We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.

dMmpL3, a resistance-nodulation-division (RND) superfamily transporter, has been implicated in the formation of the outer membrane of Mycobacterium tuberculosis; specifically, MmpL3 is required for the export of mycolic acids in the form of trehalose monomycolates (TMM) to the periplasmic space or outer membrane of M. tuberculosis. Recently, seven series of inhibitors identified by whole-cell screening against M. tuberculosis, including the antituberculosis drug candidate SQ109, were shown to abolish MmpL3-mediated TMM export. However, this mode of action was brought into question by the broad-spectrum activities of some of these inhibitors against a variety of bacterial and fungal pathogens that do not synthesize mycolic acids. This observation, coupled with the ability of three of these classes of inhibitors to kill nonreplicating M. tuberculosis bacilli, led us to investigate alternative mechanisms of action. Our results indicate that the inhibitory effects of adamantyl ureas, indolecarboxamides, tetrahydropyrazolopyrimidines, and the 1,5-diarylpyrrole BM212 on the transport activity of MmpL3 in actively replicating M. tuberculosis bacilli are, like that of SQ109, most likely due to their ability to dissipate the transmembrane electrochemical proton gradient. In addition to providing novel insights into the modes of action of compounds reported to inhibit MmpL3, our results provide the first explanation for the large number of pharmacophores that apparently target this essential inner membrane transporter.

Menaquinone (MK) with partially saturated isoprenyl moieties is found in a wide range of eubacteria and Archaea. In many Gram-positive organisms, including mycobacteria, it is the double bond found in the β-isoprene unit that is reduced. Mass spectral characterization of menaquinone from mycobacterial knockout strains and heterologous expression hosts demonstrates that Rv0561c (designated menJ) encodes an enzyme which reduces the β-isoprene unit of menaquinone in Mycobacterium tuberculosis, forming the predominant form of menaquinone found in mycobacteria. MenJ is highly conserved in mycobacteria species but is not required for growth in culture. Disruption of menJ reduces mycobacterial electron transport efficiency by 3-fold, but mycobacteria are able to maintain ATP levels by increasing the levels of the total menaquinone in the membrane; however, MenJ is required for M. tuberculosis survival in host macrophages. Thus, MK with partially hydrogenated isoprenyl moieties represents a novel virulence factor and MenJ is a contextually essential enzyme and a potential drug target in pathogenic mycobacteria and other Gram-positive pathogens.

Chapter summary The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, has inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation (OXPHOS) for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis, and the opportunities for drug discovery.

The interaction of benzoic acid and benzoate with model membrane systems was characterized to understand the molecular interactions of the two forms of a simple aromatic acid with the components of the membrane. The microemulsion system based on bis(2ethylhexyl)sulfosuccinate (AOT) allowed determination of the molecular positioning using 1D NMR and 2D NMR spectroscopic methods. Benzoic acid and benzoate were both found to penetrate the membrane/water interfaces; however, the benzoic acid was able to penetrate much deeper and thus is more readily able to traverse a membrane. The Langmuir monolayer model system, using dipalmitoylphosphatidylcholine, was used as a generic membrane lipid for a cell. Compression isotherms of monolayers demonstrated a pH dependent interaction with a lipid monolayer and confirming the pH dependent observations shown in the reverse micellar model system. These studies provide an explanation for the antimicrobial activity of benzoic acid while benzoate is inactive. Furthermore, these studies form the framework upon which we are investigating the mode of bacterial uptake of pyrazinoic acid, the active form of pyrazinamide, a front line drug used to combat tuberculosis.