Thesis title: New frontiers for epigenetics: from immunology to cardiovascular diseases and beyond
The Post-translational modifications (PTMs) have gained significant attention due to their dynamic role in determining the chromatin’s state, either open (euchromatin) or closed (heterochromatin), thereby influencing chromatin structure and functions. These modifications can occur on both histone or non-histone proteins, with acetylation and its counterpart deacetylation, being among the most extensively studied, playing a critical role in regulating gene expression. Consequently, histone acetylation is crucial for understanding the impact of (de)acetylating enzymes in the development of various diseases. HDACs are a class of epigenetic enzymes responsible for removing acetyl groups from lysine residues on histone tails, resulting in chromatin remodeling. The HDAC family is categorized into four classes: class I (HDAC 1, 2, 3, 8), class IIa (HDAC 4, 5, 7, 9) and IIb (HDAC 6, 10), class III (Sirtuins) and class IV (HDAC 11). These enzymes are central to transcriptional silencing, and their activity is essential for cell proliferation, differentiation, and maintaining cellular homeostasis. For this reason, their dysregulation has been implicated in various diseases, including cancer, making HDAC inhibition a promising therapeutic strategy. Additionally, evidences suggest that epigenetic modifications can help overcome immune cycle impairment, which are pivotal in resistance to PD-1/PD-L1 immunotherapy. Recent advances in cancer immunotherapy have increasingly focused on the PD-1/PD-L1 signaling pathway as a therapeutic target to enhance immune cell functionality. Interaction between PD-1 and its ligand PD-L1 suppresses T-cells activity, contributing to immune evasion in tumors. Notably, emerging evidence indicates a synergistic effect when combining PD-L1 antibodies with HDAC inhibitors, yielding favorable outcomes in clinical trials, including phase II studies. Hence, novel dual inhibitors could further improve survival outcomes for some patients. Class III HDACs, known as Sirtuins, consist of 7 isoforms and function via a distinct mechanism. Unlike canonical HDACs, which rely on a Zn2+-dependent process, Sirtuins require NAD+ as a cofactor for their activity. The mitochondrial SIRT3, a key deacetylating enzyme, regulates pathways related to cancer, metabolism, and hypoxia-associated diseases. Meanwhile, the mitochondrial SIRT5 displays an affinity for negatively charged acyl groups and mainly catalyzes lysine deglutarylation, desuccinylation, and demalonylation while possessing weak deacetylase activity. SIRT5 substrates are critical for metabolic processes and reactive oxygen species (ROS) detoxification, and SIRT5 activity is protective in neuronal and cardiac health. Therefore, considering the multifaceted roles of sirtuins, the development of sirtuin activators or inhibitors holds promise as a potential strategy for addressing various pathologies, including cancer and cardiovascular disorders.
In the present thesis, I describe a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates, as well as 2′-aminoanilides (compounds 5–8), as potential anticancer agents. Among these, the hydroxamate 5d demonstrated significant selectivity for HDAC3 and -6, with IC50 values of 80 and 11 nM, respectively. Conversely, its analogue 5e acted as a nanomolar inhibitor of HDAC1-3, -6, -8, and -10 (class I/IIb-selective inhibitor). Notably, 5e exhibited exceptional antiproliferative activity against both haematological and solid cancer cell lines, achieving nanomolar IC50 values. In leukaemia U937 cells, hydroxamate 5d and 2′-aminoanilide 8f induced significant cell death after 48 h, with 76% and 100% pre-G1 phase arrest, respectively, outperforming the reference compounds SAHA and MS-275. Furthermore, 2′-aminoanilide 8d demonstrated the strongest dose- and time-dependent cytodifferentiation in U937 cells, with up to 35% CD11c positive/propidium iodide-negative cells at 5 μM for 48 h. Compounds 5d, 5e and 8d also effectively upregulated p21 protein expression in the same cell line. Mechanistically, 5d, 5e, 8d and 8f were found to enhance mRNA expression of p21, BAX and BAK, downregulate cyclin D1 and BCL-2, and modulate pro- and antiapoptotic microRNAs to induce apoptosis.
Moreover, based on the discovery of the 1,4-dihydropyridines compounds 2 and 3, the latter being a SIRT3-specific activator, a new series of related compounds was developed. Among these, 3c displayed the strongest SIRT3 binding and activation, with a KD of 29 μM and 387% enzymatic activation. On the other hand, 3d proved most effective in enhancing glutamate dehydrogenase activity and deacetylating K68- and K122-acMnSOD in triple-negative MDA-MB-231 breast cancer cells. In CAL-62 thyroid cancer and MDA-MB-231 cells, 3d exhibited potent time- and dose-dependent reductions in cell viability and clonogenicity at single-digit micromolar levels, inducing cell death under both normoxia and hypoxia conditions. Furthermore, 3d downregulated hypoxia-induced factors (e.g., HIF-1α, EPAS-1, CA-IX), key regulators of epithelial−mesenchymal transition (e.g., SNAIL1, ZEB1, SLUG), and extracellular matrix components (e.g., COL1A2, MMP2, MMP9), significantly impairing MDA-MB-231 cell migration.
Building on the evidence of the demonstrated synergy of combining HDAC inhibitors with anti-PD-L1 antibiotics, we designed and synthesized dual inhibitors capable of targeting both epigenetic (HDAC) and immunological (PD-L1) pathways. To achieve this, we hybridized the PD-L1 inhibiting scaffold with well-known HDAC inhibitory moieties (Tubastatin, Entinostat, and Vorinostat). The dual inhibitors we synthesized (compounds 18–23) demonstrated significant promise in targeting both immune checkpoints and epigenetic regulators. Compounds 18–23 effectively inhibited PD-L1 and HDAC isoforms, with compound 18 standing out for its nanomolar range HDAC6 inhibition and 1000-fold selectivity compared to other isoforms. Moreover, in B16-F10 melanoma cells, compounds 18 and 19 surpassed the efficacy of single-agent reference compounds, with compound 18 effectively restoring T-cell functionality under chronic stimulation, reducing T-cell exhaustion markers (PD-1 and TIM-3) and enhancing cytokine production (IFN-γ and TNF-α). Furthermore, compound 18 induced immunogenic cell death and enhanced the expression of MHC-I, improving tumor immunogenicity, and enabling better recognition by cytotoxic T lymphocytes. These immune-stimulating effects were further validated in A375 melanoma co-culture models, where compound 18 showed superior efficacy in the presence of HLA-A2-positive PBMCs, indicating its capacity to leverage antigen presentation pathways for enhanced immune-mediated cytotoxicity.
Since the cardioprotective potential of SIRT5 in terms of infarct size (IS) reduction is elusive, we employed the newly-synthesized SIRT5 specific agonist, compound 52, to explore for the first time pharmacological activation of SIRT5 as target for cardioprotection. In in vitro screening experiments, SIRT1 and SIRT5 agonists, compounds 51 and 52, at 1-20μΜ were added to cardiomyoblasts (H9c2) and human endothelial cells (EA.hy-926) during 24h hypoxia/2h reoxygenation (H/R). SIRT1 and SIRT5 agonists mitigated H/R injury. Male C57BL/6J mice underwent 30min ischemia (I) followed by 2h or 24h reperfusion (R). They received vehicle, the SIRT1 or SIRT5 agonists at 20 and 30mg/kg at the 20th min of ischemia and IS was quantified via triphenyl-tetrazolium chloride staining (n=5-7/group). SIRT5 activation via compound 52 at 20mg/kg reduced IS at 24h R compared to controls (25.18±2.7% vs 38.80±4.7%). Targeted mass-spectrometry based metabolomic analysis in the ischemic heart at the 10th min of reperfusion pointed out precursors related to fatty acid oxidation. In the same timepoint, molecular analysis indicated that the SIRT5 agonist activated AMPKα and Reperfusion Injury Salvage Kinase (RISK) pathway. Additionally, at 3 h reperfusion, compound 52 led to increased mitofusin 2 without altering apoptosis suggesting improved mitochondrial dynamics. Co-administration of the SIRT5 inhibitor TW-37 abrogated the compound 52 IS reduction.
Finally, I assessed and implemented the technology transfer of a propafenone hydrochloride coated tablet specialty drug, utilizing High-Shear equipment, following the transfer of this manufacturing process to the pharmaceutical company ITC Production. The aim of this project was to oversee all risk control activities carried out by ITC, from the collaborative drafting of the risk assessment to prototype-scale production in the R&D laboratory, to ensure the quality of the final product and mitigate the risks associated with industrial scale-up. This approach was aimed at guaranteeing the reproducibility, quality, and safety of the manufacturing process.