Antimicrobial Activity of Gallium(III) Compounds: Pathogen-Dependent Targeting of Multiple Iron/Heme-Dependent Biological Processes.

Metals play vital roles in biological systems, with iron/heme being essential for cellular and metabolic functions necessary for survival and/or virulence in many bacterial pathogens. Given the rise of bacterial resistance to current antibiotics, there is an urgent need for the development of non-toxic and novel antibiotics that do not contribute to resistance to other antibiotics. Gallium, which mimics iron, has emerged as a promising antimicrobial agent, offering a novel approach to combat bacterial infections. Gallium does not have any known functions in biological systems. Gallium exerts its effects primarily by replacing iron in redox enzymes, effectively inhibiting bacterial growth by targeting multiple iron/heme-dependent biological processes and suppressing the development of drug resistance. The aim of this review is to highlight recent findings on the mechanisms of action of gallium and provide further insights into the development of gallium-based compounds. Understanding the mechanisms underlying gallium's biological activities is crucial for designing drugs that enhance their therapeutic therapies while minimizing side effects, offering promising avenues for the treatment of infectious diseases.

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.

Synthesis and Biological Evaluation of Salicylic Acid Analogues of Celecoxib as a New Class of Selective Cyclooxygenase-1 Inhibitor.

A series of salicylic acid analogues of celecoxib where the phenylsulfonamide moiety in the structure of celecoxib is replaced by salicylic acid moiety was synthesized and tested for in vitro cyclooxygenase (COX)-1 and COX-2 enzyme inhibition. Among the series, 5-substituted-2-hydroxy-benzoic acid analogues (7a-7h) generally showed better inhibitory activities on both enzymes than 4-substituted-2-hydroxy-benzoic acid analogues (12a-12h). In particular, the chloro analogue 7f which had the highest inhibitory effect (IC50 = 0.0057 µM) to COX-1 with excellent COX-1 selectivity (SI = 768) can be classified as a new potent and selective COX-1 inhibitor. The high inhibitory potency of 7f was rationalized through the docking simulation of this analogue in the active site of COX-1 enzyme.

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.

Regio- and chemoselective immobilization of proteins on gold surfaces.

Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by ß-mercaptoethanol than alkane-thiol SAMs.

δ-Deuterium isotope effects as probes for transition-state structures of isoprenoid substrates.

The biosynthetic pathways to isoprenoid compounds involve transfer of the prenyl moiety in allylic diphosphates to electron-rich (nucleophilic) acceptors. The acceptors can be many types of nucleophiles, while the allylic diphosphates only differ in the number of isoprene units and stereochemistry of the double bonds in the hydrocarbon moieties. Because of the wide range of nucleophilicities of naturally occurring acceptors, the mechanism for prenyltransfer reactions may be dissociative or associative with early to late transition states. We have measured δ-secondary kinetic isotope effects operating through four bonds for substitution reactions with dimethylallyl derivatives bearing deuterated methyl groups at the distal (C3) carbon atom in the double bond under dissociative and associative conditions. Computational studies with density functional theory indicate that the magnitudes of the isotope effects correlate with the extent of bond formation between the allylic moiety and the electron-rich acceptor in the transition state for alkylation and provide insights into the structures of the transition states for associative and dissociative alkylation reactions.

Novel route to chaetomellic acid A and analogues: serendipitous discovery of a more competent FTase inhibitor.

A new practical route to chaetomellic acid A (ACA), based on the copper catalysed radical cyclization (RC) of (Z)-3-(2,2-dichloropropanoyl)-2-pentadecylidene-1,3-thiazinane, is described. Remarkably, the process entailed: (i) a one-pot preparation of the intermediate N-α-perchloroacyl-2-(Z)-alkyliden-1,3-thiazinanes starting from N-(3-hydroxypropyl)palmitamide, (ii) a two step smooth transformation of the RC products into ACA and (iii) only one intermediate chromatographic purification step. The method offers a versatile approach to the preparation of ACA analogues, through the synthesis of an intermediate maleic anhydride with a vinylic group at the end of the aliphatic tail, a function that can be transformed through a thiol-ene coupling. Serendipitously, the disodium salt of 2-(9-(butylthio)nonyl)-3-methylmaleic acid, that we prepared as a representative sulfurated ACA analogue, was a more competent FTase inhibitor than ACA. This behaviour was analysed by a molecular docking study.

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.

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.

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.

Gallium Porphyrin and Gallium Nitrate Synergistically Inhibit Mycobacterial Species by Targeting Different Aspects of Iron/Heme Metabolism.

There is an urgent need for new effective and safe antibiotics active against pathogenic mycobacterial species. Gallium (Ga) nitrate (Ga(NO3)3) and Ga porphyrin (GaPP) have each been shown to inhibit the growth of a variety of mycobacterial species. The Ga(III) ion derived from Ga(NO3)3 has the potential to disrupt the mycobacterial Fe(III) uptake mechanisms and utilization, including replacing iron (Fe) in the active site of enzymes, resulting in the disruption of function. Similarly, noniron metalloporphyrins such as heme mimetics, which can be transported across the bacterial membrane via heme-uptake pathways, would potentially block the acquisition of iron-containing heme and bind to heme-utilizing proteins, making them nonfunctional. Given that they likely act on different aspects of mycobacterial Fe metabolism, the efficacy of combining Ga(NO3)3 and GaPP was studied in vitro against Mycobacterium avium, Mycobacterium abscessus, and Mycobacterium tuberculosis (M. tb). The combination was then assessed in vivo in a murine pulmonary infection model of M. abscessus. We observed that Ga(NO3)3 in combination with GaPP exhibited synergistic inhibitory activity against the growth of M. avium, M. tb, and M. abscessus, being most active against M. abscessus. Activity assays indicated that Ga(NO3)3 and GaPP inhibited both catalase and aconitase at high concentrations. However, the combination showed a synergistic effect on the aconitase activity of M. abscessus. The Ga(NO3)3/GaPP combination via intranasal administration showed significant antimicrobial activity in mice infected with M. abscessus. M. abscessus CFU from the lungs of the Ga(NO3)3/GaPP-treated mice was significantly less compared to that of nontreated or single Ga(III)-treated mice. These findings suggest that combinations of different Ga(III) compounds can synergistically target multiple iron/heme-utilizing mycobacterial enzymes. The results support the potential of combination Ga therapy for development against mycobacterial pathogens.