Fig. 2: JNJ-6440 target identification and de-risking.
From: Targeting de novo purine biosynthesis for tuberculosis treatment
a, Susceptibility of JNJ-6640-resistant clones (R1–R5). MIC90 values are shown in Extended Data Table 2. b, Induced fit docking model of JNJ-6640 (cyan) in the MtPurF-binding pocket (grey). Phe428 (pink), key interactions and resistance residues are annotated. c, Percentage 15N-stable isotope incorporation following 100 nM JNJ-6640 treatment. Control metabolite (glutamine) was the most abundant nitrogen-containing metabolite. n = 5 biological replicates, representative of two independent experiments. Significance was calculated with two-sided (Bonferroni–Dunn) Student’s t-test with Welsh correction. d, Growth kinetics of CRISPRi-mediated purF transcript knockdown strains (low, medium and high) compared with ‘empty’ vector (control) and kill (atpE high) controls after ATc induction. n = 3 independent experiments. e, CRISPRi-mediated PurF ‘low’ knockdown increases susceptibility to JNJ-6640 (MIC50 = 6.4 nM for −ATc and MIC50 = 0.8 nM for +ATc). n = 3 technical replicates, representative of two independent experiments. f, Nucleobase rescue assay with CRISPRi-mediated high, medium and no (control) purF transcript knockdown in the presence of 10 µM nucleobase or nucleoside with (+ATc) and without (−ATc) induction. Starting inoculum was approximately 1 × 105 CFU ml−1. n = 3 independent experiments. g, MtPurF (IC50 = 1 nM) and Homo sapiens PPAT (HsPPAT; IC50 = 14 µM) enzymatic assays with JNJ-6640. n = 2 biological replicates. h, Cell proliferation assay with JNJ-6640 and three known cell proliferation inhibitors using 93 different cancer cell lines derived from different tissue types. The average pIC50 shown for each tissue type: pIC50 ~ 4 (IC50 ~ 100 µM) was considered non-active. Aza, azathioprine; MMPR, 6-methyl-mercaptopurine riboside; MPA, mycophenolic acid. Representative dataset from two independent experiments. For panels c–f, data shown are mean ± s.d.
