Phosphoinositide-dependent Kinase-1 Inhibitor Research Articles

Phosphoinositide-dependent Kinase-1 (PDPK1) regulates serum/glucocorticoid-regulated Kinase 3 (SGK3) for prostate cancer cell survival.

Prostate cancer (PCa) is the most common malignancy and is the second leading cause of cancer among men globally. Using a kinome‐wide lentiviral small‐hairpin RNA (shRNA) library screen, we identified phosphoinositide‐dependent kinase‐1 (PDPK1) as a potential mediator of cell survival in PCa cells. We showed that knock‐down of endogenous human PDPK1 induced significant tumour‐specific cell death in PCa cells (DU145 and PC3) but not in the normal prostate epithelial cells (RWPE‐1). Further analyses revealed that PDPK1 mediates cancer cell survival predominantly via activation of serum/glucocorticoid‐regulated kinase 3 (SGK3). Knock‐down of endogenous PDPK1 in DU145 and PC3 cells significantly reduced SGK3 phosphorylation while ectopic expression of a constitutively active SGK3 completely abrogated the apoptosis induced by PDPK1. In contrast, no such effect was observed in SGK1 and AKT phosphorylation following PDPK1 knock‐down. Importantly, PDPK1 inhibitors (GSK2334470 and BX‐795) significantly reduced tumour‐specific cell growth and synergized docetaxel sensitivity in PCa cells. In summary, our results demonstrated that PDPK1 mediates PCa cells’ survival through SGK3 signalling and suggest that inactivation of this PDPK1‐SGK3 axis may potentially serve as a novel therapeutic intervention for future treatment of PCa.

PDK1 Inhibitor GSK-470 Exhibits Potent Anticancer Activity in a Pheochromocytoma PC12 Cell Tumor Model via Akt/mTOR Pathway.

Phosphoinositide-Dependent Kinase 1 (PDK1) is now widely studied in malignant solid tumors. Researchers have previously revealed that targeting PDK1 is thought of as a promising anticancer treatment strategy. The aim of this study was designed to evaluate the anticancer activity of GSK-470, a novel and highly specific inhibitor of PDK1, in Pheochromocytoma (PCC) tumor model. PC12 cells were xenografted into nude mice to build PCC tumor model. Animals were treated with GSK-470 vs vehicle. Mean tumor volume was calculated and compared across groups. TUNEL was used to detect apoptosis. The effects of PDK1 inhibitor GSK-470 on activation of the Akt signaling and its downstream Akt/mTOR pathway in xenotransplant tumor tissues were examined by western bolt. The mean tumor volume in GSK-470 group was significantly less than that in control group. TUNEL results found that cell apoptosis was markedly increased in GSK-470 group compared with the control group. The western bolt analysis showed that the phosphorylation of Akt at threonine 308 was significantly reduced in GSK-470 group. Also, GSK-470 strongly inhibited phosphorylation of mTOR on Ser2448, a marker for mTORC1 activity, as well as phosphorylation of p70S6K, best characterized targets of mTOR. Our results showed that GSK-470 exhibited potent anticancer activity in PC12 tumor-bearing mice. Also, we found that this effect appeared to be mediated by the inhibition of the Akt/mTOR pathway. The present study once again provides new insights into the therapeutic effects of inhibiting PDK1 in the treatment of malignant PCC. Therefore, we propose that GSK-470 might be an effective therapeutic agent for the treatment of malignant PCC.

Ligand Based Pharmacophore Modeling Followed by Biological Screening Lead to Discovery of Novel PDK1 Inhibitors as Anticancer Agents.

Phosphoinositide-Dependent Kinase-1 (PDK1) is a serine/threonine kinase, which belongs to AGC kinase family required by cancer cells. Pharmacophoric space of 86 PDK1 inhibitors using six diverse sets of inhibitors was explored to identify high-quality pharmacophores. The best combination of pharmacophoric models and physicochemical descriptors was selected by genetic algorithm-based QSAR analysis that can elucidate the variation of bioactivity within the training inhibitors. Two successful orthogonal pharmacophores emerged in the optimum QSAR equation (r2 69 = 0.90, r2 LOO= 0.86, F= 51.92, r2 PRESS against 17 test inhibitors = 0.79). Receiver Operating Characteristic (ROC) curve analyses were used to estimate the QSAR-selected pharmacophores. 5 out of 11 compounds tested had shown potential intracellular PDK1 inhibition with the highest inhibition percent for compounds 92 and 93 as follows; 90 and 92% PDK1 inhibition, respectively. PDK1 inhibitors are potential anticancer agents that can be discovered by combination method of ligand based design with QSAR and ROC analysis.

Phospho‐inositide‐dependent kinase 1 regulates signal dependent translation in megakaryocytes and platelets

BackgroundRegulated protein synthesis is essential for megakaryocyte (MK) and platelet functions, including platelet production and activation. PDK1 (phosphoinositide‐dependent kinase 1) regulates platelet functional responses and has been associated with circulating platelet counts. Whether PDK1 also directly regulates protein synthetic responses in MKs and platelets, and platelet production by MKs, remains unknown. ObjectiveTo determine if PDK1 regulates protein synthesis in MKs and platelets. MethodsPharmacologic PDK1 inhibitors (BX‐795) and mice where PDK1 was selectively ablated in MKs and platelets (PDK1−/−) were used. PDK1 signaling in MKs and platelets (human and murine) were assessed by immunoblots. Activation‐dependent translation initiation and protein synthesis in MKs and platelets was assessed by probing for dissociation of eIF4E from 4EBP1, and using m7‐GTP pulldowns and S35 methionine incorporation assays. Proplatelet formation by MKs, synthesis of Bcl‐3 and MARCKs protein, and clot retraction were employed for functional assays. ResultsInhibiting or ablating PDK1 in MKs and platelets abolished the phosphorylation of 4EBP1 and eIF4E by preventing activation of the PI3K and MAPK pathways. Inhibiting PDK1 also prevented dissociation of eIF4E from 4EBP1, decreased binding of eIF4E to m7GTP (required for translation initiation), and significantly reduced de novo protein synthesis. Inhibiting PDK1 reduced proplatelet formation by human MKs and blocked MARCKs protein synthesis. In both human and murine platelets, PDK1 controlled Bcl‐3 synthesis. Inhibition of PDK1 led to complete failure of clot retraction in vitro. ConclusionsPDK1 is a previously unidentified translational regulator in MKs and platelets, controlling protein synthetic responses, proplatelet formation, and clot retraction.

Investigation of Binding Characteristics of Phosphoinositide-dependent Kinase-1 (PDK1) Co-crystallized Ligands Through Virtual Pharmacophore Modeling Leading to Novel Anti-PDK1 Hits.

3-Phosphoinositide Dependent Protein Kinase-1 (PDK1) is being lately considered as an attractive and forthcoming anticancer target. A Protein Data Bank (PDB) cocrystallized crystal provides not only rigid theoretical data but also a realistic molecular recognition data that can be explored and used to discover new hits. This incited us to investigate the co-crystallized ligands' contacts inside the PDK1 binding pocket via a structure-based receptor-ligand pharmacophore generation technique in Discovery Studio 4.5 (DS 4.5). Accordingly, 35 crystals for PDK1 were collected and studied. Every single receptorligand interaction was validated and the significant ones were converted into their corresponding pharmacophoric features. The generated pharmacophores were scored by the Receiver Operating Characteristic (ROC) curve analysis. Consequently, 169 pharmacophores were generated and sorted, 11 pharmacophores acquired good ROC-AUC results of 0.8 and a selectivity value above 8. Pharmacophore 1UU3_2_01 was used in particular as a searching filter to screen NCI database because of its acceptable validity criteria and its distinctive positive ionizable feature. Several low micromolar PDK1 inhibitors were revealed. The most potent hit illustrated anti-PDK1 IC50 values of 200 nM with 70% inhibition against SW480 cell lines. Eventually, the active hits were docked inside the PDK1 binding pocket and the recognition points between the active hits and the receptor were analyzed that led to the discovery of new scaffolds as potential PDK1 inhibitors.

PDK1 inhibitor GSK2334470 synergizes with proteasome inhibitor MG‑132 in multiple myeloma cells by inhibiting full AKT activity and increasing nuclear accumulation of the PTEN protein.

Phosphoinositide‑dependent kinase1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

Fragment-based design, synthesis, biological evaluation, and SAR of 1H-benzo[d]imidazol-2-yl)-1H-indazol derivatives as potent PDK1 inhibitors

In this work, we describe the use of the rule of 3 fragment-based strategies from biochemical screening data of 1100 in-house, small, low molecular weight fragments. The sequential combination of in silico fragment hopping and fragment linking based on S160/Y161/A162 hinge residues hydrogen bonding interactions leads to the identification of novel 1H-benzo[d]imidazol-2-yl)-1H-indazol class of Phosphoinositide-Dependent Kinase-1 (PDK1) inhibitors. Consequent SAR and follow-up screening data led to the discovery of two potent PDK1 inhibitors: compound 32 and 35, with an IC50 of 80 nM and 94 nM, respectively. Further biological evaluation showed that, at the low nanomolar concentration, the drug had potent ability to inhibit phosphorylation of AKT and p70S6, and selectively kill the cancer cells with mutations in both PTEN and PI3K. The microarray data showed that DUSP6, DUSP4, and FOSL1 were down-regulated in the sensitive cell lines with the compound treatment. The in vivo test showed that 35 can significantly inhibit tumor growth without influencing body weight growth. Our results suggest that these compounds, especially 35, merit further pre-clinical evaluation.

Periostin suppresses in vivo invasiveness via PDK1/Akt/mTOR signaling pathway in a mouse orthotopic model of bladder cancer.

Periostin is an extracellular matrix protein involved in the regulation of intercellular adhesion. The present study investigated the in vivo tumor suppressor function of periostin in a mouse orthotopic model of bladder cancer. Retroviral vectors were used to transfect human bladder cancer UMUC-3 cell line with periostin. Bladders of nude mice that were transurethrally instilled with periostin-expressing UMUC-3 cells were revealed to weigh less compared with bladders instilled with vector control cells. In total, five (83.3%) of six vector control UMUC-3 bladder tumors exhibited histological evidence of muscle invasion. However, none of the five periostin-expressing UMUC-3 bladder tumors revealed muscle invasion. Thick edematous lesions were present in the submucosa of periostin-expressing UMUC-3 bladder tumors. The expression of periostin also suppressed in vitro cell invasiveness of UMUC-3 cells without affecting cellular proliferation. The level of phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), protein kinase B (Akt) and S6 ribosomal protein, a downstream protein of mammalian target of rapamycin (mTOR) was decreased in periostin-expressing UMUC-3 cells compared with vector control cells. Treatment with 100 ng/ml recombinant human periostin protein also suppressed cell invasiveness and phosphorylation of PDK1, Akt and S6 in UMUC-3 cells, consistent with results using periostin-expressing UMUC-3 cells. Treatment with PDK1, Akt and mTOR inhibitors significantly suppressed UMUC-3 cell invasiveness. These results demonstrate that periostin suppresses in vivo and in vitro invasiveness of bladder cancer via the PDK1/Akt/mTOR signaling pathway. Periostin may be useful as a potent chemotherapeutic agent by suppressing bladder cancer invasiveness.

Dual Inhibition of PDK1 and Aurora Kinase A: An Effective Strategy to Induce Differentiation and Apoptosis of Human Glioblastoma Multiforme Stem Cells.

The poor prognosis of glioblastoma multiforme (GBM) is mainly attributed to drug resistance mechanisms and to the existence of a subpopulation of glioma stem cells (GSCs). Multitarget compounds able to both affect different deregulated pathways and the GSC subpopulation could escape tumor resistance and, most importantly, eradicate the stem cell reservoir. In this respect, the simultaneous inhibition of phosphoinositide-dependent kinase-1 (PDK1) and aurora kinase A (AurA), each one playing a pivotal role in cellular survival/migration/differentiation, could represent an innovative strategy to overcome GBM resistance and recurrence. Herein, the cross-talk between these pathways was investigated, using the single-target reference compounds MP7 (PDK1 inhibitor) and Alisertib (AurA inhibitor). Furthermore, a new ligand, SA16, was identified for its ability to inhibit the PDK1 and the AurA pathways at once, thus proving to be a useful tool for the simultaneous inhibition of the two kinases. SA16 blocked GBM cell proliferation, reduced tumor invasiveness, and triggered cellular apoptosis. Most importantly, the AurA/PDK1 blocker showed an increased efficacy against GSCs, inducing their differentiation and apoptosis. To the best of our knowledge, this is the first report on combined targeting of PDK1 and AurA. This drug represents an attractive multitarget lead scaffold for the development of new potential treatments for GBM and GSCs.

The hVps34-SGK3 pathway alleviates sustained PI3K/Akt inhibition by stimulating mTORC1 and tumourgrowth.

We explore mechanisms that enable cancer cells to tolerate PI3K or Akt inhibitors. Prolonged treatment of breast cancer cells with PI3K or Akt inhibitors leads to increased expression and activation of a kinase termed SGK3 that is related to Akt. Under these conditions, SGK3 is controlled by hVps34 that generates PtdIns(3)P, which binds to the PX domain of SGK3 promoting phosphorylation and activation by its upstream PDK1 activator. Furthermore, under conditions of prolonged PI3K/Akt pathway inhibition, SGK3 substitutes for Akt by phosphorylating TSC2 to activate mTORC1. We characterise 14h, a compound that inhibits both SGK3 activity and activation in vivo, and show that a combination of Akt and SGK inhibitors induced marked regression of BT‐474 breast cancer cell‐derived tumours in a xenograft model. Finally, we present the kinome‐wide analysis of mRNA expression dynamics induced by PI3K/Akt inhibition. Our findings highlight the importance of the hVps34‐SGK3 pathway and suggest it represents a mechanism to counteract inhibition of PI3K/Akt signalling. The data support the potential of targeting both Akt and SGK as a cancer therapeutic.

Locking PDK1 in DFG-out conformation through 2-oxo-indole containing molecules: Another tools to fight glioblastoma

The phosphoinositide-dependent kinase-1 (PDK1) is one of the main components of the PI3K/Akt pathway. Also named the “master kinase” of the AGC family, PDK1 plays a critical role in tumorigenesis, by enhancing cell proliferation and inhibiting apoptosis, as well as in cell invasion and metastasis formation. Although there have been done huge efforts in discovering specific compounds targeting PDK1, nowadays no PDK1 inhibitor has yet entered the clinic. With the aim to pick out novel and potent PDK1 inhibitors, herein we report the design and synthesis of a new class of molecules obtained by merging the 2-oxo-indole nucleus with the 2-oxo-pyridonyl fragment, two moieties with high affinity for the PDK1 hinge region and its DFG-out binding site, respectively. To this purpose, a small series of compounds were synthesised and a tandem application of docking and Molecular Dynamic (MD) was employed to get insight into their mode of binding. The OXID-pyridonyl hybrid 8, possessing the lower IC50 (IC50 = 112 nM), was also tested against recombinant kinases involved in the PI3K/PDK1/Akt pathway and was subjected to vitro studies to evaluate the cytotoxicity and the inhibition of tumour cell migration. All together the results let us to consider 8, as a lead compound of a new generation of PDK1 inhibitors and encourage us to further studies in this direction.

14-3-3 Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells.

Akt activation has been associated with proliferation, differentiation, survival and death of epithelial cells. Phosphorylation of Thr308 of Akt by phosphoinositide-dependent kinase 1 (PDK1) is critical for optimal stimulation of its kinase activity. However, the mechanism(s) regulating this process remain elusive. Here, we report that 14-3-3 proteins control Akt Thr308 phosphorylation during intestinal inflammation. Mechanistically, we found that IFNγ and TNFα treatment induce degradation of the PDK1 inhibitor, 14-3-3η, in intestinal epithelial cells. This mechanism requires association of 14-3-3ζ with raptor in a process that triggers autophagy and leads to 14-3-3η degradation. Notably, inhibition of 14-3-3 function by the chemical inhibitor BV02 induces uncontrolled Akt activation, nuclear Akt accumulation and ultimately intestinal epithelial cell death. Our results suggest that 14-3-3 proteins control Akt activation and regulate its biological functions, thereby providing a new mechanistic link between cell survival and apoptosis of intestinal epithelial cells during inflammation.

Combined 3D QSAR Based Virtual Screening and Molecular Docking Study of Some Selected PDK-1 Kinase Inhibitors

Phosphoinositide-dependent kinase-1 (PDK-1) is an important therapeutic target for the treatment of cancer. In order to identify the important chemical features of PDK-1 inhibitors, a 3D QSAR pharmacophore model was developed based on 21 available PDK-1 inhibitors. The best pharmacophore model (Hypo1) exhibits all the important chemical features required for PDK-1 inhibitors. The correlation coefficient, root mean square deviation (RMSD), and cost difference were 0.96906, 1.0719, and 168.13, respectively, suggesting a good predictive ability of the model (Hypo1) among all the ten pharmacophore models that were analyzed. The best pharmacophore model (Hypo1) was further validated by Fisher’s randomization method (95%), test set method(r=0.87), and the decoy set with the goodness of fit (0.73). Further, this validated pharmacophore model Hypo1 was used as a 3D query to screen the molecules from databases like NCI database and Maybridge. The resultant hit compounds were subsequently subjected to filtration by Lipinski’s rule of five as well as the ADMET study. Docking study was done to refine the retrieved hits and as a result to reduce the rate of false positive. Best hits will further be subjected to in vitro study in future.

Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of BrafV600E::Pten–/– melanoma

Phosphoinositide-dependent kinase-1 (PDK-1) is a serine/threonine protein kinase that phosphorylates members of the conserved AGC kinase superfamily, including AKT and PKC, and is implicated in important cellular processes including survival, metabolism and tumorigenesis. In large cohorts of nevi and melanoma samples, PDK1 expression was significantly higher in primary melanoma, compared with nevi, and was further increased in metastatic melanoma. PDK1 expression suffices for its activity, due to auto-activation, or elevated phosphorylation by phosphoinositide 3'-OH-kinase (PI 3-K). Selective inactivation of Pdk1 in the melanocytes of BrafV600E::Pten−/− or BrafV600E::Cdkn2a−/−::Pten−/− mice delayed the development of pigmented lesions and melanoma induced by systemic or local administration of 4-HT. Melanoma invasion and metastasis were significantly reduced or completely prevented by Pdk1 deletion. Administration of the PDK1 inhibitor GSK2334470 (PDKi) effectively delayed melanomagenesis and metastasis in BrafV600E::Pten−/− mice. Pdk1−/− melanomas exhibit a marked decrease in the activity of AKT, P70S6K and PKC. Notably, PDKi was as effective in inhibiting AGC kinases and colony forming efficiency of melanoma with Pten WT genotypes. Gene expression analyses identified Pdk1-dependent changes in FOXO3a-regulated genes and inhibition of FOXO3a restored proliferation and colony formation of Pdk1−/− melanoma cells. Our studies provide direct genetic evidence for the importance of PDK1, in part through FOXO3a-dependent pathway, in melanoma development and progression.

Abstract 4413: Mechanisms of sensitivity to treatment with the PDK1 inhibitors HCI-1680 and HCI-1708.

Abstract The phosphoinositide-dependent kinase-1 (PDK1) is a serine/threonine kinase that has been considered a promising potential oncology drug target because of its role as an important regulator in the PI3K/AKT/mTOR pathway. PDK1 phosphorylates highly conserved Ser or Thr residues in the activation loop of several AGC super family kinases including PKC, SGK, PKB/Akt, p70S6K, and PDK1 itself. Approximately, 40-50% of all tumors involve mutations in the phosphatase and tensin homolog (PTEN) protein, which results in elevated levels of PIP3 and enhanced activation of PKB/AKT, p70S6K, and SGK. It has been proposed that inhibitors of PDK1 could provide a valuable therapeutic approach to targeting cancer, particularly those with PTEN deficiencies. Using a fragment-based design strategy, we screened a collection of 1100 low molecular weight (< 250 MW) fragments against the PDK1 kinase and identified 9 fragments with moderate inhibitory activity against PDK1 (IC50 values from 45-82 μM). Subsequent molecular docking studies using a crystal structure of PDK1 allowed for the structural rationalization of how these fragments bound in the ATP-binding pocket (hydrogen bonding to S160/A162 hinge residues) and provided insight for further optimization. Synthesis and follow-up screening led to the discovery of HCI-1680 and a related compound SGI-1708, as potent PDK1 inhibitors with IC50 values of 80 and 94 nM, respectively. We used a large cell line panel of over 100 cancer cell lines to examine the ability of these compounds to kill cancer cells. Both HCI-1680 and SGI-1708 demonstrated remarkable selectivity for cell killing in several cell lines (KATO3, KG-1, MV4-11, Kasumi-1, MFE296 and AN3CA) in the low nanomolar range compared to all of the remaining cell lines in which the compounds showed low micromolar activity. Based on the known mutations in these cell lines we determined that HCI-1680 and SGI-1708 were more active in cells with PTEN deletion/silencing as well as activation of PIK3CA through activating mutations in PIK3CA or Ras proteins. Our compounds from this series were also shown to inhibit the activation of AKT and other downstream signaling molecules. We have explored the effects on gene expression Moreover, the lead compounds had high ligand efficiency with promising solubility and permeability parameters. Early animal studies examining pharmacokinetics and efficacy in xenograft models of human cancers have suggested that HCI-1680 and SGI-1708 have properties and activity to be developed as potential clinical candidates. We hypothesize that tumors, which have inactivated PTEN through mutations or silencing and also harbor mutations that activate PI3K define a population of cancer cells that are uniquely sensitive to PDK1 Inhibition. By inhibiting PDK1 activity we will block signaling from the PI3K pathway and lead to inhibition of cell proliferation and survival of these cancer cells. Citation Format: Brian Walker, Sorna Venkataswamy, Steven Warner, Lee Call, Hariprasad Vankayalapati, Sunil Sharma, David J. Bearss. Mechanisms of sensitivity to treatment with the PDK1 inhibitors HCI-1680 and HCI-1708. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4413. doi:10.1158/1538-7445.AM2013-4413

Phosphoinositide-Dependent Kinase-1 and Protein Kinase CδContribute to Endothelin-1 Constriction and Elevated Blood Pressure in Intermittent Hypoxia

Obstructive sleep apnea (OSA) is associated with cardiovascular complications including hypertension. Previous findings from our laboratory indicate that exposure to intermittent hypoxia (IH), to mimic sleep apnea, increases blood pressure in rats. IH also increases endothelin-1 (ET-1) constrictor sensitivity in a protein kinase C (PKC) δ-dependent manner in mesenteric arteries. Because phosphoinositide-dependent kinase-1 (PDK-1) regulates PKCδ activity, we hypothesized that PDK-1 contributes to the augmented ET-1 constrictor sensitivity and elevated blood pressure following IH. Male Sprague-Dawley rats were exposed to either sham or IH (cycles between 21% O(2)/0% CO(2) and 5% O(2)/5% CO(2)) conditions for 7 h/day for 14 or 21 days. The contribution of PKCδ and PDK-1 to ET-1-mediated vasoconstriction was assessed in mesenteric arteries using pharmacological inhibitors. Constrictor sensitivity to ET-1 was enhanced in arteries from IH-exposed rats. Inhibition of PKCδ or PDK-1 blunted ET-1 constriction in arteries from IH but not sham group rats. Western analysis revealed similar levels of total and phosphorylated PDK-1 in arteries from sham and IH group rats but decreased protein-protein interaction between PKCδ and PDK-1 in arteries from IH- compared with sham-exposed rats. Blood pressure was increased in rats exposed to IH, and treatment with the PDK-1 inhibitor OSU-03012 [2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide] (33 mg/day) lowered blood pressure in IH but not sham group rats. Our results suggest that exposure to IH unmasks a role for PDK-1 in regulating ET-1 constrictor sensitivity and blood pressure that is not present under normal conditions. These novel findings suggest that PDK-1 may be a uniquely effective antihypertensive therapy for OSA patients.

Phosphoinositide Dependent Kinase 1 (PDK1) Contributes to Intermittent Hypoxia‐induced Hypertension

Obstructive sleep apnea (OSA) is a risk factor for hypertension. Previous studies from our laboratory demonstrate increased blood pressure in rats exposed to the intermittent hypoxia (IH) model of OSA and increased endothelin‐1 (ET‐1)‐mediated constriction in mesenteric arteries from IH‐exposed rats that is dependent on protein kinase C δ (PKCδ). Phosphoinositide dependent kinase 1 (PDK1) is an upstream regulator of PKCδ. We hypothesized that PDK1contributes to increased ET‐1 constriction and increased blood pressure in IH‐exposed rats. Rats were exposed to IH episodes (cycles between 21% O2/0% CO2 and 5% O2/5% CO2) for 7 hours/day for 14 or 21 days and compared to control rats (maintained at 21% O2/0% CO2). Blood pressure was monitored by telemetry. After 21 days of IH or sham exposure, rats were given a PDK1 inhibitor OSU‐03012 or placebo (33 mg/kg/day) for 3 days. Constriction to ET‐1 but not phenylephrine (PE) was greater in pressurized mesenteric arteries from IH rats compared to control. Inhibiting PDK1 or PKCδ diminished ET‐1 but not PE constriction in arteries from IH rats. Blood pressure was increased in IH compared to control rats. Inhibiting PDK1 in vivo decreased blood pressure in IH rats but had no effect in control rats. Our results provide evidence of a novel role for PDK1/PKCδ in blood pressure regulation in a rodent model of OSA and point to this pathway as a target for blood pressure control in OSA patients.

Novel isoquinolone PDK1 inhibitors discovered through fragment-based lead discovery

Phosphoinositide-dependent kinase-1 (PDK1) is a critical enzyme in the PI3K/AKT pathway and to the activation of AGC family protein kinases, including S6K, SGK, and PKC. Dysregulation of this pathway plays a key role in cancer cell growth, survival and tumor angiogenesis. As such, inhibitors of PDK1 offer the promise of a new therapeutic modality for cancer treatment. Fragment based drug screening has recently become a viable entry point for hit identification. In this work, NMR spectroscopy fragment screening of PDK1 afforded novel chemotypes as orthogonal starting points from HTS screening hits. Compounds identified as hits by NMR spectroscopy were tested in a biochemical assay, and fragments with activity in both assays were clustered. The Pfizer compound file was mined via substructure and 2D similarity search, and the chemotypes were prioritized by ligand efficiency (LE), SAR mining, chemical attractiveness, and chemical enablement of promising vectors. From this effort, an isoquinolone fragment hit, 5 (IC(50) 870μM, LE=0.39), was identified as a novel, ligand efficient inhibitor of PDK1 and a suitable scaffold for further optimization. Initially in the absence of crystallographic data, a fragment growing approach efficiently explored four vectors of the isoquinolone scaffold via parallel synthesis to afford a compound with crystallographic data, 16 (IC(50) 41.4μM, LE=0.33). Subsequent lead optimization efforts provided 24 (IC(50) 1.8μM, LE=0.42), with greater than fivefold selectivity against other key pathway kinases.

Abstract 3578: A novel and selective 3-phosphoinositide-dependent kinase-1 inhibitor, PF-5177624, blocks insulin-like growth factor-1 induced tumorigenesis in breast cancer cells

Abstract Phosphatidylinositol 3-kinases (PI3Ks) phosphorylate membrane phosphatidylinositides to generate phosphatidylinositol 3,4,5-triphosphate (PIP3); PIP3 serves as an important secondary messenger recruiting and activating proteins that contain a pleckstrin homology (PH) domain including AKT and 3’-phosphoinositide-dependent kinase-1 (PDK1). PDK1 is a 63-kDa Ser/Thr kinase with a catalytic domain near its N-terminus and a pleckstrin homology domain at its C-terminus. The PH domain is necessary for targeting PDK1 to the plasma membrane to phosphroylate the T-loop sites of numerous substrates such as AKT at the residue threonine-308 (T308). The phosphorylation at both T308 and the serine-473 residue by mTORC2, fully activates AKT to induce the downstream signaling pathways that are important for tumor progression. PDK1 also has been shown to phosphorylate p70S6K, isoforms of PKCs, and many other kinase substrates. The oncogenic activity of aberrant PI3K pathway signaling through PDK1 has been extensively validated. PDK1 is highly expressed in a majority of human breast cancers and cell lines, and elevated phosphorylation of PDK1 was associated with PIK3CA mutations in human breast tumors and cell lines. Therefore, targeting PDK1 in the PI3K/AKT pathway may provide an opportunity for breast cancer treatment. In this study, we demonstrate that insulin-like growth factor-1 (IGF-1) stimulates PDK1 activity and a specific and potent PDK1 inhibitor, PF-5177624, inhibits IGF-1 stimulated downstream signaling in breast cancer cells, including a reduction in phosphorylation of both AKT (T308) and p70S6K. The inhibition of PDK1 activity through the IGF-1 axis is sufficient to induce in vitro anti-tumor activity in breast cancer cells such that PF-5177624 inhibits cell proliferation and cell transformation in breast cancer cells. Our data suggest that a specific and potent PDK1 inhibitor (PF-5177624) is likely to have antitumor activity in breast cancer cells and can be developed as a cancer drug for breast cancer patients. 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 3578. doi:10.1158/1538-7445.AM2011-3578

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: CHARACTERIZATION OF A SELECTIVE ALLOSTERIC KINASE INHIBITOR

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.