Phd in LIFE SCIENCE

Thesis title: Design and synthesis of new heterocyclic compounds as HIV-1 RNase H inhibitors

Candidate: Alessandro De Leo Design and synthesis of new heterocyclic compounds as HIV-1 RNase H inhibitors Since the beginning the pandemic in the early ‘80s, 76 million people have become infected with HIV-1, and 33 million of patients died from AIDS-related complications. Around 38 million people worldwide were living with HIV at the end of 2020. After the transmission event, HIV-1 primarily targets T CD4+ cells and the resulting infection causes a progressive irreversible harm to innate and adaptive immune system, leading to AIDS in the absence of therapeutic intervention. Being a Retrovirus, HIV-1 can successfully incorporate the retrotranscribed DNA into the host cell genome, thereby causing a persistent infection which cannot be eradicated with current therapies. Over the past three decades, more than 40 FDA-approved drugs have been approved and employed in the USA. This treatment is based on a multi-pill regimen and targets the main steps of HIV-1 life cycle, from entry to maturation/egress. ART creates a high genetic barrier that dramatically discourages the development of drug resistance. It can successfully prevent the progression towards AIDS, it potently suppresses viral loads, it dramatically decreases transmission rates, it markedly reduces morbidity and mortality, and it considerably prolongs life expectancy. Despite the undeniable success of ART, some drawbacks are observed, including long-term toxicities, and drug-drug interactions. Although ART is deliberately devised to overcome HIV-1 high genetic variability, drug-resistance strains can still be selected, and the spread of such strains represents a public health concern. Since the very beginning, HIV-1 reverse transcriptase (RT) has been addressed as a feasible viral target, given the lack of a close homologue in humans. The protein is heterodimeric, composed of two different subunits: p66 and p51. p66 harbours two functional active sites: the RNA dependent DNA polymerase (RDDP) and the RNase H (RH), that digests the RNA component of RNA/DNA hybrids during retrotranscription. Both the enzymatic functions depend upon the presence of two magnesium ions within the active site. Two classes of RT inhibitors have been approved: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) that behave as active site binders and allosteric inhibitors, respectively. Conversely, no RH inhibitors (RHIs) have reached the clinical pipeline so far, despite the compelling evidence that this enzymatic function is essential for viral replication. On the basis of the mode of action, RHIs are grouped into active site, allosteric, dual inhibitors and natural products endowed with anti-RH activity. Our research team started from previously published pyrrolyl diketo acid (DKA) derivatives to design, synthetize, and biologically evaluate non-DKA pyrrole-pyrazole compounds as active site metal chelating agents with improved pharmacokinetic and stability properties compared to the DKA hit compounds. In addition, we aimed to design selective RHIs with no IN side activity. The rationale consisted in the incorporation of the typical DKA moiety into a heterocyclic ring bearing heteroatoms with metal-coordination properties. With this in mind, we biologically evaluated our library of pyrrole-pyrazole derivatives in enzymatic and cell-based assays. A molecular modelling approach was coupled with site directed mutagenesis studies to substantiate our biological data and shed light on the interactions of our compounds with the target. Finally, in vitro serum stability experiments demonstrated that our derivatives were stable in human serum, compared to the DKA counterparts. In the second part of my PhD, starting from DKA-quinolononyl derivatives with double efficacy against IN and RH, our research work consisted in the design, synthesis, and biological evaluation of non-DKA quinolononyl-based compounds as new RHIs. With the aim of ameliorating the druggability of our derivatives, we decided to shorten the DKA branch, keeping fixed the heteroatoms involved in the metal coordination. We biologically evaluated our compounds in the same assays performed before for the pyrrole-pyrazole library. We also combined computational approaches and site-directed mutagenesis, as well. In addition, spectrophotometric experiments were performed to assess the metal coordination properties of our compounds in solution. Interestingly, our derivatives proved to be selective towards RH over IN. On the other hand, they inhibited the RDDP activity of RT, as well. Taken together, these results show that the antiviral properties of our compounds can be ascribed to the combined inhibition of both these two essential enzymatic functions of RT.