Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores.

Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure-activity relationship (QSAR) derived from acid-base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

Proline Dehydrogenase and Pyrroline 5 Carboxylate Dehydrogenase from Mycobacterium tuberculosis: Evidence for Substrate Channeling.

In Mycobacterium tuberculosis, proline dehydrogenase (PruB) and ∆1-pyrroline-5-carboxylate (P5C) dehydrogenase (PruA) are monofunctional enzymes that catalyze proline oxidation to glutamate via the intermediates P5C and L-glutamate-γ-semialdehyde. Both enzymes are essential for the replication of pathogenic M. tuberculosis. Highly active enzymes were expressed and purified using a Mycobacterium smegmatis expression system. The purified enzymes were characterized using natural substrates and chemically synthesized analogs. The structural requirements of the quinone electron acceptor were examined. PruB displayed activity with all tested lipoquinone analogs (naphthoquinone or benzoquinone). In PruB assays utilizing analogs of the native naphthoquinone [MK-9 (II-H2)] specificity constants Kcat/Km were an order of magnitude greater for the menaquinone analogs than the benzoquinone analogs. In addition, mycobacterial PruA was enzymatically characterized for the first time using exogenous chemically synthesized P5C. A Km value of 120 ± 0.015 µM was determined for P5C, while the Km value for NAD+ was shown to be 33 ± 4.3 µM. Furthermore, proline competitively inhibited PruA activity and coupled enzyme assays, suggesting that the recombinant purified monofunctional PruB and PruA enzymes of M. tuberculosis channel substrate likely increase metabolic flux and protect the bacterium from methylglyoxal toxicity.

SAR study of piperidine derivatives as inhibitors of 1,4-dihydroxy-2-naphthoate isoprenyltransferase (MenA) from Mycobacterium tuberculosis.

The electron transport chain (ETC) in the cell membrane consists of a series of redox complexes that transfer electrons from electron donors to acceptors and couples this electron transfer with the transfer of protons (H+) across a membrane. This process generates proton motive force which is used to produce ATP and a myriad of other functions and is essential for the long-term survival of Mycobacterium tuberculosis (Mtb), the causative organism of tuberculosis (TB), under the hypoxic conditions present within infected granulomas. Menaquinone (MK), an important carrier molecule within the mycobacterial ETC, is synthesized de novo by a cluster of enzymes known as the classic/canonical MK biosynthetic pathway. MenA (1,4-dihydroxy-2-naphthoate prenyltransferase), the antepenultimate enzyme in this pathway, is a verified target for TB therapy. In this study, we explored structure-activity relationships of a previously discovered MenA inhibitor scaffold, seeking to improve potency and drug disposition properties. Focusing our campaign upon three molecular regions, we identified two novel inhibitors with potent activity against MenA and Mtb (IC50 = 13-22 µM, GIC50 = 8-10 µM). These analogs also displayed substantially improved pharmacokinetic parameters and potent synergy with other ETC-targeting agents, achieving nearly complete sterilization of Mtb in combination therapy within two weeks in vivo. These new inhibitors of MK biosynthesis present a promising new strategy to curb the continued spread of TB.

LdtC Is a Key l,d-Transpeptidase for Peptidoglycan Assembly in Mycobacterium smegmatis.

The peptidoglycan of mycobacteria has two types of direct cross-links, classical 4-3 cross-links that occur between diaminopimelate (DAP) and alanine residues, and nonclassical 3-3 cross-links that occur between DAP residues on adjacent peptides. The 3-3 cross-links are synthesized by the concerted action of d,d-carboxypeptidases and l,d-transpeptidases (Ldts). Mycobacterial genomes encode several Ldt proteins that can be classified into six classes based upon sequence identity. As a group, the Ldt enzymes are resistant to most ß-lactam antibiotics but are susceptible to carbapenem antibiotics, with the exception of LdtC, a class 5 enzyme. In previous work, we showed that loss of LdtC has the greatest effect on the carbapenem susceptibility phenotype of Mycobacterium smegmatis (also known as Mycolicibacterium smegmatis) compared to other ldt deletion mutants. In this work, we show that a M. smegmatis mutant lacking the five ldt genes other than ldtC has a wild-type phenotype with the exception of increased susceptibility to rifampin. In contrast, a mutant lacking all six ldt genes has pleiotropic cell envelope defects, is temperature sensitive, and has increased susceptibility to a variety of antibiotics. These results indicate that LdtC is capable of functioning as the sole l,d-transpeptidase in M. smegmatis and suggest that it may represent a carbapenem-resistant pathway for peptidoglycan biosynthesis. IMPORTANCE Mycobacteria have several enzymes to catalyze nonclassical 3-3 linkages in the cell wall peptidoglycan. Understanding the biology of these cross-links is important for the development of antibiotic therapies to target peptidoglycan biosynthesis. Our work provides evidence that LdtC can function as the sole enzyme for 3-3 cross-link formation in M. smegmatis and suggests that LdtC may be part of a carbapenem-resistant l,d-transpeptidase pathway.

Mycobacterial MenG: Partial Purification, Characterization, and Inhibition.

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C-methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C-methyltransferase specific activity by threefold. Rv0558 encodes a canonical C-methyltransferase or, more specifically, a S-adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent Km values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 µM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC50 value of 5.1 ± 0.5 µM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC50 value of 2.6 ± 0.6 µM.

Do bioactive 8-hydroxyquinolines oxidovanadium(IV) and (V) complexes inhibit the growth of M. smegmatis?

The antiproliferative effects of four series of VIVO- and VVO-based compounds containing 8-hydroxyquinoline ligands on the bacterium Mycolicibacterium smegmatis (M. smeg) were investigated. The effects on M. smeg were compared to the antiproliferative effects on the protozoan parasite Trypanosoma cruzi (T. cruzi), the causative agent for Chagas disease. In this study, we investigate the speciation of these compounds under physiological conditions as well as the antiproliferative effects on the bacterium M. smeg. We find that the complexes are more stable the less H2O is present, and that the stability increases in lipid-like environments. Only one heteroleptic complex and two homoleptic complexes were found to show similar antiproliferative effects on M. smeg as reported for T. cruzi so the responses generally observed by M.smeg. is less than observed by the pathogen. In summary, we find that M. smeg is more sensitive to the detailed structure of the V-complex but overall these complexes are less effective against M. smeg compared to T. cruzi.

Electron Transport Lipids Fold Within Membrane-Like Interfaces.

Lipoquinones, such as ubiquinones (UQ) and menaquinones (MK), function as essential lipid components of the electron transport system (ETS) by shuttling electrons and protons to facilitate the production of ATP in eukaryotes and prokaryotes. Lipoquinone function in membrane systems has been widely studied, but the exact location and conformation within membranes remains controversial. Lipoquinones, such as Coenzyme Q (UQ-10), are generally depicted simply as "Q" in life science diagrams or in extended conformations in primary literature even though specific conformations are important for function in the ETS. In this study, our goal was to determine the location, orientation, and conformation of UQ-2, a truncated analog of UQ-10, in model membrane systems and to compare our results to previously studied MK-2. Herein, we first carried out a six-step synthesis to yield UQ-2 and then demonstrated that UQ-2 adopts a folded conformation in organic solvents using 1H-1H 2D NOESY and ROESY NMR spectroscopic studies. Similarly, using 1H-1H 2D NOESY NMR spectroscopic studies, UQ-2 was found to adopt a folded, U-shaped conformation within the interface of an AOT reverse micelle model membrane system. UQ-2 was located slightly closer to the surfactant-water interface compared to the more hydrophobic MK-2. In addition, Langmuir monolayer studies determined UQ-2 resided within the monolayer water-phospholipid interface causing expansion, whereas MK-2 was more likely to be compressed out and reside within the phospholipid tails. All together these results support the model that lipoquinones fold regardless of the headgroup structure but that the polarity of the headgroup influences lipoquinone location within the membrane interface. These results have implications regarding the redox activity near the interface as quinone vs. quinol forms may facilitate locomotion of lipoquinones within the membrane. The location, orientation, and conformation of lipoquinones are critical for their function in generating cellular energy within membrane ETS, and the studies described herein shed light on the behavior of lipoquinones within membrane-like environments.

Synthetic Sansanmycin Analogues as Potent Mycobacterium tuberculosis Translocase I Inhibitors.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

Interactions of Truncated Menaquinones in Lipid Monolayers and Bilayers.

Menaquinones (MK) are hydrophobic molecules that consist of a naphthoquinone headgroup and a repeating isoprenyl side chain and are cofactors used in bacterial electron transport systems to generate cellular energy. We have previously demonstrated that the folded conformation of truncated MK homologues, MK-1 and MK-2, in both solution and reverse micelle microemulsions depended on environment. There is little information on how MKs associate with phospholipids in a model membrane system and how MKs affect phospholipid organization. In this manuscript, we used a combination of Langmuir monolayer studies and molecular dynamics (MD) simulations to probe these questions on truncated MK homologues, MK-1 through MK-4 within a model membrane. We observed that truncated MKs reside farther away from the interfacial water than ubiquinones are are located closer to the phospholipid tails. We also observed that phospholipid packing does not change at physiological pressure in the presence of truncated MKs, though a difference in phospholipid packing has been observed in the presence of ubiquinones. We found through MD simulations that for truncated MKs, the folded conformation varied, but MKs location and association with the bilayer remained unchanged at physiological conditions regardless of side chain length. Combined, this manuscript provides fundamental information, both experimental and computational, on the location, association, and conformation of truncated MK homologues in model membrane environments relevant to bacterial energy production.

PtIV- or MoVI-substituted decavanadates inhibit the growth of Mycobacterium smegmatis.

Inhibitory effects of two monosubstituted decavanadates by PtIV in monoplatino(IV)nonavanadate(V) ([H2PtIVV9O28]5-, V9Pt), and by MoIV in monomolybdo(VI)nonavanadate(V) ([MoVIV9O28]5-,V9Mo) were investigated against the growth of Mycobacterium smegmatis with the EC50 values of 0.0048 mM and 0.015 mM, respectively. These compare to the reported inhibitory value for decavanadate ([V10O28]6-/[HV10O28]5-, V10) on Mycobacterium smegmatis (EC50 = 0.0037 mM). Time-dependent 51V NMR spectroscopic studies were carried out for all three polyanions in aqueous solution, biological medium (7H9), heated and non-heated supernatant to evaluate their stability in their respective media, monitor their hydrolysis to form various oxovanadates over time and calculate the EC50 values. These studies allow us to calculate adjusted and maximum EC50 for the polyoxovanadate (POV) present in solution at the beginning of the study when there is most intact anion in the media and thus the EC50 values represent the initial effects of the POVs. The results have shown that V10 is 1.3 times more potent than V9Pt and 4 times more potent than V9Mo, indicating that the inhibitory effects of monosubstituted polyanions are related to the V10 structure. We attributed the minor differences in the growth inhibitory effects to the differences in charges (5- vs 6-) of V9Pt and V9Mo compared to V10 and/or the differences in the chemical composition. We concluded that the potency of the growth inhibition by V10 is mainly due to the chemical properties of the vanadium and the decametalate's unique structure even though the presence of the Mycobacterium smegmatis facilitate hydrolysis of the anions. SYNOPSIS: Two decavanadate derivatives, monoplatino(IV)nonavanadate(V) ([H2PtIVV9O28]5-), monomolybdo(VI)nonavanadate(V) ([MoVIV9O28]5-) and decavanadate are more potent growth inhibitors of Mycobacterium smegmatis than monomeric vanadate. The spectroscopic characterization carried out in the growth medium led to the conclusion that both the decavanadate structure and its properties are important for its growth effects.

The Acid-Base Equilibrium of Pyrazinoic Acid Drives the pH Dependence of Pyrazinamide-Induced Mycobacterium tuberculosis Growth Inhibition.

Pyrazinamide, a first-line antibiotic used against Mycobacterium tuberculosis, has been shown to act in a pH-dependent manner in vitro. Why pyrazinamide, an antitubercle prodrug discovered more than 65 years ago, exhibits this pH-dependent activity was unclear. Upon entering mycobacterial cells, pyrazinamide is deamidated to pyrazinoate by an enzymatic process and exists in an acid-base equilibrium with pyrazinoic acid. Thus, the effects of total pyrazinoic acid (pyrazinoic acid + pyrazinoate) on M. tuberculosis growth, pH homeostasis, and proton motive force over a range of pH values found in host tissues were investigated. Although M. tuberculosis was able to maintain pH homeostasis over an external pH range of 7.0 to 5.5, total pyrazinoic acid induced growth inhibition increased as culture medium pH was decreased from 7.3 to 6.4. Consistent with growth inhibition, total pyrazinoic acid increased both acidification of the bacterial cytoplasm and dissipation of membrane potential as the environmental pH decreased when added to the bacterial suspensions. The results suggest pyrazinoic acid is the active form of the drug, which acts as an uncoupler of proton motive force, likely a protonophore, providing a mechanistic explanation for the pH dependence of the drug activity.

Mycobacterium tuberculosis Survival in J774A.1 Cells Is Dependent on MenJ Moonlighting Activity, Not Its Enzymatic Activity.

MenJ, a flavoprotein oxidoreductase, is responsible for the saturation of the ß-isoprene unit of mycobacterial menaquinone, resulting in the conversion of menaquinone with nine isoprene units (MK-9) to menaquinone with nine isoprene units where the double bond in the second unit is reduced [MK-9(II-H2)]. The hydrogenation of MK-9 increases the efficiency of the mycobacterial electron transport system, whereas the deletion of MenJ results in decreased survival of the bacteria inside J774A.1 macrophage-like cells but is not required for growth in culture. Thus, it was suggested that MenJ may represent a contextual drug target in M. tuberculosis, that is, a drug target that is valid only in the context of an infected macrophage. However, it was unclear if the conversion of MK-9 to MK-9(II-H2) or the MenJ protein itself was responsible for bacterial survival. In order to resolve this issue, a plasmid expressing folded, full-length, inactive MenJ was engineered. Primary sequence analysis data revealed that MenJ shares conserved FAD binding, NADH binding, and catalytic and C-terminal motifs with archaeal geranylgeranyl reductases. A MenJ mutant deficient in any one of these motifs is devoid of reductase activity. Therefore, point mutations of highly conserved amino acids in the conserved motifs were generated and the recombinant proteins were monitored for conformational changes by circular dichroism and oxidoreductase activity. The mutational analysis indicates that amino acids tryptophan 215 (W215) and cysteine 46 (C46) of M. tuberculosis MenJ, conserved in known archaeal geranylgeranyl reductases and putative menaquinone saturases, are essential to the hydrogenation of MK-9. The mutation of either C46 to serine (C46S) or W215 to leucine (W215L) in MenJ completely abolishes the catalytic activity in vitro, and menJ knockout strains of M. tuberculosis expressing either the C46S or W215L mutant protein are unable to convert MK-9 to MK-9(II-H2) but survive inside the J774A.1 cells. Thus, surprisingly, the survival of M. tuberculosis in J774A.1 cells is dependent on the expression of MenJ rather than its oxidoreductase activity, the conversion of MK-9 to MK-9(II-H2) as previously hypothesized. Overall, the current data suggest that MenJ is a moonlighting protein.

L,D-Transpeptidase Specific Probe Reveals Spatial Activity of Peptidoglycan Cross-Linking.

Peptidoglycan (PG) is a cross-linked, meshlike scaffold endowed with the strength to withstand the internal pressure of bacteria. Bacteria are known to heavily remodel their peptidoglycan stem peptides, yet little is known about the physiological impact of these chemical variations on peptidoglycan cross-linking. Furthermore, there are limited tools to study these structural variations, which can also have important implications on cell wall integrity and host immunity. Cross-linking of peptide chains within PG is an essential process, and its disruption thereof underpins the potency of several classes of antibiotics. Two primary cross-linking modes have been identified that are carried out by D,D-transpeptidases and L,D-transpeptidases (Ldts). The nascent PG from each enzymatic class is structurally unique, which results in different cross-linking configurations. Recent advances in PG cellular probes have been powerful in advancing the understanding of D,D-transpeptidation by Penicillin Binding Proteins (PBPs). In contrast, no cellular probes have been previously described to directly interrogate Ldt function in live cells. Herein, we describe a new class of Ldt-specific probes composed of structural analogs of nascent PG, which are metabolically incorporated into the PG scaffold by Ldts. With a panel of tetrapeptide PG stem mimics, we demonstrated that subtle modifications such as amidation of iso-Glu can control PG cross-linking. Ldt probes were applied to quantify and track the localization of Ldt activity in Enterococcus faecium, Mycobacterium smegmatis, and Mycobacterium tuberculosis. These results confirm that our Ldt probes are specific and suggest that the primary sequence of the stem peptide can control Ldt cross-linking levels. We anticipate that unraveling the interplay between Ldts and other cross-linking modalities may reveal the organization of the PG structure in relation to the spatial localization of cross-linking machineries.

Characterization of MenA (isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate isoprenyltransferase) from Mycobacterium tuberculosis.

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48-8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 µM, respectively. Ro 48-8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate.

Investigating Substrate Analogues for Mycobacterial MenJ: Truncated and Partially Saturated Menaquinones.

Menaquinones (MKs) are essential for electron transport in prokaryotes, and importantly, partially saturated MKs represent a novel virulence factor. However, little is known regarding how the degree of saturation in the isoprenyl side chain influences conformation or quinone redox potential. MenJ is an enzyme that selectively reduces the second isoprene unit on MK-9 and is contextually essential for the survival of Mycobacterium tuberculosis in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target for pathogenic mycobacteria. Therefore, fundamental information about the properties of this system is important, and we synthesized the simplest MKs, unsaturated MK-1 and the saturated analogue, MK-1(H2). Using two-dimensional nuclear magnetic resonance spectroscopy, we established that MK-1 and MK-1(H2) adopted similar folded-extended conformations (i.e., the isoprenyl side chain folds upward) in each solvent examined but the folded-extended conformations differed slightly between organic solvents. Saturation of the isoprenyl side chain slightly altered the MK-1 analogue conformation in each solvent. We used molecular mechanics to illustrate the MK-1 analogue conformations. The measured quinone redox potentials of MK-1 and MK-1(H2) differed between organic solvents (presumably due to differences in dielectric constants), and remarkably, an ∼20 mV semiquinone redox potential difference was observed between MK-1 and MK-1(H2) in pyridine, acetonitrile, and dimethyl sulfoxide, demonstrating that the degree of saturation in the isoprenyl side chain of MK-1 influences the quinone redox potential. Finally, MK-1 and MK-1(H2) interacted with Langmuir phospholipid monolayers and Aerosol-OT reverse micelle (RM) model membrane interfaces, where MK-1 adopted a slightly different folded conformation within the RM model membrane interface.

Mechanism of Fluorinated Anthranilate-Induced Growth Inhibition in Mycobacterium tuberculosis.

The biosynthesis of tryptophan in Mycobacterium tuberculosis is initiated by the transformation of chorismate to anthranilate, catalyzed by anthranilate synthase (TrpE/TrpG). Five additional enzymes are required to complete tryptophan biosynthesis. M. tuberculosis strains auxotrophic for tryptophan, an essential amino acid in the human diet, are avirulent. Thus, tryptophan synthesis in M. tuberculosis has been suggested as a potential drug target, and it has been reported that fluorinated anthranilate is lethal to the bacillus. Two mechanisms that could explain the cellular toxicity were tested: (1) the inhibition of tryptophan biosynthesis by a fluorinated intermediate or (2) formation of fluorotryptophan and its subsequent effects. Here, M. tuberculosis mc2 6230 cultures were treated with anthranilates fluorinated at positions 4, 5, and 6. These compounds inhibited bacterial growth on tryptophan-free media with 4-fluoroanthranilate being more potent than 5-fluoroanthranilate or 6-fluoroanthranilate. LC-MS based analysis of extracts from bacteria treated with these compounds did not reveal accumulation of any of the expected fluorinated intermediates in tryptophan synthesis. However, in all cases, significant levels of fluorotryptophan were readily observed, suggesting that the enzymes involved in the conversion of fluoro-anthranilate to fluorotryptophan were not being inhibited. Inclusion of tryptophan in cultures treated with the fluoro-anthranilates obviated the cellular toxicity. Bacterial growth was also inhibited in a dose-dependent manner by exposure to tryptophan substituted with fluorine at positions 5 or 6. Thus, the data suggest that fluorotryptophan rather than fluoro-anthranilate or intermediates in the synthesis of fluorotryptophan causes the inhibition of M. tuberculosis growth.

Decavanadate Inhibits Mycobacterial Growth More Potently Than Other Oxovanadates.

51V NMR spectroscopy is used to document, using speciation analysis, that one oxometalate is a more potent growth inhibitor of two Mycobacterial strains than other oxovanadates, thus demonstrating selectivity in its interaction with cells. Historically, oxometalates have had many applications in biological and medical studies, including study of the phase-problem in X-ray crystallography of the ribosome. The effect of different vanadate salts on the growth of Mycobacterium smegmatis (M. smeg) and Mycobacterium tuberculosis (M. tb) was investigated, and speciation was found to be critical for the observed growth inhibition. Specifically, the large orange-colored sodium decavanadate (V10 O 28 6 - ) anion was found to be a stronger inhibitor of growth of two mycobacterial species than the colorless oxovanadate prepared from sodium metavanadate. The vanadium(V) speciation in the growth media and conversion among species under growth conditions was monitored using 51V NMR spectroscopy and speciation calculations. The findings presented in this work is particularly important in considering the many applications of polyoxometalates in biological and medical studies, such as the investigation of the phase-problem in X-ray crystallography for the ribosome. The findings presented in this work investigate the interactions of oxometalates with other biological systems.

Stearoly-CoA desaturase 1 differentiates early and advanced dengue virus infections and determines virus particle infectivity.

Positive strand RNA viruses, such as dengue virus type 2 (DENV2) expand and structurally alter ER membranes to optimize cellular communication pathways that promote viral replicative needs. These complex rearrangements require significant protein scaffolding as well as changes to the ER chemical composition to support these structures. We have previously shown that the lipid abundance and repertoire of host cells are significantly altered during infection with these viruses. Specifically, enzymes in the lipid biosynthesis pathway such as fatty acid synthase (FAS) are recruited to viral replication sites by interaction with viral proteins and displayed enhanced activities during infection. We have now identified that events downstream of FAS (fatty acid desaturation) are critical for virus replication. In this study we screened enzymes in the unsaturated fatty acid (UFA) biosynthetic pathway and found that the rate-limiting enzyme in monounsaturated fatty acid biosynthesis, stearoyl-CoA desaturase 1 (SCD1), is indispensable for DENV2 replication. The enzymatic activity of SCD1, was required for viral genome replication and particle release, and it was regulated in a time-dependent manner with a stringent requirement early during viral infection. As infection progressed, SCD1 protein expression levels were inversely correlated with the concentration of viral dsRNA in the cell. This modulation of SCD1, coinciding with the stage of viral replication, highlighted its function as a trigger of early infection and an enzyme that controlled alternate lipid requirements during early versus advanced infections. Loss of function of this enzyme disrupted structural alterations of assembled viral particles rendering them non-infectious and immature and defective in viral entry. This study identifies the complex involvement of SCD1 in DENV2 infection and demonstrates that these viruses alter ER lipid composition to increase infectivity of the virus particles.

Mycobacterial MenJ: An Oxidoreductase Involved in Menaquinone Biosynthesis.

MenJ, annotated as an oxidoreductase, was recently demonstrated to catalyze the reduction (saturation) of a single double bond in the isoprenyl side-chain of mycobacterial menaquinone. This modification was shown to be essential for bacterial survival in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target in Mycobacterium tuberculosis and other pathogenic mycobacteria. Recombinant protein was expressed in a heterologous host, and the activity was characterized. Although highly regiospecific in vivo, the activity is not absolutely regiospecific in vitro; in addition, the enzyme is not specific for naphthoquinones vs benzoquinones. Coenzyme Q-1 (a benzoquinone, UQ-1) was used as the lipoquinone substrate, and NADH oxidation was followed spectrophotometrically as the activity readout. NADPH could not be substituted for NADH in the reaction mixture. The enzyme contains a FAD binding site that was 72% occupied in the purified recombinant protein. Enzyme activity was maximal at 37 °C and pH 7.0; addition of divalent cations, EDTA, and reducing agents such as dithiothreitol to the reaction mixture had no effect on activity. The addition of detergents did not stimulate activity, and addition of saturating levels of FAD had relatively little effect on the observed kinetic parameters. These properties allowed the development of a facile assay needed to study this potential drug target, which is also amenable to high throughput screening. The Km values for UQ-1 using recombinant MenJ from Mycobacterium smegmatis or M. tuberculosis without saturating concentrations of FAD were found to be 52 ± 9.6 and 44 ± 4.8 µM, respectively, while the KmNADH values were determined to be 59 ± 14 and 64 ± 15 µM. The Km for MK-1, the menaquinone analogue of UQ-1, using recombinant MenJ from M. tuberculosis without saturating concentrations of FAD but in the presence of 0.5% Tween 80 was shown to be 30 ± 2.9 µM. Thus, this is the first report of a kinetic characterization of a member of the geranylgeranyl reductase family of enzymes.

Structure Dependence of Pyridine and Benzene Derivatives on Interactions with Model Membranes.

Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, 1H one-dimensional NMR and 1H-1H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.