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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 -
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 -
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 -
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 -
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 -
Frontiers in Plant Science 2019The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of...
UNLABELLED
The vegetative phase change in flowering plants is controlled by microRNA156 (miR156) under transcriptional regulation. However, the developmental signals upstream of miR156 are not well understood. The glutathione/glutathione disulfide (GSH/GSSG) ratios and GSH levels decline significantly during phase change, which is consistent with miR156 expression in apple ( Borkh.). Here, we found that the content of protein conjugated glutathione was remarkably higher in chloroplasts and nuclei of adult than juvenile phase apple hybrids. The decrease in miR156 expression was most relevant to the activities of serine acetyltransferase (SAT) and soluble γ-glutamyl transpeptidase (GGT), and the expressions of or . Transgenic apples over-expressing or miR156-mimetic (MIM156) did not alter expression or the soluble GGT activity. Inhibition of GGT activity with serine-borate complex or acivicin led to significant reduction in GSH content, the GSH/GSSG ratio, and the expressions of , , and miR156. Depletion of GSH with diethyl maleate without altering GGT activity caused a dramatic decrease in the expression of , , and miR156. Manipulating GGT activity and GSH homeostasis by transgenic over-expressing or RNAi increased or decreased and levels, respectively. These data provided novel evidence that participates in transcriptional level of transcription regulation of miR156 precursors during ontogenesis.
HIGHLIGHTS
- MdGGT1 affects thiol redox status and indirectly participates in the regulation of miR156 expression during vegetative phase change.
PubMed: 31417600
DOI: 10.3389/fpls.2019.00994