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1.  A Targeted Library Screen Reveals a New Inhibitor Scaffold for Protein Kinase D 
PLoS ONE  2012;7(9):e44653.
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.
PMCID: PMC3445516  PMID: 23028574
2.  Synthesis and Structure-Activity Relationships of Benzothienothiazepinone Inhibitors of Protein Kinase D 
ACS medicinal chemistry letters  2011;2(2):154-159.
Protein kinase D (PKD) is a member of a novel family of serine/threonine kinases that regulate fundamental cellular processes. PKD is implicated in the pathogenesis of several diseases, including cancer. Progress in understanding the biological functions and therapeutic potential of PKD has been hampered by the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently identified as the first potent and selective PKD inhibitor. The study of structure-activity relationships (SAR) of this lead structure led to further improvements in PKD1 potency. We describe herein the synthesis and biological evaluation of novel benzothienothiazepinone analogs. We achieved a ten-fold increase in the in vitro PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accomplished a transition to an almost equally potent novel pyrimidine scaffold, while maintaining excellent target selectivity. These promising results will guide the design of pharmacological tools to dissect PKD function and pave the way for the development of potential anti-cancer agents.
PMCID: PMC3100199  PMID: 21617763
Protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673
3.  Synthesis and Structure−Activity Relationships of Benzothienothiazepinone Inhibitors of Protein Kinase D 
ACS Medicinal Chemistry Letters  2010;2(2):154-159.
Protein kinase D (PKD) is a member of a novel family of serine/threonine kinases that regulate fundamental cellular processes. PKD is implicated in the pathogenesis of several diseases, including cancer. Progress in understanding the biological functions and therapeutic potential of PKD has been hampered by the lack of specific inhibitors. The benzoxoloazepinolone CID755673 was recently identified as the first potent and selective PKD inhibitor. The study of structure−activity relationships (SAR) of this lead compound led to further improvements in PKD1 potency. We describe herein the synthesis and biological evaluation of novel benzothienothiazepinone analogues. We achieved a 10-fold increase in the in vitro PKD1 inhibitory potency for the second generation lead kb-NB142-70 and accomplished a transition to an almost equally potent novel pyrimidine scaffold, while maintaining excellent target selectivity. These promising results will guide the design of pharmacological tools to dissect PKD function and pave the way for the development of potential anticancer agents.
PMCID: PMC3100199  PMID: 21617763
Protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673
4.  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 
PLoS ONE  2015;10(3):e0119346.
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.
PMCID: PMC4352033  PMID: 25747583
5.  New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells 
PLoS ONE  2013;8(9):e75601.
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, in vitro 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 PKD1M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.
PMCID: PMC3781056  PMID: 24086585
6.  Protein Kinase D Regulates Cell Death Pathways in Experimental Pancreatitis 
Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-κB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK) in pancreatic acinar cells. Conversely, up-regulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins, caspases, receptor-interacting protein kinase 1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.
PMCID: PMC3313474  PMID: 22470346
pancreatic acinar cells; CCK; CID755673; CRT0066101; apoptosis; necrosis
7.  Discovery of Diverse Small Molecule Chemotypes with Cell-Based PKD1 Inhibitory Activity 
PLoS ONE  2011;6(10):e25134.
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.
PMCID: PMC3187749  PMID: 21998636
8.  CID755673 enhances mitogenic signaling by phorbol esters, bombesin and EGF through a protein kinase D-independent pathway 
Recently, CID755673 was reported to act as a highly selective inhibitor of protein kinase D (PKD). In the course of experiments using CID755673, we noticed that it exerted unexpected stimulatory effects on [3H]thymidine incorporation and cell cycle progression in Swiss 3T3 cells stimulated by bombesin, a Gq-coupled receptor agonist, phorbol 12,13-dibutyrate (PDBu), a biologically active tumor promoting phorbol ester and epidermal growth factor (EGF). These stimulatory effects could be dissociated from the inhibitory effect of CID755673 on PKD activity, since enhancement of DNA synthesis was still evident in cells with severely down-regulated PKD1 after transfection of siRNA targeting PKD1. A major point raised by our study is that CID755673 can not be considered a specific inhibitor of PKD and it should be used with great caution in experiments attempting to elucidate the role of PKD family members in cellular regulation, particularly cell cycle progression from G1/Go to S phase.
PMCID: PMC2812606  PMID: 19896460
Swiss 3T3 cells; PDGF; PKD knock down; cell cycle; DNA synthesis
9.  In vitro Cytotoxicity, Pharmacokinetics, Tissue Distribution, and Metabolism of Small-Molecule Protein Kinase D Inhibitors, kb-NB142-70 and kb-NB165-09, in Mice bearing Human Cancer Xenografts 
Protein kinase D (PKD) mediates diverse biological responses including cell growth and survival. Therefore, PKD inhibitors may have therapeutic potential. We evaluated the in vitro cytotoxicity of two PKD inhibitors, kb-NB142-70 and its methoxy analog, kb-NB165-09, and examined their in vivo efficacy and pharmacokinetics.
The in vitro cytotoxicities of kb-NB142-70 and kb-NB165-09 were evaluated by MTT assay against PC-3, androgen independent prostate cancer cells, and CFPAC-1 and PANC-1, pancreatic cancer cells. Efficacy studies were conducted in mice bearing either PC-3 or CPFAC-1 xenografts. Tumor-bearing mice were euthanized between 5 and 1440 min after iv dosing, and plasma and tissue concentrations were measured by HPLC-UV. Metabolites were characterized by LC-MS/MS.
kb-NB142-70 and kb-NB165-09 inhibited cellular growth in the low-mid μM range. The compounds were inactive when administered to tumor-bearing mice. In mice treated with kb-NB142-70, the plasma Cmax was 36.9 nmol/mL and the PC-3 tumor Cmax was 11.8 nmol/g. In mice dosed with kb-NB165-09, the plasma Cmax was 61.9 nmol/mL while the PANC-1 tumor Cmax was 8.0 nmol/g. The plasma half-lives of kb-NB142-70 and kb-NB165-09 were 6 and 14 min, respectively. Both compounds underwent oxidation and glucuronidation.
kb-NB142-70 and kb-NB165-09 were rapidly metabolized, and concentrations in tumor were lower than those required for in vitro cytotoxicity. Replacement of the phenolic hydroxyl group with a methoxy group increased the plasma half-life of kb-NB165-09 2.3-fold over that of kb-NB142-70. Rapid metabolism in mice suggests that next-generation compounds will require further structural modifications to increase potency and/or metabolic stability.
PMCID: PMC3557573  PMID: 23108699
Protein Kinase D (PKD) inhibitors; pharmacokinetics; prostate cancer; pancreatic cancer; kb-NB142-70; kb-NB165-09
10.  Design, Synthesis, and Biological Evaluation of PKD Inhibitors 
Pharmaceutics  2011;3(2):186-228.
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.
PMCID: PMC3261798  PMID: 22267986
protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673; thiazepinothiophenopyrimidinone
11.  Design, Synthesis, and Biological Evaluation of PKD Inhibitors 
Pharmaceutics  2011;3(2):186-228.
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.
PMCID: PMC3261798  PMID: 22267986
protein kinase D; small molecule inhibitor; benzothienothiazepinone; pyrimidines; CID755673; thiazepinothiophenopyrimidinone
12.  A Novel Small Molecule Inhibitor of Protein Kinase D Blocks Pancreatic Cancer Growth In Vivo 
Protein kinase D (PKD) is a novel family (PKD1, PKD2, and PKD3) of serine-threonine kinases with diverse biologic functions including cell proliferation and growth. Pancreatic cancer (PaCa) is a devastating disease with few therapeutic options. We showed earlier that PKD signaling pathways promote mitogenesis in multiple PaCa cell lines. However, nothing is known about targeting biologic functions of PKD family in PaCa. Our PKD inhibitor discovery program yielded CRT0066101, which specifically blocks activation of PKD family.
The aim of this study was to determine the effects of CRT0066101 in PaCa, both in vitro and in vivo.
Methods and Results:
Immunohistochemical analysis showed that activated PKD1 (pS916-PKD1) is significantly upregulated in PaCa as compared with normal ducts (91% vs. 22%; P < .001). We also showed that PKD1 and PKD2 are over-expressed in multiple PaCa cell lines including Panc-1. Using Panc-1 as a model system, we demonstrated that CRT0066101 blocked proliferation and BrdU incorporation with an IC50 of 1 μM, and also blocked PKD1-dependent NF-κB activation using luciferase reporter assays. CRT0066101 given orally (80 mg/kg/d) for 4 weeks significantly abrogated growth in a subcutaneous Panc-1 xenograft model (n=8; P < .01). The expression of activated PKD1 (pS916-PKD1) in the treated tumor explants was significantly inhibited (P < .05), with peak plasma CRT0066101 concentration (12 μM) achieved within 6 hours of oral administration. Further, CRT0066101 given orally (80 mg/kg/d) for 21 days in an orthotopic model potently blocked Panc-1 tumor growth (n=7; P < .01). CRT0066101 significantly reduced Ki-67+ proliferation index (P < .01), increased apoptosis (measured by in situ TUNEL assay) of PaCa tumors (P < .05), and potently abrogated expression of NF-κB-regulated multiple proliferative and pro-survival proteins, including cyclin D1, survivin, Bcl-2, Bcl-xL, activated PKD1 (pS916-PKD1), and activated PKD2 (pS876-PKD2).
These results demonstrate for the first time that the PKD-specific small molecule inhibitor CRT0066101 blocks PaCa growth both in vitro and in vivo. Thus, PKD is a novel therapeutic target in PaCa.
PMCID: PMC3047024
13.  A novel small molecule inhibitor of protein kinase D blocks pancreatic cancer growth in vitro and in vivo 
Molecular cancer therapeutics  2010;9(5):1136-1146.
Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer (PaCa). However, nothing is known about the effects of targeting PKD in PaCa. Our PKD-inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in PaCa. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in PaCa and that PKD1/2 are expressed in multiple PaCa cell-lines. Using Panc-1 as a model system, we demonstrated that CRT0066101 reduced BrdU incorporation, increased apoptosis, blocked neurotensin (NT)-induced PKD1/2 activation, reduced NT-induced PKD-mediated Hsp27 phosphorylation, attenuated PKD1-mediated NF-κB activation, and abrogated expression of NF-κB-dependent-dependent proliferative and pro-survival proteins. We showed that CRT0066101 given orally (80 mg/kg/day) for 28 days significantly abrogated PaCa growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor-explants was significantly inhibited with peak tumor concentration (12 µM) of CRT0066101 achieved within 2 h after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/day) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67+ proliferation index (p< 0.01), increased TUNEL+ apoptotic cells (p<0.05), and abrogated expression of NF-κB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results demonstrate for the first time that a PKD-specific small molecule inhibitor CRT0066101 blocks PaCa growth in vivo and show that PKD is a novel therapeutic target in PaCa.
PMCID: PMC2905628  PMID: 20442301
protein kinase D; small molecule inhibitor; pancreatic cancer; tumor xenografts
14.  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 
Carcinogenesis  2011;32(8):1198-1206.
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.
PMCID: PMC3149210  PMID: 21665893
15.  The PKD Inhibitor CID755673 Enhances Cardiac Function in Diabetic db/db Mice 
PLoS ONE  2015;10(3):e0120934.
The development of diabetic cardiomyopathy is a key contributor to heart failure and mortality in obesity and type 2 diabetes (T2D). Current therapeutic interventions for T2D have limited impact on the development of diabetic cardiomyopathy. Clearly, new therapies are urgently needed. A potential therapeutic target is protein kinase D (PKD), which is activated by metabolic insults and implicated in the regulation of cardiac metabolism, contractility and hypertrophy. We therefore hypothesised that PKD inhibition would enhance cardiac function in T2D mice. We first validated the obese and T2D db/db mouse as a model of early stage diabetic cardiomyopathy, which was characterised by both diastolic and systolic dysfunction, without overt alterations in left ventricular morphology. These functional characteristics were also associated with increased PKD2 phosphorylation in the fed state and a gene expression signature characteristic of PKD activation. Acute administration of the PKD inhibitor CID755673 to normal mice reduced both PKD1 and 2 phosphorylation in a time and dose-dependent manner. Chronic CID755673 administration to T2D db/db mice for two weeks reduced expression of the gene expression signature of PKD activation, enhanced indices of both diastolic and systolic left ventricular function and was associated with reduced heart weight. These alterations in cardiac function were independent of changes in glucose homeostasis, insulin action and body composition. These findings suggest that PKD inhibition could be an effective strategy to enhance heart function in obese and diabetic patients and provide an impetus for further mechanistic investigations into the role of PKD in diabetic cardiomyopathy.
PMCID: PMC4370864  PMID: 25798941
16.  Protein Kinase D as a Potential Chemotherapeutic Target for Colorectal Cancer 
Molecular cancer therapeutics  2014;13(5):1130-1141.
Protein kinase D (PKD) signaling plays a critical role in the regulation of DNA synthesis, proliferation, cell survival, adhesion, invasion/migration, motility, and angiogenesis. To date, relatively little is known about the potential role of PKD in the development and/or progression of human colorectal cancer (CRC). We evaluated the expression of different PKD isoforms in CRC and investigated the antitumor activity of PKD inhibitors against human CRC. PKD2 was the dominant isoform expressed in human colon cancer cells. PKD3 expression was also observed but PKD1 expression, at both the RNA and protein levels, was not detected. Suppression of PKD using the small molecule inhibitors, CRT0066101 and kb-NB142-70, resulted in low micromolar in vitro antiproliferative activity against multiple human CRC cell lines. Drug treatment was associated with dose-dependent suppression of PKD2 activation. Incubation with CRT0066101 resulted in G2/M phase arrest and induction of apoptosis in human CRC cells. Further studies showed that CRT0066101 treatment gave rise to a dose-dependent increase in expression of cleaved PARP and activated caspase-3, in addition to inhibition of AKT and ERK signaling, and suppression of NF-κB activity. Transfection of PKD2-targeted siRNAs resulted in similar effects on downstream pathways as observed with small molecule inhibitors. Daily administration of CRT0066101 resulted in significant inhibition of tumor growth in HCT116 xenograft nude mice. Taken together, our studies show that PKD plays a significant role in mediating growth signaling in CRC and may represent a novel chemotherapeutic target for the treatment of CRC.
PMCID: PMC4019967  PMID: 24634417
protein kinase D; human colorectal cancer; apoptosis; NF-κB; chemotherapy target
17.  Differential PKC-dependent and -independent PKD activation by G protein α subunits of the Gq family: selective stimulation of PKD Ser748 autophosphorylation by Gαq 
Cellular Signalling  2011;24(4):914-921.
Protein kinase D (PKD) is activated within cells by stimulation of multiple G protein coupled receptors (GPCR). Earlier studies demonstrated a role for PKC to mediate rapid activation loop phosphorylation-dependent PKD activation. Subsequently, a novel PKC-independent pathway in response to Gαq-coupled GPCR stimulation was identified. Here, we examined further the specificity and PKC-dependence of PKD activation using COS-7 cells cotransfected with different Gq-family Gα and stimulated with aluminum fluoride (AlF4−). PKD activation was measured by kinase assays, and Western blot analysis of activation loop sites Ser744, a prominent and rapid PKC transphosphorylation site, and Ser748, a site autophosphorylated in the absence of PKC signaling. Treatment with AlF4− potently induced PKD activation and Ser744 and Ser748 phosphorylation, in the presence of cotransfected Gαq, Gα11, Gα14 or Gα15. These treatments achieved PKD activation loop phosphorylation similar to the maximal levels obtained by stimulation with the phorbol ester, PDBu. Preincubation with the PKC inhibitor GF1 potently blocked Gα11-, Gα14-, and Gα15-mediated enhancement of Ser748 phosphorylation induced by AlF4−, and largely abolished Ser744 phosphorylation. In contrast, Ser748 phosphorylation was almost completely intact, and Ser744 phosphorylation was significantly activated in cells cotransfected with Gαq. Importantly, the differential Ser748 phosphorylation was also promoted by treatment of Swiss 3T3 cells with Pasteurella multocida toxin, a selective activator of Gαq but not Gα11. Taken together, our results suggest that Gαq, but not the closely related Gα11, promotes PKD activation in response to GPCR ligands in a unique manner leading to PKD autophosphorylation at Ser748.
PMCID: PMC3286641  PMID: 22227248
18.  Inducible silencing of protein kinase D3 inhibits secretion of tumor-promoting factors in prostate cancer 
Molecular Cancer Therapeutics  2012;11(7):1389-1399.
Protein kinase D acts as a major mediator of several signaling pathways related to cancer development. Aberrant PKD expression and activity have been demonstrated in multiple cancers, and novel PKD inhibitors show promising anti-cancer activities. Despite these advances, the mechanisms through which PKD contributes to the pathogenesis of cancer remain unknown. Here, we establish a novel role for PKD3, the least studied member of the PKD family, in the regulation of prostate cancer cell growth and motility through modulation of secreted tumor-promoting factors. Using both a stable inducible knockdown cell model and a transient knockdown system employing multiple siRNAs, we demonstrate that silencing of endogenous PKD3 significantly reduces prostate cancer cell proliferation, migration, and invasion. Additionally, conditioned medium from PKD3 knockdown cells exhibits less migratory potential compared to that from control cells. Further analysis indicated that depletion of PKD3 blocks secretion of multiple key tumor-promoting factors including MMP-9, IL-6, IL-8, and GROα, but does not alter mRNA transcript levels for these factors, implying impairment of the secretory pathway. More significantly, inducible depletion of PKD3 in a subcutaneous xenograft model suppresses tumor growth and decreases levels of intratumoral GROα in mice. These data validate PKD3 as a promising therapeutic target in prostate cancer and shed light on the role of secreted tumor-promoting factors in prostate cancer progression.
PMCID: PMC3392457  PMID: 22532599
Protein kinase D; prostate cancer; cytokines; secretion; inducible knockdown
19.  Protein Kinase Inhibitors CK59 and CID755673 Alter Primary Human NK Cell Effector Functions 
Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.
PMCID: PMC3600540  PMID: 23508354
NK cells; immune modulation; signaling pathways; PKD; CaMKII; effector function; Fyn; CID755673
20.  Role of protein kinase D signaling in pancreatic cancer 
Biochemical pharmacology  2010;80(12):1946-1954.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with dismal survival rates. Its intransigence to conventional therapy renders PDAC an aggressive disease with early metastatic potential. Thus, novel targets for PDAC therapy are urgently needed. Multiple signal transduction pathways are implicated in progression of PDAC. These pathways stimulate production of intracellular messengers in their target cells to modify their behavior, including the lipid-derived diacylglycerol (DAG). One of the prominent intracellular targets of DAG is the protein kinase C (PKC) family. However, the mechanisms by which PKC-mediated signals are decoded by the cell remain incompletely understood. Protein kinase D1 (PKD or PKD1, initially called atypical PKCµ), is the founding member of a novel protein kinase family that includes two additional protein kinases that share extensive overall homology with PKD, termed PKD2, and PKD3. The PKD family occupies a unique position in the signal transduction pathways initiated by DAG and PKC. PKD lies downstream of PKCs in a novel signal transduction pathway implicated in the regulation of multiple fundamental biological processes. We and others have shown that PKD-mediated signaling pathways promote mitogenesis and angiogenesis in PDAC. Our recent observations demonstrate that PKD also potentiates chemoresistance and invasive potential of PDAC cells. This review will briefly highlight diverse biological roles of PKD family in multiple neoplasias including PDAC. Further, this review will underscore our latest advancement with the development of a potent PKD family inhibitor and its effect both in vitro and in vivo in PDAC.
PMCID: PMC2974013  PMID: 20621068
Protein kinase D; Protein kinase C; Pancreatic cancer; Signal transduction
21.  Protein Kinase D Protects Against Oxidative Stress-Induced Intestinal Epithelial Cell Injury via Rho/ROK/PKC-δ Pathway Activation 
Protein kinase D (PKD) is a novel protein serine kinase which has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was: (i) to define the activation of PKD in intestinal epithelial cells treated with hydrogen peroxide (H2O2), an agent which induces oxidative stress, and (ii) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H2O2 in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective protein kinase C (PKC)-δ inhibitor, and small interfering RNA (siRNA) directed against PKC-δ, suggesting the regulation of PKD activity by upstream PKC-δ. Activation of PKD was also blocked by a Rho kinase (ROK) specific inhibitor, Y27632, as well as C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H2O2-induced PKC-δ phosphorylation was inhibited by C3 treatment, further suggesting that PKC-δ is downstream of Rho/ROK. Interestingly, H2O2-induced intestinal cell apoptosis was enhanced by PKD siRNA. Taken together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells, and this activation is regulated by upstream PKC-δ and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications to intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants).
PMCID: PMC2613753  PMID: 16421204
Intestinal epithelial injury; oxidative stress; PKD; PKC-δ; Rho/ROK
22.  Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells 
PLoS ONE  2014;9(12):e115146.
The flower buds of Daphne genkwa Sieb. et Zucc. have been used as a traditional Chinese medicine although their functional mechanisms have not been discovered yet. We have studied the potential effects of the plant extracts on natural killer (NK) cell activation, and isolated an active fraction. Genkwadaphnin (GD-1) displayed a potent efficacy to induce IFN-γ transcription in NK cells with concentration- and time-dependent manners. GD-1 treatment triggered the phosphorylation of PKD1, a member of PKC family, MEK and ERK, resulting in IKK activation to induce IκB degradation, and the nuclear localization of p65, an NF-κB subunit, which regulates IFN-γ transcription. GD-1 effect on IFN-γ production was blocked by the addition of Rottlerin, a PKC inhibitor, CID 755673, a PKD inhibitor, or Bay11-7082, an IKKα inhibitor. The nuclear localization of p65 was also inhibited by the kinase inhibitors. Secreted IFN-γ activates STAT1 phosphorylation as autocrine-loops to sustain its secretion. GD-1 induced the phosphorylation of STAT1 probably through the increase of IFN-γ. STAT1 inhibitor also abrogated the sustained IFN-γ secretion. These results suggest that GD-1 is involved in the activation of PKD1 and/or ERK pathway, which activate NK-κB triggering IFN-γ production. As positive feedback loops, secreted IFN-γ activates STAT1 and elongates its production in NK-92 cells.
PMCID: PMC4269520  PMID: 25517939
23.  Protein Kinase Cδ mediates the activation of Protein Kinase D2 in Platelets 
Biochemical pharmacology  2011;82(7):720-727.
Protein Kinase D (PKD) is a subfamily of serine/threonine specific family of kinases, comprised of PKD1, PKD2 and PKD3 (PKCμ, PKD2 and PKCν in humans). It is known that PKCs activate PKD, but the relative expression of isoforms of PKD or the specific PKC isoform/s responsible for its activation in platelets is not known. This study is aimed at investigating the pathway involved in activation of PKD in platelets. We show that PKD2 is the major isoform of PKD that is expressed in human as well as murine platelets but not PKD1 or PKD3. PKD2 activation induced by AYPGKF was abolished with a Gq inhibitor YM-254890, but was not affected by Y-27632, a RhoA/p160ROCK inhibitor, indicating that PKD2 activation is Gq-, but not G12/13-mediated Rho-kinase dependent. Calcium-mediated signals are also required for activation of PKD2 as dimethyl BAPTA inhibited its phosphorylation. GF109203X, a pan PKC inhibitor abolished PKD2 phosphorylation but Go6976, a classical PKC inhibitor had no effect suggesting that novel PKC isoforms are involved in PKD2 activation. Importantly, Rottlerin, a non-selective PKCδ inhibitor, inhibited AYPGKF-induced PKD2 activation in human platelets. Similarly, AYPGKF- and Convulxin-induced PKD2 phosphorylation was dramatically inhibited in PKCδ-deficient platelets, but not in PKCθ– or PKCε–deficient murine platelets compared to that of wild type platelets. Hence, we conclude that PKD2 is a common signaling target downstream of various agonist receptors in platelets and Gq-mediated signals along with calcium and novel PKC isoforms, in particular, PKCδ activate PKD2 in platelets.
PMCID: PMC3156373  PMID: 21736870
Protein kinase C; Calcium; Protein kinase D; Protease activated receptor; Platelet; Gq
24.  Protein kinase D2 induces invasion of pancreatic cancer cells by regulating matrix metalloproteinases 
Molecular Biology of the Cell  2014;25(3):324-336.
Protein kinase D2 (PKD2) expression is up-regulated in pancreatic cancer. PKD1 expression is low. Both kinases differentially control pancreatic cancer cell invasion. PKD2 enhances invasion and angiogenesis in 3D ECM, as well as in chorioallantois membrane models, by stimulating expression and secretion of matrix metalloproteinase 7/9.
Pancreatic cancer cell invasion, metastasis, and angiogenesis are major challenges for the development of novel therapeutic strategies. Protein kinase D (PKD) isoforms are involved in controlling tumor cell motility, angiogenesis, and metastasis. In particular PKD2 expression is up-regulated in pancreatic cancer, whereas PKD1 expression is lowered. We report that both kinases control pancreatic cancer cell invasive properties in an isoform-specific manner. PKD2 enhances invasion in three-dimensional extracellular matrix (3D-ECM) cultures by stimulating expression and secretion of matrix metalloproteinases 7 and 9 (MMP7/9), by which MMP7 is likely to act upstream of MMP9. Knockdown of MMP7/9 blocks PKD2-mediated invasion in 3D-ECM assays and in vivo using tumors growing on chorioallantois membranes. Furthermore, MMP9 enhances PKD2-mediated tumor angiogenesis by releasing extracellular matrix–bound vascular endothelial growth factor A, increasing its bioavailability and angiogenesis. Of interest, specific knockdown of PKD1 in PKD2-expressing pancreatic cancer cells further enhanced the invasive properties in 3D-ECM systems by generating a high-motility phenotype. Loss of PKD1 thus may be beneficial for tumor cells to enhance their matrix-invading abilities. In conclusion, we define for the first time PKD1 and 2 isoform–selective effects on pancreatic cancer cell invasion and angiogenesis, in vitro and in vivo, addressing PKD isoform specificity as a major factor for future therapeutic strategies.
PMCID: PMC3907273  PMID: 24336522
25.  Protein Kinase D isoforms – New targets for therapy in invasive Breast Cancers? 
Expert review of anticancer therapy  2013;13(8):10.1586/14737140.2013.816460.
The Protein Kinase D (PKD) family of serine/threonine kinases consists of three members-PKD1, PKD2, and PKD3. While PKD1 in many cancers has been identified as a suppressor of the invasive phenotype, the two other PKD subtypes, PKD2 and PKD3, have been attributed oncogenic functions. In invasive Breast Cancer cells PKD1 expression is downregulated by methylation of the PRKD1 promoter. On the other hand, PKD2 and PKD3 are not silenced, and drive proliferation, invasion, and mediate chemoresistance. Two strategies emerge to utilize this knowledge for novel treatment opportunities. First, pan PKD inhibitors could be developed and used for these aggressive cancers. An alternative approach to obtain similar effects would be to induce the re-expression of PKD1.
PMCID: PMC3856925  PMID: 23944680
Protein Kinase D; PKD; isoforms; breast cancer; therapy

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