Project Funding Details

NADPH oxidases (NOXs) are the only human enzymes generating ROS as a prime product and not a byproduct. NOXs are part of the redox signaling that sustains cell proliferation, oncoprotein-driven cell transformation, and tumor the microenvironment. Among the seven isoforms, NOX1 and NOX4 sustain proliferative activity in colon carcinoma cells by amplifying the Ras oncogene signaling, while NOX2 is overexpressed in hematopoietic and lymphoid cancers, and its deregulated activity in microglia can have neurotoxic effects. Anticancer therapies induce excessive levels of oxidative stress to kill tumor cells. Our hypothesis is that ROS fine modulation can function as a specific weapon to halt tumors, thus decreasing drug toxicity in healthy cells. By combining structural information and virtual screening of ultra-large libraries, along with a tight workflow for biochemical assays to discard false positives, we identified the first "bona fide" covalent and non-covalent NOX inhibitors. Notably, few of them displayed encouraging results in cancer cells. We aim to develop selective NOX inhibitors that will be assessed in cancer mouse models after iterative cycles of extensive biochemical, biophysical, and cellular characterization. Specific aims: - Design and synthesis of novel covalent NOX inhibitors and chemical probes, and identification of possible NOX interactor proteins; - Identification of true non-covalent NOX inhibitors, from in silico screenings to extensive hit-to-lead processes; - In vitro biochemical, biophysical, and cellular characterization against hNOX1/2/4/5. Evaluation of selected compounds in cancer cells, alone and in combination with known anticancer drugs; - Preliminary in vivo studies in cancer mouse models performed on the 2-3 best candidates. WP1. Design and synthesis of novel covalent NOX inhibitors and chemical probes. Task 1a. Development of novel dual Cys/FAD trappers. Task 1b. Development of clickable and biotinylated chemical probes and identification of NOX interactor proteins. WP2. Hit-to lead processes for non-covalent NOX inhibitors. Task 2a. Optimization of Molport-based compounds. Task 2b. Optimization of the Atomwise compounds. WP3. Biochemical, biophysical, and cellular evaluation of the new NOX inhibitors. Task 3a. Biochemical and biophysical characterization of the new compounds. Insight on their MoA. Task 3b. Evaluation of selected NOX inhibitors in both solid and blood cancer cell lines. Effects of combination with focused anticancer agents. WP4. In vivo ADMET studies and evaluation in cancer mouse models. Task 4a. In vivo safety, toxicity, and ADMET profiling of 10 selected molecules. Task 4b. Evaluation of 2-3 best NOX-targeting molecules in CDX mouse models. - Discovery of the first NOX-targeting compounds through hit-to-lead development on the validated scaffolds to improve their potency and isoform-selectivity. - Validation in selected cancer cells. - Identification of 2-3 candidates active against cancer mouse models. This 5-year project aims to produce "bona fide" NOX inhibitors as new therapeutic strategies, alone or in combination with known chemotherapeutics, for the treatment of cancers with an unfavorable prognosis. The achievement of this objective will enhance cooperation networks between industries and Universities in order to address the demand for innovative therapies for poorly prognostic cancers, which may be further expanded at the clinical level as well as for technology transfer.