Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

Abstract

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa(3) system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics.

Fig. 2.

The reduced and the CO-binding pigments of H37Rv cell extracts. (A) NADH-reduced minus air-oxidized difference spectrum. Reduction was accomplished 5 min after the addition of 10 mM NADH. (B) (NADH-reduced plus CO) minus (NADH-reduced) difference spectrum. To show the spectral perturbation due to CO binding, CO was bubbled through the sample before reduction.

Fig. 1.

Proposed pathway of aerobic electron flow in mycobacteria. Complexes are shown in boxes, with corresponding gene names and GenBank accession nos. given outside of the boxes.

Fig. 3.

Structures of analogs of CPZ.

Fig. 4.

Phenothiazine inhibition of Mtb respiration. (A) TPZ inhibition of NADH-dependent oxygen consumption by Mtb membranes. Mtb membrane protein was added to phosphate buffer in a vessel equipped with a Clark-type oxygen electrode. Respiration was initiated by the addition of 10 mM NADH and arrested upon the addition of 1 mM TPZ. Addition of 10 mM ascorbate and 1 mM TMPD produced an immediate resumption of respiration. (B) [NADH-reduced] minus [air-oxidized Mtb] membranes in the absence (solid line) or presence (dotted and hashed lines) of 100 μM TPZ. For each trace, 10 mM NADH was added to a 1-ml quartz semimicrocuvette. For the samples containing TPZ, drug was added to the sample and incubated for 5 min before the addition of NADH. Samples were suspended in 50 mM Hepes buffer (pH 7.4) at a protein concentration of 21.3 mg/ml.

Fig. 5.

Phenothiazines inhibit recombinant Ndh and NdhA. (A) The Mtb Ndh (Left) and NdhA (Right) were expressed in E. coli with an N-terminal His-6-tag. Lane 1 shows a Coomassie brilliant blue-stained polyacrylamide gel, and lane 2 shows Western blotting with anti-His tag IgG. The Ndh and NdhA products are indicated by arrows. Their apparent molecular mass is 51 kDa. (B) CPZ titration of NADH-Q₂ oxidoreductase activity of the purified recombinant Mtb Ndh (Left) and NdhA (Right). The IC₅₀ is 10 μm for both Ndh and NdhA. Specific activity is ≈0.12 μmol of oxidized NADH per min/mg protein.

Fig. 6.

Test of phenothiazine efficacy in a BALB/c mouse model of acute Mtb infection. Each treatment group consists of five female mice intranasally infected with 100 cfu of H₃₇R_v Mtb. After 11 days of the indicated treatment, mice were killed, and the levels of Mtb in the lung and spleen were determined.