Identification of 2‑Aryl-Quinolone Inhibitors of Cytochrome <i>bd</i> and Chemical Validation of Combination Strategies for Respiratory Inhibitors against Mycobacterium tuberculosis

Mycobacterium tuberculosis cytochrome <i>bd</i> quinol oxidase (cyt <i>bd</i>), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome <i>bd</i>. The heterologous M. tuberculosis cytochrome <i>bd</i> expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome <i>bd</i> inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt <i>bd</i> inhibitor CK-2-63 with either cyt <i>bcc</i>-<i>aa</i>₃ inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and <i>in vitro</i> sterilization kinetics. <i>In vivo</i> combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the <i>in vivo</i> experiments compared to <i>in vitro</i> experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt <i>bd</i> inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.