Tricyclic SpiroLactams Kill Mycobacteria In Vitro and In Vivo by Inhibiting Type II NADH Dehydrogenases

Abstract

It is critical that novel classes of antituberculosis drugs are developed to combat the increasing burden of infections by multidrug-resistant strains. To identify such a novel class of antibiotics, a chemical library of unique 3-D bioinspired molecules was explored revealing a promising, mycobacterium specific Tricyclic SpiroLactam (TriSLa) hit. Chemical optimization of the TriSLa scaffold delivered potent analogues with nanomolar activity against replicating and nonreplicating Mycobacterium tuberculosis. Characterization of isolated TriSLa-resistant mutants, and biochemical studies, found TriSLas to act as allosteric inhibitors of type II NADH dehydrogenases (Ndh-2 of the electron transport chain), resulting in an increase in bacterial NADH/NAD+ ratios and decreased ATP levels. TriSLas are chemically distinct from other inhibitors of Ndh-2 but share a dependence for fatty acids for activity. Finally, in vivo proof-of-concept studies showed TriSLas to protect zebrafish larvae from Mycobacterium marinum infection, suggesting a vulnerability of Ndh-2 inhibition in mycobacterial infections.

Rapid and Efficient Access to Novel Bio-Inspired 3-Dimensional Tricyclic SpiroLactams as Privileged Structures via Meyers’ Lactamization

“…A focused library of new three-dimensional tricyclic spirolactams is currently produced and screened on a variety of biological targets. Among them, several compounds have already been identified as lead compounds against mycobacteria [ 47 ]. The structure-activity relationship studies will be reported soon.…”

Synthetic lethality ofMycobacterium tuberculosisNADH dehydrogenases is due to impaired NADH oxidation

“…Importantly, expression of Nox rescued wild-type Mtb from Ndh-1/Ndh-2 synthetic lethality, further confirming that NADH oxidation is the main biological function of NADH dehydrogenases regarding bacterial viability. Several potent Ndh-2 inhibitors have been identified (10)(11)(12)(13)(14)(15)(16). ndh-2 susceptibility to long-chain fatty acids in the medium, an important carbon source during infection, and to hypoxic conditions (9) argues for Ndh-2 inhibitors to be effective in specific microenvironments like the necrotic center of caseating granulomas.…”

“…In addition to phenothiazines, which we later showed to inhibit Mtb's in vitro growth independently of Ndh-2 (9), multiple small molecules have been proposed to specifically inhibit Ndh-2 in Mtb: quinolones (10), quinolinyl pyrimidines (11), molecules with thioquinazoline and tetrahydroindazole cores (12), diphenyleneiodonium analogues (13), 2-Mercapto-Quinazolinones (14), 7-phenyl benzoxaborole compound series (15), and, more recently, tricyclic spirolactams (16). However, the activity of these compounds against Mtb was only demonstrated in in vitro conditions and an Mtb strain in which both genes encoding Ndh-2 enzymes have been deleted (Mtb ndh-2) is only mildly attenuated in a mouse model of infection, suggesting that inhibition of Ndh-2 alone will not kill Mtb during infection (9).…”

Antitubercular activity of 2-mercaptobenzothiazole derivatives targeting Mycobacterium tuberculosis type II NADH dehydrogenase

“…Electrons enter this metabolic pathway via NADH dehydrogenase, thereby oxidizing NADH to NAD + . [16][17][18][19] Mtb possesses two types of NADH dehydrogenase, type I and type II (ndh and ndhA). From a genetic knockout study of ndh and ndhA, the indispensable role of ndh in the unaltered functioning of the oxidative phosphorylation pathway has been reported.…”