Novel bona-fide NOXs inhibitors and dual EZH2/HDACs epigenetic modulators: innovative strategies to fight cancerous and non-cancerous diseases

In the present thesis, I describe the core projects undertaken during my PhD period, with the support and collaboration of my research team and under the guidance of my Scientific Supervisor, Professor Antonello Mai. Firstly, I dealt with the design and synthesis of novel NADPH oxidases (NOXs) inhibitors, pursued through two parallel project lines. The first line of investigation focused on the development of bona-fide triazolopyrimidine-based NOXs inhibitors, while the second aimed in the obtaining of quinoline-based NOXs inhibitors. Secondly, in addition to NOX inhibitors, I turned my focus to the design and synthesis of novel multi-targeting agents capable of simultaneously inhibiting two crucial epigenetic targets: the Enhancer of Zeste Homolog 2 (EZH2) and the Histone Deacetylases (HDACs). This dual-target approach is pivotal, given the complex role these proteins play in cancer progression and epigenetic regulation. The successful outcomes of this research were significantly enhanced by collaborations with various research groups and my enriching visiting PhD experience at the Institute of Oncology Research in Bellinzona, Switzerland. These collaborations allowed me to obtain important biochemical and biological data, enabling a comprehensive evaluation of the inhibitory activities of the newly synthesized compounds. Furthermore, these compounds were assessed for their anticancer potential against both solid and hematological tumors, as well as for their neuroprotective properties. Through this multifaceted approach, my work not only advances our understanding of NOX inhibition and epigenetic modulation but also contributes to the broader quest for effective therapeutic agents in cancer treatment, and beyond. Development of VAS2870 analogues as triazolopyrimidine-based NOXs inhibitors NADPH oxidases (NOXs) are the only known human enzymes solely in charge of ROS production. In addition to their roles in innate immunity and response to stressful conditions, NOXs are part of the redox signaling pathways that sustain cell proliferation, oncoprotein (e.g. RAS) driven cell transformation, and neuroinflammation. Recently, our group identified VAS2870 as a covalent inhibitor of mainly NOX1 and NOX5, by alkylating the conserved Cys668 in the active site, blocking productive substrate binding. To enhance potency and selectivity towards NOXs, we conducted a series of chemical modifications, leading to the discovery of compound 9a that preferentially inhibited NOX2 with an IC50 of 0.155 μM, and only upon its pre-activation. Tested against NOX2-overexpressing PLB-985 cells, compound 9a proved to be the most effective, with IC50 of 0.135 μM. Surprisingly, we considered that 9a bears the pargyline (N-benzyl-N-methylprop-2-yn-1-amine) moiety, a well-known irreversible and selective monoamine oxidase B (MAO-B) inhibitor. When evaluated towards human MAO-A and MAO-B, 9a proved to be a selective MAO-B inhibitor (SI MAO-B/MAO-A = 465), that could be considered the first-inclass dual NOX2/MAO-B irreversible inhibitor, with a balanced potency against both targets. Finally, given the involvement of NOX2 and MAO-B in neuroinflammatory pathways, we further investigated the effects of 9a in the BV2 microglia neuroinflammation cell model. It reduced ROS production and downregulated mRNA transcripts of pro-inflammatory genes, demonstrating more potent effects than either single-target or their combined effects. Development of M41 analogues as quinoline-based NOXs inhibitors In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. NOXs present a therapeutic target, yet current drugs often lack selectivity: there is a need for isoenzyme-selective inhibitors. Our project focused on the molecular simplification of M41, a lead-compound discovered through two ultra-large in silico screens of 350 million compounds, which stood out for its promising Ki values towards NOX isoforms. Given the adaptable binding mode of M41, we investigated the contributions of the various chemical groups, obtaining a first series of simplified derivatives. Particularly, the quinoline derivatives 10t and 10s exhibited single-digit micromolar IC50 values and isoform selectivity for NOX2 and NOX5, respectively. Building on these results, a second series of derivatives (based on the two just mentioned lead-compounds) was developed, with the bisquinoline compound 15a identified as the best candidate for targeting NOX2. The study also assessed 15a’s potential in KRAS-mutant colorectal cancer, where NOX2 is upregulated. Compound 15a enhanced the effectiveness of the KRASG12D inhibitor MRTX1133 in these cancer cells. The findings underscore the importance of structural features for NOX selectivity and potency and suggest potential therapeutic benefits in KRAS-driven cancers. Development of novel dual EZH2/HDAC inhibitors Since the histone-modifying enzymes EZH2 and HDACs synergistically control several epigenetic-dependent carcinogenic pathways, we synthesized novel dual EZH2/HDAC inhibitors. This was achieved by merging the structure of Tazemetostat, the only FDA-approved EZH2 inhibitor, with pharmacophore moieties from various established HDAC inhibitors. In biochemical assay, most of our hybrid compounds exhibited nanomolar inhibition of EZH2, with some achieving even picomolar potency, while displaying sub-micromolar to nanomolar inhibition of HDACs. When tested across diffuse large B cell lymphomas (DLBCL) cell lines, a histological subtype that comprises a defined genetic subset of EZH2 mutated tumors, these hybrid inhibitors showed significant antiproliferative effects, with low micromolar concentrations sufficient to inhibit cell proliferation. In both DOHH2 cell line (wild-type EZH2) and WSU-DLCL2 cell line (mutated EZH2), the dual inhibitors modulated specific histone marks, confirming their targets engagement. Additionally, these compounds induced G1 phase cell cycle arrest, leading to a cytostatic effect and an accumulation of cells in sub-G1, indicating a cytotoxic impact. Finally, we further evaluated the two most promising dual compounds, 18a and 19a, based on the just-mentioned assays, by analyzing their effects on the transcriptomes of DOHH2 and WSU-DLCL2 cell lines. These dual inhibitors significantly downregulated transcriptional targets of the oncogene MYC, as well as genes involved in mTORC1 signaling, DNA repair, G2-M cell cycle checkpoint, and oxidative phosphorylation. Overall, the transcriptome analyses revealed that 18a and 19a were at least as effective, if not more so, than single-target agents in modulating the expression of genes associated with lymphomagenesis.