Abstract
Compounds that target energy generation in many bacterial pathogens are effective inhibitors of growth, however their precise molecular targets and interactions are poorly understood. Type II (non-proton-translocating) NADH dehydrogenase is a monotopic membrane protein (40 – 70 kDa) that catalyses electron transfer from NADH to quinone, via a flavin cofactor. The absence of mammalian homologs and its essentiality in many pathogenic microorganisms such as Mycobacterium tuberculosis makes NDH-2 an attractive target for drug development. Despite this potential, no potent nanomolar inhibitors of NDH-2 are in the development pipeline and our mechanistic and biochemical understanding of the bacterial enzyme is limited. The aim of the current study was to improve our fundamental understanding of bacterial NDH-2, namely its biochemical features, catalytic mechanism and substrate binding interactions, and apply this information to the development of potent inhibitory molecules. A combinational approach using the recent crystal structure of Caldalkalibacillus thermarum NDH-2 and kinetic analysis revealed that this enzyme conducts catalysis by a two-site ping-pong mechanism. The active sites of NDH-2 were further explored, where the nucleotide-binding site was shown to have a strong (nanomolar) affinity for NADH and residues involved in NADH/NADPH substrate specificity were identified. Structural and sequence analysis revealed a conserved quinone-binding motif (316 –AQXAXQ-321). The enzymatic environment of NDH-2 for inhibitor screening was investigated by comparing detergent-solubilized and lipid-reconstituted NDH-2 systems. The detergent-solubilized system was shown to detect more drug-like compounds and inhibitor scaffolds than the lipid-reconstituted system, however lipid-reconstitution significantly reduced the number insoluble (i.e. highly lipophilic) compounds detected. Conjugation of the alkyltriphenylphosphonium cation delivery functionality to a previously reported NDH-2 inhibitor class (the phenothiazines) caused a 130-fold increase in potency against growing cultures of M. tuberculosis. The conjugate was also shown to target NDH-2 linked process, such as NADH oxidation and oxygen consumption. Overall, the findings of this study provide valuable insight into the biochemistry and catalysis of bacterial NDH-2, where this information is utilized to develop methods for the identification of potent inhibitors of this enzyme.