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Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain "MmpL3-like" (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of "active-site" residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan, and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe > His residue change, and a Ser/Thr H-bond network, involved in H+ -transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in "lipid" (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.

SQ109 is a tuberculosis drug candidate that has high potency against Mycobacterium tuberculosis and is thought to function at least in part by blocking cell wall biosynthesis by inhibiting the MmpL3 transporter. It also has activity against bacteria and protozoan parasites that lack MmpL3, where it can act as an uncoupler, targeting lipid membranes and Ca2+ homeostasis. Here, we synthesized 19 analogs of SQ109 and tested them against bacteria: M. smegmatis, M. tuberculosis, M. abscessus, Bacillus subtilis and Escherichia coli, as well as against the protozoan parasites, Trypanosoma brucei, T. cruzi, Leishmania donovani, L. mexicana and Plasmodium falciparum. Activity against the mycobacteria was generally less than with SQ109 and was reduced by increasing the size of the alkyl adduct, but two analogs were ~4-8 fold more active than was SQ109 against M. abscessus, including a highly drug resistant strain harboring a A309P mutation in MmpL3. There was also better activity than found with SQ109 with other bacteria and protozoa. Of particular interest, we found that the adamantyl C-2 ethyl, butyl, phenyl and benzyl analogs had 4-10x increased activity against P. falciparum asexual blood stages, together with low toxicity to a human HepG2 cell line, making them of interest as new anti-malarial drug leads. We also used surface plasmon resonance to investigate the binding of inhibitors to MmpL3, and differential scanning calorimetry to investigate binding to lipid membranes. There was no correlation between MmpL3 binding and M. tuberculosis or M. smegmatis cell activity, suggesting that MmpL3 is not a major target, in mycobacteria. However, some of the more active species decreased lipid phase transition temperatures, indicating increased accumulation in membranes, expected to lead to enhanced uncoupler activity.

Terpenes and isoprenoids are the most diverse small organic molecules on Earth. Here, we obtained the structures of sesquisabinene and santalene synthases from Santalum album L. in the presence or ...

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

There is interest in the development of drugs to treat fungal infections due to the increasing threat of drug resistance, and here, we report the first crystallographic structure of the catalytic domain of a fungal squalene synthase (SQS)

Tumor cells and pathogen-infected cells are presented to human γδ T cells based on “inside-out” signaling in which metabolites called phosphoantigens (pAgs) inside target cells are recognized by the intracellular domain of a butyrophilin protein (BTN3A1), leading to an extracellular conformational change. Here, we report that pAgs function as molecular “glues” that initiate a heteromeric association between the intracellular domains of BTN3A1 and the structurally similar BTN2A1. Working with both exogenous and endogenous pAgs, we used x-ray crystallography, mutational studies, cellular assays, synthetic probe as well as molecular dynamics investigations to determine how pAgs glue intracellular BTN3A1 and BTN2A1 together for the “inside-out” signaling that triggers γδ T cell activation. This γδ T cell-specific mode of antigen sensing creates opportunities for the development of alternative immunotherapies against cancer and infectious diseases that do not involve αβ T cells.One Sentence SummaryThe responses of gamma-delta T cells to cancer cells or pathogens are initiated via the intracellular association of heteromeric butyrophilins that are glued together by isoprenoid metabolites.

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC50 ~ 11 nM). The proposed mechanism of action on L. mexicana involves the disruption of the parasite intracellular Ca2+ homeostasis through the collapse of the mitochondrial electrochemical potential (ΔΨm). In the present work, we show a potent effect of SQ109 on L. donovani, the parasite responsible for visceral leishmaniasis, the more severe and uniquely lethal form of these infections, obtaining a toxic effect on amastigotes inside macrophages even lower to that obtained in L. mexicana (IC50 of 7.17 ± 0.09 nM) and with a selectivity index > 800, even higher than in L. mexicana. We also demonstrated for first time that SQ109, besides collapsing ΔΨm of the parasite, induced a very rapid damage to the parasite acidocalcisomes, essential organelles involved in the bioenergetics and many other important functions, including Ca2+ homeostasis. Both effects of the drug on these organelles generated a dramatic increase in the intracellular Ca2+ concentration, causing parasite death.

Human farnesyl pyrophosphate synthase (Homo sapiens FPPS, HsFPPS) is a target for treating bone resorption diseases and some cancers. HsFPPS is potently inhibited by bisphosphonates, but due to poor cell penetration and distribution in soft tissue, there is currently interest in the development of non-bisphosphonate inhibitors as cancer therapeutics. Here, we report the discovery and development of HsFPPS inhibitors based on the phenolic diterpene carnosic acid (CA), an antimicrobial found in rosemary and sage, which showed better cellular anticancer activities than the bisphosphonate drug zoledronate in pancreatic cancer cell lines, as well as an HsFPPS-dependent mechanism of action. Hit-to-lead optimization of CA improved HsFPPS inhibition by >100-fold. A slow dissociation inhibition pattern and a noncompetitive allosteric binding mode were found, and cellular mechanism-of-action studies showed that these inhibitors inhibit tumor cell growth primarily by inhibiting HsFPPS, leading to downregulation of Ras prenylation and cell apoptosis. The discovery of this series of compounds together with proof-of-mechanism in pancreatic cancer cells may pave the way for targeting HsFPPS in soft tissue cancers using natural-product-derived inhibitors.

Lipid transporters play an important role in most if not all organisms, ranging from bacteria to humans. For example, in Mycobacterium tuberculosis, the trehalose monomycolate transporter MmpL3 is involved in cell wall biosynthesis, while in humans, cholesterol transporters are involved in normal cell function as well as in disease. Here, using structural and bioinformatics information, we propose that there are proteins that also contain “MmpL3-like” (MMPL) transmembrane (TM) domains in many protozoa, including Trypanosoma cruzi, as well as in the bacterium Staphylococcus aureus, where the fatty acid transporter FarE has the same set of “active-site” residues as those found in the mycobacterial MmpL3s, and in T. cruzi. We also show that there are strong sequence and predicted structural similarities between the TM proton-translocation domain seen in the X-ray structures of mycobacterial MmpL3s and several human as well as fungal lipid transporters, leading to the proposal that there are similar proteins in apicomplexan parasites, and in plants. The animal, fungal, apicomplexan and plant proteins have larger extra-membrane domains than are found in the bacterial MmpL3, but they have a similar TM domain architecture, with the introduction of a (catalytically essential) Phe>His residue change, and a Ser/Thr H-bond network, involved in H -transport. Overall, the results are of interest since they show that MMPL-family proteins are present in essentially all life-forms: archaea, bacteria, protozoa, fungi, plants and animals and, where known, they are involved in “lipid” (glycolipid, phospholipid, sphingolipid, fatty acid, cholesterol, ergosterol) transport, powered by transmembrane molecular pumps having similar structures.

SQ109 is a drug candidate for the treatment of tuberculosis (TB). It is thought to target primarily the protein MmpL3 in Mycobacterium tuberculosis, but it also inhibits the growth of some other bacteria. SQ109 is metabolized by the liver, and it has been proposed that some of its metabolites might be responsible for its activity against TB. Here, we synthesized six potential P450 metabolites of SQ109 and used these as well as 10 other likely metabolites as standards in a mass spectrometry study of M. tuberculosis-infected rabbits treated with SQ109, in addition to testing all 16 putative metabolites for antibacterial activity. We found that there were just two major metabolites in lung tissue: a hydroxy-adamantyl analog of SQ109 and N′-adamantylethylenediamine. Neither of these, or the other potential metabolites tested, inhibited the growth of M. tuberculosis or of M. smegmatis, Bacillus subtilis, or E. coli, making it unlikely that an SQ109 metabolite contributes to its antibacterial activity. In the rabbit TB model, it is thus the gradual accumulation of nonmetabolized SQ109 in tissues to therapeutic levels that leads to good efficacy. Our results also provide new insights into how SQ109 binds to its target MmpL3, based on our mass spectroscopy results which indicate that the charge in SQ109 is primarily localized on the geranyl nitrogen, explaining the very short distance to a key Asp found in the X-ray structure of SQ109 bound to MmpL3.

SQ109 is a drug candidate for the treatment of tuberculosis (TB). It is thought to target primarily the protein MmpL3 in Mycobacterium tuberculosis, but it also inhibits the growth of some other bacteria, as well as fungi and protozoa. SQ109 is metabolized by the liver, and it has been proposed that some of its metabolites might be responsible for its activity against TB. Here, we synthesized six potential P450 metabolites of SQ109 and used these as well as 10 other likely metabolites as standards in a mass spectrometry study of M. tuberculosis-infected rabbits treated with SQ109, in addition to testing all 16 putative metabolites for anti-bacterial activity. We found that there were just two major metabolites in lung tissue: a hydroxy-adamantyl analog of SQ109 and N’-adamantylethylenediamine. Neither of these, or the other potential metabolites tested, inhibited the growth of M. tuberculosis, or of M. smegmatis, Bacillus subtilis or E. coli, making it unlikely that an SQ109 metabolite contributes to its anti-bacterial activity. In the rabbit TB model, it is thus the gradual accumulation of non-metabolized SQ109 in tissues to therapeutic levels that leads to good efficacy. Our results also provide new insights into how SQ109 binds to its target MmpL3, based on our mass spectroscopy results which indicate that the charge in SQ109 is primarily localized on the geranyl nitrogen, explaining the very short distance to a key Asp found in the X-ray structure of SQ109 bound to MmpL3. Our results also suggest that it is intact SQ109 that is likely to target some of the other bacteria, fungi and protozoa in which MmpL3-like proteins have recently been reported.

The tuberculosis drug candidate SQ109 targets the trehalose monomycolate transporter MmpL3 in Mycobacterium tuberculosis and also has activity against other pathogens. We found related proteins in 22 protozoa, including Trypanosoma cruzi and Entamoeba histolytica, as well as in archaea and other bacteria, including the fatty acid transporter, FarE. We show these proteins, alpha-MMPL proteins, adopt similar structures to that of MsMmpL3 having two sets (P1 and P2) of conserved active site/H+-transporter Asp, Tyr and Phe residues in “pentad” motifs (DYxxF) that can bind to the SQ109 ethylenediamine and adamantyl moieties. Based on structural comparisons with MsMmpL3, we find that there are superimposable transmembrane and H+-transporter structures in much larger proteins, beta-MMPLs, found in apicomplexan parasites, fungi, plants and animals. They also contain double “pentad” motifs in which the P1 Asp is totally conserved, but the P2 Asp may also be a Glu, and the P2 Phe seen in the alpha-MMPLs is a His that H-bonds to the P1 and P2 Asp/Glu residues. There are also 5 conserved Ser/Thr residues that extend the H-bond/H+-transporter network with 2 interacting directly with the P1 Asp, and the His. We propose that all MMPL proteins are involved in proton motive force-mediated lipid (phospholipid, glycolipid, sterol, fatty acid) transport, and that SQ109 may target some pathogens directly, by binding to the P1/P2 motifs. Overall, the results are of general interest since they indicate that there are two major classes of lipid transporters: alpha-MMPL proteins found in many bacteria and protozoa, and much larger, beta-MMPL proteins, found in fungi, apicomplexa, plants and animals and, in some cases, they are potential drug targets.

The tuberculosis drug candidate SQ109 targets the trehalose monomycolate transporter MmpL3 in Mycobacterium tuberculosis and also has activity against other pathogens. We found related proteins in 22 protozoa, including Trypanosoma cruzi and Entamoeba histolytica, as well as in archaea and other bacteria, including the fatty acid transporter, FarE. We show these proteins, alpha-MMPL proteins, adopt similar structures to that of MsMmpL3 having two sets (P1 and P2) of conserved active site/H+-transporter Asp, Tyr and Phe residues in “pentad” motifs (DYxxF) that can bind to the SQ109 ethylenediamine and adamantyl moieties. Based on structural comparisons with MsMmpL3, we find that there are superimposable transmembrane and H+-transporter structures in much larger proteins, beta-MMPLs, found in apicomplexan parasites, fungi, plants and animals. They also contain double “pentad” motifs in which the P1 Asp is totally conserved, but the P2 Asp may also be a Glu, and the P2 Phe seen in the alpha-MMPLs is a His that H-bonds to the P1 and P2 Asp/Glu residues. There are also 5 conserved Ser/Thr residues that extend the H-bond/H+-transporter network with 2 interacting directly with the P1 Asp, and the His. We propose that all MMPL proteins are involved in proton motive force-mediated lipid (phospholipid, glycolipid, sterol, fatty acid) transport, and that SQ109 may target some pathogens directly, by binding to the P1/P2 motifs. Overall, the results are of general interest since they indicate that there are two major classes of lipid transporters: alpha-MMPL proteins found in many bacteria and protozoa, and much larger, beta-MMPL proteins, found in fungi, apicomplexa, plants and animals and, in some cases, they are potential drug targets.

We report that alkyl-subsituted bisphosphonates have activity against Bacillus anthracis Sterne (0.40 µg/mL), Mycobacterium smegmatis (1.4 µg/mL), Bacillus subtilis (1.0 µg/mL) and Staphylococcus aureus (13 µg/mL). In many cases, there is no effect of serum binding, as well as low activity against a human embryonic kidney cell line. Targeting of isoprenoid biosynthesis is involved with74 having IC(50) values of ~100 nM against heptaprenyl diphosphate synthase and 200 nM against a farnesyl diphosphate synthase. Bacillus subtilis growth inhibition was rescued by addition of farnesyl diphosphate, menaquinone-4 (MK-4) or undecaprenyl diphosphate (UP) and the combination of MK-4 plus UP resulted in a 25x increase in ED(50), indicating targeting of both quinone and cell wall biosynthesis. Clostridioides difficile was inhibited by 74 and since this organism does not synthesize quinones, cell wall biosynthesis is the likely target. We also solved three X-ray structures of inhibitors bound to octaprenyl diphosphate and/or undecaprenyl diphosphate synthases.

Summary Human Vγ9/Vδ2 T cells, mucosal-associated invariant T (MAIT) cells, and other unconventional T cells are specialised in detecting microbial metabolic pathway intermediates that are absent in humans. The recognition by such semi-invariant innate-like T cells of compounds like (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), the penultimate metabolite in the MEP isoprenoid biosynthesis pathway, and intermediates of the riboflavin biosynthesis pathway and their metabolites allows the immune system to rapidly sense pathogen-associated molecular patterns that are shared by a wide range of micro-organisms. Given the essential nature of these metabolic pathways for microbial viability, they have emerged as promising targets for the development of novel antibiotics. Here, we review recent findings that link enzymatic inhibition of microbial metabolism with alterations in the levels of unconventional T cell ligands produced by treated micro-organisms that have given rise to the concept of ‘immuno-antibiotics’: combining direct antimicrobial activity with an immunotherapeutic effect via modulation of unconventional T cell responses.

An oligoamidine with two bacteria-specific mechanisms of action showed broad-spectrum, resistance-resistant antimicrobial effect., Antibiotic resistance is now a major threat to human health, and one approach to combating this threat is to develop resistance-resistant antibiotics. Synthetic antimicrobial polymers are generally resistance resistant, having good activity with low resistance rates but usually with low therapeutic indices. Here, we report our solution to this problem by introducing dual-selective mechanisms of action to a short amidine-rich polymer, which can simultaneously disrupt bacterial membranes and bind to bacterial DNA. The oligoamidine shows unobservable resistance generation but high therapeutic indices against many bacterial types, such as ESKAPE strains and clinical isolates resistant to multiple drugs, including colistin. The oligomer exhibited excellent effectiveness in various model systems, killing extracellular or intracellular bacteria in the presence of mammalian cells, removing all bacteria from Caenorhabditis elegans, and rescuing mice with severe infections. This “dual mechanisms of action” approach may be a general strategy for future development of antimicrobial polymers.

We report the structures of the terpene cyclases Santalum album L. sesquisabinene synthases 1 and 2 and of santalene synthase, in apo forms, and with the sesquisabinene synthases, bound to either farnesyl diphosphate (FPP), farnesyl S-thiolo-diphosphate, FPP containing a POP bridging O-to-CCl2 substitution, or to sabinene, leading to a sequential mechanism for substrate binding and catalysis. We trapped early pre-catalytic inactive open forms that show how ligands initially bind to the apo-proteins, then when the pocket closes, catalysis can proceed. We also show that there are strong structural similarities between the most highly conserved residues in class I cyclases and those in head-to-tail (aHT) trans-prenyl transferases—outside the well-known DDXXD-like and NSE/DTE-like domains. In the aHT prenyltransferases there is a highly conserved Thr>Gln>Asp>Tyr motif and in the cyclases, a similar Thr>Arg>Asp>Tyr domain, these residues forming very similar, extended H-bond networks (rmsd ~1.4 Å) that are involved in catalysis, leading to the proposal that there are 3 key domains in both the cyclases and the aHT prenyltransferases: The AC-domain that binds MgA and MgC; the B domain that binds MgB and leads to pocket closure, ionization, and condensation or cyclization; and the D-domain H-bond network, involved in H+ elimination. In aHH prenyltransferases the overall folds and MgABC motifs are similar to those found in the cyclase and aHT proteins, but the full Thr>Arg/Gln>Asp>Tyr domain is absent and instead there are Tyr/Asp or Tyr/Glu residues that bind to MgC and are highly conserved. Overall, the results are of general interest since they show unexpected similarities between the enzymes that produce the most diverse molecules on Earth: aHT and aHH prenyltransferases, and terpenoid cyclases.

We report the results of an investigation into the catalytic role of highly conserved amide (asparagine, glutamine) and OH-containing (serine, tyrosine) residues in several prenyltransferases. We first obtained the X-ray structure of cyclolavandulyl diphosphate synthase containing two molecules of the substrate analog dimethylallyl (S)-thiolodiphosphate (DMASPP). The two molecules have similar diphosphate group orientations to those seen in other ζ-fold (cis- head-to-tail and head-to-middle) prenyltransferases with one diphosphate moiety forming a bidentate chelate with Mg(2+) in the so-called S1 site (which is typically the allylic binding site in ζ-fold proteins) while the second diphosphate binds to Mg(2+) in the so-called S2 site (which is typically the homoallylic binding site in ζ-fold proteins) via a single P1O1 oxygen. The latter interaction can facilitate direct phosphate-mediated proton abstraction via P1O2, or more likely by an indirect mechanism in which P1O2 stabilizes a basic asparagine species that removes H(+), which is then eliminated via an Asn-Ser shuttle. The universal occurrence of Asn-Ser pairs in ζ-fold proteins leads to the idea that the highly conserved amide (Asn, Gln) and OH-containing (Tyr) residues seen in many “head-to-head” prenyltransferases such as squalene and dehydrosqualene synthase might play similar roles, in H(+) elimination. Structural, bioinformatics and mutagenesis investigations indeed indicate an important role of these residues in catalysis, with the results of density functional theory calculations showing that Asn bound to Mg(2+) can act as a general (imine-like) base, while Gln, Tyr and H(2)O form a proton channel that is adjacent to the conventional (Asp-rich) “active site”. Taken together, our results lead to mechanisms of proton-elimination from carbocations in numerous prenyltransferases in which neutral species (Asn, Gln, Ser, Tyr, H(2)O) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu) that bind to Mg(2+), and basic groups (primarily Arg) that bind to diphosphates, in isoprenoid biosynthesis.

The promising activity of phenylthiazoles against multidrug-resistant bacterial pathogens, in particular MRSA, has been hampered by their limited systemic applicability, due to their rapid metabolism by hepatic microsomal enzymes, resulting in short half-lives. Here, we investigated a series of phenylthiazoles with alkynyl side-chains that were synthesized with the objective of improving stability to hepatic metabolism, extending the utility of phenylthiazoles from topical applications to treatment of a more invasive, systemic MRSA infections. The most promising compounds inhibited the growth of clinically-relevant isolates of MRSA in vitro at concentrations as low as 0.5 μg/mL, and exerted their antibacterial effect by interfering with bacterial cell wall synthesis via inhibition of undecaprenyl diphosphate synthase and undecaprenyl diphosphate phosphatase. We also identified two phenylthiazoles that successfully eradicated MRSA inside infected macrophages. In vivo PK analysis of compound 9 revealed promising stability to hepatic metabolism with a biological half-life of ∼4.5 h. In mice, compound 9 demonstrated comparable potency to vancomycin, and at a lower dose (20 mg/kg versus 50 mg/kg), in reducing the burden of MRSA in a systemic, deep-tissue infection, using the neutropenic mouse thigh-infection model. Compound 9 thus represents a new phenylthiazole lead for the treatment of MRSA infections that warrants further development.

We report the first X-ray crystallographic structure of the "head-to-middle" prenyltransferase, isosesquilavandulyl diphosphate synthase, involved in biosynthesis of the merochlorin class of antibiotics. The protein adopts the ζ or cis-prenyl transferase fold but remarkably, unlike tuberculosinol adenosine synthase and other cis-prenyl transferases (e.g. cis-farnesyl, decaprenyl, undecaprenyl diphosphate synthases), the large, hydrophobic side chain does not occupy a central hydrophobic tunnel. Instead, it occupies a surface pocket oriented at 90° to the hydrophobic tunnel. Product chain-length control is achieved by squeezing out the ligand from the conventional allylic S1 binding site, with proton abstraction being achieved using a diphosphate-Asn-Ser relay. The structures revise and unify our thinking as to the mechanism of action of many other prenyl transferases and may also be of use in engineering new merochlorin-class antibiotics.

Lysyl oxidase (LOX) is a copper amine oxidase that cross-links collagens and elastin in connective tissue and plays an important role in fibrosis, cancer development, and formation of the "metastatic niche". Despite its important biological functions, the structure of human LOX remains unknown (unlike that of an unrelated LOX, from Pichia pastoris). Here, we expressed active LOX from Drosophila melanogaster, DmLOXL1, a close homologue of human LOX, and characterized it by MS, UV-vis, activity, and inhibition assays. We then used bioinformatics, electron paramagnetic resonance, electron spin-echo envelope modulation, and hyperfine sublevel-correlation (HYSCORE) spectroscopies to probe Cu-ligand bonding finding direct evidence for pH-dependent Cu-His interactions. At pH = 9.3, the spectroscopic data indicated primarily a single His bound to Cu, but at pH = 7.5, there was evidence for a ∼ 1:1 mixture of species containing 1 and 3 His ligands. We then used HYSCORE to probe possible interactions between the LOX inhibitor BAPN (β-aminopropionitrile; 1-[

We synthesized a series of polyoxometalate-bisphosphonate complexes containing MoVIO6 octahedra, zoledronate or an N-alkyl (n-C6 or n-C8) zoledronate analog, and in two cases, Mn as a heterometal. Mo6L2 (L = Zol, ZolC6, ZolC8) and Mo4L2Mn (L = Zol, ZolC8) were characterized by using single-crystal X-ray crystallography and/or IR spectroscopy, elemental and EDX analysis and 31P NMR. We found promising activity against human non-small cell lung cancer (NCI-H460) cells with IC50 values for growth inhibition of ~5 μM per bisphosphonate ligand. The effects of bisphosphonate complexation on IC50 decreased with increasing bisphosphonate chain length: C0 ~6.1x; C6 ~3.4× and C8 ~1.1x. We then determined the activity of one of the most potent compounds in the series, Mo4Zol2Mn(III), against SK-ES-1 sarcoma cells in a mouse xenograft system finding a ~5× decrease in tumor volume than found with the parent compound zoledronate at the same compound dosing (5 μg/mouse). Overall, the results are of interest since we show for the first time that heteropolyoxomolybdate-bisphosphonate hybrids kill tumor cells in vitro and significantly decrease tumor growth, in vivo, opening up new possibilities for targeting both Ras as well as EGFR (epidermal growth factor receptor) driven cancers.

Many organisms contain head-to-head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor-bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP-stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head-to-head prenyl synthases, although their biological functions remain to be elucidated.

Isoprenoid biosynthesis is an important area for anti-infective drug development. One isoprenoid target is (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (HMBPP) reductase (IspH), which forms isopentenyl diphosphate and dimethylallyl diphosphate from HMBPP in a 2H+ /2e- reduction. IspH contains a 4 Fe-4 S cluster, and in this work, we first investigated how small molecules bound to the cluster by using HYSCORE and NRVS spectroscopies. The results of these, as well as other structural and spectroscopic investigations, led to the conclusion that, in most cases, ligands bound to IspH 4 Fe-4 S clusters by η1 coordination, forming tetrahedral geometries at the unique fourth Fe, ligand side chains preventing further ligand (e.g., H2 O, O2 ) binding. Based on these ideas, we used in silico methods to find drug-like inhibitors that might occupy the HMBPP substrate binding pocket and bind to Fe, leading to the discovery of a barbituric acid analogue with a Ki value of ≈500 nm against Pseudomonas aeruginosa IspH.

We report that the tuberculosis drug SQ109 [ N -adamantan-2-yl- N ′-(( E )-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine] has potent activity against the intracellular amastigote form of Leishmania mexicana (50% inhibitory concentration [IC 50 ], ∼11 nM), with a good selectivity index (>500). It is also active against promastigotes (IC 50 , ∼500 nM) and acts as a protonophore uncoupler, in addition to disrupting Ca 2+ homeostasis by releasing organelle Ca 2+ into the cytoplasm, and as such, it is an interesting new leishmaniasis drug hit candidate.

Leishmania donovani is the causative agent of visceral leishmaniasis. Annually, 500 million new cases of infection are reported mainly in poor communities, decreasing the interest of the pharmaceutical industries. Therefore, the repositioning of new drugs is an ideal strategy to fight against these parasites. SQ109, a compound in phase IIb/III of clinical trials to treat resistant Mycobacterium tuberculosis, has a potent effect against Trypanosoma cruzi, responsible for Chagas' disease, and on Leishmania mexicana, the causative agent of cutaneous and muco-cutaneous leishmaniasis. In the latter, the toxic dose against intramacrophagic amastigotes is very low (IC

The antibiotic moenomycin A is a phosphoglycerate derivative with a C25-moenocinyl chain and a branched oligosaccharide. Formation of the C25-chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2-Z,E-farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans-type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C2O lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

We report the first structure of heptaprenyl diphosphate synthase from Staphylococcus aureus (SaHepPPS), together with an investigation of its mechanism of action and inhibition. The protein is involved in the formation of menaquinone, a key electron transporter in many bacteria, including pathogens. SaHepPPS consists of a "catalytic " subunit (SaHepPPS-2) having two "DDXXD" motifs and a "regulatory" subunit (SaHepPPS-1) that lacks these motifs. High concentrations of the substrates, isopentenyl diphosphate and farnesyl diphosphate, inhibit the enzyme, which is also potently inhibited by bisphosphonates. The most active inhibitors (Ki ∼200 nm) were N-alkyl analogues of zoledronate containing ∼C6 alkyl side chains. They were modestly active against S. aureus cell growth, and growth inhibition was partially "rescued" by the addition of menaquinone-7. Because SaHepPPS is essential for S. aureus cell growth, its structure is of interest in the context of the development of menaquinone biosynthesis inhibitors as potential antibiotic leads.

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are "bent" and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics.

Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis.

Bisphosphonates are a major class of drugs used to treat osteoporosis, Paget’s disease, and cancer. They have been proposed to act by inhibiting one or more targets including protein prenylation, the epidermal growth factor receptor, or the adenine nucleotide translocase. Inhibition of the latter is due to formation in cells of analogs of ATP: the isopentenyl ester of ATP (ApppI) or an AppXp-type analog of ATP, such as AMP-clodronate (AppCCl(2)p). We screened both ApppI as well as AppCCl(2)p against a panel of 369 kinases finding potent inhibition of some tyrosine kinases by AppCCl(2)p, attributable to formation of a strong hydrogen bond between tyrosine and the terminal phosphonate. We then synthesized bisphosphonate preprodrugs that are converted in cells to other ATP-analogs, finding low nM kinase inhibitors that inhibited cell signaling pathways. These results help clarify our understanding of the mechanisms of action of bisphosphonates, potentially opening up new routes to the development of bone resorption, anticancer, and anti-inflammatory drug leads.

Summary Motivated by the clinical observation that interruption of the mevalonate pathway stimulates immune responses, we hypothesized that this pathway may function as a druggable target for vaccine adjuvant discovery. We found that lipophilic statin drugs and rationally designed bisphosphonates that target three distinct enzymes in the mevalonate pathway have potent adjuvant activities in mice and cynomolgus monkeys. These inhibitors function independently of conventional “danger sensing.” Instead, they inhibit the geranylgeranylation of small GTPases, including Rab5 in antigen-presenting cells, resulting in arrested endosomal maturation, prolonged antigen retention, enhanced antigen presentation, and T cell activation. Additionally, inhibiting the mevalonate pathway enhances antigen-specific anti-tumor immunity, inducing both Th1 and cytolytic T cell responses. As demonstrated in multiple mouse cancer models, the mevalonate pathway inhibitors are robust for cancer vaccinations and synergize with anti-PD-1 antibodies. Our research thus defines the mevalonate pathway as a druggable target for vaccine adjuvants and cancer immunotherapies.

Malaria, mainly caused by Plasmodium falciparum and Plasmodium vivax, has been a growing cause of morbidity and mortality. P. falciparum is more lethal than is P. vivax, but there is a vital need for effective drugs against both species. Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme involved in the biosynthesis of quinones and in protein prenylation, and has been proposed to be a malaria drug target. However, the structure of P. falciparum GGPPS (PfGGPPS) has not been determined, due to difficulties in crystallization. Here, we created a PfGGPPS model using the homologous P.vivax GGPPS X-ray structure as a template. We simulated the modeled PfGGPPS as well as PvGGPPS using conventional and Gaussian accelerated molecular dynamics in both apo- and GGPP -bound states. The MD simulations revealed a striking similarity in the dynamics of both enzymes with loop 9–10 controlling access to the active site. We also found that GGPP stabilizes PfGGPPS and PvGGPPS into closed conformations and via similar mechanisms. Shape-based analysis of the binding sites throughout the simulations suggest that the two enzymes will be readily targeted by the same inhibitors. Finally, we produced three MD-validated conformations of PfGGPPS to be used in future virtual screenings for potential new anti-malarial drugs acting on both PvGGPPS and PfGGPPS.

We synthesized a library of 48 analogs of the Mycobacterium tuberculosis cell growth inhibitor SQ109 in which the ethylene diamine linker was replaced by oxa-, thia- or heterocyclic species, and in some cases, the adamantyl group was replaced by a 1,2-carborane or the N-geranyl group by another hydrophobic species. Compounds were tested against Mycobacterium tuberculosis (H37Rv and/or Erdman), Mycobacterium smegmatis, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Trypanosoma brucei and two human cell lines (human embryonic kidney, HEK293T, and the hepatocellular carcinoma, HepG2). Most potent activity was found against T. brucei, the causative agent of human African trypanosomiasis, and involved targeting of the mitochondrial membrane potential with 15 SQ109 analogs being more active than was SQ109 in cell growth inhibition, having IC50 values as low as 12 nM (5.5 ng/mL) and a selectivity index of ~300.

Farnesyl diphosphate synthase (FPPS) is an important drug target for bone resorption, cancer, and some infectious diseases. Here, we report five new structures including two having unique bound ligand geometries. The diamidine inhibitor 7 binds to human FPPS close to the homoallylic (S2) and allosteric (S3) sites and extends into a new site, here called S4. With the bisphosphonate inhibitor 8, two molecules bind to Trypanosoma brucei FPPS, one molecule in the allylic site (S1) and the other close to S2, the first observation of two bisphosphonate molecules bound to FPPS. We also report the structures of apo-FPPS from T. brucei, together with two more bisphosphonate-bound structures (2,9), for purposes of comparison. The diamidine structure is of particular interest because 7 could represent a new lead for lipophilic FPPS inhibitors, while 8 has low micromolar activity against T. brucei, the causative agent of human African trypanosomiasis.