Translational Research have been developed, including one to permit high throughput screening. Novel, irreversible, inhibitors of a lysine demethylase have been designed, synthesized and evaluated: the new derivatives, besides having inhibitor potency in the nanomolar range, showed target-related gene modulation in cells. Further, several compounds were profiled for in vitro ADME and in vivo PK characteristics and one molecule was selected for further analysis. This compound is currently undergoing assessment in an in vivo efficacy study. In addition, an alternative strategy to identify reversible inhibitors of the lysine demethylase was established, In collaboration with a Contract Research Organization, a high through put screening campaign against a proprietary library of commercially available compounds was conducted and several interesting hits belonging to different chemical classes were identified (in coll. with S. Minucci, P.G. Pelicci e G. Natoli). Cancer metabolism Altered cancer metabolism is now considered one of the hallmarks of cancer and strategies to target these alternations have become the focus for novel targeted therapies. Many tumors increase glucose uptake and, even in the presence of abundant oxygen, switch from mitochondrial oxidative phosphorylation to perform anaerobic glycolysis, converting pyruvate to lactate (known as the “Warburg effect”). Many of the genes involved in this altered metabolism are regulated by tumor hypoxia and oncogenes, such as Kras and myc. Lactate dehydrogenase is a key enzyme in the glycolytic switch and converts pyruvate to lactate with the subsequent production of 2 molecules of ATP and the regeneration of nicotinamide adenine dinucleotide. LDH-A is up-regulated in many tumor types and its high expression correlates with poor prognosis. Gene ablation studies have shown that the LDH-A gene is required for the proliferation of several cancers in vitro and in vivo. In Collaboration with Prof F. Minutolo (University
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of Pisa) the DDP is developing small molecular weight inhibitors of LDH-A. These inhibitors are competitive with LDH-A substrates in biochemical assays and demonstrate anti-proliferative activity against a panel of tumor cell lines (including, breast, pancreatic and non small cell lung cancer (NSCLC)). In addition, consistent with attenuation of LDH-A activity, these inhibitors are able to reduce the production of lactate in tumor cells. Further, when administered orally once daily to mice, LDH-A inhibitors exhibit good tumor growth inhibitory properties in xenograft models of NSCLC, with no observable toxicity. A potential clinical candidate has been identified and is being advanced as rapidly as possible towards an IND submission and clinical trials. In the meantime, novel chemical classes are being evaluated for LDH-A inhibitory activity in order to develop a second generation of inhibitors. Cancer stem cells: Numb modulation Loss of Numb expression concomitantly results in two major effects: deregulation of a potent oncogene (the Notch receptor) and loss of function of a tumor suppressor (the p53 protein). The combined dysfunction of the Numb/Notch and Numb/p53 axes most likely accounts for the particularly aggressive phenotypes displayed by Numb-defective tumors (e.g breast and lung cancers). Numb expression in tumors is most likely determined at the post-transcriptional level where posttranslational modifications (ubiquitination, phosphorylation) target the protein for degradation. Hence there is an urgent need to identify these upstream mechanisms as their inhibition could restore Numb levels and counter the imbalance in both the Numb/ Notch and Numb/p53 axes. These potential Numb regulators present ideal pharmacological targets for the stabilization of Numb levels and the restoration of its tumor suppressor activity. Two strategies have initially been considered to identify the “upstream regulators” of Numb: “target identification” by siRNA screening
(forward strategy-ongoing); “phenotypic screening” against a small molecule library (reverse strategy). The ”forward” strategy has been implemented and candidates of different gene families have been identified and are currently undergoing validation. In order to facilitate the “reverse strategy” a cellular model, suitable to run a phenotypic High Content Screening campaign, has been established. By evaluating several protocols and reagents to obtain correct image segmentation, optimal experimental conditions in terms of good dynamic range, have been identified. Moreover, the data obtained during uniformity and signal variability assessment were all in the range of acceptance criteria defined by the NIH Chemical Genomic Center guidelines (in coll. with S. Pece and P.P. Di Fiore). Exploratory activities are also developed internally or through external collaboration in order to create a machinery ready to feed the running project pipeline. Exploratory activities include, e.g., approaches to restore functional p53, design of novel scaffolds for innovative chemical libraries and the preparation of kinase-targeted libraries.
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