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PLoS Neglected Tropical Diseases Dec 2021Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. The...
Studies of drug resistance in the protozoan parasites of the genus Leishmania have been helpful in revealing biochemical pathways as potential drug targets. The chlorinated glutamine analogue acivicin has shown good activity against Leishmania cells and was shown to target several enzymes containing amidotransferase domains. We selected a Leishmania tarentolae clone for acivicin resistance. The genome of this resistant strain was sequenced and the gene coding for the amidotransferase domain-containing GMP synthase was found to be amplified. Episomal expression of this gene in wild-type L. tarentolae revealed a modest role in acivicin resistance. The most prominent defect observed in the resistant mutant was reduced uptake of glutamate, and through competition experiments we determined that glutamate and acivicin, but not glutamine, share the same transporter. Several amino acid transporters (AATs) were either deleted or mutated in the resistant cells. Some contributed to the acivicin resistance phenotype although none corresponded to the main glutamate transporter. Through sequence analysis one AAT on chromosome 22 corresponded to the main glutamate transporter. Episomal expression of the gene coding for this transporter in the resistant mutant restored glutamate transport and acivicin susceptibility. Its genetic knockout led to reduced glutamate transport and acivicin resistance. We propose that acivicin binds covalently to this transporter and as such leads to decreased transport of glutamate and acivicin thus leading to acivicin resistance.
Topics: Amino Acid Transport Systems; Antiprotozoal Agents; Biological Transport; Drug Resistance; Glutamic Acid; Humans; Isoxazoles; Leishmania; Leishmaniasis; Protozoan Proteins
PubMed: 34914690
DOI: 10.1371/journal.pntd.0010046 -
Nature Immunology May 2020Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells...
Oxidative stress is a central part of innate immune-induced neurodegeneration. However, the transcriptomic landscape of central nervous system (CNS) innate immune cells contributing to oxidative stress is unknown, and therapies to target their neurotoxic functions are not widely available. Here, we provide the oxidative stress innate immune cell atlas in neuroinflammatory disease and report the discovery of new druggable pathways. Transcriptional profiling of oxidative stress-producing CNS innate immune cells identified a core oxidative stress gene signature coupled to coagulation and glutathione-pathway genes shared between a microglia cluster and infiltrating macrophages. Tox-seq followed by a microglia high-throughput screen and oxidative stress gene network analysis identified the glutathione-regulating compound acivicin, with potent therapeutic effects that decrease oxidative stress and axonal damage in chronic and relapsing multiple sclerosis models. Thus, oxidative stress transcriptomics identified neurotoxic CNS innate immune populations and may enable discovery of selective neuroprotective strategies.
Topics: Animals; Antioxidants; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gene Expression Profiling; Gene Regulatory Networks; High-Throughput Screening Assays; Humans; Immunity, Innate; Isoxazoles; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microglia; Multiple Sclerosis; Neurogenic Inflammation; Oxidative Stress; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 32284594
DOI: 10.1038/s41590-020-0654-0 -
Molecules (Basel, Switzerland) Apr 2023Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we...
Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we investigated the significance of chirality for nature-inspired 3-Br-acivicin (3-BA) and its derivatives. The three unnatural isomers of 3-BA and its ester and amide derivatives were prepared and characterized for their antimalarial activity. Only the (5, α) isomers displayed significant antiplasmodial activity, revealing that their uptake might be mediated by the L-amino acid transport system, which is known to mediate the acivicin membrane's permeability. In addition, we investigated the inhibitory activity towards glyceraldehyde 3-phosphate dehydrogenase (GAPDH) since it is involved in the multitarget mechanism of action of 3-BA. Molecular modeling has shed light on the structural and stereochemical requirements for an efficient interaction with GAPDH, leading to covalent irreversible binding and enzyme inactivation. While stereochemistry affects the target binding only for two subclasses (- and -), it leads to significant differences in the antimalarial activity for all subclasses, suggesting that a stereoselective uptake might be responsible for the enhanced biological activity of the (5, α) isomers.
Topics: Antimalarials; Isoxazoles; Plasmodium falciparum; Models, Molecular
PubMed: 37049935
DOI: 10.3390/molecules28073172 -
Proceedings of the National Academy of... Feb 2019Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes...
Glutamate is the most abundant excitatory neurotransmitter, present at the bulk of cortical synapses, and participating in many physiologic and pathologic processes ranging from learning and memory to stroke. The tripeptide, glutathione, is one-third glutamate and present at up to low millimolar intracellular concentrations in brain, mediating antioxidant defenses and drug detoxification. Because of the substantial amounts of brain glutathione and its rapid turnover under homeostatic control, we hypothesized that glutathione is a relevant reservoir of glutamate and could influence synaptic excitability. We find that drugs that inhibit generation of glutamate by the glutathione cycle elicit decreases in cytosolic glutamate and decreased miniature excitatory postsynaptic potential (mEPSC) frequency. In contrast, pharmacologically decreasing the biosynthesis of glutathione leads to increases in cytosolic glutamate and enhanced mEPSC frequency. The glutathione cycle can compensate for decreased excitatory neurotransmission when the glutamate-glutamine shuttle is inhibited. Glutathione may be a physiologic reservoir of glutamate neurotransmitter.
Topics: Animals; Cells, Cultured; Excitatory Postsynaptic Potentials; Glutamic Acid; Glutathione; Homeostasis; Neurons; Rats, Sprague-Dawley; Synapses; Synaptic Transmission
PubMed: 30692251
DOI: 10.1073/pnas.1817885116 -
Translational Vision Science &... Jul 2020To investigate whether human donor lenses are capable of exporting reduced glutathione.
PURPOSE
To investigate whether human donor lenses are capable of exporting reduced glutathione.
METHODS
Human lenses of varying ages were cultured in artificial aqueous humor for 1 hour under hypoxic conditions to mimic the physiologic environment and reduced glutathione (GSH) and oxidized glutathione (GSSG) levels measured in the media and in the lens.
RESULTS
Human donor lenses released both GSH and GSSG into the media. Donor lenses cultured in the presence of acivicin, a γ-glutamyltranspeptidase inhibitor, exhibited a significant increase in GSSG levels ( < 0.05), indicating that GSSG undergoes degradation into its constituent amino acids. Screening of GSH/GSSG efflux transporters revealed Mrp1, Mrp4, and Mrp5 to be present at the transcript level, but only Mrp5 was expressed at the protein level. Blocking Mrp5 function with the Mrp inhibitor MK571 led to a significant decrease in GSSG efflux ( < 0.05), indicating that Mrp5 is likely to be involved in mediating GSSG efflux. Measurements of efflux from the anterior and posterior surface of the lens revealed that GSH and GSSG efflux occurs at both surfaces but predominantly at the anterior surface.
CONCLUSIONS
Human lenses export GSH and GSSG into the surrounding ocular humors, which can be recycled by the lens to maintain intracellular GSH homeostasis or used by neighboring tissues to maintain GSH levels.
TRANSLATIONAL RELEVANCE
Early removal of a clear lens, as occurs to treat myopia and presbyopia, would eliminate this GSH reservoir and reduce the supply of GSH to other tissues, which, over time, may have clinical implications for the progression of other ocular diseases associated with oxidative stress.
Topics: Biological Transport; Glutathione; Glutathione Disulfide; Humans; Lens, Crystalline; Oxidative Stress
PubMed: 32855883
DOI: 10.1167/tvst.9.8.37 -
Pharmaceuticals (Basel, Switzerland) Aug 2021Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel...
Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel mechanisms of action are urgently needed. Previously, we identified CACNA2D1 as a novel modulator of intraocular pressure (IOP) and demonstrated that a topically applied CACNA2D1 antagonist-pregabalin (PRG)-lowered IOP in a dose-dependent manner. To further validate this novel IOP modulator as a drug target for IOP-lowering pharmaceutics, a homology model of CACNA2D1 was built and docked against the NCI library, which is one of the world's largest and most diverse compound libraries of natural products. Acivicin and zoledronic acid were identified using this method and together with PRG were tested for their plausible IOP-lowering effect on Dutch belted rabbits. Although they have inferior potency to PRG, both of the other compounds lower IOP, which in turn validates CACNA2D1 as a valuable drug target in treating glaucoma.
PubMed: 34577587
DOI: 10.3390/ph14090887 -
BMC Microbiology Jun 2021The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although...
BACKGROUND
The global rise in the incidence of non-tuberculosis mycobacterial infections is of increasing concern due their high levels of intrinsic antibiotic resistance. Although integrated viral genomes, called prophage, are linked to increased antibiotic resistance in some bacterial species, we know little of their role in mycobacterial drug resistance.
RESULTS
We present here for the first time, evidence of increased antibiotic resistance and expression of intrinsic antibiotic resistance genes in a strain of Mycobacterium chelonae carrying prophage. Strains carrying the prophage McProf demonstrated increased resistance to amikacin. Resistance in these strains was further enhanced by exposure to sub-inhibitory concentrations of the antibiotic, acivicin, or by the presence of a second prophage, BPs. Increased expression of the virulence gene, whiB7, was observed in strains carrying both prophages, BPs and McProf, relative to strains carrying a single prophage or no prophages.
CONCLUSIONS
This study provides evidence that prophage alter expression of important mycobacterial intrinsic antibiotic resistance genes and additionally offers insight into the role prophage may play in mycobacterial adaptation to stress.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Drug Resistance, Bacterial; Mycobacterium chelonae; Prophages; Virulence Factors
PubMed: 34107872
DOI: 10.1186/s12866-021-02224-z -
Cellular Microbiology Mar 2019Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated...
BACKGROUND
Helicobacter saguini is a novel enterohepatic Helicobacter species isolated from captive cotton top tamarins with chronic colitis and colon cancer. Monoassociated H. saguini infection in gnotobiotic IL-10 mice causes typhlocolitis and dysplasia; however, the virulent mechanisms of this species are unknown. Gamma-glutamyltranspeptidase (GGT) is an enzymatic virulence factor expressed by pathogenic Helicobacter and Campylobacter species that inhibits host cellular proliferation and promotes inflammatory-mediated gastrointestinal pathology. The aim of this study was to determine if H. saguini expresses an enzymatically active GGT homologue with virulence properties.
EXPERIMENTAL PROCEDURES
Two putative GGT paralogs (HSGGT1 and HSGGT2) identified in the H. saguini genome were bioinformatically analysed to predict enzymatic functionality and virulence potential. An isogenic knockout mutant strain and purified recombinant protein of HSGGT1 were created to study enzymatic activity and virulence properties by in vitro biochemical and cell culture experiments.
RESULTS
Bioinformatic analysis predicted that HSGGT1 has enzymatic functionality and is most similar to the virulent homologue expressed by Helicobacter bilis, whereas HSGGT2 contains putatively inactivating mutations. An isogenic knockout mutant strain and recombinant HSGGT1 protein were successfully created and demonstrated that H. saguini has GGT enzymatic activity. Recombinant HSGGT1 protein and sonicate from wild-type but not mutant H. saguini inhibited gastrointestinal epithelial and lymphocyte cell proliferation without evidence of cell death. The antiproliferative effect by H. saguini sonicate or recombinant HSGGT1 protein could be significantly prevented with glutamine supplementation or the GGT-selective inhibitor acivicin. Recombinant HSGGT1 protein also induced proinflammatory gene expression in colon epithelial cells.
CONCLUSIONS
This study shows that H. saguini may express GGT as a potential virulence factor and supports further in vitro and in vitro studies into how GGT expression by enterohepatic Helicobacter species influences the pathogenesis of gastrointestinal inflammatory diseases.
Topics: Animals; Cell Survival; Chronic Disease; Colitis; Computational Biology; Epithelial Cells; Gene Expression; Gene Knockout Techniques; Helicobacter; Interleukin-10; Mice, Knockout; Recombinant Proteins; Saguinus; Virulence Factors; gamma-Glutamyltransferase
PubMed: 30365223
DOI: 10.1111/cmi.12968 -
Molecules (Basel, Switzerland) Apr 2020is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian...
is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to survival. Recently, we described that can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.
Topics: Animals; Azaserine; CHO Cells; Cell Cycle; Cell Proliferation; Cricetulus; Energy Metabolism; Glutamic Acid; Glutamine; Isoxazoles; Life Cycle Stages; Molecular Structure; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 32252252
DOI: 10.3390/molecules25071628 -
ChemMedChem Apr 2017The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of...
The natural product acivicin inhibits the glutaminase activity of cytidine triphosphate (CTP) synthetase and is a potent lead compound for drug discovery in the area of neglected tropical diseases, specifically trypanosomaisis. A 2.1-Å-resolution crystal structure of the acivicin adduct with the glutaminase domain from Trypanosoma brucei CTP synthetase has been deposited in the RCSB Protein Data Bank (PDB) and provides a template for structure-based approaches to design new inhibitors. However, our assessment of that data identified deficiencies in the model. We now report an improved and corrected inhibitor structure with changes to the chirality at one position, the orientation and covalent structure of the isoxazoline moiety, and the location of a chloride ion in an oxyanion binding site that is exploited during catalysis. The model is now in agreement with established chemical principles and allows an accurate description of molecular recognition of the ligand and the mode of binding in a potentially valuable drug target.
Topics: Bacillus subtilis; Carbon-Nitrogen Ligases; Catalytic Domain; Glutaminase; Helicobacter pylori; Hydrogen Bonding; Isoxazoles; Ligands; Trypanocidal Agents; Trypanosoma brucei brucei; gamma-Glutamyltransferase
PubMed: 28333400
DOI: 10.1002/cmdc.201700118