Combination Therapy with Gallium Protoporphyrin and Gallium Nitrate Exhibits Enhanced Antimicrobial Activity In Vitro and In Vivo against Methicillin-Resistant Staphylococcus aureus.

There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.

Synthesis and in vitro analysis of novel gallium tetrakis(4-methoxyphenyl)porphyrin and its long-acting nanoparticle as a potent antimycobacterial agent.

Bacterial heme uptake pathways offer a novel target for antimicrobial drug discovery. Recently, gallium (Ga) porphyrin complexes were found to be effective against mycobacterial heme uptake pathways. The goal of the current study is to build on this foundation and develop a new Ga(III) porphyrin and its nanoparticles, formulated by a single emulsion-evaporation technique to inhibit the growth of Mycobacterium avium complex (MAC) with enhanced properties. Gallium 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin chloride (GaMeOTP) was synthesized from 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin and GaCl3. GaMeOTP showed enhanced antimicrobial activity against MAC104 and some clinical M. avium isolates. The synthesized Ga(III) porphyrin antimicrobial activity resulted in the overproduction of reactive oxygen species. Our study also demonstrated that F127 nanoparticles encapsulating GaMeOTP exhibited a smaller size than GaTP nanoparticles and a better duration of activity in MAC-infected macrophages compared to the free GaMeOTP. The nanoparticles were trafficked to endosomal compartments within MAC-infected macrophages, likely contributing to the antimicrobial activity of the drug.

Synthesis, optimization, in vitro and in vivo study of bicyclic substituted amine as MenA inhibitor.

Menaquinone (MK) plays essential role in the electron transport chain (ETC), suggesting MK biosynthesis enzymes as potential targets for drug development. Previously, we demonstrated that Methicillin-resistant Staphylococcus aureus (MRSA) is susceptible to naphthol-based compounds which were developed by mimicking demethylmenaquinone, a product of MenA enzymatic reaction. Here, a series of new MenA inhibitors (4-19) were synthesized and evaluated as MenA inhibitors in this study. The inhibitors were designed to improve growth inhibitory activity against MRSA. Among the MenA inhibitors, bicyclic substituted amine 3 showed MIC of 3 µg/mL, and alkenyl substituted amine 11 showed MIC of 8 µg/mL against USA300. Regrowth of MRSA was observed on addition of MK when exposed to 8 µg/mL of inhibitor 11, supporting inhibition of MK biosynthesis. However, inhibitor 11 did not show efficacy in treating USA300 infected C. elegans up to 25 µg/mL concentration. However, all infected C. elegans survived when exposed to a bicyclic substituted amine 3. Hence, a bicyclic substituted amine was tested in mice for tolerability and biodistribution and observed 100% tolerable and high level of compound accumulation in lungs.

Iron/Heme Metabolism-Targeted Gallium(III) Nanoparticles Are Active against Extracellular and Intracellular Pseudomonas aeruginosa and Acinetobacter baumannii.

Iron/heme acquisition systems are critical for microorganisms to acquire iron from the human host, where iron sources are limited due to the nutritional immune system and insolubility of the ferric form of iron. Prior work has shown that a variety of gallium compounds can interfere with bacterial iron acquisition. This study explored the intra- and extracellular antimicrobial activities of gallium protoporphyrin (GaPP), gallium mesoporphyrin (GaMP), and nanoparticles encapsulating GaPP or GaMP against the Gram-negative pathogens Pseudomonas aeruginosa and Acinetobacter baumannii, including clinical isolates. All P. aeruginosa and A. baumannii isolates were susceptible to GaPP and GaMP, with MICs ranging from 0.5 to ∼32 µg/ml in iron-depleted medium. Significant intra- and extracellular growth inhibition was observed against P. aeruginosa cultured in macrophages at a gallium concentration of 3.3 µg/ml (5 µM) of all Ga(III) compounds, including nanoparticles. Nanoparticle formulations showed prolonged activity against both P. aeruginosa and A. baumannii in previously infected macrophages. When the macrophages were loaded with the nanoparticles 3 days prior to infection, there was a 5-fold decrease in growth of P. aeruginosa in the presence of single emulsion F127 copolymer nanoparticles encapsulating GaMP (eFGaMP). In addition, all Ga(III) porphyrins and nanoparticles showed significant intracellular and antibiofilm activity against both pathogens, with the nanoparticles exhibiting intracellular activity for 3 days. Ga nanoparticles also increased the survival rate of Caenorhabditis elegans nematodes infected by P. aeruginosa and A. baumannii Our results demonstrate that Ga nanoparticles have prolonged in vitro and in vivo activities against both P. aeruginosa and A. baumannii, including disruption of their biofilms.

Novel MenA Inhibitors Are Bactericidal against Mycobacterium tuberculosis and Synergize with Electron Transport Chain Inhibitors.

Mycobacterium tuberculosis is the leading cause of morbidity and death resulting from infectious disease worldwide. The incredible disease burden, combined with the long course of drug treatment and an increasing incidence of antimicrobial resistance among M. tuberculosis isolates, necessitates novel drugs and drug targets for treatment of this deadly pathogen. Recent work has produced several promising clinical candidates targeting components of the electron transport chain (ETC) of M. tuberculosis, highlighting this pathway's potential as a drug target. Menaquinone is an essential component of the M. tuberculosis ETC, as it functions to shuttle electrons through the ETC to produce the electrochemical gradient required for ATP production for the cell. We show that inhibitors of MenA, a component of the menaquinone biosynthetic pathway, are highly active against M. tuberculosis MenA inhibitors are bactericidal against M. tuberculosis under both replicating and nonreplicating conditions, with 10-fold higher bactericidal activity against nutrient-starved bacteria than against replicating cultures. MenA inhibitors have enhanced activity in combination with bedaquiline, clofazimine, and inhibitors of QcrB, a component of the cytochrome bc1 oxidase. Together, these data support MenA as a viable target for drug treatment against M. tuberculosis MenA inhibitors not only kill M. tuberculosis in a variety of physiological states but also show enhanced activity in combination with ETC inhibitors in various stages of clinical trial testing.

In Vitro Efficacy of Free and Nanoparticle Formulations of Gallium(III) meso-Tetraphenylporphyrine against Mycobacterium avium and Mycobacterium abscessus and Gallium Biodistribution in Mice.

The nontuberculous mycobacterial (NTM) pathogens, M. avium complex (MAC) and M. abscessus, can result in severe pulmonary infections. Current antibiotics confront significant challenges for treatment of these NTM infections due to emerging multidrug-resistance. Thus, development of new antibiotics targeted against these agents is needed. We examined the inhibitory activities of Ga(NO3)3, GaCl3, gallium meso-tetraphenylporphyrine (GaTP), and gallium nanoparticles (GaNP) against intra- and extracellular M. avium and M. abscessus. GaTP, an analogue of natural heme, inhibited growth of both M. avium and M. abscessus with MICs in Fe-free 7H9 media of 0.5 and 2 µg/mL, respectively. GaTP was more active than Ga(NO3)3 and GaCl3. Ga(NO3)3 and GaCl3 were not as active in Fe-rich media compared to Fe-free media. However, GaTP was much less impacted by exogenous Fe, with MICs against M. avium and M. abscessus of 2 and 4 µg/mL, respectively, in 7H9 OADC media (Fe rich). Confocal microscopy showed that GaNP penetrates the M. avium cell wall. As assessed by determining colony forming units, GaNP inhibited the growth of NTM growing in THP-1 macrophages up to 15 days after drug-loading of the cells, confirming a prolonged growth inhibitory activity of the GaNP. Biodistribution studies of GaNP conducted in mice showed that intraperitoneal injection is more effective than intramuscular injection in delivering Ga(III) into lung tissue. GaTP exhibits potential as a lead compound for development of anti-NTM agents that target heme-bound iron uptake mechanisms by mycobacteria and inhibit growth by disrupting mycobacterial iron acquisition/utilization.

Ga(III) Nanoparticles Inhibit Growth of both Mycobacterium tuberculosis and HIV and Release of Interleukin-6 (IL-6) and IL-8 in Coinfected Macrophages.

Treatment of individuals coinfected with human immunodeficiency virus (HIV) type 1 and Mycobacterium tuberculosis is challenging due to the prolonged treatment requirements, drug toxicity, and emergence of drug resistance. Mononuclear phagocytes (MP; macrophages) are one of the natural reservoirs for both HIV and M. tuberculosis Here, the treatment of HIV and M. tuberculosis coinfection was studied by preloading human macrophages with MP-targeted gallium (Ga) nanoparticles to limit subsequent simultaneous infection with both HIV and M. tuberculosis Ga nanoparticles provided sustained drug release for 15 days and significantly inhibited the replication of both HIV and M. tuberculosis Addition of Ga nanoparticles to MP already infected with M. tuberculosis or HIV resulted in a significant decrease in the magnitude of these infections, but the magnitude was less than that achieved with nanoparticle preloading of the MP. In addition, macrophages that were coinfected with HIV and M. tuberculosis and that were loaded with Ga nanoparticles reduced the levels of interleukin-6 (IL-6) and IL-8 secretion for up to 15 days after drug loading. Ga nanoparticles also reduced the levels of IL-6 and IL-8 secretion by ionomycin- and lipopolysaccharide-induced macrophages, likely by modulating the IκB kinase-ß/NF-κB pathway. Delivery of Ga nanoparticles to macrophages is a potent long-acting approach for suppressing HIV and M. tuberculosis coinfection of macrophages in vitro and sets the stage for the development of new approaches to the treatment of these important infections.

Development of potential broad spectrum antimicrobials using C2-symmetric 9-fluorenone alkyl amine.

DNA-dependent RNA primase is essential for de novo primer synthesis during DNA replication in all living organisms. Bacterial DnaG primase is an attractive target for inhibition because it is essential, low in copy number and structurally distinct from eukaryotic and archaeal primases. DnaG primase is sensitive to known inhibitors including suramin and doxorubicin. Recently, tilorone was discovered by high throughput screening to be an inhibitor of Bacillus anthracis primase DnaG but it failed to reduce the growth of B. anthracis in vitro. In this study we determined that tilorone also inhibited DnaG primase from Staphylococcus aureus. C2-Symmetric fluorenone-based compounds, similar to tilorone chemical structure were synthesized and tested to identify potential lead compounds that inhibit bacterial growth in B. anthracis, MRSA and Burkholderia thailandensis. These compounds were evaluated by determining the minimum inhibitory concentration (MIC) against several different bacterial species which demonstrated 17.5 and 16 µg/ml MIC profiles. Importantly, some of the fluorenone-based compounds with a long carbon chain showed a relatively low MIC against B. anthracis, S. aureus, MRSA, Francisella tularensis, and B. thailandensis, suggesting it may be a promising lead compound.

Long-acting antituberculous therapeutic nanoparticles target macrophage endosomes.

Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 µg/10(6) cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 µg/10(6) cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.

Biochemical and biologic characterization of exosomes and microvesicles as facilitators of HIV-1 infection in macrophages.

Exosomes and microvesicles (MV) are cell membranous sacs originating from multivesicular bodies and plasma membranes that facilitate long-distance intercellular communications. Their functional biology, however, remains incompletely understood. Macrophage exosomes and MV isolated by immunoaffinity and sucrose cushion centrifugation were characterized by morphologic, biochemical, and molecular assays. Lipidomic, proteomic, and cell biologic approaches uncovered novel processes by which exosomes and MV facilitate HIV-1 infection and dissemination. HIV-1 was "entrapped" in exosome aggregates. Robust HIV-1 replication followed infection with exosome-enhanced fractions isolated from infected cell supernatants. MV- and exosome-facilitated viral infections are affected by a range of cell surface receptors and adhesion proteins. HIV-1 containing exosomes readily completed its life cycle in human monocyte-derived macrophages but not in CD4(-) cells. The data support a significant role for exosomes as facilitators of viral infection.

Synthesis of azide derivative and discovery of glyoxalase pathway inhibitor against pathogenic bacteria.

A glyoxalase inhibitor was synthesized and tested against Staphylococcus aureus for first time and showed MIC90 of 20 µg/ml. Henceforth, we synthesized unnatural azide derivative of the same inhibitor to improve the biological activity. In that order, an azide carboxylate was synthesized from dimethyl tartrate by tosylation and azide substitution. The synthesized, azide compound was coupled with glutathione derivative in high yield and tested against S. aureus and showed improved MIC90 of 5 µg/ml. In general, it can be also easily converted to unnatural ß-amino acid in good yield. The shown methodology will be extended to study induced suicide in Burkholderia mallei, Francisella tularensis and Mycobacterium tuberculosis in future.

In Vitro and In Vivo Antimicrobial Activity of an Oxidative Stress-Mediated Bicyclic Menaquinone Biosynthesis Inhibitor against MRSA.

Menaquinone (MK) is an essential component in the oxidative phosphorylation pathway of Gram-positive bacteria. Drugs targeting enzymes involved in MK biosynthesis can prevent electron transfer, which leads to ATP starvation and thereby death of microorganisms. Previously, we reported a series of MenA inhibitors and demonstrated their antimicrobial activity against Gram-positive bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA) and mycobacteria. These inhibitors were developed by mimicking demethylmenaquinone, a product of MenA enzymatic reaction in MK biosynthesis. In this study, compound NM4, MK biosynthesis inhibitor, inhibited the formation of MRSA biofilm and it was screened against 1952 transposon mutants to elucidate mechanisms of action; however, no resistant mutants were found. Also, compound NM4 induced the production of reactive oxygen species (ROS) by blocking electron transfer in the oxidative phosphorylation pathway as observed by MRSA growth recovery using various ROS scavengers. An oxygen consumption assay also showed that NM4 blocks the oxygen consumption by MRSA, but the addition of menaquinone (MK) restores growth of MRSA. The NM4-treated MRSA induced the expression of catalase by more than 25%, as quantified by the native gel. A pulmonary murine model exhibited that NM4 significantly reduced bacterial lung load in mice without toxicity. An NM4-resistant USA300 strain was developed to attempt to identify the targets participating in the mechanism of resistance. Our results support that respiration and oxidative phosphorylation are potential targets for developing antimicrobial agents against MRSA. Altogether, our findings suggest the potential use of MK biosynthesis inhibitors as an effective antimicrobial agent against MRSA.

Investigating Novel IspE Inhibitors of the MEP Pathway in Mycobacterium.

In a recent effort to mitigate harm from human pathogens, many biosynthetic pathways have been extensively evaluated for their ability to inhibit pathogen growth and to determine drug targets. One of the important products/targets of such pathways is isopentenyl diphosphate. Isopentenyl diphosphate is the universal precursor of isoprenoids, which are essential for the normal functioning of microorganisms. In general, two biosynthetic pathways lead to the formation of isopentenyl diphosphate: (1) the mevalonate pathway in animals; and (2) the non-mevalonate or methylerythritol phosphate (MEP) in many bacteria, and some protozoa and plants. Because the MEP pathway is not found in mammalian cells, it is considered an attractive target for the development of antimicrobials against a variety of human pathogens, including Mycobacterium tuberculosis (M.tb). In the MEP pathway, 4-diphosphocytidyl-2-c-methyl-d-erythritol kinase (IspE) phosphorylates 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDPME2P). A virtual high-throughput screening against 15 million compounds was carried out by docking IspE protein. We identified an active heterotricyclic compound which showed enzymatic activity; namely, IC50 of 6 µg/mL against M.tb IspE and a MIC of 12 µg/mL against M.tb (H37Rv). Hence, we designed and synthesized similar new heterotricyclic compounds and tested them against mycobacterium, observing a MIC of 5 µg/mL against M. avium. This study will provide the critical insight necessary for developing novel antimicrobials that target the MEP pathways in pathogens.

Development of Gallium(III) as an Antimicrobial Drug Targeting Pathophysiologic Iron Metabolism of Human Pathogens.

The treatment of infections is becoming more difficult due to emerging resistance of pathogens to existing drugs. As such, alternative druggable targets, particularly those that are essential for microbe viability and thus make it harder to develop resistance, are desperately needed. In turn, once identified, safe and effective agents that disrupt these targets must be developed. Microbial acquisition and use of iron is a promising novel target for antimicrobial drug development. In this Review we look at the various facets of iron metabolism critical to human infection with pathogenic microbes and the various ways in which it can be targeted, altered, disrupted, and taken advantage of to halt or eliminate microbial infections. Although a variety of agents will be touched upon, the primary focus will be on the potential use of one or more gallium complexes as a new class of antimicrobial agents. In vitro and in vivo data on the activity of gallium complexes against a variety of pathogens including ESKAPE pathogens, mycobacteria, emerging viruses, and fungi will be discussed in detail, as well as pharmacokinetics, novel formulations and delivery approaches, and early human clinical results.

Effect of Cannabidiol on the Neural Glyoxalase Pathway Function and Longevity of Several C. elegans Strains Including a C. elegans Alzheimer's Disease Model.

Cannabidiol is a nonpsychoactive phytocannabinoid produced by the Cannabis sativa plant and possesses a wide range of pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective activities. Cannabidiol functions in a neuroprotective manner, in part through the activation of cellular antioxidant pathways. The glyoxalase pathway detoxifies methylglyoxal, a highly reactive metabolic byproduct that can accumulate in the brain, and contributes to the severity of neurodegenerative diseases, including Alzheimer's disease. While cannabidiol's antioxidant properties have been investigated, it is currently unknown how it may modulate the glyoxalase pathway. In this research paper, we examine the effects of Cannabidiol on cerebellar neurons and in several Caenorhabditis elegans strains. We determined that a limited amount of Cannabidiol can prevent methylglyoxal-mediated cellular damage through enhancement of the neural glyoxalase pathway and extend the lifespan and survival of C. elegans, including a transgenic C. elegans strain modeling Alzheimer's disease.

Combining Gallium Protoporphyrin and Gallium Nitrate Enhances In Vitro and In Vivo Efficacy against Pseudomonas aeruginosa: Role of Inhibition of Bacterial Antioxidant Enzymes and Resultant Increase in Cytotoxic Reactive Oxygen Species.

Pseudomonas aeruginosa is a highly antibiotic-resistant opportunistic pathogenic bacteria that is responsible for thousands of deaths each year. Infections with P. aeruginosa disproportionately impact individuals with compromised immune systems as well as cystic fibrosis patients, where P. aeruginosa lung infection is a leading cause of morbidity and mortality. In previous work, we showed that a combination of gallium (Ga) nitrate and Ga protoporphyrin worked well in several bacterial infection models but its mechanism of action (MOA) is unknown. In the current work, we have investigated the MOA of Ga combination therapy in P. aeruginosa and its analysis in the in vivo model. In P. aeruginosa treated with Ga combination therapy, we saw a decrease in catalase and superoxide dismutase (SOD) activity, key antioxidant enzymes, which could correlate with a higher potential for oxidative stress. Consistent with this hypothesis, we found that, following combination therapy, P. aeruginosa demonstrated higher levels of reactive oxygen species, as measured using the redox-sensitive fluorescent probe, H2DCFDA. We also saw that the Ga combination therapy killed phagocytosed bacteria inside macrophages in vitro. The therapy with low dose was able to fully prevent mortality in a murine model of P. aeruginosa lung infection and also significantly reduced lung damage. These results support our previous data that Ga combination therapy acts synergistically to kill P. aeruginosa, and we now show that this may occur through increasing the organism's susceptibility to oxidative stress. Ga combination therapy also showed itself to be effective at treating infection in a murine pulmonary-infection model.

Nanoparticulate ß-Cyclodextrin with Gallium Tetraphenylporphyrin Demonstrates in Vitro and in Vivo Antimicrobial Efficacy against Mycobacteroides abscessus and Mycobacterium avium.

The emergence of drug-resistant pathogens causes the greatest challenge for drug development research. Recently, gallium(III)-based compounds have received great attention as novel antimicrobial agents against drug-resistant pathogens. Here, we synthesized a new ß-cyclodextrin Ga nanoparticle (CDGaTP) using Ga tetraphenylporphyrin (GaTP, a hemin analogue) and ß-cyclodextrin. The newly synthesized nanoparticle was nontoxic and efficient at a single dose, showing sustained drug release for 15 days in vitro. CDGaTP's activity with transferrin or lactoferrin was tested, and synergism in activity was observed against nontuberculosis mycobacteria (NTM), Mycobacterium avium (M. avium), and Mycobacteroides abscessus. Human serum albumin (HSA) decreased the efficacy of both GaTP and CDGaTP in a concentration-dependent manner. The NTMs incubated with GaTP or CDGaTP significantly produced reactive oxygen species (ROS), indicating potential inhibition of antioxidant enzymes, such as catalase. The single-dose CDGaTP displayed a prolonged intracellular inhibitory activity in an in vitro macrophage infection model against both NTMs. In addition, CDGaTP, not GaTP, was effective in a murine lung M. avium infection model when delivered via intranasal administration. These results suggest that CDGaTP provides new opportunities for the development of gallium-porphyrin based antibiotics.

DosS responds to a reduced electron transport system to induce the Mycobacterium tuberculosis DosR regulon.

The DosR regulon in Mycobacterium tuberculosis is involved in respiration-limiting conditions, its induction is controlled by two histidine kinases, DosS and DosT, and recent experimental evidence indicates DosS senses either molecular oxygen or a redox change. Under aerobic conditions, induction of the DosR regulon by DosS, but not DosT, was observed after the addition of ascorbate, a powerful cytochrome c reductant, demonstrating that DosS responds to a redox signal even in the presence of high oxygen tension. During hypoxic conditions, regulon induction was attenuated by treatment with compounds that occluded electron flow into the menaquinone pool or decreased the size of the menaquinone pool itself. Increased regulon expression during hypoxia was observed when exogenous menaquinone was added, demonstrating that the menaquinone pool is a limiting factor in regulon induction. Taken together, these data demonstrate that a reduced menaquinone pool directly or indirectly triggers induction of the DosR regulon via DosS. Biochemical analysis of menaquinones upon entry into hypoxic/anaerobic conditions demonstrated the disappearance of the unsaturated species and low-level maintenance of the mono-saturated menaquinone. Relative to the unsaturated form, an analog of the saturated form is better able to induce signaling via DosS and rescue inhibition of menaquinone synthesis and is less toxic. The menaquinone pool is central to the electron transport system (ETS) and therefore provides a mechanistic link between the respiratory state of the bacilli and DosS signaling. Although this report demonstrates that DosS responds to a reduced ETS, it does not rule out a role for oxygen in silencing signaling.

Menaquinone synthesis is critical for maintaining mycobacterial viability during exponential growth and recovery from non-replicating persistence.

Understanding the basis of bacterial persistence in latent infections is critical for eradication of tuberculosis. Analysis of Mycobacterium tuberculosis mRNA expression in an in vitro model of non-replicating persistence indicated that the bacilli require electron transport chain components and ATP synthesis for survival. Additionally, low microM concentrations of aminoalkoxydiphenylmethane derivatives inhibited both the aerobic growth and survival of non-replicating, persistent M. tuberculosis. Metabolic labelling studies and quantification of cellular menaquinone levels suggested that menaquinone synthesis, and consequently electron transport, is the target of the aminoalkoxydiphenylmethane derivatives. This hypothesis is strongly supported by the observations that treatment with these compounds inhibits oxygen consumption and that supplementation of growth medium with exogenous menaquinone rescued both growth and oxygen consumption of treated bacilli. In vitro assays indicate that the aminoalkoxydiphenylmethane derivatives specifically inhibit MenA, an enzyme involved in the synthesis of menaquinone. Thus, the results provide insight into the physiology of mycobacterial persistence and a basis for the development of novel drugs that enhance eradication of persistent bacilli and latent tuberculosis.

Neural Glyoxalase Pathway Enhancement by Morin Derivatives in an Alzheimer's Disease Model.

The glyoxalase pathway (GP) is an antioxidant defense system that detoxifies metabolic byproduct methylglyoxal (MG). Through sequential reactions, reduced glutathione (GSH), glyoxalase I (glo-1), and glyoxalase II (glo-2) convert MG into d-lactate. Spontaneous reactions involving MG alter the structure and function of cellular macromolecules through the formation of inflammatory advanced glycation endproducts (AGEs). Accumulation of MG and AGEs in neural cells contributes to oxidative stress (OS), a state of elevated inflammation commonly found in neurodegenerative diseases including Alzheimer's disease (AD). Morin is a common plant-produced flavonoid polyphenol that exhibits the ability to enhance the GP-mediated detoxification of MG. We hypothesize that structural modifications to morin will improve its inherent GP enhancing ability. Here we synthesized a morin derivative, dibromo-morin (DBM), formulated a morin encapsulated nanoparticle (MNP), and examined their efficacy in enhancing neural GP activity. Cultured mouse primary cerebellar neurons and Caenorhabditis elegans were induced to a state of OS with MG and treated with morin, DBM, and MNP. Results indicated the morin derivatives were more effective compared to the parent compound in neural GP enhancement and preventing MG-mediated OS in an AD model.