Q. Jane Wang

Potent and Selective Disruption of Protein Kinase D Functionality by a Benzoxoloazepinolone

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 (PKCμ)-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), CAMKIIα, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cell-based 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 as a potential new target for cancer therapy.

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.

Novel protein kinase D inhibitors cause potent arrest in prostate cancer cell growth and motility

Background Protein 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. Results After 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. Conclusions Throughout 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.

Design, Synthesis, and Biological Evaluation of PKD Inhibitors

Protein kinase D (PKD) belongs to a family of serine/threonine kinases that play an important role in basic cellular processes and are implicated in the pathogenesis of several diseases. Progress in our understanding of the biological functions of PKD has been limited due to the lack of a PKD-specific inhibitor. The benzoxoloazepinolone CID755673 was recently reported as the first potent and kinase-selective inhibitor for this enzyme. For structure-activity analysis purposes, a series of analogs was prepared and their in vitro inhibitory potency evaluated.

A protein kinase C/protein kinase D pathway protects LNCaP prostate cancer cells from phorbol ester-induced apoptosis by promoting ERK1/2 and NF-κB activities

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) induce apoptosis in many tumor cells including the androgen-sensitive LNCaP prostate cancer cells. Although phorbol ester-induced apoptotic pathways have been well characterized, little is known of the pro-survival pathways modulated by these agents. We now provide experimental evidence to indicate that protein kinase D (PKD) promotes survival signals in LNCaP cells in response to PMA treatment. Knockdown of endogenous PKD1 or PKD2 decreased extracellular signal-regulated kinase (ERK) 1/2 and nuclear factor-kappaB (NF-κB)-dependent transcriptional activities and potentiated PMA-induced apoptosis, whereas overexpression of wild-type PKD1 enhanced ERK1/2 activity and suppressed PMA-induced apoptosis. PMA caused rapid activation, followed by progressive downregulation of endogenous PKD1 in a time- and concentration-dependent manner. The downregulation of PKD1 was dependent on the activity of protein kinase C (PKC), but not that of PKD. Selective depletion of endogenous PKC isoforms revealed that both PKCδ and PKCε were required for PKD1 activation and subsequent downregulation. Further analysis showed that the downregulation of PKD1 was mediated by a ubiquitin-proteasome degradation pathway, inhibition of which correlated to increased cell survival. In summary, our data indicate that PKD1 is activated and downregulated by PMA through a PKC-dependent ubiquitin-proteasome degradation pathway, and the activation of PKD1 or PKD2 counteracts PMA-induced apoptosis by promoting downstream ERK1/2 and NF-κB activities in LNCaP prostate cancer cells.

SD-208, a Novel Protein Kinase D Inhibitor, Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.

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.

Androgen suppresses protein kinase D1 expression through fibroblast growth factor receptor substrate 2 in prostate cancer cells

In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment.

Abstract 3829: Inducible silencing of PKD3 reveals a critical role for PKD3 in prostate cancer growth and metastasis

Protein kinase D is a family of novel serine/threonine kinases that act as major mediators of several signaling pathways related to cancer development. Increasing evidence has demonstrated that specific PKD isoforms are deregulated in human cancers and that PKD plays an active role in cancer cell proliferation, migration, and invasion. Here, we present evidence on the development of a PC3 tetracycline-inducible PKD3 knockdown prostate cancer cell line and its application in both the study of PKD3 function in cells and the validation of PKD3 as a therapeutic target in vivo. We show that tetracycline treatment results in >50% knockdown of endogenous PKD3 expression in two distinct clones expressing PKD3 shRNA (shPKD3-C7 and shPKD3-C26), but has no effect on cells expressing scrambled shRNA (shSCR). Furthermore, endogenous PKD1 or PKD2 expression levels are not altered in PKD3 knockdown cells treated with tetracycline, supporting the specific targeting of the shRNA to the PKD3 isoform. Phenotypic analysis revealed that, similar to studies utilizing PKD siRNAs or novel PKD inhibitors, tetracycline-induced silencing of PKD3 causes potent arrest in cell growth and motility. Additionally, we found that conditioned media collected from tetracycline-treated shPKD3-C7 cells causes a reduction in PC3 cell migration, and we observed increased secretion of the tissue inhibitor of metalloproteinases-2 (TIMP-2) protein, which has important anti-angiogenic functions, from tetracycline-treated shPKD3-C7 cells. Western blotting revealed that both siRNA-mediated transient knockdown of PKD2 or PKD3 or treatment with novel PKD inhibitors led to decreased levels of the matrix metalloproteinases MMP-9, MMP-11, and MMP-14 in PC3 cells. Finally, we found that reduction of PKD3 expression by tetracycline-inducible shRNA significantly retarded both tumor growth in a subcutaneous xenograft model as well as tumor growth and metastasis in an orthotopic prostate cancer xenograft model. Taken together, these data suggest that PKD is a critical signaling protein that modulates prostate cancer cell growth and motility through a pathway involving multiple secreted factors, and that PKD3 is a potential therapeutic target in the treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2011-3829

Abstract 2532: In vitro cytotoxicity, and pharmacokinetics, tissue distribution, and metabolism of the protein kinase D inhibitors, kb-NB142-70 and kb-NB184-43, in mice bearing human cancer xenografts

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Protein kinase D (PKD) contributes to prostate and pancreatic cancer cell growth and survival. Therefore, PKD inhibitors are potential anticancer agents. kb-NB142-70 (NB142) and its methoxy-analogue, kb-NB184-43 (NB184), inhibit PKD in vitro. Methods: The in vitro inhibition of growth of Pc 3 prostate cells and Panc-1 pancreatic cells by NB142 and NB184 was evaluated by MTT assay. We evaluated the pharmacokinetics of NB142 in SCID mice bearing Pc 3 human prostate cancer xenografts and of NB184 in SCID mice bearing Panc-1 cancer xenografts. NB142 or NB184 was administered i.v. at 25 mg/kg. Three mice per time point were euthanized at 5, 15, 30, 60, 120, 240, 360, 960 and 1440 min after dosing. Plasma, tissues and urine were collected, and concentrations of NB142 and NB184 were quantitated by HPLC-UV. Metabolites in plasma and urine were characterized by LC-MS/MS. Results: The IC50s of NB142 and NB184 were 21.0 µM and 24.3 µM, respectively, against Pc 3; and 33.7 µM and 27.4 µM, respectively, against Panc-1. After dosing NB142, the plasma NB142 Cmax (at 5 min) was 36.9 µM, and the NB142 Pc 3 tumor Cmax was 11.8 µM. NB142 was not detected in plasma, liver, kidney, spleen, or heart beyond 30 min. After dosing NB184, the plasma, liver, and kidney NB184 Cmax, (at 5 min) were 61.9 µM, 42.6 µM, and 94.8 µM, respectively. The NB184 Cmax in Panc-1 tumors (at 15 min) was 8.0 µM. Areas under the concentration vs time curves of NB142 and NB184 in plasma were 409 and 782 nmol·min/ml, respectively. The plasma half-lives of NB142 and NB184 were 6 and 14 min, respectively. The major metabolite of NB142 was a glucuronide. Between 0-6 h, urinary excretion of NB142 and metabolites accounted for 2.5 % and 7.6%, respectively, of the administered dose. NB184 underwent oxidation and glucuronidation, and more than 10 metabolites were observed. NB142 was not detected in plasma after administration of NB184. Between 0-16 h, urinary excretion of NB184 and metabolites accounted for 2.1 % and 5.3%, respectively, of the administered dose. Conclusions: Although the inclusion of a methoxy group in NB184 increased its plasma half-life approximately 3-fold compared to NB142, metabolic and urinary elimination of both compounds was relatively rapid. Peak tumor concentrations of NB142 and NB184 were of the same order of magnitude as the concentrations required in vitro for cytotoxicity. (Support: P30-CA47904 and P01-[CA078039][1]) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2532. [1]: /lookup/external-ref?link_type=GEN&access_num=CA078039&atom=%2Fcanres%2F70%2F8_Supplement%2F2532.atom