Cholesterol Analogs with Degradation-resistant Alkyl Side Chains Are Effective Mycobacterium tuberculosis Growth Inhibitors

Abstract

Cholest-4-en-3-one, whether added exogenously or generated intracellularly from cholesterol, inhibits the growth ofMycobacterium tuberculosiswhen CYP125A1 and CYP142A1, the cytochrome P450 enzymes that initiate degradation of the sterol side chain, are disabled. Here we demonstrate that a 16-hydroxy derivative of cholesterol, which was previously reported to inhibit growth ofM. tuberculosis, acts by preventing the oxidation of the sterol side chain even in the presence of the relevant cytochrome P450 enzymes. The finding that (25R)-cholest-5-en-3β,16β,26-triol (1) (and its 3-keto metabolite) inhibit growth suggests that cholesterol analogs with non-degradable side chains represent a novel class of anti-mycobacterial agents. In accord with this, two cholesterol analogs with truncated, fluorinated side chains have been synthesized and shown to similarly block the growth in culture ofM. tuberculosis.

Keywords: Mycobacterium tuberculosis; cholesterol metabolism; cytochrome P450; dehydrogenase; enzyme inhibitor.

FIGURE 1.

Effect of cholest-4-en-3-one and 1 on growth of wild-type (A) or Δcyp125A1 (B, D, and E) strains of CDC1551, and Erdmann (C) Mtb on defined media with glycerol (A–C), acetate (D), or glucose (E) as the sole carbon source. Data shown are the average of three independent growths.

FIGURE 2.

Reverse type I binding of 1 to CYP125A1 (2 μm) as determined by difference spectroscopy. The fit of the data to the quadratic binding equation gives K_s = 15.4 ± 1.1 μm. Data shown are the average of three independent titrations, error is the standard error. AU, absorbance units.

SCHEME 1.

Synthesis of difluoromethyl analogs 2 and 3. Reagents and conditions: (a) i, see Ref. ; ii, PCC, NaOAc, CH₂Cl₂, 74%. (b) DeoxoFluor, CH₂Cl_2, reflux, 5: 59%, 9: 76%. (c) i, ethyl 2-(triphenylphosphoranylidene)acetate (20), CH₂Cl_2, reflux, 87%; ii, H₂, PtO₂, 1,4-dioxane/AcOH; iii, LiAlH₄, THF, reflux (80% over 2 steps); iv, PCC, NaOAc, CH₂Cl₂, 64%. (d) HF/pyridine, THF, 2: 68%, 3: 73%. The structure of compound 1, independently synthesized by a literature procedure (17), is also shown.

FIGURE 3.

Inhibition of the growth on 0.1% glycerol of wild-type CDC1551 Mtb and its Δcyp125A1 mutant by 0.1 mm compound 2 (A) and 0.1 mm compound 3 (B). Data shown are the average of three independent growths.

FIGURE 4.

Oxidation of 1, 2, and 3 by 3β-HSD to the 3-keto derivatives. HPLC analysis of the oxidation of 1 by 3β-HSD (A) and cholesterol oxidase (B); and the oxidation of 2 (C) and 3 (D) by 3β-HSD.

FIGURE 5.

GC-MS analysis of the oxidation of compound 3 (solid line) to the carboxylic acid (retention time 26.5 min) by CYP125A1 (small dashes) and CYP142A1 (large dashes). A second unidentified metabolite is seen at retention time 24.5 min.

SCHEME 2.

Oxidation of difluoromethyl analog 3 by CYP125A1 and CYP142A1 to an acyl fluoride that can be trapped by water or methanol.