Evaluation of Piperazine-Based Compounds Against Flaviviruses and SARS-CoV-2, and In Vitro/In Vivo Selection of Benchmark Antivirals Against SARS-CoV-2

Emerging and re-emerging RNA viruses represent one of the most pressing challenges in modern infectious disease medicine. Despite significant advances in therapeutic development over recent years, the absence of approved direct-acting antivirals for flaviviruses such as Dengue virus (DENV) and Zika virus (ZIKV), combined with growing concerns about resistance to existing SARS-CoV-2 treatments, underscores the need for continued drug discovery and virological surveillance efforts. This doctoral thesis presents two interconnected lines of research. The first focused on the identification and characterization of novel piperazine-based compounds as potential broad-spectrum antiviral agents against ZIKV, DENV, and SARS-CoV-2. A library of 22 structurally diverse molecules, synthesized by the Department of Organic and Medicinal Chemistry at the University of Seville through a privileged structure-based design approach, was evaluated using live-virus cell-based assays in HuH-7 and A549-AT cell lines. Cytotoxicity profiling revealed that the majority of compounds exhibited favorable safety profiles, with CC₅₀ values exceeding 200 µM in both cell lines. Among the compounds tested, eleven showed meaningful anti-ZIKV activity, with IC₅₀ values ranging from 1.6 to 48.1 µM. Notably, compounds 9, 13, 20, and 21 achieved inhibitory concentrations in the low micromolar range, with compound 13 (IC₅₀ = 1.6 µM) outperforming the reference antiviral Sofosbuvir. Several of these ZIKV-active molecules also retained activity against DENV-2, pointing to a mechanism likely involving the conserved NS3 protease of the Flaviviridae family. Only compound 21 showed borderline activity against SARS-CoV-2 (IC₅₀ = 22.5 µM), indicating that while the piperazine scaffold holds clear promise for flavivirus targeting, further structural optimization will be required to extend its antiviral spectrum to coronaviruses. These findings support the continued development of piperazine derivatives as a versatile platform for multi-pathogen antiviral discovery. The second research line addressed a clinically relevant and increasingly important question: what is the genetic barrier of SARS-CoV-2 to the two principal direct-acting antivirals currently used in clinical practice, Remdesivir (RDV) and Nirmatrelvir (NRM)? This question was approached through an integrated strategy combining longitudinal genomic analysis of treated patients with systematic in vitro viral resistance selection experiments, using both the ancestral B.1 lineage and the currently circulating Omicron KP.3 subvariant. The clinical cohort comprised 101 paired nasopharyngeal samples from 98 patients, predominantly elderly individuals with multiple comorbidities, collected at baseline and after approximately five days of treatment at three Italian hospital centers. Whole-genome sequencing revealed that genotypic resistance to DAAs was rare in vivo, with resistance-associated RdRp mutations (V792I and M794I) detected in only three immunocompromised patients treated with RDV. No 3CLpro mutations were observed in patients receiving NRM. In parallel, in vitro resistance selection experiments confirmed that both drugs have a high genetic barrier, though with notable strain-dependent differences. Under prolonged drug pressure, B.1 developed RdRp substitutions conferring up to a 35-fold reduction in RDV susceptibility, and accumulated multiple 3CLpro mutations, including the L50F/E166V combination, resulting in a 277-fold increase in NRM resistance. By contrast, the KP.3 variant showed significantly constrained evolutionary flexibility: it reached only a 4-fold increase in RDV resistance and failed entirely to develop NRM resistance before undergoing viral extinction, suggesting that the extensive mutational burden carried by advanced Omicron lineages limits their capacity for further adaptive evolution. Crucially, combining RDV and NRM markedly suppressed the emergence of resistance in B.1 and completely prevented it in KP.3, providing strong molecular rationale for dual DAA therapy, particularly in immunocompromised patients at highest risk of persistent infection and resistance development. Taken together, these studies contribute to two complementary dimensions of antiviral research: the discovery of new chemical scaffolds with activity against neglected viral pathogens, and the characterization of resistance dynamics for approved therapeutics against a pathogen of ongoing global concern.