Reverse type I inhibitor of Mycobacterium tuberculosis CYP125A1

Abstract

Cytochrome P450 CYP125A1 of Mycobacterium tuberculosis, a potential therapeutic target for tuberculosis in humans, initiates degradation of the aliphatic chain of host cholesterol and is essential for establishing M. tuberculosis infection in a mouse model of disease. We explored the interactions of CYP125A1 with a reverse type I inhibitor by X-ray structure analysis and UV-vis spectroscopy. Compound LP10 (α-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide), previously identified as a potent type II inhibitor of Trypanosomacruzi CYP51, shifts CYP125A1 to a water-coordinated low-spin state upon binding with low micromolar affinity. When LP10 is present in the active site, the crystal structure and spectral characteristics both demonstrate changes in lipophilic and electronic properties favoring coordination of the iron axial water ligand. These results provide an insight into the structural requirements for developing selective CYP125A1 inhibitors.

Fig. 1. Chemical structure of α-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide, LP10

Fig. 2. UV-visible absorption spectra of M. tuberculosis CYP125A1_C429L

The Soret (A) and enlarged visible (B) regions are shown. Spectra of the resting ferric (thin line), ferric LP10 (thick line), and ferric imidazole (dashed line) forms were recorded at the total hemoprotein concentration of 6.45 μM in 50 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA at 23 °C.

Fig. 3. Binding of LP10 to M. tuberculosis CYP125A1_C429L

A. Concentration dependence of LP10 binding deduced from difference absorption changes obtained from titration of protein with increasing concentrations of inhibitor. A representative set of the non-corrected difference spectra obtained using the two-chamber cuvettes is shown in the inset. B, Inhibition of CYP125A1 enzymatic activity by LP10. Concentration of the substrate cholest-4-en-3-one used for the inhibition assay was 50 μM.

Fig. 4. Structure of the CYP125A1_C429L-LP10 complex

A. Overall ribbon structure of the CYP125A1_C429L-LP10 complex (2XC3) with B, G and I helices highlighted in purple. LP10 (yellow), heme (orange) and the iron axial water ligand (red) are shown in stick mode. The pyridinyl ring of LP10 points toward heme. The indole and methylcyclohexyl moieties point toward the viewer between the B and G helices. B. A view of LP10 emphasizing the H-bonding interactions in the active site. Selected residues within 4 Å of LP10 are in purple. Alternate conformations of Lys214 interact with both indole and methylcyclohexyl moieties of LP10. C. Interactions of the methylcyclohexyl moiety within 5 Å of the methyl group. D. Catalytic chamber filled with three water molecules. In B, C and D, fragments of the 2F_o-F_c electron density map (calculated with LP10 coordinates omitted from the input file) are shown for heme, LP10, selected amino acid residues (blue mesh) and water molecules (pink mesh). Black lines highlight H-bonding interactions.

Fig. 5. Structure of CYP125A1_C429L in the ferric resting state

Catalytic chamber of CYP125A1_C429L (2X5L) surrounded by residues within 6 Å of the heme iron axial water ligand (purple sticks). Two water molecules (red spheres) with partial occupancy of 0.5 are placed into elongated electron density (blue mesh) 1.46 Å away from each other. The line connecting both molecules runs at a 45° angle to the heme plane. A fragment of the 2F_o-F_c map was calculated with the water coordinates omitted from the input file. Heme is in orange. The yellow arrow suggests an oscillation of the water ligand between ligating and non-ligating positions.