Venkat R. Pallela

Design, synthesis, and biological evaluation of 1-(4-sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines as cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) inhibitors

A series of 20 novel 1-(4-sulfamylphenyl)-3-trifluoromethyl-5-indolyl pyrazolines were designed, synthesized, and screened in vitro for anti-inflammatory activity. These compounds were designed for evaluation as dual inhibitors of cyclooxygenases (COX-1 and COX-2) and lipoxygenases (LOX-5, LOX-12, and LOX-15) that are responsible for inflammation and pain. All pyrazoline molecules prepared are optically active and compounds that are more potent in COX-2 inhibitory activity (5a and 5f) were resolved by chiral column and each enantiomer was tested for cyclooxygenase inhibitory activity. Molecular modeling and comparison of molecular models of 5a enantiomers with that of celecoxib model shows that 5a (enantiomer-1) and 5a (enantiomer-2) have more hydrogen bonding interactions in the catalytic domain of COX-2 enzyme than celecoxib. Compounds 5a, 5e, and 5f showed moderate to good LOX-5 and LOX-15 inhibitory activity and this is comparable to that of celecoxib and more potent than rofecoxib.

Discovery of 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x) as a Potent Inhibitor of Cyclin-Dependent Kinase 4 (CDK4) and AMPK-Related Kinase 5 (ARK5)

The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multikinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyano pyridopyrimidine compounds and identified structure–activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x), was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30–100 nM. In vitro kinase profiling revealed that 7x is a multikinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure–activity relationship, kinase inhibitory profile, in vitro cytotoxicity, and in vivo tumor regression studies by this lead compound.

(Z)-1-aryl-3-arylamino-2-propen-1-ones, highly active stimulators of tubulin polymerization: synthesis, structure-activity relationship (SAR), tubulin polymerization, and cell growth inhibition studies.

Tubulin, the major structural component of microtubules, is a target for the development of anticancer agents. A series of (Z)-1-aryl-3-arylamino-2-propen-1-one (10) were synthesized and evaluated for antiproliferative activity in cell-based assay. The most active compound (Z)-1-(2-bromo-3,4,5-trimethoxyphenyl)-3-(3-hydroxy-4-methoxyphenylamino)prop-2-en-1-one (10ae) was tested in 20 tumor cell lines including multidrug resistant phenotype and was found to induce apoptosis in all these cell lines with similar GI(50) values. Flow cytometry studies showed that 10ae arrested the cells in G2/M phase of cell cycle. In addition to G2/M block, these compounds caused microtubule stabilization like paclitaxel and induced apoptosis via activation of the caspase family. The observations made in this investigation demonstrate that (Z)-1-Aryl-3-arylamino-2-propen-1-one (10) represents a new class of microtubule-stabilizing agents.

Design, synthesis and evaluation of (E)-α-benzylthio chalcones as novel inhibitors of BCR-ABL kinase

Novel (E)-alpha-benzylthio chalcones are reported with preliminary in vitro activity data indicating that several of them are potent inhibitors (comparable to imatinib, the reference compound) of BCR-ABL phosphorylation in leukemic K562 cells, known to express high levels of BCR-ABL. The ability of such compounds to significantly inhibit K562 cell proliferation suggests that this scaffold could be a promising lead for the development of anticancer agents that are able to block BCR-ABL phosphorylation in leukemic cells.

Hydrothiolation of benzyl mercaptan to arylacetylene: application to the synthesis of (E) and (Z)-isomers of ON 01910·Na (Rigosertib®), a phase III clinical stage anti-cancer agent

A stereoselective and efficient method for free radical addition of benzyl thiol to aryl acetylene in the presence of Et3B-hexane has been developed for the synthesis of (Z) and (E)-styryl benzyl sulfides where base catalyzed hydrothiolations have failed. The scope of this reaction was successfully extended for the synthesis of (E)-ON 01910·Na, a phase III clinical stage anti-cancer agent and its inactive geometrical isomer (Z)-ON 01910·Na. It is interesting to note that all the E-isomers synthesized have shown better cytotoxicity profile on cancer cells compared to the Z-isomers.

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.

Abstract 4453: The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells

Triple negative breast cancer (TNBC) is associated with a poor prognosis and high frequency of recurrence. Because the molecular mechanisms that are deregulated in this tumor type are not well understood, there is a lack of targeted therapies that can effectively treat this disease. An unfortunate limitation of existing TNBC therapies is the frequency of relapse, which is highly resistant and metastatic and has been attributed to tumor-initiating stem cells (T-ICs). In patients with relapsed TNBC, T-ICs with a CD44high/CD24-/low antigenic phenotype are enriched in the tumor cell population. Our previous data illustrated that a small molecule kinase inhibitor, ON108600, potently inhibited the survival and growth of TNBC cell lines and mouse xenografts. To investigate whether ON108600 has a similar inhibitory effect on T-ICs, we performed clonogenic survival assays using sorted CD44high CD24-/low cells isolated from TNBC cell lines. ON108600 potently inhibited the stem cell activity and self-renewal ability of these T-ICs. Although paclitaxel (PTX) treatment improves survival of TNBC patients, acquired resistance to this drug is a common occurrence. Furthermore, strategies that target PTX resistant cells remain elusive. To investigate the molecular mechanisms underlying acquired PTX-resistance in TNBC and evaluate the efficacy of ON108600, we generated PTX-resistant cell lines using the MDA-MB-231 and BT-20 TNBC cell lines. Drug-resistant cells were established by exposure to increasing concentrations of Paclitaxel, and resistance was validated by cell viability and colony formation in the presence of high concentrations of PTX. PTX-resistant MDA-MB-231 and BT-20 cells exhibited an approximate 1000-fold increase in resistance to PTX as compared to the parental cells. Importantly, PTX-resistant TNBC cells displayed a stem-like phenotype characterized by loss of epithelial differentiation markers (e-Cadherin, CD24) and a gain of epithelial-mesenchymal transition markers (N-Cadherin, CD44, Oct4, Snail). Kinase profiling studies have indicated that ON108600 targets kinases CK2, and Traf2- and Nck-interacting kinase, TNIK. Although CK2 subunit levels in resistant cells were unchanged, PTX-resistant cells showed a marked upregulation of TNIK, an activating kinase for T-cell factor-4 (TCF-4) and consequently, a marked increase in β-catenin and Wnt target genes Axin-2 and Cyclin D1. We therefore examined whether dual inhibition of CK2 and TNIK was effective in killing PTX-resistant cells. Treatment of PTX-resistant TNBC cells with ON108600 resulted in marked increase in apoptosis and inhibition of clonogenic survival and mamosphere forming ability of these cells, suggesting that dual CK2/TNIK inhibition may be an effective way to overcome PTX-resistance in TNBC. We are currently testing the efficacy of ON108600 in paclitaxel resistant xenograft models. Citation Format: Amol Padgaonkar, Stephen Cosenza, Venkat Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, E Premkumar Reddy. The dual CK2/TNIK inhibitor, ON108600 targets cancer stem cells and induces apoptosis of paclitaxel resistant triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4453. doi:10.1158/1538-7445.AM2015-4453

Abstract 3239: Discovery and biological characterization of ON108600, a small molecule inhibitor of protein kinase CK2.

Protein kinase CK2 (Casein kinase 2) is a highly conserved, constitutively active Ser/Thr protein kinase implicated in cellular transformation and tumorigenesis. CK2 regulates multiple oncogenic pathways involved in cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA repair. Unlike other signaling molecules such as PI3K, RAF, RAS, where genetic alterations lead to deregulated signaling pathways, in the case of CK2, only its high expression levels have been associated with a disease state and no mutations have been found to date. Here we describe the mechanism of action of ON108600 a potent small molecule inhibitor of both catalytic subunits of protein kinase CK2. ON108600 showed broad-spectrum anti-proliferative and cytotoxic activity in multiple cancer cell lines while having little or no effect on normal cells. CK2 regulates Akt signaling through phosphorylation and inactivation of PTEN and via direct and specific phosphorylation of Akt at S129. Treatment with ON108600 resulted in dramatic reductions of phosphorylation of PTEN and Akt S129 and downstream targets of Akt in multiple cancer cell lines. Further, phosphorylation of p21Cip1/Waf1 at Thr145 and its cytoplasmic localization has been shown to be a poor prognostic marker in breast cancer. ON108600 showed a dose and time dependant dephosphorylation of p21 at Thr145 and induced a potent mitotic cell cycle arrest in most cancer cell lines. CK2 has also been implicated in regulating Caspase signaling via phosphorylation of caspase cleavage sites and thus exerting its pro-survival effects. ON108600 treatment induced potent apoptosis by activating the Caspase 3/7 signaling cascade. To understand the structural basis of CK2 inhibition by ON108600 we performed x-ray crystallographic studies of ON108600-CK2. The co-crystal structure of ON108600-CK2 revealed that ON108600 binds in the active site pocket of CK2α wherein it mimics the binding of GTP in the CK2 active site. Structural studies also revealed that ON108600 induces a conformational change in the β4-β5 loop of the catalytic subunit which is known to interact with the β-regulatory subunit of CK2 and critical for substrate recognition and activation. CK2α has been reported to be a structural microtubule associated protein and the CK2 holoenzyme has been shown to regulate microtubule dynamics. Interestingly, ON108600 was found to inhibit CK2 holoenzyme mediated polymerization of β-tubulins in in-vitro assays. CK2 has emerged as novel druggable target and selective inhibitors of CK2 such as ON108600 may prove as a potential anti-cancer therapy. Our ongoing studies are focused towards identifying novel combinations of ON108600 with existing chemotherapeutic agents in multiple myeloma, prostate and breast cancer. Citation Format: Amol Padgaonkar, Olga Rechkoblit, Stephen Cosenza, Venkat R. Pallela, Venkata Subbaiah DRC, MV Ramana Reddy, Aneel Aggarwal, E Premkumar Reddy. Discovery and biological characterization of ON108600, a small molecule inhibitor of protein kinase CK2. [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 3239. doi:10.1158/1538-7445.AM2013-3239 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.