γ-Monoalkyl triphosphate analogues of acyclic nucleoside phosphonates inhibit SARS-CoV-2 replication in vitro by inducing dissociation of the minimal replication-transcription complex

Nucleoside triphosphate analogues in which the gamma-phosphate has two different non-cleavable lipophilic alkyl residues were observed to potently inhibit HIV-1 replication in vitro. We report that a series of gamma-monoalkyl triphosphate analogues of acyclic nucleoside phosphonates inhibits 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 gamma-phosphonate. The enzymatic PAGEbased assay of SARS-CoV-2 nsp12 with cofactors nsp7/8 revealed IC50 values in the low micromolar range, whose potency depended on the length of the alkyl group, 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 nonnucleoside inhibitors. By multiple techniques, we demonstrated that the inhibitors induce the dissociation of the nsp7 cofactor from the nsp12 subunit. Molecular docking and molecular dynamic simulations supported this mechanism identifying a putative binding site in an interface cavity between nsp12-nsp8 and nsp7, where the compound forms both hydrogen bonds and hydrophobic interactions. Different prodrugs of the most potent nucleotide analogues were synthesized and showed inhibitory activity against SARS-CoV-2 replication in cell culture.