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.

The significance of fever in response to a bacterial infection has been investigated using the lizard Dipsosaurus dorsalis as an animal model. These lizards develop a fever of about 2 degrees C after injection with the bacterium Aeromonas hydrophila. To determine whether this elevation in body temperature increases the resistance of the host to this infection, as measured by survival, lizards were infected with the live bacteria and placed in a neutral (38 degrees C), low (34 degrees or 36 degrees C), or high (40 degrees or 42 degrees C) ambient temperature. An elevation in temperature following experimental bacterial infection results in a significant increase in host survival.

Phosphatidylinositol (PI) and metabolically derived products such as the phosphatidylinositol mannosides and linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium sp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. To determine if PI is an essential phospholipid of mycobacteria, we identified the pgsA gene of Mycobacterium tuberculosis encoding the phosphatidylinositol synthase enzyme and constructed a pgsA conditional mutant of Mycobacterium smegmatis. The ability of this mutant to synthesize phosphatidylinositol synthase and subsequently PI was dependent on the presence of a functional copy of the pgsA gene carried on a thermosensitive plasmid. The mutant grew like the control strain under permissive conditions (30°C), but ceased growing when placed at 42°C, a temperature at which the rescue plasmid is lost. Loss of cell viability at 42°C was observed when PI and phosphatidylinositol dimannoside contents dropped to ϳ30 and 50% of the wild-type levels, respectively. This work provides the first evidence of the essentiality of PI to the survival of mycobacteria. PI synthase is thus an essential enzyme of Mycobacterium that shows promise as a drug target for anti-tuberculosis therapy.

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.

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks.

The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of D-arabinofuranose (Araf) residues. The immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-Araf, which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Araf is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-␤-D-5-phosphoribose. (ii) Decaprenylphosphoryl-␤-D-5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-␤-D-ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-␤-D-erythro-pentofuranose. (iv) Decaprenylphosphoryl-2-keto-␤-D-erythro-pentofuranose is reduced to form decaprenylphosphoryl-␤-D-Araf.Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

The compositional complexity of the mycobacterial cell envelope differentiates Mycobacterium species from most other prokaryotes. Historically, research in this area has focused on the elucidation of the structure of the mycobacterial cell envelope with the result that the structures of the mycolic acid-arabinogalactan-peptidoglycan complex from M. tuberculosis are fairly well understood. However, the current impetus for studying M. tuberculosis and other pathogenic mycobacteria is the need to identify targets for the development of new drugs. Therefore, emphasis has been shifting to the study of cell envelope biosynthesis and the identification of enzymes that are essential to the viability of M. tuberculosis. The publication of the complete M. tuberculosis genome in 1998 has greatly aided these studies. To date, thirteen enzymes involved in the synthesis of the arabinogalactan-peptidoglycan complex of M. tuberculosis have been identified and at least partially characterized. Eleven of these enzymes were reported subsequent to the publication of the M. tuberculosis genome, a clear indication of the rapid evolution of knowledge stimulated by the sequencing of the genome. In this article we review the current understanding of M. tuberculosis arabinogalactan-peptidoglycan structure and biosynthesis.

There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5-2 μg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.T here is a need for new antibiotics, due to the increase in drug resistance (1, 2). For example, some studies report that by 2050, absent major improvements in drug discovery and use, more individuals will die from drug-resistant bacterial infections than from cancer, resulting in a cumulative effect on global gross domestic product of as much as 100 trillion dollars (3, 4). To discover new drugs, new targets, leads, concepts, and implementations are needed (5, 6).Currently, one major cause of death from bacterial infections is tuberculosis (TB) (7), where very highly drug-resistant strains have been found (8). Therapy is lengthy, even with drug-sensitive strains, and requires combination therapies with four drugs. Two recent TB drugs/drug leads (9-11) are TMC207 [bedaquiline (1); Sirturo] and SQ109 (2) (Fig. 1). Bedaquiline (1) targets the Mycobacterium tuberculosis ATP synthase (9) whereas SQ109 (2) has been proposed to target MmpL3 (mycobacterial membrane protein large 3), a trehalose monomycolate transporter essential for cell wall biosynthesis (12). SQ109 (2) is a lipophilic base containing an adamantyl "headgroup" connected via an ethylene diamine "linker" to a geranyl (C 10 ) "side chain," and in recent work (13), we synthesized a series of 11 analogs of SQ109 (2) finding that the ethanolamine (3) was more potent th...