Planejamento de moléculas bioativas contra proteases de SARS-COV-2: estudos in silico e potencial biológico

Abstract

As of January 25, 2023, approximately 664.8 million SARS-CoV-2 infections have been reported worldwide, with over 6.72 million deaths notified over the last three years. Whilst non-pharmacological interventions, vaccines’ development and distribution, direct antiviral drugs approval (e.g., nirmatrelvir and remdesivir), as well as monoclonal antibodies have been employed against COVID-19, low vaccine coverage and new SARS-CoV-2 mutations negatively impact the control of the pandemic scenario. In addition, the possibility of resistant strains can limit therapeutic options. In this sense, it is still necessary to design and develop bioactive molecules against SARS-CoV-2. Therefore, the present work aims to obtain new inhibitors against SARS-CoV-2 proteases, Mpro and PLpro, employing in silico and in vitro approaches. Initially, an HQSAR model (q2 = 0.964 and r2 = 0.975) was selected to predict ligands with potential inhibitory activity against Mpro. Here, 24 potential inhibitors obtained followed to enzymatic inhibition activity determination, all of which were inactive (less than 50% inhibition) at 10 µM. Subsequently, crystallographic structures of the proteases were selected for binding sites predictions and molecular docking analysis (redocking) to build pharmacophoric models based on SARS-CoV-2 and other coronaviruses Mpro and PLpro. These were used in virtual screening campaigns to select potential inhibitors in chemical libraries (BraCoLi, DrugBank, GMR, NuBBE and ZINC15), from a total of 6.8 million compounds. Virtual screening was conducted to both targets obtaining 46 compounds from two available libraries (DrugBank and GMR). Among the screened compounds (14 available in house), none showed inhibitory activity (over 50% inhibition) against Mpro and PLpro up to 10 µM. A series of 127 synthetic structural analogues of the 46 unavailable virtual hits were evaluated in enzymatic assays, and five out of 127 compounds had their 50% inhibitory concentrations (IC50) determined for SARS-CoV-2 Mpro (0.086 ± 0.0015 to 16.42 ± 0.095 µM). Two compounds were also active against SARS-CoV and MERS-CoV Mpro (0.11 ± 0.0175 to 3.786 ± 0.64 µM), and all 127 were inactive against PLpro. In addition, plant extracts and triterpene isolates designed against SARS-CoV-2 were evaluated for their cytotoxicity and antiviral activity by MTT assay, determining 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) against a murine coronavirus (MHV-3). Four compounds showed antiviral activity, with selectivity indexes (SI) between 1.08 and 34.36. Taken together, the computational strategies employed were able to obtain inhibitors predicted in silico and determined in vitro, demonstrating the potential and success in the planning of bioactive molecules against SARS-CoV-2.