Development of therapeutics for SARS-CoV-2 infection

Division Of Basic Sciences - Nci

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Abstract

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome virus 2 (SARS-CoV-2, SCoV2), has been one of the most devastating pandemics of recent times. More serious global health crises would have occurred if the lack of antivirals against COVID-19 continued. However, the emergence and approval of potent inhibitors of the viral main protease (Mpro), such as nirmatrelvir (NIR), offered hope not only on the therapeutic front but also in the context of prophylaxis against severe COVID-19. By their nature, RNA viruses including human immunodeficiency virus (HIV) inherently have high mutation rates, and lessons learnt from previous and currently ongoing pandemics have taught us that these viruses can promptly develop drug resistance through mutations of vital target amino acid residues, although the actual clinical contributions of such phenotypic SCoV2 resistance against Mpro inhibitors (MPIs) remain to be studied. Ritonavir-boosted nirmatrelvir targeting SARS-CoV-2's main protease (Mpro) shows efficacy in patients with mild-to-moderate COVID-19. However, ritonavir causes drug-drug-interactions that warrant cautions due to CYP3A4 inhibition, thus limiting nirmatrelvir's clinical utility. A novel Mpro inhibitor, TKB272, achieves higher intracellular concentrations, more potently inhibits Mpro, and more effectively blocks the infectivity and replication of SARS-CoV-2 strains than nirmatrelvir. When orally administered in mice, TKB272 potently blocked SARS-CoV-2XBB1.5 without ritonavir. When propagated with nirmatrelvir in vitro, highly nirmatrelvir-resistant E166V-carrying SARS-CoV-2E166V readily emerged; however, when propagated with TKB272, no variants emerged. TKB272 showed substantial activity against SARS-CoV-2E166V. TKB272 may serve as a therapeutic for COVID-19 with a potential to overcome resistance and its potency may pave the way for the prevention of long COVID. Here, we also generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors, nirmatrelvir (NIR), TKB245, and 5h, by propagating the ancestral SARS-CoV-2WK521 in highly SARS-CoV-2-susceptible VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined the structural and virological profiles of the variants obtained. A predominant variant E166V (SARS-CoV-2WK521E166V), which emerged when selected with NIR and TKB245, proved to be resistant to NIR and TKB245. E166V-containing recombinant Mpro (MproE166V) was resistant to all three inhibitors. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed doing so. Native mass spectrometry confirmed that NIR and TKB245 promoted the dimerization of the wild-type Mpro (MproWT), but compromised the enzymatic integrity and activity; while E166V substitution disrupted the binding of NIR and TKB245 to MproE166V. SARS-CoV-2WK521 selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and well-replicated in the presence of 5h, dominating over SARS-CoV-2WK521. However, no significant enzymatic or structural changes in MproA191T were observed. The competitive SARS-CoV-2 replication assay showed that the replicability of SARS-CoV-2WK521E166V was compromised compared to SARS-CoV-2WK521 but predominated over SARS-CoV-2WK521 in the presence of NIR in replicability. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521 in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2's drug resistance acquisition and the development of novel resistance-repellant COVID-19 therapeutics.