Anti-HIV therapy has dramatically progressed from palliative care to high effectiveness, with virus replication and disease progression halted in most patients yet not eradicated. However, the need for lifelong therapy has kept anti-HIV drug development at high pace and novel strategies, drugs and drug classes have been regularly introduced over time. Issues to be tackled during prolonged antiretroviral therapy include adherence, toxicity, drug-drug interactions and the development of drug resistance. During my PhD course at the HIV Monitoring Laboratory (HML) of the Department of Medical Biotechnology at the University of Siena, I have been given the opportunity to focus my efforts in this area along two different lines. The first activity has been done in the field of drug discovery within the EU FP7 funded THINPAD project. THINPAD is the acronym of “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance”, thus the objective of this project was to discover and develop a novel class of anti-HIV agents targeting NCp7, a small nucleocapsid protein that plays a number of different roles in HIV replication. The project used a funnel strategy from virtual screening to biochemical testing, then cell based assays and finally humanized mice as the animal model to validate the candidate NCp7 inhibitors (NCIs). My unit measured the antiviral activity of the candidate NCIs through cell based assays developed at the HML. The compounds with the highest selectivity indexes were next demonstrated to retain comparable efficacy against wild-type virus and virus resistant to licensed drug classes, thus confirming NCp7 as a specific target suitable to block the replication of currently circulating drug resistant viruses. The same compounds were used to perform experiments aiming at the definition of the mechanism of action through the measurement of viral nucleic acids intermediates produced in infected cells in presence of inhibitory concentration of NCIs at different time points. The results of this analysis were in agreement with antiviral activity exerted at the early and/or late steps of viral replication, suggesting that NCIs are able to interfere with variable impact on the different functions of NCp7 during HIV life cycle. While preliminary efficacy tests in the humanized mouse model were not successful, in vitro data support further NCI development. The second activity has investigated the role of natural HIV-1 variability or specific HIV-1 mutants in the susceptibility or genetic barrier to resistance to licensed or upcoming antiretrovirals. Completed studies of this kind have shown (i) a role for the natural polymorphisms E138A in the reverse transcriptase in lowering the genetic barrier to resistance to the non-nucleoside reverse transcriptase inhibitor etravirine, (ii) a minimal impact of the integrase E157Q polymorphism in resistance to integrase inhibitors and (iii) the infrequent occurrence of natural resistance to the novel entry inhibitor fostemsavir in the HIV-1 CRF02_AG, an evolutionary lineage of HIV-1 originated in Africa and substantially represented in Italy. Although different, the three topics shared the need to clarify uncertain areas and derive indications for optimal drug use in the clinic. Both activities have been using cell based systems developed at the HML and further refined for the specific application. Phenotypic drug susceptibility testing is a fairly complex task, usually assigned to specialized companies in collaborative research projects and not used at all in the clinic. Among academic systems proposed over time, our assay has been uniquely validated through comparison with the de facto reference commercially available Phenosense assay from Monogram Biosciences. Overall, contributing to expanding and using the laboratory portfolio of systems required for advanced investigation of anti-HIV drug was perfectly in line with my expectations as a biotechnologist.
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|Titolo:||HIV drug discovery and resistance testing through cell based assays|
|Citazione:||Giannini, A. (2019). HIV drug discovery and resistance testing through cell based assays.|
|Appare nelle tipologie:||8.1 Tesi Dottorato|