we disclose structures and biological activity of the entire library, many of which exhibited potent in vitro activity against drug resistant strains, and detailed profiling of 172 representative candidates. A reverse chemical genetic study identified 19 new inhibitors of 4 validated drug targets and 15 novel binders among 61 malarial proteins. Phylochemogenetic profiling in multiple organisms revealed similarities between Toxoplasma gondii and mammalian cell lines and dissimilarities between P. falciparum and related protozoans. One exemplar compound displayed efficacy in a murine model. Overall, our findings provide the scientific community with new starting points for malaria drug discovery.

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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...

Cancer, more than any other human disease, now has a surfeit of potential molecular targets poised for therapeutic exploitation. Currently, a number of attractive and validated cancer targets remain outside of the reach of pharmacological regulation. Some have been described as undruggable, at least by traditional strategies. In this article, we outline the basis for the undruggable moniker, propose a reclassification of these targets as undrugged, and highlight three general classes of this imposing group as exemplars with some attendant strategies currently being explored to reclassify them. Expanding the spectrum of disease-relevant targets to pharmacological manipulation is central to reducing cancer morbidity and mortality.

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Protein kinase D is a novel family of serine/threonine kinases and diacylglycerol receptors that belongs to the calcium/calmodulin-dependent kinase superfamily. Evidence has established that specific PKD isoforms are dysregulated in several cancer types, and PKD involvement has been documented in a variety of cellular processes important to cancer development, including cell growth, apoptosis, motility, and angiogenesis. In light of this, there has been a recent surge in the development of novel chemical inhibitors of PKD. This review focuses on the potential of PKD as a chemotherapeutic target in cancer treatment and highlights important recent advances in the development of PKD inhibitors.

The protease-activated receptor 2, expressed on keratinocytes but not on melanocytes, has been ascribed functional importance in the regulation of pigmentation by phagocytosis of melanosomes. Inhibition of protease-activated receptor 2 activation by synthetic serine protease inhibitors requires keratinocyte-melanocyte contact and results in depigmentation of the dark skinned Yucatan swine, suggesting a new class of depigmenting mechanism and agents. We therefore examined natural agents that could exert their effect via the protease-activated receptor 2 pathway. Here we show that soymilk and the soybean-derived serine protease inhibitors soybean trypsin inhibitor and Bowman-Birk inhibitor inhibit protease-activated receptor 2 cleavage, affect cytoskeletal and cell surface organization, and reduce keratinocyte phagocytosis. The depigmenting activity of these agents and their capability to prevent ultraviolet-induced pigmentation are demonstrated both in vitro and in vivo. These results imply that inhibition of the protease-activated receptor 2 pathway by soymilk may be used as a natural alternative to skin lightening.

BackgroundProtein kinase D (PKD) has been implicated in a wide range of cellular processes and pathological conditions including cancer. However, targeting PKD therapeutically and dissecting PKD-mediated cellular responses remains difficult due to lack of a potent and selective inhibitor. Previously, we identified a novel pan-PKD inhibitor, CID755673, with potency in the upper nanomolar range and high selectivity for PKD. In an effort to further enhance its selectivity and potency for potential in vivo application, small molecule analogs of CID755673 were generated by modifying both the core structure and side-chains.ResultsAfter initial activity screening, five analogs with equal or greater potencies as CID755673 were chosen for further analysis: kb-NB142-70, kb-NB165-09, kb-NB165-31, kb-NB165-92, and kb-NB184-02. Our data showed that modifications to the aromatic core structure in particular significantly increased potency while retaining high specificity for PKD. When tested in prostate cancer cells, all compounds inhibited PMA-induced autophosphorylation of PKD1, with kb-NB142-70 being most active. Importantly, these analogs caused a dramatic arrest in cell proliferation accompanying elevated cytotoxicity when applied to prostate cancer cells. Cell migration and invasion were also inhibited by these analogs with varying potencies that correlated to their cellular activity.ConclusionsThroughout the battery of experiments, the compounds kb-NB142-70 and kb-NB165-09 emerged as the most potent and specific analogs in vitro and in cells. These compounds are undergoing further testing for their effectiveness as pharmacological tools for dissecting PKD function and as potential anti-cancer agents in the treatment of prostate cancer.