Combination of antiviral drugs inhibits SARS-CoV-2 polymerase and exonuclease and demonstrates COVID-19 therapeutic potential in viral cell culture.

SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment.

Identification of recurrent mutations in exonuclease (nsp14); a potential drug target in SARS-CoV-2.

CONTEXT: The rapid outbreak of SARS-CoV-2 has become a significant global health concern, highlighting the dire need for antiviral therapeutic agents. RNA-dependent RNA polymerase (RdRp) of coronavirus plays crucial roles in RNA synthesis, and hence remains the druggable target for the treatment of this disease. The most potent broad-spectrum inhibitors of viral RdRp are members of nucleoside analogs (NAs). However, SARS-CoV-2 proved to be a challenging one for the novel NA drug designing strategy because coronavirus possesses an exonuclease (ExoN) domain that is capable of excising NAs, thus showing resistance to existing antiviral drugs. AIM: The objective of our study was to compare the SARS-CoV-2 exonuclease (nsp14) protein sequence of Wuhan-type virus with those of Indian SARS-Cov-2 isolates and to study the effect of multiple mutations on the secondary structure alterations of proteins. SUBJECTS AND METHODS: Multiple-sequence alignment of exonuclease amino-acid sequences followed by phylogenetic analysis and prediction of its secondary structure of the protein was performed. RESULTS: Altogether, seven mutations were detected in the nsp14 of Indian SARS-CoV-2 isolates. Subsequently, prediction of their secondary structures revealed that mutations altered the structural stability of exonuclease proteins. CONCLUSIONS: Present findings, therefore, further suggest that evolvability of SARS-CoV-2 is primarily associated with the onset of multiple novel mutations that rapidly spread at several new locations of the viral genome and also provides important insight to develop specific control strategies to fight against COVID-19 infections.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global COVID-19 pandemic. Nonstructural protein 14 (NSP14), which features exonuclease (ExoN) and guanine N7 methyltransferase activity, is a critical player in SARS-CoV-2 replication and fidelity and represents an attractive antiviral target. Initiating drug discovery efforts for nucleases such as NSP14 remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for NSP14 ExoN activity. The assay was used to measure NSP14 activity and gain insight into substrate specificity and the reaction mechanism. Next, the assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z factor > 0.8) and a significant assay window (signal-to-background ratio > 200). Screening 10,240 small molecules from a diverse library revealed candidate inhibitors, which were counterscreened for NSP14 selectivity and RNA intercalation. The assay methodology described here will enable, for the first time, a label-free and high-throughput assay for NSP14 ExoN activity to accelerate drug discovery efforts and, due to the assay flexibility, can be more broadly applicable for measuring other enzyme activities from other viruses or implicated in various pathologies.

Structural analysis of viral ExoN domains reveals polyphyletic hijacking events.

Nidoviruses and arenaviruses are the only known RNA viruses encoding a 3'-5' exonuclease domain (ExoN). The proofreading activity of the ExoN domain has played a key role in the growth of nidoviral genomes, while in arenaviruses this domain partakes in the suppression of the host innate immune signaling. Sequence and structural homology analyses suggest that these proteins have been hijacked from cellular hosts many times. Analysis of the available nidoviral ExoN sequences reveals a high conservation level comparable to that of the viral RNA-dependent RNA polymerases (RdRp), which are the most conserved viral proteins. Two highly preserved zinc fingers are present in all nidoviral exonucleases, while in the arenaviral protein only one zinc finger can be identified. This is in sharp contrast with the reported lack of zinc fingers in cellular ExoNs, and opens the possibility of therapeutic strategies in the struggle against COVID-19.

Isolation and genetic characterization of SARS-CoV-2 from Indian patients in a single family without H/O travel abroad.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.