Background: Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.Methods: Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.Results: Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of similar to 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.Conclusion: In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further predinical studies.General significance: Since 3b (6-[(3-acetylphenyl)aminolquinazoline-5,8-dione) exerts Alct-dependent ROSmediated cell death, it might provide potential therapeutic options for chemoresistant and Alct-overexpressing cancers. (C) 2013 Elsevier B.V. All rights reserved.

Abstract Pancreatic cancer is a complex disease. Late stage detection, poor prognosis, and resistance to available chemotherapeutics highlight the need to discover new and potent anticancer agents for its effective treatment. Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most forms of cancer including pancreatic cancer. Cancer cells are more prone to reactive oxygen species (ROS)-mediated cell death due to their inherent elevated basal oxidative stress as compared to normal cells. Increased basal ROS levels could represent the Achilles’ heel of cancer cells. Even though cancer cells have adapted to survive in an oxidative stress environment, exogenous oxidative insults can overwhelm the adaptive antioxidant capacity of cancer cells and trigger ROS-mediated cell death We have identified a novel class of compounds that act as redox modulators and specifically induce cell death in cancer cells. Compound 3a was identified through a medium throughput screen of ∼1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics using XF 24 extracellular flux analyzer (Seahorse Bioscience, Billerica, MA). Further structural optimizations led to the discovery of a more potent analog, 3b that displayed anti-proliferative activities at low micromolar levels in both drug-sensitive and drug-resistant cancer cell lines. Treatment with compound 3b causes Akt activation resulting in increased cellular oxygen consumption, oxidative stress and depletion of cellular antioxidant pool in pancreatic cancer cells. Akt is a pro-survival kinase that is upregulated in several forms of human cancers. Hyperactive Akt inhibits apoptosis induced by numerous stimuli. However, Akt is unable to inhibit ROS-mediated cell death, and in fact, Akt aids in ROS-directed cell death by inducing cellular oxygen consumption, promoting ROS generation, and impairing ROS degeneration. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) and resulted in cancer cell apoptosis. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. In conclusion, our novel class of compounds are promising leads that by exacerbating oxygen consumption, ROS production, and reducing the antioxidant capacity of cancer cells tip the balance towards activation of stress kinases that ultimately leads to cell death. Citation Format: Divya Pathania, Mario Sechi, Michele Palomba, Vanna Sanna, Francesco Berrettini, Angela Sias, Laleh Taheri, Nouri Neamati. Design and discovery of novel small molecule redox modulators as therapies for pancreatic cancer. [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 4555. doi:10.1158/1538-7445.AM2013-4555

Abstract Oxidative stress is an important hallmark of cancer. Elevated levels of intrinsic reactive oxygen species (ROS) make tumor cells more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm the adaptive antioxidant capacity of cancer cells and trigger ROS-mediated cell death. Increasing ROS production or decreasing ROS scavengers shows potential for selectively targeting tumor cells that are under persistent oxidative stress. Recently, we have discovered novel class of quinazolinediones that exert their anticancer effects by modulating ROS-mediated cell signaling.The sub-micromolar anti-proliferative properties of these compounds were demonstrated by MTT and colony formation assays in a panel of cancer cell lines of different origins. Treatment with these agents induced cell cycle arrest, and increased superoxide production in cancer cells. Furthermore, proteomics analysis of 600 target antibody array revealed that these molecules significantly affected several cell signaling pathways implicated in carcinogenesis, cancer progression, redox signaling, and cell death.In conclusion, quinazolinediones are promising lead compounds that selectively enhance ROS production and induce ROS-mediated cell death in cancer cells, and warrant further preclinical studies. 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 682. doi:10.1158/1538-7445.AM2011-682

Abstract Proliferation under selective pressures exerted by an unstable microenvironment, requires tumor cells employ adaptive mechanisms that confer growth advantage. Adaptation to adverse conditions results in cellular phenotypes that typify neoplastic transformation and offer unique opportunities for selective targeting of cancer cells. Agents that target tumor cell mitochondria with high selectivity hold clinical significance due to the adaptive, modulatory and essential role of this organelle in cancer cell energy production, metabolism and apoptosis. To this end, we have identified a series of novel, mitochondriotropic phosphonium salts, (TP compounds), that have shown broad-spectrum anti-cancer and anti-angiogenic activity in preclinical evaluation. A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 and numerous close analogues as having IC50 concentrations in the sub-micromolar range. TP treatment induced cell cycle arrest, lowered oxygen consumption, and increased mitochondrial superoxide production. Administered as single agents in a mouse model of human breast cancer, TP compounds significantly decreased tumor growth with no observed toxicities. Protein microarray data demonstrated significant down-regulation of integrin and growth factor mediated signaling pathways governing key processes including cancer cell survival, proliferation and tumor angiogenesis. At low micromolar concentrations, TP compounds prevented integrin-mediated cell adhesion to fibronectin and vitronectin coated substrates and tumor cell haptotaxsis on vitronectin coated boyden chambers. Similar concentrations of TP compound also prevented growth factor induced endothelial cell tube formation in 3-D culture using basement membrane extracts. Taken together, these results suggest that as mitochondria-targeted agents, TP compounds act to inhibit tumor cell proliferation and angiogenic capacity. 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 2542. doi:10.1158/1538-7445.AM2011-2542

Background Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. Methodology/Principal Findings A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC50 concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation. Conclusions/Significance Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.

Quinoxolinehydrazines represent a novel class of compounds with excellent potency in a panel of cancer cell lines. A prototype compound, SC144, showed significant in vivo efficacy in mice xenogroft models of human breast cancer cells. The subsequent structure-activity relationship study resulted in the discovery of SC161 with better potency in cancer cell lines. Further exploring the possible conformational space by a 10 ns molecular dynamics simulation as presented herein, resulted in various pharmacophore orientations. The trajectory analysis indicated that in most of the simulation time, the molecule stays favorably in a compact planarlike orientation. We therefore built a pharmacophore model based on the cluster containing the highest number of frames to represent the most probable orientation. The model was used to screen a subset of our small molecule database containing 350,000 compounds. We selected 35 compounds for the initial cytotoxicity screen. Seventeen compounds belonging to oxadiazolopyrazine and quinoline class displayed cytotoxicity in various cancer cell lines. Five of them, compounds 2 6, 15, 16, and 19, all bearing an oxadiazolopyrazine scaffold, showed IC(50) values <3 mu M in certain tumor cell lines. The most potent compound, 2, showed IC(50) values <2 mu M in HCT116 p53(+/+), HCT116 p53(-/-), and HEY cells, and 8 mu M in NIH3T3 cells. This study shows that conformational sampling of a lead small molecule followed by representative pharmocophore model development is an efficient approach for the rational design of novel anticancer agents with similar or better potency than the original lead but with different physicochemical properties.

Previously, we discovered linomide analogues as novel HIV-1 integrase (IN) inhibitors. Here, to make possible structure-activity relationships, we report on the design and synthesis of a series of substituted dihydroquinoline-3-carboxylic acids. The crystal structure of the representative compound 2c has also been solved. Among the eight new analogues, 2e showed a potency in inhibiting IN strand transfer catalytic activity similar to the reference diketo acid inhibitor L-731,988 (IC(50)=0.9 microM vs. 0.54 microM, for 2e and L-731,988, respectively). Furthermore, none of the compounds showed significant cytotoxicity in two tested cancer cell lines. These compounds represent an interesting prototype of IN inhibitors, potentially involved in a metal chelating mechanism, and further optimization is warranted.

The previously discovered salicylhydrazide class of compounds displayed potent HIV-1 integrase (IN) inhibitory activity. The development of this class of compounds as antiretroviral agents was halted due to cytotoxicity in the nanomolar to sub-micromolar range. We identified a novel class of non-cytotoxic hydrazide IN inhibitors utilizing the minimally required salicylhydrazide substructure as a template in a small-molecule database search. The novel hydrazides displayed low micromolar IN inhibitory activity and are several hundred-fold less cytotoxic than previously disclosed salicylhydrazide IN inhibitors.