Fig. 1: JNJ-9676 targets the M protein.
From: A small-molecule SARS-CoV-2 inhibitor targeting the membrane protein
a, The structure of JNJ-9676, (S)-N-(3-cyanophenyl)-5-(4-(difluoro(phenyl)methyl)phenyl)-6-methyl-4-oxo-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine-3-carboxamide. b, The mean EC50 values of JNJ-9676 against sarbecoviruses (SARS-CoV-2 B1 strain, n = 21 (A549-hACE2 cells), n = 6 (VeroE6-eGFP cells); SARS-CoV-2 B1.617.1, n = 2 (VeroE6-eGFP cells); SARS-CoV-2 B1.1.529, n = 12 (A549-hACE2 cells), n = 11 (VeroE6-eGFP cells); SARS-CoV, n = 12 (A549-hACE2 cells); SHC014, n = 1 (A549-hACE2 cells); WIV1, n = 1 (A549-hACE2 cells); and Pg-CoV Guangdong, n = 1 (A549-hACE2 cells)) assessed in A549-hACE2 cells (asterisks) or VeroE6-eGFP cells (circles). The EC50 for nirmatrelvir in SARS-CoV-2-infected A549-hACE2 cells is also shown. The box plots show the 25th–75th percentile (box limits), median (centre line) and the whiskers show the spread between minimum and maximum values. All replicates listed are biological replicates. c, The effect of JNJ-9676 and nirmatrelvir on RNA copy numbers in nasal epithelial cultures infected with SARS-CoV-2 (48 h.p.i., apical). n = 3, biological replicates. Data are mean ± s.d. d, The transmembrane structure of the SARS-CoV-2 M protein. IVRS mutation residues (black), important residues in the cryo-EM structure (blue), the intravirion domain (green), the extravirion domain (red) and transmembrane domains (grey) are indicated. e, The M protein structure annotated with mutations identified in IVRS. Left, mutations identified in more than 2 IVRS samples. Right, mutations in the binding site. The asterisks indicate mutations potentially altering the equilibrium between the long and short forms of the dimer. f, The mean EC50 fold changes in drug resistance potency of site-directed mutants (SDMs). n = 3, biological replicates. g, ASMS evaluation of M protein with compound (black) and buffer control with breakthrough (pink). n = 3 technical replicates. Data are mean ± s.e.m. h, M-protein-enriched extracted ion chromatogram (EIC) with a 3 ppm mass error tolerance window and the corresponding MS spectrum at the M protein EIC peak apex (inset). i, Buffer control EIC with a 3 ppm mass error tolerance window with the corresponding MS spectrum at the M protein EIC peak apex (inset). j, NanoDSF melting profile with the fluorescence ratio (350 nm/320 nm) and the first derivative plotted against temperature; the table indicates technical replicates (n = 3).
