RNA-Dependent RNA Polymerase as a Target for COVID-19 Drug Discovery

Abstract

COVID-19 respiratory disease caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global health issue since it emerged in December 2019. While great global efforts are underway to develop vaccines and to discover or repurpose therapeutic agents for this disease, as of this writing only the nucleoside drug remdesivir has been approved under Emergency Use Authorization to treat COVID-19. The RNA-dependent RNA polymerase (RdRP), a viral enzyme for viral RNA replication in host cells, is one of the most intriguing and promising drug targets for SARS-CoV-2 drug development. Because RdRP is a viral enzyme with no host cell homologs, selective SARS-CoV-2 RdRP inhibitors can be developed that have improved potency and fewer off-target effects against human host proteins and thus are safer and more effective therapeutics for treating COVID-19. This review focuses on biochemical enzyme and cell-based assays for RdRPs that could be used in high-throughput screening to discover new and repurposed drugs against SARS-CoV-2.

Keywords: COVID-19; RNA-dependent RNA polymerase; RdRP assays; RdRP inhibitors; SARS-CoV-2; coronavirus infection.

Figure 1.

(A) Illustration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that consists of four structural proteins and RNA genome. (B) Schematic illustration of SARS-CoV-2 RNA genome and virus life cycle in host cell. The SARS-CoV-2 RNA genome encodes 16 nsps, 4 structural proteins, and 9 putative accessory factors. In the virus life circle, SARS-CoV-2 binds to angiotensin-converting enzyme-2 (ACE2) receptor and then releases RNA genome to cytosol to initiate the RNA replication and the formation of new virions. Created with Biorender.com.

Figure 2.

(A) The catalytic mechanism of RNA-dependent RNA polymerase (RdRP) in RNA replication. (B) The intervention of nucleotide analog as an inhibitor (an insertion of the nucleotide analog stops the RNA elongation after a few nucleotides that is catalyzed by RdRP).

Figure 3.

Schematic of biomedical assay designs. (A) Polymerase elongation template element (PETE) assay. An RNA oligonucleotide template is labeled with a fluorophore that can undergo rapid molecular rotation. The addition of nucleoside triphosphates (NTPs) to the RNA template mediated by RNA-dependent RNA polymerase (RdRP) sterically hinders the rotation of the fluorophore, resulting in an increase of the fluorescence polarization (FP) signal. (B) Fluorescence-based alkaline phosphatase–coupled polymerase assay (FAPA). The incorporation of (2-[2-benzothiazoyl]-6-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) into the RNA strand by RdRP results in the generation of BBT pyrophosphate (BBT_PPi) that can be further catalyzed by reacting with the calf intestinal alkaline phosphatase (CIP) to produce fluorescent BBT anion. (C) Fluorometric RdRP activity assay. Using a single-stranded RNA (ssRNA) template, RdRP catalyzes the formation of double-stranded RNA (dsRNA) that can be detected by PicoGreen. (D) Scintillation proximity assay (SPA). SPA beads bind to the synthesized RNA containing [H]-GTPs (guanosine triphosphates), leading to the close proximity that generates the SPA signal.