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MBio Jun 2022Metabolic and growth arrest are primary drivers of antibiotic tolerance and persistence in clinically diverse bacterial pathogens. We recently showed that adenosine...
Metabolic and growth arrest are primary drivers of antibiotic tolerance and persistence in clinically diverse bacterial pathogens. We recently showed that adenosine (ADO) suppresses bacterial growth under nutrient-limiting conditions. In the current study, we show that despite the growth-suppressive effect of ADO, extracellular ADO enhances antibiotic killing in both Gram-negative and Gram-positive bacteria by up to 5 orders of magnitude. The ADO-potentiated antibiotic activity is dependent on purine salvage and is paralleled with a suppression of guanosine tetraphosphate synthesis and the massive accumulation of ATP and GTP. These changes in nucleoside phosphates coincide with transient increases in rRNA transcription and proton motive force. The potentiation of antibiotic killing by ADO is manifested against bacteria grown under both aerobic and anaerobic conditions, and it is exhibited even in the absence of alternative electron acceptors such as nitrate. ADO potentiates antibiotic killing by generating proton motive force and can occur independently of an ATP synthase. Bacteria treated with an uncoupler of oxidative phosphorylation and NADH dehydrogenase-deficient bacteria are refractory to the ADO-potentiated killing, suggesting that the metabolic awakening induced by this nucleoside is intrinsically dependent on an energized membrane. In conclusion, ADO represents a novel example of metabolite-driven but growth-independent means to reverse antibiotic tolerance. Our investigations identify the purine salvage pathway as a potential target for the development of therapeutics that may improve infection clearance while reducing the emergence of antibiotic resistance. Antibiotic tolerance, which is a hallmark of persister bacteria, contributes to treatment-refractory infections and the emergence of heritable antimicrobial resistance. Drugs that reverse tolerance and persistence may become part of the arsenal to combat antimicrobial resistance. Here, we demonstrate that salvage of extracellular ADO reduces antibiotic tolerance in nutritionally stressed Escherichia coli, Salmonella enterica, and Staphylococcus aureus. ADO potentiates bacterial killing under aerobic and anaerobic conditions and takes place in bacteria lacking the ATP synthase. However, the sensitization to antibiotic killing elicited by ADO requires an intact NADH dehydrogenase, suggesting a requirement for an energized electron transport chain. ADO antagonizes antibiotic tolerance by activating ATP and GTP synthesis, promoting proton motive force and cellular respiration while simultaneously suppressing the stringent response. These investigations reveal an unprecedented role for purine salvage stimulation as a countermeasure of antibiotic tolerance and the emergence of antimicrobial resistance.
Topics: Adenosine; Adenosine Triphosphate; Anti-Bacterial Agents; Escherichia coli; Guanosine Triphosphate; Microbial Sensitivity Tests; NADH Dehydrogenase; Nucleosides; Salmonella enterica
PubMed: 35575513
DOI: 10.1128/mbio.00480-22 -
Human Molecular Genetics Dec 2017Approximately 50% of cystic fibrosis (CF) patients are heterozygous with a rare mutation on at least one allele. Several mutants exhibit functional defects, correctable...
Approximately 50% of cystic fibrosis (CF) patients are heterozygous with a rare mutation on at least one allele. Several mutants exhibit functional defects, correctable by gating potentiators. Long-term exposure (≥24 h) to the only available potentiator drug, VX-770, leads to the biochemical and functional downregulation of F508del-CFTR both in immortalized and primary human airway cells, and possibly other CF mutants, attenuating its beneficial effect. Based on these considerations, we wanted to determine the effect of chronic VX-770 exposure on the functional and biochemical expression of rare CF processing/gating mutants in human airway epithelia. Expression of CFTR2 mutants was monitored in the human bronchial epithelial cell line (CFBE41o-) and in patient-derived conditionally reprogrammed bronchial and nasal epithelia by short-circuit current measurements, cell surface ELISA and immunoblotting in the absence or presence of CFTR modulators. The VX-770 half-maximal effective (EC50) concentration for G551D-CFTR activation was ∼0.63 μM in human nasal epithelia, implying that comparable concentration is required in the lung to attain clinical benefit. Five of the twelve rare CFTR2 mutants were susceptible to ∼20-70% downregulation by chronic VX-770 exposure with an IC50 of ∼1-20 nM and to destabilization by other investigational potentiators, thereby diminishing the primary functional gain of CFTR modulators. Thus, chronic exposure to VX-770 and preclinical potentiators can destabilize CFTR2 mutants in human airway epithelial models in a mutation and compound specific manner. This highlights the importance of selecting potentiator drugs with minimal destabilizing effects on CF mutants, advocating a precision medicine approach.
Topics: Aminophenols; Bronchi; Cell Line; Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Drug Synergism; Epithelial Cells; Humans; Ion Channel Gating; Lung; Models, Molecular; Mutation; Quinolones; Respiratory Mucosa
PubMed: 29040544
DOI: 10.1093/hmg/ddx367 -
ACS Infectious Diseases Oct 2023By illuminating key 6-azasteroid-protein interactions in both () and the closely related model organism (), we sought to improve the antimycobacterial potency of...
By illuminating key 6-azasteroid-protein interactions in both () and the closely related model organism (), we sought to improve the antimycobacterial potency of 6-azasteroids and further our understanding of the mechanisms responsible for their potentiation of the antituberculosis drug bedaquiline. We selected a newly developed 6-azasteroid analog and an analog reported previously ( , (7), 1239-1251) to study their phenotypic effects on and , both alone and in combination with bedaquiline. The 6-azasteroid analog, 17β-[-(4-trifluoromethoxy-diphenylmethyl)carbamoyl]-6-propyl-azaandrostan-3-one, robustly potentiated bedaquiline-mediated antimycobacterial activity, with a nearly 8-fold reduction in bedaquiline minimal inhibitory concentration (85 nM alone versus 11 nM with 20 μM 6-azasteroid). This analog displayed minimal inhibitory activity against recombinant mycobacterial 3β-hydroxysteroid dehydrogenase, a previously identified target of several 6-azasteroids. Dose-dependent potentiation of bedaquiline by this analog reduced mycobacterial intracellular ATP levels and impeded the ability of to neutralize exogenous oxidative stress in culture. We developed two 6-azasteroid photoaffinity probes to investigate azasteroid-protein interactions in whole cells. Using bottom-up mass spectrometric profiling of the cross-linked proteins, we identified eight potential / protein targets for 6-azasteroids. The nature of these potential targets indicates that proteins related to oxidative stress resistance play a key role in the BDQ-potentiating activity of azasteroids and highlights the potential impact of inhibition of these targets on the generation of drug sensitivity.
Topics: Azasteroids; Antitubercular Agents; Mycobacterium tuberculosis; Bacterial Proteins; Mycobacterium marinum
PubMed: 37774412
DOI: 10.1021/acsinfecdis.3c00296 -
IScience Jan 2022Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator...
Characterization of I37R, a mutation located in the lasso motif of the CFTR chloride channel, was conducted by theratyping several CFTR modulators from both potentiator and corrector classes. Intestinal current measurements in rectal biopsies, forskolin-induced swelling (FIS) in intestinal organoids, and short circuit current measurements in organoid-derived monolayers from an individual with I37R/F508del CFTR genotype demonstrated that the I37R-CFTR results in a residual function defect amenable to treatment with potentiators and type III, but not type I, correctors. Molecular dynamics of I37R using an extended model of the phosphorylated, ATP-bound human CFTR identified an altered lasso motif conformation which results in an unfavorable strengthening of the interactions between the lasso motif, the regulatory (R) domain, and the transmembrane domain 2 (TMD2). Structural and functional characterization of the I37R- mutation increases understanding of CFTR channel regulation and provides a potential pathway to expand drug access to CF patients with ultra-rare genotypes.
PubMed: 35072004
DOI: 10.1016/j.isci.2021.103710 -
FEMS Yeast Research Aug 2018Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase...
Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.
Topics: Aspartic Acid; Glutamic Acid; Heat-Shock Proteins; Models, Molecular; Mutation, Missense; Phosphorylation; Protein Folding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 29788207
DOI: 10.1093/femsyr/foy042 -
Neuropharmacology Jan 2023Ketamine exerts rapid and long-lasting antidepressant effects in patients with treatment-resistant depression. However, its clinical use is limited by its undesirable... (Review)
Review
Ketamine exerts rapid and long-lasting antidepressant effects in patients with treatment-resistant depression. However, its clinical use is limited by its undesirable psychotomimetic side effects. Accumulating evidence from preclinical studies has shown that the antidepressant effects of ketamine are dependent on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R) activation, which triggers activation of the mechanistic target of rapamycin pathway and brain-derived neurotrophic factor release. Thus, AMPA-R has emerged as a promising new target for novel antidepressants with a rapid onset of action. However, almost all known AMPA-R potentiators carry the risk of a narrow bell-shaped dose-response curve and a poor safety margin against seizures. Our data suggest that agonistic activity is not only related to the risks of bell-shaped dose-response curves and seizures but also to the reduced synaptic transmission and procognitive effects of AMPA-R potentiators. In this review, we describe our original screening approach that led to the discovery of an investigational AMPA-R potentiator with low agonistic activity, TAK-653. We further review the in vitro and in vivo profiles of TAK-653, including its procognitive and antidepressant-like effects, as well as its safety profile, in comparison with known AMPA-R potentiators with agonistic activity and AMPA, an AMPA-R agonist. The low agnostic activity of TAK-653 may overcome limitations of known AMPA-R potentiators. This article is part of the Special Issue on 'Ketamine and its Metabolites'.
Topics: Humans; Ketamine; Receptors, AMPA; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Antidepressive Agents; Seizures
PubMed: 36341809
DOI: 10.1016/j.neuropharm.2022.109308 -
Frontiers in Pharmacology 2018The psoralen-related compound, 4,6,4'-trimethylangelicin (TMA) potentiates the cAMP/PKA-dependent activation of WT-CFTR and rescues F508del-CFTR-dependent chloride...
The psoralen-related compound, 4,6,4'-trimethylangelicin (TMA) potentiates the cAMP/PKA-dependent activation of WT-CFTR and rescues F508del-CFTR-dependent chloride secretion in both primary and secondary airway cells homozygous for the F508del mutation. We recently demonstrated that TMA, like lumacaftor (VX-809), stabilizes the first membrane-spanning domain (MSD1) and enhances the interface between NBD1 and ICL4 (MSD2). TMA also demonstrated anti-inflammatory properties, via reduction of IL-8 expression, thus making TMA a promising agent for treatment of cystic fibrosis. Unfortunately, TMA was also found to display potential phototoxicity and mutagenicity, despite the fact that photo-reactivity is absent when the compound is not directly irradiated with UVA light. Due to concerns about these toxic effects, new TMA analogs, characterized by identical or better activity profiles and minimized or reduced side effects, were synthesized by modifying specific structural features on the TMA scaffold, thus generating compounds with no mutagenicity and phototoxicity. Among these compounds, we found TMA analogs which maintained the potentiation activity of CFTR in FRT-YFP-G551D cells. Nanomolar concentrations of these analogs significantly rescued F508del CFTR-dependent chloride efflux in FRT-YFP-F508del, HEK-293 and CF bronchial epithelial cells. We then investigated the ability of TMA analogs to enhance the stable expression of varying CFTR truncation mutants in HEK-293 cells, with the aim of studying the mechanism of their corrector activity. Not surprisingly, MSD1 was the smallest domain stabilized by TMA analogs, as previously observed for TMA. Moreover, we found that TMA analogs were not effective on F508del-CFTR protein which was already stabilized by a second-site mutation at the NBD1-ICL4 interface. Altogether, our findings demonstrate that these TMA analogs mediate correction by modifying MSD1 and indirectly stabilizing the interface between NBD1 and CL4.
PubMed: 30022950
DOI: 10.3389/fphar.2018.00719 -
Journal of Applied Physiology... Sep 2022This study was conducted to examine the effects of an acute bout of eccentric muscle contraction (ECC) on titin stiffness-related contractile properties in rat...
This study was conducted to examine the effects of an acute bout of eccentric muscle contraction (ECC) on titin stiffness-related contractile properties in rat fast-twitch skeletal muscles. Intact gastrocnemius muscles were electrically stimulated in situ to undergo 200 repeated ECCs. Immediately after the cessation of the stimulation, the superficial regions of the muscles were dissected and subjected to biochemical and skinned fiber analyses. Small heat shock protein αB-crystallin in the muscle fraction enriched for myofibrillar proteins was increased by ECC. ECC resulted in an increase in the titin-based passive force. Protein kinase A-treatment decreased the passive force only in ECC-subjected but not in rested fibers. ECC decreased the maximum Ca-activated force at a sarcomere length (SL) of 2.4 μm and had no effect on myofibrillar-Ca sensitivity at 2.6-μm SL. In both rested and ECC-subjected fibers, these two variables were higher at 3.0-μm SL than at 2.4- or 2.6-μm SL. The differences in the two variables between the short and long SLs were greater in ECC-subjected than in rested fibers. These results indicate that an acute bout of ECC potentiates titin-based passive force, maximum active force at long SLs, and length-dependent activation and suggest that this potentiation may resist muscle fatigue in the muscles of the exercising body. It remains unclear whether eccentric contraction of skeletal muscle affects titin stiffness-related contractile properties. Here, we provide evidence that an acute bout of eccentric contraction can potentiate titin-based passive force, maximum active force at long sarcomere lengths, and length-dependent activation. This potentiation may resist muscle fatigue in the muscles of the exercising body.
Topics: Animals; Calcium; Connectin; Muscle Contraction; Muscle, Skeletal; Myofibrils; Rats
PubMed: 35981734
DOI: 10.1152/japplphysiol.00327.2022 -
Journal of Cystic Fibrosis : Official... May 2021The c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation...
BACKGROUND
The c.3700A>G mutation, a rare cystic fibrosis (CF)-causing CFTR mutation found mainly in the Middle East, produces full-length transcript encoding a missense mutation (I1234V-CFTR), and a cryptic splice site that deletes 6 amino acids in nucleotide binding domain 2 (I1234del-CFTR).
METHODS
FRT cell models expressing I1234V-CFTR and I1234del-CFTR were generated. We also studied an I1234del-CFTR-expressing gene-edited human bronchial (16HBE14o-) cell model, and primary cultures of nasal epithelial cells from a c.3700A>G homozygous subject. To identify improved mutation-specific CFTR modulators, high-throughput screening was done using I1234del-CFTR-expressing FRT cells. Motivated by the in vitro findings, Trikafta was tested in two c.3700A>G homozygous CF subjects.
RESULTS
FRT cells expressing full-length I1234V-CFTR had similar function to that of wildtype CFTR. I1234del-CFTR showed reduced activity, with modest activation seen with potentiators VX-770 and GLPG1837, correctors VX-809, VX-661 and VX-445, and low-temperature incubation. Screening identified novel arylsulfonyl-piperazine and spiropiperidine-quinazolinone correctors, which when used in combination with VX-445 increased current ~2-fold compared with the VX-661/VX-445 combination. The combination of VX-770 with arylsulfonamide-pyrrolopyridine, piperidine-pyridoindole or pyrazolo-quinoline potentiators gave 2-4-fold greater current than VX-770 alone. Combination potentiator (co-potentiator) efficacy was also seen in gene-edited I1234del-CFTR-expressing human bronchial epithelial cells. In two CF subjects homozygous for the c.3700A>G mutation, one subject had a 27 mmol/L decrease in sweat chloride and symptomatic improvement on Trikafta, and a second subject showed a small improvement in lung function.
CONCLUSIONS
These results support the potential benefit of CFTR modulators, including co-potentiators, for CF caused by the c.3700A>G mutation.
Topics: Aminophenols; Aminopyridines; Benzodioxoles; Cells, Cultured; Chloride Channel Agonists; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Humans; Indoles; Mutant Proteins; Mutation, Missense; Pyrazoles; Pyridines; Pyrrolidines; Quinolones
PubMed: 32674984
DOI: 10.1016/j.jcf.2020.07.003 -
Microbiology Spectrum Jun 2022Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health requiring the development of novel therapies. TP0586532 is a novel non-hydroxamate...
Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent threat to public health requiring the development of novel therapies. TP0586532 is a novel non-hydroxamate LpxC inhibitor that inhibits the synthesis of lipopolysaccharides, which are components of the outer membranes of Gram-negative bacteria. Based on the mechanism of action of TP0586532, we hypothesized that it might enhance the antibacterial activity of other antibiotics by increasing the permeability of the outer bacterial membrane. The combination of TP0586532 with meropenem, amikacin, cefepime, piperacillin, and tigecycline showed synergistic and additive effects against carbapenem-susceptible Klebsiella pneumoniae and Escherichia coli. Checkerboard experiments against 21 carbapenem-resistant K. pneumoniae and E. coli strains (13 +, 5 +, 2 +, and 1 +) showed that the combination of TP0586532 with meropenem yielded synergistic and additive effects against 9 and 12 strains, respectively. In a time-kill assay examining 12 CRE strains, synergistic effects were observed when TP0586532 was combined with meropenem against many of the strains. A membrane permeability assay using ethidium bromide (EtBr) was performed to investigate the mechanism of the potentiating effect. TP0586532 increased the influx of EtBr into a CRE strain, suggesting that TP0586532 increased membrane permeability and facilitated intracellular access for the antibiotics. Our study demonstrates that TP0586532 potentiates the antibacterial activity of meropenem against CRE. Combination therapy consisting of TP0586532 and meropenem has potential as a treatment for CRE infections. Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent public health threat, as therapeutic options are limited. TP0586532 is a novel LpxC inhibitor that inhibits the synthesis of lipopolysaccharides in the outer membranes of Gram-negative bacteria. Here, we demonstrated the potentiating effects of TP0586532 on the antibacterial activity of meropenem against CRE harboring various types of carbapenemase genes (+, + +, and +). TP0586532 also augmented the bactericidal effects of meropenem against CRE strains, even against those with a high level of resistance to meropenem. The potentiating effects were suggested to be mediated by an increase in bacterial membrane permeability. Our study revealed that a combination therapy consisting of TP0586532 and meropenem has the potential to be a novel therapeutic option for CRE infections.
Topics: Humans; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Butanols; Carbapenem-Resistant Enterobacteriaceae; Carbapenems; Enterobacteriaceae Infections; Escherichia coli; Gram-Negative Bacteria; Imidazoles; Klebsiella pneumoniae; Meropenem; Microbial Sensitivity Tests
PubMed: 35647694
DOI: 10.1128/spectrum.00828-22