Betulonic acid derivatives inhibiting coronavirus replication in cell culture via the nsp15 endoribonuclease

Abstract

The lack of medication to suppress coronavirus infections is a main reason for the dramatic course of the COVID-19 pandemic. There is an urgent need to identify suitable coronavirus drug targets and corresponding lead molecules. Here we describe the discovery of a class of coronavirus inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. SAR exploration of these 1,2,3-triazolo fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 μM) of human coronavirus 229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity

“…Next, a pocket-selection tool (Qvina-W for blind docking [29]) was used to identify the most likely ligand binding pockets in the C-terminal catalytic domain surrounded by His235 and His250 in the nsp15 monomer. This approach is consistent with previous studies where a similar region was used for the nsp15 docking calculations [16]. Using Exebryl-1 and the identified pockets, docking scores were generated.…”

“…Indeed, the intermolecular bonds of the hexamer appear to stabilize the active site of nsp15 endoU. A recent preprint demonstrates nsp15 function in alpha coronavirus isolate, HCoV-229E, and chemical-genetic validation with a betulonic acid derivative (lead molecule 5h) that inhibited replication of Coronavirus 229E at 0.6 µM EC50 [16]. It was shown that a nsp15 deficient coronavirus was much less sensitive to 5h and selecting for mutants with 5h led to mutations in the N terminus of nsp15 that map to residues important in the hexameric macromolecule of CoV nsp15.…”

“…It is made The copyright holder for this preprint this version posted January 21, 2021. ; https://doi.org/10.1101/2021.01.21.427657 doi: bioRxiv preprint 5 activity, this compound exhibits only minimal antiviral activity at 50 µM concentrations, thus more potent inhibitors are desired [15]. Recently, a preprint chemically-genetically validated alpha-coronavirus nsp15 endoU as a target for antiviral activity [16]. Taken together, nsp15 endoU RNase appears to be an attractive therapeutic target for high-throughput screening (HTS) and structure-guided drug discovery (SGDD).…”

Prediction of Ciclesonide Binding Site on Middle-East Respiratory Syndrome Coronavirus Nsp15 Multimer by Molecular Dynamics Simulations

“…The present result suggests that in addition to the enzyme active center, the multimer interface may become a good target of inhibition. Indeed, a recent preprint article [26] has reported that Betulonic acid derivatives can inhibit nsp15 via the binding to multimer interface site. The development of a systematic screening procedure targeting these multimer interfaces may be beneficial to stand against different pathogens.…”

Main Chemotypes of SARS-CoV-2 Reproduction Inhibitors

“…Natural and semisynthetic tetra-and pentacyclic triterpenoids (compounds 225-234 [57,90,[151][152][153][154][155]) were found capable of inhibiting SARS-CoV-2 replication (Fig. 25 Natural and semisynthetic antibiotics 235-240 [57,85,90,156,157] showed quite a high in vitro activity aganst coronaviruses.…”