Development of Histone Deacetylase Inhibitors and Degraders with Antiplasmodial and Anticancer Activity

Abstract

Histone deacetylases (HDACs) are key targets in epigenetic drug discovery, as they catalyze the removal of acetyl and acyl groups from lysine side chains of histones and other proteins, thereby influencing gene expression and protein function. In humans, 18 HDAC isoforms have been identified: eleven are Zn²⁺-dependent, while seven use NAD⁺ as a cofactor, the latter are also known as sirtuins. Efforts in drug discovery have led to the FDA approval of five HDAC inhibitors: vorinostat, romidepsin, belinostat, panobinostat, and givinostat. HDAC inhibitors have also shown activity against <em>Plasmodium falciparum</em>, the deadliest malaria parasite. Malaria remains a major global health problem, with the WHO estimating 263 million cases and 597,000 deaths in 2023, coupled with rising resistance to current antimalarial drugs. <br/> The first project in this thesis focused on developing novel antiplasmodial HDAC inhibitors through structure-activity relationship studies of heteroaryl-decorated peptoid-based inhibitors. These inhibitors were synthesized using a straightforward approach involving the Ugi four-component reaction (U-4CR). A set of 16 compounds was synthesized and screened for activity against asexual blood-stage <em>P. falciparum</em> parasites, followed by testing against liver-stage parasites and assessment of the metabolic stability. Compound 6i was identified as the most potent inhibitor, showing promise as a starting point for antimalarial drug development. <br/> The second project aimed at modifying the clinical-stage anticancer HDAC inhibitor quisinostat, which had previously shown potent antiplasmodial activity but exhibited significant toxicity to human cells. To improve its selectivity profile, multicomponent syntheses were used to generate small sets of compounds based on three different chemotypes. These compounds were evaluated for antiplasmodial activity and for antiproliferative activity against solid cancer cell lines. 18b emerged as the most potent antiplasmodial HDAC inhibitor, while 9b, 9d, and 13f showed promising antiproliferative effects against different cancer cell lines. <br/> The third project focused on modifying an existing HDAC inhibitor cap group by combining it with different linkers and zinc-binding groups (ZBGs) to create new HDAC inhibitors with alternative ZBGs. <br/> Sixteen compounds were synthesized and screened for their inhibition of HDAC1-3 and HDAC6, as well as for antiplasmodial activity against <em>P. falciparum</em> and antiproliferative activity against selected cancer cells. Although most of the compounds showed low HDAC inhibitory properties or weak activity in the phenotypic assays, compound 20 (named DS-103) emerged as a potent ethyl hydrazide-based HDAC inhibitor. Further studies showed that DS-103 was effective in reversing cisplatin resistance in cancer cells and exhibited strong synergism with cisplatin. <br/> Building on these results, the fourth project aimed at the development of HDAC proteolysis-targeting chimeras (PROTACs) using the ethyl hydrazide moiety as the ZBG. The subsequent screening of the synthesized PROTACs featuring VHL- and CRBN-targeting ligands identified several compounds that selectively degraded HDAC6, with 17c being the most potent degrader (D_max = 91%; DC₅₀ = 14 nM). Mechanistic studies showed that 17c-induced HDAC6 degradation is dependent on neddylation and binding to both HDAC6 and CRBN. Chemoproteomics further confirmed the selectivity of 17c for HDAC6.