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.

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...

With the rise in resistance to antibiotics such as methicillin, there is a need for new drugs. We report here the discovery and X-ray crystallographic structures of 10 chemically diverse compounds (benzoic, diketo, and phosphonic acids, as well as a bisamidine and a bisamine) that inhibit bacterial undecaprenyl diphosphate synthase, an essential enzyme involved in cell wall biosynthesis. The inhibitors bind to one or more of the four undecaprenyl diphosphate synthase inhibitor binding sites identified previously, with the most active leads binding to site 4, outside the catalytic center. The most potent leads are active against Staphylococcus aureus [minimal inhibitory concentration (MIC) 90 ∼0.25 μg/mL], and one potently synergizes with methicillin (fractional inhibitory concentration index = 0.25) and is protective in a mouse infection model. These results provide numerous leads for antibacterial development and open up the possibility of restoring sensitivity to drugs such as methicillin, using combination therapies.drug discovery | in silico high-throughput screening | peptidoglycan | protein structure

We report the results of an in vitro screening assay targeting the intraerythrocytic form of the malaria parasite Plasmodium falciparum using a library of 560 prenyl-synthase inhibitors. Based on "growth-rescue" and enzyme-inhibition experiments, geranylgeranyl diphosphate synthase (GGPPS) is shown to be a major target for the most potent leads, BPH-703 and BPH-811, lipophilic analogs of the bone-resorption drugs zoledronate and risedronate. We determined the crystal structures of these inhibitors bound to a Plasmodium GGPPS finding that their head groups bind to the ½Mg 2þ 3 cluster in the active site in a similar manner to that found with their more hydrophilic parents, whereas their hydrophobic tails occupy a long-hydrophobic tunnel spanning both molecules in the dimer. The results of isothermal-titration-calorimetric experiments show that both lipophilic bisphosphonates bind to GGPPS with, on average, a ΔG of −9 kcal mol −1 , only 0.5 kcal mol −1 worse than the parent bisphosphonates, consistent with the observation that conversion to the lipophilic species has only a minor effect on enzyme activity. However, only the lipophilic species are active in cells. We also tested both compounds in mice, finding major decreases in parasitemia and 100% survival. These results are of broad general interest because they indicate that it may be possible to overcome barriers to cell penetration of existing bisphosphonate drugs in this and other systems by simple covalent modification to form lipophilic analogs that retain their enzyme-inhibition activity and are also effective in vitro and in vivo.M alaria, caused by Plasmodium spp., causes approximately 1 million deaths each year (1), and there are ever-present problems due to drug resistance (2). There is, therefore, a need for new drugs and drug leads. In earlier work, we and others found that the bisphosphonate class of drugs (3) used to treat bonerelated diseases-osteoporosis, Paget disease, and hypercalcemia due to malignancy-also inhibited the growth of a range of parasitic protozoa, including Trypanosoma cruzi (4, 5), Trypanosoma brucei (4, 6), Leishmania spp. (4,7,8), Toxoplasma gondii (4, 9), Cryptosporidium parvum (10, 11), Entamoeba histolytica (4, 12, 13), and Plasmodium spp. (4, 13-15). In the case of Plasmodium spp., the most potent inhibitors were not, however, the nitrogencontaining bisphosphonates such as zoledronate or risedronate (Scheme 1) used to treat bone diseases, but more lipophilic n-alkyl bisphosphonates (13). Their target in Plasmodium falciparum was not determined. However, more recently, a Plasmodium vivax geranylgeranyl diphosphate synthase (PvGGPPS) has been cloned, expressed, purified, and crystallized, and its three-dimensional structure determined (16). The enzyme is inhibited by bisphosphonates (16), so it seemed possible that it might be a target for the inhibitors discovered earlier. To investigate this possibility, we recently determined the IC 50 values for 25 bisphosphonates against PvGGPPS and compared the results for enzyme inhi...

We report the synthesis and characterization of five novel Mo-containing polyoxometalate (POM) -bisphosphonate complexes with nuclearities ranging from 4 to 12 and with either fully reduced, fully oxidized or mixed valence (Mo V , Mo VI ) molybdenum in which the bisphosphonates bind to the POM cluster via their two phosphonate groups as well as a deprotonated 1-OH group. (5) are alkali salts of, respectively, fully reduced octanuclear and fully oxidized dodecanuclear POMs. The activities of 2-5 (which are water soluble) against three human tumor cell lines were investigated in vitro. 2-4 had weak but measurable activity but 5 had IC 50 values of ~10 μM, about four times the activity of the parent alendronate molecule, on a per-alendronate basis, opening up the possibility of the development of novel drug leads based on Mo-bisphosphonate clusters.

We report the synthesis and characterization of eight new Mo, W, or V-containing polyoxometalate (POM) bisphosphonate complexes with metal nuclearities ranging from 1 to 6. The compounds were synthesized in water by treating MoVI, WVI, VIV or VV precursors with biologically active bisphosphonates H2O3PCR(OH)PO3H2 (R = C3H6NH2, Ale; R = CH2S(CH3)2, Sul and R = C4H5N2, Zol, where Ale=alendronate, Sul=(2-Hydroxy-2,2-bis-phosphono-ethyl)-dimethyl-sulfonium and Zol=zoledronate.). Mo6(Sul)2 and Mo6(Zol)2 contain two trinuclear MoVI cores which can rotate around a central oxo group while Mo(Ale)2 and W(Ale)2 are mononuclear species. In V5(Ale)2 and V5(Zol)2 a central VIV ion is surrounded by two VV dimers bound to bisphosphonate ligands. V6(Ale)4 can be viewed as the condensation of one V5(Ale)2 with one additional VIV ion and two Ale ligands, while V3(Zol)3 is a triangular VIV POM. These new POM bisphosphonates complexes were all characterized by single-crystal X-ray diffraction. The stability of the Mo and W POMs was studied by 31P NMR spectroscopy and showed that all compounds except the mononuclear Mo(Ale)2 and W(Ale)2 were stable in solution. EPR measurements performed on the vanadium derivatives confirmed the oxidation state of the V ions and evidenced their stability in aqueous solution. Electrochemical studies on V5(Ale)2 and V5(Zol)2 showed reduction of VV to VIV and magnetic susceptibility investigations on V3(Zol)3 enabled a detailed analysis of the magnetic interactions. The presence of zoledronate or vanadium correlated with the most potent activity (IC50~1–5 μM) against three human tumor cell lines.

IspG is a 4Fe4S protein involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites, there is a large insert domain (A*) whose structure and function are unknown. We show that bacterial IspGs function in solution as (AB) 2 dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, because there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer. We propose, using computational modeling and electron microscopy, that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product, and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest because they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design.

We report the results of an investigation of the activity of a series of amidine and bisamidine compounds against Staphylococcus aureus and Escherichia coli. The most active compounds bound to an AT-rich DNA dodecamer (CGCGAATTCGCG)2, and using DSC were found to increase the melting transition by up to 24 °C. Several compounds also inhibited undecaprenyl diphosphate synthase (UPPS) with IC50 values of 100–500 nM and we found good correlations (R2 = 0.89, S. aureus; R2 = 0.79, E. coli)) between experimental and predicted cell growth inhibition by using DNA ΔTm and UPPS IC50 experimental results together with 1 computed descriptor. We also solved the structures of three bisamidines binding to DNA as well as three UPPS structures. Overall, the results are of general interest in the context of the development of resistance-resistant antibiotics that involve multi-targeting.