Extended Data Fig. 1: Time-of-addition (TOA) and in vitro resistance selection experiments with JNJ-9676.
From: A small-molecule SARS-CoV-2 inhibitor targeting the membrane protein
a, Representation of ToA study with CPE scoring as a readout showing the presence of infectious progeny after compound treatment. b, ToA assay with 5 µM JNJ-9676 (n = 1) and nirmatrelvir (n = 2 for 5 h.p.i. and n = 3 for 0 and 3 h.p.i., error bars representing the standard deviation) reading out infectious virus. The dotted horizontal line represents the reinfection with supernatant directly collected after washing away the virus input at 1 h.p.i. (n = 3). c, Schematic representation of the IVRS procedure. d, Whole genome sequencing was utilized to investigate the emergence of mutations in various SARS-CoV-2 lineages/variants under selection of JNJ-9676 (n = 3 for B1 and B.1.617.2 lineages and n = 2 for the omicron lineage; error bars showing error of the mean). Each coloured line represents the appearance dynamics of a specific mutation during virus passaging in the presence of JNJ-9676, with the mutation colour-coded accordingly. Sequencing was conducted on SARS-CoV-2 variants collected at intervals of every 2nd to 6th passage and at the end of the experiment. The experiment involved two passages per week. The dotted line on the graph represents the 15% threshold for variant detection compared to the WT in the virus population. The resistance dynamics for each viral strain was plotted. e, The average number of IVRS mutations per replicate (AMR) was defined. Proteins with an AMR ≥ 1 were considered potential targets. f, AMR values normalized for protein size. g, Effect of resistance mutations on replication fitness (n = 3 with 8 technical replicates per experiment, error bars showing the standard deviation). h, Plaque assay showing representative images (n = 3, independent experiments) of plaque sizes from the site directed mutants in g. ToA, time-of-addition; CPE, cytopathic effect; h.p.i., hours post-infection; IVRS, in vitro resistance selection; WT, wild-type; AMR, average number of IVRS mutations per replicate.
