BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir

Abstract

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC 50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128’s primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently—but complementary—with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect—a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Systematic Studies on the Anti-SARS-CoV-2 Mechanisms of Tea Polyphenol-Related Natural Products

“…We proceeded to evaluate the inhibitory activities of all of the natural products against RdRp using a commercial assay kit and our prepared RdRp complex . We found that theaflavin, theaflavin-3′-gallate, TF3, and proanthocyanidin exhibited inhibition toward RdRp with IC 50 values of 40.2 ± 2.3, 23.2 ± 0.4, 2.3 ± 0.6, and 23.4 ± 1.3 μM, respectively (Figure k–n).…”

“…The procedure involved using both E. coli for expressing nsp7L8 and baculovirus-infected insect cells for expressing nsp12. , Subsequently, nsp12, nsp7L8, and nsp8 were combined at a molar ratio of 1:3:3 and preincubated on ice for 10 min to facilitate the formation of an active complex following a reported protocol. , In vitro RdRp activity was monitored using a fluorescence plate reader (Synergy H1 Hybrid multimode Reader, BioTek) with a commercial kit (ProFoldin, Huson, MA). The initial rates of the inhibited reactions were plotted against different inhibitor concentrations to determine the IC 50 value by fitting with the equation: A­( I ) = A(0) × {1–[ I /( I + IC 50 )]}.…”