A Targeted Library Screen Reveals a New Inhibitor Scaffold for Protein Kinase D

Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD. © 2012 Tandon et al

Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity

Protein kinase D (PKD) is a novel family of serine/threonine kinases regulated by diacylglycerol, which is involved in multiple cellular processes and various pathological conditions. The limited number of cell-active, selective inhibitors has historically restricted biochemical and pharmacological studies of PKD. We now markedly expand the PKD1 inhibitory chemotype inventory with eleven additional novel small molecule PKD1 inhibitors derived from our high throughput screening campaigns. The in vitro IC50s for these eleven compounds ranged in potency from 0.4 to 6.1 µM with all of the evaluated compounds being competitive with ATP. Three of the inhibitors (CID 1893668, (1Z)-1-(3-ethyl-5-methoxy-1,3-benzothiazol-2-ylidene)propan-2-one; CID 2011756, 5-(3-chlorophenyl)-N-[4-(morpholin-4-ylmethyl)phenyl]furan-2-carboxamide; CID 5389142, (6Z)-6-[4-(3-aminopropylamino)-6-methyl-1H-pyrimidin-2-ylidene]cyclohexa-2,4-dien-1-one) inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. The specificity of these compounds for PKD1 inhibitory activity was supported by kinase assay counter screens as well as by bioinformatics searches. Moreover, computational analyses of these novel cell-active PKD1 inhibitors indicated that they were structurally distinct from the previously described cell-active PKD1 inhibitors while computational docking of the new cell-active compounds in a highly conserved ATP-binding cleft suggests opportunities for structural modification. In summary, we have discovered novel PKD1 inhibitors with in vitro and cell-based inhibitory activity, thus successfully expanding the structural diversity of small molecule inhibitors available for this important pharmacological target

MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension.

BACKGROUND: Vascular hyperproliferative disorders are characterized by excessive smooth muscle cell (SMC) proliferation leading to vessel remodeling and occlusion. In pulmonary arterial hypertension (PAH), SMC phenotype switching from a terminally differentiated contractile to synthetic state is gaining traction as our understanding of the disease progression improves. While maintenance of SMC contractile phenotype is reportedly orchestrated by a MEF2C-myocardin (MYOCD) interplay, little is known regarding molecular control at this nexus. Moreover, the burgeoning interest in microRNAs (miRs) provides the basis for exploring their modulation of MEF2C-MYOCD signaling, and in turn, a pro-proliferative, synthetic SMC phenotype. We hypothesized that suppression of SMC contractile phenotype in pulmonary hypertension is mediated by miR-214 via repression of the MEF2C-MYOCD-leiomodin1 (LMOD1) signaling axis. METHODS AND RESULTS: In SMCs isolated from a PAH patient cohort and commercially obtained hPASMCs exposed to hypoxia, miR-214 expression was monitored by qRT-PCR. miR-214 was upregulated in PAH- vs. control subject hPASMCs as well as in commercially obtained hPASMCs exposed to hypoxia. These increases in miR-214 were paralleled by MEF2C, MYOCD and SMC contractile protein downregulation. Of these, LMOD1 and MEF2C were directly targeted by the miR. Mir-214 overexpression mimicked the PAH profile, downregulating MEF2C and LMOD1. AntagomiR-214 abrogated hypoxia-induced suppression of the contractile phenotype and its attendant proliferation. Anti-miR-214 also restored PAH-PASMCs to a contractile phenotype seen during vascular homeostasis. CONCLUSIONS: Our findings illustrate a key role for miR-214 in modulation of MEF2C-MYOCD-LMOD1 signaling and suggest that an antagonist of miR-214 could mitigate SMC phenotype changes and proliferation in vascular hyperproliferative disorders including PAH

Genetic Divergences and Phylogenetic Relationships Among the Fejervarya limnocharis Complex in Thailand and Neighboring Countries Revealed by Mitochondrial and Nuclear Genes

To clarify the genetic divergence in the F. limnocharis complex from Thailand and neighboring countries and to elucidate the phylogenetic problems of this taxon, we analyzed partial sequences of the mitochondrial 12S and 16S rRNA genes and the nuclear CXCR4, NCX1, RAG-1, and tyrosinase genes. The F. limnocharis complex from Thailand had three distinct haplotypes for 12S and 16S rRNA genes. Nucleotide similarities and the phylogenetic relationships indicated that the haplotype 1 group corresponded to the real “F. limnocharis”, the haplotype 2 group was F. orissaensis or closely related to it, and the haplotype 3 group was possibly an undescribed species. Mitochondrial gene data also showed two major clades of the genus Fejervarya, the Southeastern and South Asian groups. Although F. orissaensis is so far known only from Orissa in India, the haplotype 2 group was observed in Thailand. This distribution pattern and the phylogeny suggested that the origin of F. orissaensis and the haplotype 2 group might lie in Southeast Asia. There was also evidence suggesting that the haplotype 3 group originated in the South Asian area and has spread to northern Thailand. The nuclear gene data did not support the monophyly of the haplotypes recognized by mitochondrial genes. This incongruence between the mitochondrial and nuclear data seems to be caused by ancestral polymorphic sites contained in nuclear genes. Although neither the mitochondrial nor the nuclear data clarified intergeneric relationships, the nuclear data rejected the monophyly of the genus Fejervarya

New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells

<div><p>The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification, <i>in vitro</i> characterization, structure-activity analysis, and biological evaluation of a novel PKD inhibitory scaffold exemplified by 1-naphthyl PP1 (1-NA-PP1). 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors in a small-scale targeted kinase inhibitor library assay. Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1^M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.</p></div

Molecular modeling of compound 139 in the active site of a PKD1 homology model.

<p><b>A</b>. The docking result of the bioactive compound <b>139</b> in the ATP binding site of the PKD1 kinase domain. <i>carton ribbon and thick line</i>, PKD1; <i>ball and stick</i>, Compound <b>139</b>; <i>thin line</i>, residues in the binding pocket; <i>magenta line</i>, hydrogen bond. <b>B</b>. The proposed key contacts in the active site. <i>purple line</i>, hydrogen bond; residues in different colors: <i>purple</i>, basic; <i>pink</i>, acidic; <i>green</i>, hydrophobic; <i>gray</i>, hydrophilic.</p

Primary hits identified in a PKD1 inhibitor screen of a targeted library.

<p>A targeted protein kinase inhibitor library of 80 compounds was screened for PKD1 inhibitory activity at 1 µM using an <i>in vitro</i> radiometric PKD1 kinase assay. Sixteen compounds were selected as primary hits based on their ability to inhibit PKD1 at or above 50% at 1 µM. The % PKD1 inhibition referred to the percent inhibition of the total kinase activity measured in the absence of inhibitors (DMSO). Kb-NB142-70, a previously validated PKD inhibitor, was used as a positive control. Experiments were performed with triplicate determinations at 1 µM for each compound.</p

Selectivity profiling of compounds 122 and 140.

<p>Listed in this table are all the protein kinases that bound compound <b>122</b> at over 50% at 10 µM. Their competition by compound <b>140</b> is listed in parallel. Compound <b>140</b> exhibited greater selectivity as compared to compound <b>122</b>. The data were obtained from profiling of a total of 353 kinases in the kinome. Enzymes competed by compound <b>140</b> at 99–100% are bolded.</p

Structures of 1-NA-PP1 analogs.

<p>Structures of 1-NA-PP1 analogs.</p

IKK-16 and 1-NA-PP1 were ATP-competitive inhibitors of PKD.

<p>PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of 1-NA-PP1 (<b>A</b>) and IKK-16 (<b>B</b>). Lineweaver-Burke plots of the data are shown. Data presented were representative of three independent experiments.</p