Bases bioquímicas e funcionais dos mecanismos de resistência do ensitrelvir e nirmatrelvir contra polimorfismos naturais protease principal de SARS-CoV-2.

Abstract

The pandemic caused by SARS-CoV-2 has brought various challenges to society, affecting the world socially and economically. After the first cases of COVID-19 were recorded in China, the virus spread rapidly, leading millions of people to death. Due to the accelerated spread of the virus, the scientific community in various countries turned to the development of vaccines and antivirals, resulting in the rapid approval of several vaccine candidates. Even in the context of vaccine existence, the search for therapeutic alternatives such as antivirals is necessary and essential, as vaccine coverage and efficacy suggest that cases of COVID-19 will still continue to occur. The main viral protease (Mpro) is a key enzyme for the viral cycle and one of the most promising targets for drug development. Mpro inhibitors, nirmatrelvir and ensitrelvir, demonstrate significant potential in the treatment of viral infection. Despite their high efficiencies against wild-type Mpro, very little is known about how mutations in the enzyme's active site could generate resistance to these antivirals. In this context, we identified fourteen mutations in genomic databases that exist within the range of nirmatrelvir and ensitrelvir action and may affect their activity. Using molecular biology and biochemical techniques, we produced the recombinant version of all these unique mutants. All proteins were purified using standard techniques. We performed enzymatic kinetic characterization for all Mpro mutants and compared them to the wild type. Additionally, we tested the potency of the antivirals against each of the mutants to identify possible resistant mutants. Nirmatrelvir maintained its efficacy against most of the evaluated polymorphisms, although mutants Q189K and G143S showed significant resistance, with an increase in Ki of 16.4 and 147.7, respectively, compared to the wild type. Regarding ensitrelvir, a decrease in inhibition constants was observed for the M49I, G143S, and R188S polymorphisms, but not for Q189K, indicating a differentiated resistance pattern between the inhibitors. Thus, these results are crucial both for monitoring the emergence of resistant variants of SARS-CoV-2 and for the development of more effective treatment strategies if these mutations evolve into variants of concern.