Naval Kapuriya

Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells

Despite recent advances in the clinical evaluation of various poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer (TNBC) patients, data defining potential anti-tumor mechanisms beyond PARP inhibition for these agents are lacking. To address this issue, we investigated the effects of four different PARP inhibitors (AG-014699, AZD-2281, ABT-888, and BSI-201) in three genetically distinct TNBC cell lines (MDA-MB-468, MDA-MB-231, and Cal-51). Assays of cell viability and colony formation and flow cytometric analysis were used to determine effects on cell growth and cell cycle progression. PARP-dependent and -independent signaling mechanisms of each PARP inhibitor were investigated by western blotting and shRNA approaches. Potential synergistic interactions between PARP inhibitors and cisplatin in suppressing TNBC cell viability were assessed. These PARP inhibitors exhibited differential anti-tumor activities, with the relative potencies of AG-014699xa0>xa0AZD-2281xa0>xa0ABT-888xa0>xa0BSI-201. The higher potencies of AG-014699 and AZD-2281 were associated with their effects on G2/M arrest and DNA damage as manifested by γ-H2AX formation and, for AG-014699, its unique ability to suppress Stat3 phosphorylation. Abilities of individual PARP inhibitors to sensitize TNBC cells to cisplatin varied to a great extent in a cell context- and cell line-specific manner. Differential activation of signaling pathways suggests that the PARP inhibitors currently in clinical trials have different anti-tumor mechanisms beyond PARP inhibition and these PARP-independent mechanisms warrant further investigation.

Water Mediated Construction of Trisubstituted Pyrazoles/Isoxazoles Library Using Ketene Dithioacetals

A small molecule library of alkyl, sulfone, and carboxamide functionalized pyrazoles and isoxazoles has been developed via a rapid sequential condensation of various alpha-acylketene dithioacetals (1a-o) with hydrazine hydrate or hydroxylamine hydrochloride, followed by oxidation of sulfide to sulfone using water as the reaction medium. An efficient and safe oxidation of sulfides (4/5a-o) to the corresponding sulfones (6/7a-o) using sodium per borate system in aqueous medium is reported. The concise and two step synthesis of trisubstituted pyrazoles and isoxazoles was investigated under variety of reaction condition. The newly developed methodology has the advantage of excellent yield and chemical purity with short reaction time using water as a solvent.

Vitamin E Facilitates the Inactivation of the Kinase Akt by the Phosphatase PHLPP1

Vitamin E suppresses the proliferation of prostate cancer cells by inhibiting the growth-promoting kinase Akt. Suppressing Cancer Growth with Vitamin E Vitamin E has well-known health benefits, including an anticancer effect. Tocopherols, a dietary form of vitamin E, induce the dephosphorylation of the kinase Akt, thereby inhibiting Akt-mediated signals that promote cell metabolism, proliferation, and motility. Huang et al. found that tocopherols, which integrate into cell membranes, stimulated a site-specific dephosphorylation of Akt by recruiting both Akt and the phosphatase PHLPP1 to the cell membrane through their respective pleckstrin homology (PH) domains. Tocopherol-derived synthetic compounds showed more potent effects than natural tocopherols in mediating this inactivation of Akt and reducing the growth of xenograft prostate tumors in mice, indicating potential for drug development. Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain–dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain–containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain–targeted Akt inhibitors.

Development of novel antibacterial agents against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health because of its resistance to multiple antibiotics most commonly used to treat infection. In this study, we report the unique ability of the cyclooxygenase-2 (COX-2) inhibitor celecoxib to kill Staphylococcus aureus and MRSA with modest potency. We hypothesize that the anti-Staphylococcus activity of celecoxib could be pharmacologically exploited to develop novel anti-MRSA agents with a distinct mechanism. Examination of an in-house, celecoxib-based focused compound library in conjunction with structural modifications led to the identification of compound 46 as the lead agent with high antibacterial potency against a panel of Staphylococcus pathogens and different strains of MRSA. Moreover, this killing effect is bacteria-specific, as human cancer cells are resistant to 46. In addition, a single intraperitoneal administration of compound 46 at 30 mg/kg improved the survival of MRSA-infected C57BL/6 mice. In light of its high potency in eradicating MRSA in vitro and its in vivo activity, compound 46 and its analogues warrant continued preclinical development as a potential therapeutic intervention against MRSA.

BX795, a TBK1 inhibitor, exhibits antitumor activity in human oral squamous cell carcinoma through apoptosis induction and mitotic phase arrest

TANK-binding kinase 1 (TBK1), a member of IκB Kinase (IKK)-related kinases, plays a role in regulating innate immunity, inflammation and oncogenic signaling. This study aims to investigate the role of BX795, an inhibitor of TBK1, in a panel of oral squamous cell carcinoma (OSCC) cell lines. The antitumor effects and mechanisms of BX795 were assessed by MTT assays, flow cytometry, Western blotting, and confocal microscopy. BX795 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound caused apoptosis as evidenced by PARP cleavage, the presence of pyknotic nuclei in the TUNEL assay, and fragmented DNA tails in the Comet assay. BX795 inhibits Akt and NF-κB signaling, arrests cells in the mitotic phase, and increases generation of autophagy in oral cancer cells. Interestingly, the antiproliferative activity of BX795 does not correlate with TBK1 protein expression level in OSCC cells. We propose that the TBK1-independet effect is related to mitotic phase arrest. Pleiotropic anticancer activity with relative sparing of normal oral keratinocytes underscores the potential value of BX795 and warrants its further study in oral squamous cell carcinoma therapy.

Design and synthesis of novel anti-tuberculosis agents from the celecoxib pharmacophore.

The identification of compounds with anti-mycobacterial activity within classes of molecules that have been developed for other purposes is a fruitful approach for the development of anti-tuberculosis (TB) agents. In this study we used the scaffold of celecoxib which exhibits several activities against different pathogens, for the design and focused synthesis of a library of 64 compounds. For the primary screen, we used a bioluminescence-based method by constructing a luciferase-expressing reporter M.tb strain which contains the entire bacterial Lux operon cloned in a mycobacterial integrative expression vector. Through the screening of this library, we identified 6 hit compounds with high in vitro anti-mycobacterial activity (IC₅₀ ∼0.18-0.48 μM). In particular, compounds 41, 51 and 53 were capable of inhibiting M.tb as effectively as the anti-TB drug isoniazid (INH) at 5 μM over a 72-h period, as analyzed by both bioluminescence- and colony forming unit (CFU)-based assays. All hit compounds also showed anti-M.tb activities against several multi-drug-resistant (MDR) strains. Most of the hit compounds showed no cytotoxicity for human macrophages at concentrations as high as 40 μM, setting the stage for further optimization and development of these anti-TB hit compounds both ex vivo and in vivo.

Antitumor effects of BI-D1870 on human oral squamous cell carcinoma

PurposeAmong the signaling pathways implicated in the tumorigenesis of oral squamous cell carcinoma (OSCC) is the extracellular signal-regulated kinase mitogen-activated protein kinase pathway, a downstream target of which is a family of serine/threonine kinases known as the 90xa0kDa ribosomal S6 kinases (RSKs). This study aims to investigate the role of BI-D1870, a specific inhibitor of p90 RSKs, in a panel of OSCC cell lines.MethodsThe antitumor effects and mechanisms of BI-D1870 were assessed by MTT assays, flow cytometry, Western blotting, transfection, and confocal microscopy.ResultsBI-D1870 exhibited a dose-responsive antiproliferative effect on OSCC cells with relative sparing of normal human oral keratinocytes. The compound inhibited the downstream RSK target YB-1 and caused apoptosis as evidenced by PARP cleavage, activation of the caspase cascade, and the presence of pyknotic nuclei in the 4,6-diamidino-2-phenylindole assay. In addition, BI-D1870 also induced G2/M arrest by modulating the expression of p21 and other cell cycle regulators. Other newly discovered anticancer attributes of BI-D1870 included the generation of reactive oxygen species and increases in endoplasmic reticulum stress and autophagy.ConclusionsTogether, these results suggest the translational value of BI-D1870 in oral squamous cell carcinoma therapy.

Exploitation of the Ability of γ-Tocopherol to Facilitate Membrane Co-localization of Akt and PHLPP1 to Develop PHLPP1-Targeted Akt Inhibitors

Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.

Abstract 3505: Energy restriction-mimetic agents (ERMAs) decrease BRCA1 expression levels in triple-negative breast cancer cells: A rationale for combining ERMAs with poly(ADP ribose) polymerase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnPurpose: Triple-negative (ER-negative, PR-negative, HER2/neu-negative) breast cancer (TNBC) is a clinical challenge because of the lack of a specific therapeutic target. Poly (ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in the detection and repair of DNA damage. In DNA repair-defective tumors, inhibition of PARP can cause genomic instability and cell death. Among the PARP inhibitors currently being assessed in clinical trials, recent results showed that, in a randomized phase II clinical trial of TNBC patients, BSI-201 when added to platinum-containing chemotherapy significantly improved the outcome of metastatic breast cancer patients relative to chemotherapy alone. Previously, we reported that the thiazolidinedione family of peroxisome proliferation-activated receptor-γ agonist could induce apoptosis in cancer cells by eliciting cellular responses indicative of energy restriction. Consequently, we used ciglitazone as a scaffold to develop novel energy restriction-mimetic agents (ERMAs) through structural optimization, which has yielded two lead compounds, CG-5 and CG-12. In prostate cancer cells, ERMAs could induce cell death and reduce the expression of BRCA1 protein. BRCA1 helps repair DNA double-stranded breaks through homologous recombination. BRCA1 deficiency has been reported to sensitize cells to PARP inhibition through synthetic lethality. We hypothesize that ERMAs can potentiate the anti-proliferative activity of PARP inhibitors (AZD-2281, AG-014699, ABT-888 and BSI-201) through the down-regulation of BRCA1 expression in TNBC cells.nnMethods: TNBC cell lines, MDA-MB-468, MDA-MB-231 and Cal-51 (all of which have functional BRCA1) were obtained from ATCC and DSMZ and maintained in low-glucose complete growth medium. Drug effects on cell viability were assessed by MTT assays. The expression of BRCA1, phosphorylated AMP-activated kinase (AMPK), and PARP cleavage in treated cells were examined by western blotting.nnResults: The ERMAs CG-5 and CG-12 exhibited dose-dependent anti-proliferative activity against all TNBC cell lines tested with IC50 values of about 3 and 4 μM, respectively, after 72 h of treatment. These anti-proliferative effects were attributed to apoptosis as both compounds induced the cleavage of PARP. Moreover, AMPK was activated by both drugs indicative of energy restriction. BRCA1 levels were reduced dose-dependently in all three TNBC cell lines after treatment with CG-5 or CG-12.nnConclusion: The ERMAs CG-5 and CG-12 can reduce BRCA1 levels in TNBC cells. These findings provide a rationale for combining the ERMA-mediated ablation of BRCA1 with PARP inhibitors to induce TNBC cell death through synthetic lethality. Further study to address this hypothesis could lead to a new therapeutic approach for TNBC patients for whom available options are limited.nnCitation 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 3505. doi:10.1158/1538-7445.AM2011-3505