Figure 8
MJM170 targets apicomplexan cytochrome bc1 Qi : modelling, yeast surrogate assays, target validation, co-crystallography and nanoM inhibition of P. falciparum and T. gondii. (a) Modeling: MJM170 (yellow) modelled within cytochrome b Qi site (grey) highlighting residues (green) involved in binding. (b) Mutations for yeast, P. falciparum, predicted for T. gondii and bovine enzyme. Relevant mutations are indicated by colored dots in Qi domains on the bottom of the image of mitochondrion membrane for S. cerevesiae and P. falciparum, and where those amino acids are in T. gondii, human and bovine enzymes. Red dot marks G33A/V in Qi domain of P. falciparum. (c) Cytochrome b mutants and sequence accession numbers. (d) MJM 170 inhibits wild-type but not mutant yeast. Compounds MJM 170 and ELQ 271 with wild type and mutant yeast validate predictions that M221 K/Q would create a steric clash and resistance. (e) MJM170 is a potent low nM inhibitor of Plasmodium falciparum. In Table 2, wild type P. falciparum also are tested and is inhibited at <50 nM by this scaffold. D6 is a drug sensitive strain from Sierra Leone, C235 is a multi-drug resistant strain from Thailand, W2 is a chloroquine resistant strain from Thailand, and C2B has resistance to a variety of drugs including atovaquone. Mutant G33V did not confirm prediction of a steric clash. (f) MJM170 binds within Qi site of bovine cytochrome bc1 as shown by X-ray crystallography. (f(i)). An omit Fo-Fc electron density map (green) at 5σ allows unambiguous positioning of MJM170 (magenta) within the Qi site with the tetrahydroquinolone group near heme bH (white) and diphenyl ether directed out of the channel. (f(ii)) MJM 170 molecule is included into the structure, the 2Fo-Fc electron density map at 1σ (grey) allows placement of the planar head between heme bH and Phe220 with the carbonyl group positioned in a polar region surrounded by Ser35 and Asp228. (g) A stereo picture of the 2Fo–Fc electron density map. Electron density at 1σ level around cytochrome b α-helixes 118–128 and 183–199 close to MJM180 compound in X-ray structure of cytochromebc1. Inset: Crystal of mammalian cytochrome bc1 complex.
