Alphavirus antivirals targeting RNA‐dependent RNA polymerase domain of nsP4 divulged using surface plasmon resonance

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 (lM) 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 EC 50 values of 6.68, 27.88, 36.26, and 53.62 µM, respectively. This study paves the way towards the development of novel broadspectrum alphavirus antivirals targeting nsP4 RdRp.

Repurposing efavirenz, the HIV antiretroviral drug for Chikungunya virus infection

“…The alphavirus RNA synthesis inhibition can be due to the direct effect of compounds on nsPs or indirectly by affecting host factors involved in viral RNA replication. Out of four nsPs, the N-7-methyltransferase activity of CHIKV nsP1 (residues 1 to 509) (Mudgal et al, 2020), the protease activity of nsP2 (residue 471-791) (Singh et al, 2018) and TATase activity of active core catalytic domain of the SINV nsP4 (N-terminal 97 residue truncated) (Pareek et al, 2022; Tomar et al, 2006) has been reported previously from our and other research groups. Among the four nsPs, the CHIKV nsP1, CHIKV nsP2, and SINV nsP4 expressed and purified from bacterial expression are soluble, stable and enzymatically active in aqueous buffer, thus can be utilized to check interaction with efavirenz with the help of biophysical techniques such as ITC and SPR.…”

“…These non-structural protein and CHIKV CP were expressed in the bacterial expression system and recombinant proteins were purified by Immobilized metal ion affinity chromatography (IMAC) ( Supplementary Fig. 1 ) (Kaur et al, 2018; Pareek et al, 2022; Sharma et al, 2016; Singh et al, 2018). Isothermal titration calorimetry (ITC) and Surface plasmon resonance (SPR) are standard biophysical techniques for investigating the label-free molecular interactions of drug molecules with the target proteins.…”

“…As all nsPs have some attributed and indispensable roles to play in the replication of viral genome (Lulla et al, 2006), hence nsPs are potential targets for developing effective antivirals (Mudgal et al, 2022(Mudgal et al, , 2020Pareek et al, 2022;Singh et al, 2018). The RNA genome of CHIKV comprises of two open reading frames (ORFs), of which the 5ˊ two-thirds of the RNA encodes for two overlapping polyproteins, P123 and P1234 (Khan et al, 2002;Solignat et al, 2009).…”

Multi-target direct-acting SARS-CoV-2 antivirals against the nucleotide-binding pockets of virus-specific proteins

“…Furthermore, considering the high likelihood that this will not be the last pandemic we face, as the other infectious viral diseases are circulating worldwide. It is critical that we develop these strategies to combat the other viral diseases such as chikungunya and dengue fevers, for which till date there are no antivirals available in the market ( Aggarwal et al, 2017 ; Mudgal et al, 2020 ; Pareek et al, 2022 ; Sharma et al, 2018 , 2016 ; Singh et al, 2018 ).…”

Viral methyltransferase inhibitors: berbamine, venetoclax, and ponatinib as efficacious antivirals against Chikungunya virus

“…The quantified 400 ng of RNA used for cDNA preparation using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). The forward and reverse primers used for amplification are used as previously described (32). β-actin was utilised as an internal control.…”