In vitro molecular identification and characterization of investigational and licensed antivirals against viral pathogens

Multidrug-resistant (MDR) HIV-1 and emerging viral pathogens represent two of the most important challenges in current antiviral research. This doctoral thesis integrates multiple traits of in vitro antiviral investigation addressing different aspects. In the context of the drug discovery against emerging pathogens, a panel of piperazine-based compounds was evaluated against Zika virus (ZIKV), Dengue virus (DENV) and SARS-CoV-2, identifying several broad-spectrum candidates with IC₅₀ values below 5 μM and favorable selectivity indices. Proteomic analysis of the pyridobenzothiazolone derivative HeE1-17Y in West Nile Virus-infected cells revealed a multimodal antiviral mechanism involving reduction of viral NS3 and NS1 proteins and dysregulation of key host pathways. In the HIV field, the combinatorial in vitro activity of doravirine and islatravir antiretrovirals was assessed against MDR isolates from heavily-treatment experienced (HTE) people with HIV (PWH), showing additive-to-synergistic interactions in the majority of cases. A parallel study comparing islatravir and tenofovir alafenamide in viruses harboring the M184V/I mutation highlighted greater residual susceptibility to tenofovir alafenamide, reinforcing the value of phenotypic testing in therapy optimization. HIV-1 reservoir dynamics were monitored over 18 months in virologically suppressed patients simplifying from 3-drug to 2-drug regimens, showing overall marker stability with a modest but significant reduction of intact proviruses in the simplification arm. The central focus of this work, however, is the comprehensive in vitro characterization of lenacapavir, the first-in-class capsid inhibitor approved for MDR HIV-1, across clinically relevant strains differing in subtype and treatment history. Despite its picomolar-range potency and twice-yearly injectable formulation, its low genetic barrier to resistance remains a key clinical concern. Baseline phenotypic susceptibility was assessed using recombinant viruses harboring clinically derived GAG-PR sequences from both treatment-naïve (TN) and HTE PWH, including subtype B and non-B isolates. Lenacapavir demonstrated comparable high antiviral activity across all tested genetic backgrounds, indicating that neither natural genetic variability nor prior drug exposure meaningfully impairs its potency. In vitro resistance selection experiments conducted under escalating drug pressure led to the emergence of resistance-associated mutations (RAMs) at known capsid positions in both subtypes, confirming the low genetic barrier of the drug regardless of viral background. Subtype B viruses showeda shorter median time to viral breakthrough compared to non-B strains, with the Q67H substitution emerging more frequently in subtype B isolates. Site-directed mutagenesis further confirmed that non-polymorphic p24 substitutions at codons not known to be involved in enhancing resistance to lenacapavir act as compensatory mutations, reducing lenacapavir susceptibility and enhancing viral fitness. These findings demonstrate that lenacapavir retains broad antiviral potency across different HIV-1 subtypes and treatment backgrounds, while reinforcing the importance of constructing a robust optimized background regimen to minimize resistance emergence in clinical practice.