Journal of Biological ChemistryVolume 301, Issue 8, August 2025, 110409Author links open overlay panel, , , , , , , ,

During infection, the coronavirus nonstructural protein 15 (Nsp15), a uridine-specific endoribonuclease, suppresses the host cell’s antiviral response. Recently, researchers have paid more attention to this relatively underexplored yet potentially viable drug target. In this study, we employed FRET-based screening assays to identify potent Nsp15 inhibitors. Subsequently, we used active-site in silico docking methods to design new molecules with enhanced inhibitory properties. Solution assays were used to measure the potency and determine the mechanism of these inhibitors. We identified a novel class of thiazolidinedione and rhodanine analogs that inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Nsp15. Docking these compounds into the uridine-binding site shows that most analogs form two hydrogen bonds with Ser294. The most potent inhibitors are compounds 5-(3-quinolin-4-yl-allylidene)-thiazolidine-2,4-dione (KCO237) and 5-(3-isoquinolin-4-yl-allylidene)-2-thioxo-thiazolidin-4-one (KCO251) (IC50: 0.304 μM and 0.931 μM, respectively). The inhibition kinetics of KCO237 and KCO251 best align with a reversible mixed inhibition model. Mutating Ser294 did not completely abolish Nsp15 activity or the inhibitory effect of KCO237 or KCO251. These findings suggest that thiazolidinedione and rhodanine analogs likely inhibit Nsp15 by binding to the uridine active site while also implicating a possible secondary allosteric-binding site. The ability of these compounds to inhibit VERO 6 cell infection with SARS-CoV-2 at subtoxic levels highlights their potential for development as novel antiviral treatments for SARS-CoV-2 and other coronavirus-related diseases.

Keywords

docking

endoribonuclease

enzyme mutation

FRET

inhibition mechanism

inhibitor

Nsp15

rhodanine

SARS-CoV-2

thiazolidinedione

AbbreviationsDMEM

Dulbecco's modified Eagle's medium

KCO35

5-(3-anthracen-9-yl-allylidene)-thiazolidine-2,4-dione

KCO36

5-(3-phenyl-1-yl-allylidene)-thiazolidine-2,4-dione

KCO048 full name

5-(3-quinolin-4-yl-allylidene)-thiazolidine-2,4-dione

KCO230

5-(3-anthracen-9-yl-allylidene)-2-thioxo-thiazolidin-4-one

KCO231

5-(3-naphthalen-1-yl-allylidene)-thiazolidine-2,4-dione

KCO234

5-(3-isoquinolin-4-yl-allylidene)-thiazolidine-2,4-dione

KCO235

5-(3-isoquinolin-8-yl-allylidene)-thiazolidine-2,4-dione

KCO236

5-(3-isoquinolin-6-yl-allylidene)-thiazolidine-2,4-dione

KCO237 and 238

5-(3-quinolin-4-yl-allylidene)-2-thioxo-thiazolidin-4-one and 3-methyl-5-(3-anthracen-9-yl-allylidene)-thiazolidine-2,4-dione

KCO249

5-(3-naphthalen-1-yl-allylidene)-2-thioxo-thiazolidin-4-one

KCO250

3-methyl-5-(3-naphthalen-1-yl-allylidene)-thiazolidine-2,4-dione

KCO251

5-(3-isoquinolin-4-yl-allylidene)-2-thioxo-thiazolidin-4-one

MDA5

melanoma differentiation–associated protein 5

MOE

molecular operating environment

qRT–PCR

quantitative RT–PCR

RTC

replication–transcription complex

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

TAMRA

carboxytetramethylrhodamine

© 2025 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biologyé