Stefanelli, Irina (2022) Discovery of novel Direct-Acting Antiviral Agents to counteract emerging viruses. [Tesi di dottorato]

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Abstract

Emerging and re-emerging infections have threatened the humanity from several decades. The globalization, the international trade and the expansion of human population increase the risk to contracting infections and contribute to speed-up their spreading. In particular, RNA viruses such as Flaviviruses and Coronaviruses, represent critical zoonotic agents, which are involved in inter-species transmission and periodically cause epidemic outbreaks around the World. The most recent COVID-19 pandemic, caused by novel coronavirus Severe Acute Respiratory Syndrome (SARS-CoV)-2, highlights the main role of RNA viruses as etiological agents of human diseases and the major gaps in the control of new pathogens. In this context, this thesis work is focused on the discovery of novel Direct-Acting Antivirals (DAAs) to counteract emerging RNA viruses. DAAs are defined as small molecules that specifically target viral proteins with essential roles in the replication and no counterparts in the human host, thus resulting in potent and safe antiviral activity. Usually, a combination of at least two DAAs with different mode of actions ensures a higher efficacy and limits the occurrence of viral resistant mutants. Indeed, the introduction of DAAs in the antiviral therapy allowed to successfully treat or cure viral infections, such as Human Immunodeficiency Virus (HIV) and Hepatitis C virus (HCV), respectively. Thus, DAAs represent efficacious therapeutic agents against viral infections. Among RNA viruses, ZIKA virus (ZIKV) is an arising flavivirus that causes neurodevelopmental congenital diseases and the neuroinflammatory Guillain−Barré syndrome. In 2016 the World Health Organization (WHO) declared ZIKV a “Public Health Emergency of International Concern”. There are neither vaccines nor drugs for ZIKV, thus European Union invested millions of Euros to finance research against ZIKV infection. According to the aim of my doctoral project, I focused on the discovery of new small molecules as DAAs active against ZIKA virus. In chapter 3, two strategies are described for the ZIKV DAAs identification: • rational design and synthesis of non-nucleoside inhibitors of ZIKV polymerase, that is an ideal target for the development of effective and safe DAAs; • synthesis of potential polypharmacologic ZIKV candidates, selected through an ultra-fast multitargeting virtual screening by an innovative drug discovery platform, developed by Dompè Pharmaceuticals to address pandemic crisis. After biological evaluation in antiviral phenotypic assays performed by our collaborators, two promising candidates (34, 37) were identified, active in the nanomolar range and not toxic for cells. As consequence of the COVID-19 pandemic, in the early 2020, my efforts were turned immediately to identify novel DAAs active against SARS-CoV-2, when no drugs or vaccines were available. Moreover, despite the recent approval of vaccines, monoclonal antibodies and few DAAs, the demand for new efficacious and safe antivirals is compelling to complete the arsenal against the COVID-19. The viral 3-Chymotrypsin-Like Protease (3CLpro) is an essential enzyme for replication with high homology in the active site across CoVs and variants, showing an almost unique specificity for Leu-Gln as P2-P1 residues and allowing the development of broad-spectrum inhibitors. The design, synthesis, biological evaluation and cocrystal structural information of newly conceived peptidomimetic covalent reversible inhibitors are described in chapter 4. A series of designed inhibitors displayed an aldehyde warhead, while another series was endowed with a nitrile. Both series featured a Gln mimetic at P1 and modified P2-P3 residues. Particularly, functionalized proline residues were inserted at P2. The most potent compounds displayed low/sub-nM potency against the 3CLpro of SARS-CoV-2 and MERS-CoV and inhibited SARS-CoV-2 replication in cells, with derivative 46 exhibiting nM EC50 and the highest selectivity index. Some compounds were co-crystallized with SARS-CoV-2 3CLpro, confirming our design. Altogether, these results foster future work toward broad-spectrum 3CLpro inhibitors to challenge CoVs related pandemics. To date, Nirmatrelvir is the first-in-class 3CLpro inhibitor to receive approval by FDA and EMA in combination with the metabolic booster Ritonavir (Paxlovid). Nirmatrelvir has been shown to reduce the risk of progression to severe COVID-19, but a rebound of the infection with the current treatment regimen has been reported in several recent studies. The in-house multigram synthesis of Nirmatrelvir, thereafter called compound 12, and its evaluation in in vitro and in vivo assays developed by our collaborators were reported in chapter 5. In particular, it was described: • an efficient synthetic route to afford Nirmatrelvir in gram scale for in vitro/in vivo evaluation, performed prior the publication of synthesis and antiviral activity by Pfizer team; • validation of biochemical and phenotypic assays developed by our collaborators at the University of Cagliari and the “Instituto Nazionale di Genetica Molecolare” of Milan for the screening of new 3CLpro inhibitors, through the use of Nirmatrelvir as benchmarker; • validation of in vivo efficacy in an innovative and tractable mouse model of SARS-CoV-2 infection developed by our collaborators at the “Vita-Salute San Raffaele” University of Milan, and identification of a more suitable dosage and vehicle of po (oral administration) formulations of Nirmatrelvir respect to data published; • in vivo investigation of impact of Nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses, which was still unknown.

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