γ-Phosphonate monoalkyl nucleotide analogues inhibit SARS-CoV-2 replication in vitro by inducing dissociation of the minimal replication–transcription complex

Nucleoside triphosphate analogues in which the γ-phosphate has two different non-cleavable lipophilic alkyl residues were observed to potently inhibit HIV-1 replication in vitro. In the present work, a series of γ-phosphonate monoalkyl nucleotides were found to inhibit the RNA-dependent RNA polymerase (RdRp) function of the SARS-CoV-2 nsp12 protein. These nucleotide analogues were synthesized with multiple nucleobase derivatives, sharing one alkyl group of two different lengths on the γ-phosphonate. The enzymatic PAGE-based assay of SARS-CoV-2 nsp12 in the presence of cofactors nsp7/8 revealed IC₅₀ values in the low micromolar range, whose potency appeared to be dependent on the length of the alkyl group. Notably, the assay displayed an unexpected mechanism of action, with no incorporation of the analogues and complete loss of enzymatic function. Competition assay revealed that the tested nucleotide analogues do not compete with the natural triphosphate nucleotides independently of sequence complementarity, hence acting as non-nucleoside inhibitors. Using multiple techniques, we were able to identify the mechanism of action in the compound-induced dissociation of the nsp7 cofactor from the nsp12 subunit. Molecular docking confirmed the binding site, identifying putative amino acid residues relevant for compound interaction. Different prodrugs of the most potent nucleotide analogues were synthesized and showed inhibitory activity against SARS-CoV-2 replication in cell culture.