Fig 1.
Susceptibility of M. and cytotoxicity of HepG2 cells to quinazoline derivatives.
a Toxic dose that inhibits 99% of cell growth.b Toxic dose that inhibits 50% of cell growth.c Intrinsic Clearance (Clint) is expressed in μL/min/mg protein. Abbreviation: N.D. not determined.
Table 1.
Susceptibility of different organisms to quinazoline derivatives.
Fig 2.
Mutation L356V in QcrA is responsible for quinazoline resistance in M. tb.
Susceptibility to (A) 11626141, (B) 11626142, (C) 11626252 and (D) Rifampicin (RIF) was assessed in strains H37Rv-pYUB412 (black circle), H37Rv-Rv1777(Arg145Ser) (blue triangle), H37Rv-Rv2195(Leu356Val) (red square), and QuinR-M1 (green circle) by REMA. The graph represents the percentage of bacterial viability ± s.d. according to the drug concentrations. The experiment was performed in biological triplicates.
Fig 3.
Quinazoline derivatives target cytochrome bc1.
(A) QcrA and QcrB of M. tb were modelled using the homology modelling webserver (Swissmodel) and homology based on chains A and M from the CryoEM structure of the III2IV2 respiratory supercomplex (PDB code 6HWH) from M. smegmatis. Clustering of mutations associated with resistance to quinazoline derivatives, AX-35, Q203, and LPZs occurs around the quinol oxidation site of QcrB and QcrA. (B) Cross-resistance of QuinR mutants to QcrB inhibitors. The MIC of the quinazoline derivatives, Q203, LPZs and AX-35 was determined and expressed as the fold-change of MIC of each compound compared to M. tb H37Rv. (C) Bioenergetic analysis of M. tb upon Q203 and 11626252 treatment followed by the uncoupler CCCP to stimulate maximum respiration. The oxygen consumption rates of wild-type H37Rv, H37Rv ΔcydAB, and H37Rv Δcyd KO with a QcrB (A317V) mutation (Q203 resistance SNP) was monitored as described in the Material and methods section. (D) Intracellular ATP levels in H37Rv and quinazoline resistant strains were measured in the absence and presence of BDQ, Q203, and 11626252 at 2.5× MIC after 24 h using BacTiterGlo (Promega). Data from two independent experiments are presented as mean ± SD. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests (*, P < 0.03; ****, P < 0.0001).
Fig 4.
Transcriptome analysis of H37Rv exposed to 11626252.
(A) Heat map representing top significantly differentially regulated M. tb genes (Padj ≤ 0.05) after exposure of two independent cultures of M. tb H37Rv to 11626252 at 10X and 30X MIC for 4 h. The colour scale indicates differential regulation as log2 fold-change of H37Rv with 11626252 treatment relative to H37Rv with vehicle control, DMSO. Upregulation is indicated in red, downregulation is in green, and insignificant log2 fold change values for the condition are in black. Data are from two independent experiments. (B) Global transcriptome response and involvement of different metabolic responses, based on TubercuList classification (https://mycobrowser.epfl.ch/). (C) Fold-change expression of cydB and lipU determined by qRT-PCR. Bars in black, light grey and white represent the gene expression compared to H37Rv-DMSO, QuinR1-M1 in the presence of DMSO, and QuinR-M2 with DMSO, respectively. Data from two independent cultures are presented as the mean relative expression ± s.d. Statistical analysis was performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison tests (*, P <0.03; ****, P <0.0001).
Fig 5.
11626252 is bacteriostatic but bactericidal in the absence of the bd oxidase.
H37Rv, ΔcydAB and the complemented strain were exposed to increasing concentrations (1X MIC-32X MIC) of 11626252 for 7 days. Bacteria were plated on 7H10-OADC on day 0 (D0) to determine the initial number of CFU. After 7 days of exposure, untreated bacteria (D7) were plated for CFU enumeration. Results are expressed as the mean log 10 CFU/mL ± s.d.of two independent experiments. <d.l., below limit of detection.
Fig 6.
In vivo activity of 11726148 in an acute model of murine TB.
Compounds were administered by gavage at the following doses: 11726148, 150mg/kg; isoniazid (INH), 25mg/kg; lansoprazole sulfide (LPZS), 300mg/kg and Q203, 25mg/kg. 11726148 and LZPS were prepared in 20% TPGS and Q203 in 20% TPGS+1% DMSO. Bars represent the mean ± s.d. of CFUs from 5 Balb/c mice per group. Significance in difference relative to untreated groups (TPGS and TPGS+DMSO) were calculated using a Student t-test. *P< 0.05; **P< 0.005; ***P> 0.0001.<d.l., below limit of detection.