Sviluppo di nuove strategie terapeutiche contro il papillomavirus umano.

Human papillomaviruses (HPVs) are small double-stranded DNA viruses with an epithelial tropism. Low-risk genotypes commonly induce cutaneous warts, whereas the high-risk genotypes (HR-HPVs), such as HPV16 and HPV18, are responsible for the majority of cervical carcinomas worldwide, but also other types of cancers, such as ano-genital and head-and-neck cancers. Although an extensive vaccination campaign has been started to prevent viral infection, there is still an urgent need of specific treatments for patients already infected. Unfortunately, no specific anti-HPV drugs exist in the clinics for the treatment of HPV-associated cancers. Current treatments are based on surgical ablation of the tumor mass, radiotherapy, and chemotherapy, and do not exclude the possibility of relapses. The oncogenic properties of HR-HPVs are related to E6 and E7 viral oncoproteins. E7 can interact with a wide variety of cellular partners. Among them, E7 interacts with and induces the proteasomal degradation of PTPN14, an oncosuppressor and inhibitor of YAP, a strong oncogenic determinant in solid tumors. By analyzing the co-crystal structure of HPV18 E7 bound to PTPN14, the C-terminal domain of E7 appeared to be crucial for this interaction. Previously, our group conducted an in-silico screening of small-molecule compounds directed against some key residues of E7 in order to find a compound able to block this interaction. Compound 20 was identified as a promising candidate for further studies, and its characterization was one of the objectives of this work. E6 oncoprotein can dimerize through its alpha-helix and interact with E6AP (E6-associated protein) ubiquitin ligase, which triggers the proteasome-mediated degradation of several proteins, including p53, the main cellular oncosuppressor. Recently, our group identified a compound (Cpd12) able to inhibit the interaction between E6 and p53, leading to the rescue of p53 levels and restoring its antitumoral activity (Celegato et al., 2020). In addition, Cpd12 was able to synergize with standard chemotherapeutics and showed a broad-spectrum activity against HR-HPV genotypes (Celegato et al., 2022). Our research group recently exploited the idea to synthesize and characterize PROteolysis TArgeting Chimeras (PROTACs) to selectively degrade E6 oncoprotein. For this purpose, some analogs of Cpd12 were developed and tested. Among them, Cp-05 resulted the most promising candidate and was further modified in order to obtain four PROTACs. In this work, by means of cell viability assays and Western Blot experiments, we determined their toxicity and investigated the rescue of p53 and the degradation of E6 protein levels, respectively. In conclusion, my thesis work is focused on the characterization of small-molecules compounds active against the two major oncoproteins of HPV, with the aim of finding a novel therapeutic strategy to combat HPV-induced tumors.