ABSTRACT
BACKGROUNDThe role of humoral immunity in the coronavirus disease 2019 (COVID-19) is not fully understood owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome. METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution. RESULTSWe identified 54 linear epitopes from the Spike (S) and Nucleocapsid (N) protein and showed that epitopes enabled higher resolution antibody profiling than protein antigens. Specifically, we found that antibody responses to the S(811-825), S(881-895) and N(156-170) epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant B.1.1.7 altered the specificity of the corresponding epitopes. CONCLUSIONSEpitope-resolved antibody testing not only offers a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, it may also be used as predictor of clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants in both the peptide array and latex agglutination formats. FUNDINGOntario Research Fund (ORF)-COVID-19 Rapid Research Fund, the Toronto COVID-19 Action Fund, Western University, the Lawson Health Research Institute, the London Health Sciences Foundation, and the AMOSO Innovation Fund.
ABSTRACT
The COVID-19 pandemic has clearly brought the healthcare systems world-wide to a breaking point along with devastating socioeconomic consequences. The SARS-CoV-2 virus which causes the disease uses RNA capping to evade the human immune system. Non-structural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small molecule inhibitors of nsp14 methyltransferase (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 {+/-} 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 {+/-} 0.2 M showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.
ABSTRACT
SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process including the nonstructural protein 16 (nsp16) which is an S-adenosyl-L-methionine (SAM)-dependent 2-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in 384-well format with a Z'-Factor of 0.6, suitable for high throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, product of the reaction, SAH, and a common methyltransferase inhibitor, sinefungin using Isothermal Titration Calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high throughput method for screening nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.
