Alphavirus antivirals targeting RNA‐dependent RNA polymerase domain of nsP4 divulged using surface plasmon resonance. (25th February 2022) - Pareek, Akshay
Abstract : Alphaviruses are continuously re‐emerging and pose a global threat to human health and currently no antiviral drug is commercially available for alphaviral infections. Alphavirus non‐structural protein nsP4, which possesses RNA‐dependent RNA polymerase (RdRp) activity, is a potential antiviral target. To date, no antiviral drug is commercially available against alphaviruses. Since RdRp is the key virus‐specific enzyme involved in viral genome replication, this study identifies and validates the antiviral efficacy of small molecules targeting alphavirus RdRp. Purified nsP4 was characterized using the surface plasmon resonance (SPR) assay, and the binding affinities of divalent metal ions, ribonucleotides, and in vitro transcribed viral RNA oligonucleotides were obtained in the micromolar (μm ) range. Further, four potential inhibitors, piperine (PIP), 2‐thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA), were identified against nsP4 RdRp using a molecular docking approach. The SPR assay validated the binding of PIP, 2TU, PZA, and CGA to purified nsP4 RdRp with K D of 0.08, 0.13, 0.66, and 9.87 µm, respectively. Initial testing of these molecules as alphavirus replication inhibitors was done using SINV‐IRES‐Luc virus. Detailed assessment of antiviral efficacy of molecules against CHIKV was performed by plaque reduction assay, qRT‐PCR, and immunofluorescence assay. PIP, 2TU, PZA, and CGA showed antiviral potency against CHIKV with EC50 values of 6.68, 27.88, 36.26, and 53.62 µm, respectively. This study paves the way towards the development of novel broad‐spectrum alphavirus antivirals targeting nsP4 RdRp. Abstract : RNA‐dependent RNA polymerase (RdRp) is the key replication enzyme in the life cycle of alphavirus and there are no vaccines or therapeutics available against the alphavirus. In this study, new potential antiviral inhibitors of CHIKV RdRp have been identified and their antiviral efficacies have been validated. The identified inhibitors piperine, 2‐thiouridine, pyrazinamide, and chlorogenic acid can potentially be developed as effective antiviral therapy for the treatment of alphavirus disease.