Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

Medicinal chemistry strategies towards the development of effective SARS-CoV-2 inhibitors

“…12 ) present the best activity with IC 50 of 9.7 μmol/L. Therefore, nitazoxanide can be further optimized and modified as an nsp14 inhibitor 73 .

Figure 12 The structures of nitazoxanide and PF-03882845.

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Enzymatic Assays to Explore Viral mRNA Capping Machinery

“…For example, RapidFire MS technology exploit nsp14 N7-methyltransferase by quantification of nascent SAH products. [131] All MTase assays described so far were performed in vitro using recombinant proteins. A more advanced approach was reported by Sun et al [116] who proposed the application of yeast cells to study SARS-CoV, MERS-CoV, transmissible gastroenteritis virus (TGEV), murine hepatitis virus (MEV), and infectious bronchitis virus (IBV) N7-MTase nsp14.…”

“…[144] To increase inhibitor selectivity, some approaches utilize the specific properties of the binding pocket. [71] B) Pyrophosphate; [77] C) Tripolyphosphate; [77] D) Irigenol; [81,82] E) 2',2'-Bisepigallocatechin monogallate; [81,82] F) Suramin; [74,133] G) Ellagic acid; [81,82,115] H) Chembridge3 5660163; [103] I) Maybridge5 GK 02514; [103] J) Chembridge3 7871678; [103] K) Benzbromarone; [104] L) Pyrantel pamoate; [104] M) Pyrimethamine; [106] N) (3-Fluorobenzyl)-N6-SAH (X=F), (3-chlorobenzyl)-N6-SAH (X=Cl) and (3-methylbenzyl)-N6-SAH (X=CH 3 ); [122] O) NF 023; [133] P) Aurintricarboxylic acid; [133] Q) Reactive Blue 2; [133] R) Myricetin; [115,133,134] S) Quercetin; [115,133,134] T) SCH 202676 HBr; [134] U) Thimerosal; [134] V) Nitazoxanide; [131] W) Adenosine dinucleotide/SAM analogue (bisubstrate inhibitor); [145] X) 2-Hydroxy-4-oxo-4-phenyl-2-butenoic acid; [137] Y) Baloxavir acid (BXA); [138] Z) P-30; [67] and AA) PA-48. [67] For example, Lim et al [122] designed a series of SAH analogues that have been modified in the N6 adenosine position with substituents that can extend into an uncovered flavivirusconserved cavity, which is located next to the cofactor binding site of the NS5 enzyme.…”

“…Although many computational approaches have been employed to screen compound libraries against SARS-CoV-2 N7-MTase nsp14, only a few inhibitor examples have been identified with experimental assays. For example, using another HTS assay based on RapidFire Mass Spectrometry, Pearson et al [131] 2021 screened the FDA-approved drug library with 1771 compounds and identified that nitaxozanide (Figure 8V) exhibits inhibitory properties against the SARS-CoV-2 nsp14 enzyme.…”

“…Figure8. Structures of RNA-capping inhibitors identified by assays dedicated for monitoring the activity of enzymes involved in cap biosynthesis: A) ATP γ S;[71] B) Pyrophosphate;[77] C) Tripolyphosphate;[77] D) Irigenol;[81,82] E) 2',2'-Bisepigallocatechin monogallate;[81,82] F) Suramin;[74,133] G) Ellagic acid;[81,82,115] H) Chembridge3 5660163;[103] I) Maybridge5 GK 02514;[103] J) Chembridge3 7871678;[103] K) Benzbromarone;[104] L) Pyrantel pamoate;[104] M) Pyrimethamine;[106] N) (3-Fluorobenzyl)-N6-SAH (X=F), (3-chlorobenzyl)-N6-SAH (X=Cl) and (3-methylbenzyl)-N6-SAH (X=CH 3 );[122] O) NF 023;[133] P) Aurintricarboxylic acid;[133] Q) Reactive Blue 2;[133] R) Myricetin;[115,133,134] S) Quercetin;[115,133,134] T) SCH 202676 HBr;[134] U) Thimerosal;[134] V) Nitazoxanide;[131] W) Adenosine dinucleotide/SAM analogue (bisubstrate inhibitor);[145] X) 2-Hydroxy-4-oxo-4-phenyl-2-butenoic acid;[137] Y) Baloxavir acid (BXA);[138] Z) P-30;[67] and AA) PA-48 [67]. …”