Identification of novel imidazo[1,2-a]pyridine inhibitors targeting M. tuberculosis QcrB

Abstract

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. Through the use of high throughput whole cell screening of an extensive compound library a number of imidazo[1,2-a]pyridine (IP) compounds were obtained as potent lead molecules active against M. tuberculosis and Mycobacterium bovis BCG. The IP inhibitors (1-4) demonstrated minimum inhibitory concentrations (MICs) in the range of 0.03 to 5 µM against a panel of M. tuberculosis strains. M. bovis BCG spontaneous resistant mutants were generated against IP 1, 3, and 4 at 5× MIC and subsequent whole genome sequencing identified a single nucleotide polymorphism (937)ACC>(937)GCC (T313A) in qcrB, which encodes the b subunit of the electron transport ubiquinol cytochrome C reductase. This mutation also conferred cross-resistance against IP 1, 3 and 4 demonstrating a common target. Gene dosage experiments confirmed M. bovis BCG QcrB as the target where over-expression in M. bovis BCG led to an increase in MIC from 0.5 to >8 µM for IP 3. An acute murine model of TB infection established bacteriostatic activity of the IP series, which await further detailed characterization.

Figure 1. Compounds from the IP series active against M. tuberculosis.

Structures of the initial IP hits (1 and 2) identified in the HTS campaign against M. bovis BCG (activity later confirmed in M. tuberculosis) and of optimized compounds 3 and 4.

Figure 2. Whole blood pharmacokinetic profile and main parameters of IP 3 after oral administration of a 50 mg/kg suspension in 1% aqueous methylcellulose.

Main pharmacokinetic parameters were established after non-compartmental analysis: Cmax (maximum concentration observed in whole blood), 3.4 µg/ml; AUC (Area Under the Curve) (0–8 hours), 4.36 µg.h/ml; F (percentage bioavailability), 61%.

Figure 3. Therapeutic efficacy of anti-tubercular IP 3 against M. tuberculosis H37Rv in vivo.

B6 mice were infected by intra-tracheal instillation with 10⁵ cfu M. tuberculosis H37Rv per mouse. The mice were treated with the anti-tubercular orally once a day from day 1 to day 8. Efficacy results are expressed as log cfu reduction in the lungs of infected mice and are the mean±SD of five mice. The shaded line indicates the levels of administered mycobacterial inoculum.

Figure 4. Cross-resistance of M. bovis BCG mutants resistant to IP compounds.

M. bovis BCG resistant mutants raised against IP 1, 3 and 4 were plated at 5× MIC of each of the respective compounds. A wild-type control demonstrates the resistant nature of the mutants.

Figure 5. M. bovis BCG genome region harboring the genes for the cytochrome bc ₁ complex and ctaE.

The DNA regions cloned into the pMV261 vector are indicated.

Figure 6. Effect on the MIC of IP 3 during the over-expression of QcrB in M. bovis BCG.

The over-expression constructs pMV261, pMV261::qcrB, pMV261::qcrCAB and pMV261::ctaE-qcrCAB were electroporated into M. bovis BCG and the MIC of IP 3 was evaluated. The plate lay-out is shown (M. bovis BCG containing the construct detailed) and the MIC of IP 3 with reference to wild-type M. bovis BCG is stated along with the corresponding concentration analyzed. Kanamycin was present at 25 µg/ml to select for the pMV261 vector.