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The Journal of General Physiology Jun 2024Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify...
Cannabidiol (CBD), the main non-psychotropic phytocannabinoid produced by the Cannabis sativa plant, blocks a variety of cardiac ion channels. We aimed to identify whether CBD regulated the cardiac pacemaker channel or the hyperpolarization-activated cyclic nucleotide-gated channel (HCN4). HCN4 channels are important for the generation of the action potential in the sinoatrial node of the heart and increased heart rate in response to β-adrenergic stimulation. HCN4 channels were expressed in HEK 293T cells, and the effect of CBD application was examined using a whole-cell patch clamp. We found that CBD depolarized the V1/2 of activation in holo-HCN4 channels, with an EC50 of 1.6 µM, without changing the current density. CBD also sped activation kinetics by approximately threefold. CBD potentiation of HCN4 channels occurred via binding to the closed state of the channel. We found that CBD's mechanism of action was distinct from cAMP, as CBD also potentiated apo-HCN4 channels. The addition of an exogenous PIP2 analog did not alter the ability of CBD to potentiate HCN4 channels, suggesting that CBD also acts using a unique mechanism from the known HCN4 potentiator PIP2. Lastly, to gain insight into CBD's mechanism of action, computational modeling and targeted mutagenesis were used to predict that CBD binds to a lipid-binding pocket at the C-terminus of the voltage sensor. CBD represents the first FDA-approved drug to potentiate HCN4 channels, and our findings suggest a novel starting point for drug development targeting HCN4 channels.
Topics: Cannabidiol; Humans; Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; HEK293 Cells; Potassium Channels; Ion Channel Gating; Muscle Proteins
PubMed: 38652080
DOI: 10.1085/jgp.202313505 -
European Journal of Applied Physiology May 2024Post-activation potentiation (PAP) describes the enhancement of twitch torque following a conditioning contraction (CC) in skeletal muscle. In adults, PAP may be related...
BACKGROUND
Post-activation potentiation (PAP) describes the enhancement of twitch torque following a conditioning contraction (CC) in skeletal muscle. In adults, PAP may be related to muscle fibre composition and is accompanied by a decrease in motor unit (MU) firing rates (MUFRs). Muscle fibre composition and/or activation is different between children and adults. This study examined PAP and MU firing patterns of the potentiated knee extensors in boys and men.
METHODS
Twenty-three boys (10.5 ± 1.3 years) and 20 men (23.1 ± 3.3 years) completed familiarization and experimental sessions. Maximal isometric evoked-twitch torque and MU firing patterns during submaximal contractions (20% and 70% maximal voluntary isometric contraction, MVIC) were recorded before and after a CC (5 s MVIC). PAP was calculated as the percent-increase in evoked-twitch torque after the CC. MU firing patterns were examined during submaximal contractions before and after the CC using Trigno Galileo surface electrodes (Delsys Inc) and decomposition algorithms (NeuroMap, Delsys Inc). MU action potential amplitudes (MUAPamp) and MUFRs were calculated for each MU and exponential MUFR-MUAPamp relationships were calculated for each participant and trial.
RESULTS
PAP was higher in men than in boys (98.3 ± 37.1% vs. 68.8 ± 18.3%, respectively; p = 0.002). Following potentiation, the rate of decay of the MUFR-MUAPamps relationship decreased in both contractions, with a greater decrease among boys during the high-intensity contractions.
CONCLUSION
Lower PAP in the boys did not coincide with smaller changes in potentiated MU firing patterns, as boys had greater reductions in MUFRs with potentiation compared with men in high-intensity contractions.
Topics: Humans; Male; Child; Isometric Contraction; Muscle, Skeletal; Torque; Adult; Young Adult; Action Potentials; Motor Neurons
PubMed: 38159138
DOI: 10.1007/s00421-023-05377-z -
Journal of Cystic Fibrosis : Official... Sep 2021Trikafta, the combination of elexacaftor (VX-445), tezacaftor (VX-661) and ivacaftor (VX-770), was approved for therapy of cystic fibrosis (CF) patients with at least...
Trikafta, the combination of elexacaftor (VX-445), tezacaftor (VX-661) and ivacaftor (VX-770), was approved for therapy of cystic fibrosis (CF) patients with at least one allele of the CFTR mutation F508del. While the corrector function of VX-445 is well established, here we investigated the putative potentiator activity of VX-445 alone and in combination with VX-770. Acute addition of VX-445 increased the VX-770-potentiated F508del- and G551D-CFTR current by ~24% and >70%, respectively, in human bronchial and nasal epithelia. Combinatorial profiling and cluster analysis of G551D- and G1244E-CFTR channel activation with potentiator pairs indicated a distinct VX-445 mechanism of action that is, at least, additive to previously identified potentiator classes, including the VX-770. Since VX-770 only partially normalizes the G551D-CFTR channel function and adult G551D patients still experience progressive loss of lung function, VX-445+VX-770 combination therapy could provide clinical benefit to CF patients with the G551D and other dual potentiator responsive mutants.
Topics: Cells, Cultured; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Genotype; Humans; Mutation; Nasal Mucosa; Pyrazoles; Pyridines; Pyrrolidines
PubMed: 33775603
DOI: 10.1016/j.jcf.2021.03.011 -
Antibiotics (Basel, Switzerland) Jul 2023Metal ions, including Fe, affect the target site binding of some antibiotics and control the porin- and siderophore-mediated uptake of antibiotics. Amphiphilic...
Metal ions, including Fe, affect the target site binding of some antibiotics and control the porin- and siderophore-mediated uptake of antibiotics. Amphiphilic tobramycins are an emerging class of antibiotic potentiators capable of synergizing with multiple classes of antibiotics against Gram-negative bacteria, including . To study how the antibiotic-potentiating effect of amphiphilic tobramycins is affected by the presence of intermolecular iron chelators, we conjugated the FDA-approved iron chelator deferiprone (DEF) to tobramycin (TOB). Three TOB-DEF conjugates differing in the length of the carbon tether were prepared and tested for antibacterial activity and synergistic relationships with a panel of antibiotics against clinical isolates of . While all TOB-DEF conjugates were inactive against , the TOB-DEF conjugates strongly synergized with outer-membrane-impermeable antibiotics, such as novobiocin and rifampicin. Among the three TOB-DEF conjugates, containing a C tether showed a remarkable and selective potentiating effect to improve the susceptibility of multidrug-resistant isolates to tetracyclines when compared with other antibiotics. However, the antibacterial activity and antibiotic-potentiating effect of the optimized conjugate was not enhanced under iron-depleted conditions, indicating that the function of the antibiotic potentiator is not affected by the Fe concentration.
PubMed: 37627681
DOI: 10.3390/antibiotics12081261 -
Molecules (Basel, Switzerland) Feb 2022The burkholdines are a family of cyclic lipopeptides reported to exhibit antifungal activity. We synthesized a series of 18 burkholdine analogues in good yield by...
The burkholdines are a family of cyclic lipopeptides reported to exhibit antifungal activity. We synthesized a series of 18 burkholdine analogues in good yield by conventional Fmoc-SPPS followed by cyclization with DIPCI/HOBt in the solution phase. Although none of the synthesized peptides exhibited antifungal activity, several did potentiate the antibiotic effect of the antibiotic G418, including the Thr-bearing Bk analogue () and the tartaramide-bearing Bk analogue (). This work exemplifies the potential of burkholdine analogues as potentiating agents.
Topics: Antifungal Agents; Hydrophobic and Hydrophilic Interactions; Lipopeptides
PubMed: 35208979
DOI: 10.3390/molecules27041191 -
Mutation Research. Reviews in Mutation... Jul 2017Hydrogen peroxide (HO) is unique among general toxins, because it is stable in abiotic environments at ambient temperature and neutral pH, yet rapidly kills any type of... (Review)
Review
Hydrogen peroxide (HO) is unique among general toxins, because it is stable in abiotic environments at ambient temperature and neutral pH, yet rapidly kills any type of cells by producing highly-reactive hydroxyl radicals. This life-specific reactivity follows the distribution of soluble iron, Fe(II) (which combines with HO to form the famous Fenton's reagent),Fe(II) is concentrated inside cells, but is virtually absent outside them. Because of the immediate danger of HO, all cells have powerful HO scavengers, the equally famous catalases, which enable cells to survive thousand-fold higher concentrations of HO and, in combination with adequate movement of HO across membranes, make the killing HO concentrations virtually impractical to generate in vivo. And yet, low concentrations of HO are somehow used as an efficient biological weapon. Here we review several examples of how cells potentiate HO toxicity with other chemicals. At first, these potentiators were thought to simply inhibit catalases, but recent findings with cyanide suggest that potentiators mostly promote the other side of Fenton's reaction, recruiting iron from cell depots into stable DNA-iron complexes that, in the presence of elevated HO, efficiently break duplex DNA, pulverizing the chromosome. This multifaceted potentiation of HO toxicity results in robust and efficient killing.
Topics: Animals; Bacteria; Catalase; DNA; Humans; Hydrogen Peroxide; Hydrogen-Ion Concentration; Iron
PubMed: 28927535
DOI: 10.1016/j.mrrev.2016.08.006 -
Journal of Applied Physiology... Jan 2022Whole body vibration (WBV) is often applied as an alternative method for strength training or to prevent muscle force decrease. In this study, we evaluated the influence...
Whole body vibration (WBV) is often applied as an alternative method for strength training or to prevent muscle force decrease. In this study, we evaluated the influence of WBV on Ia monosynaptic input from muscle spindles because the tonic vibration reflex is responsible for the enhancement of muscle activity observed after WBV. The aim was to investigate whether repeated activation of muscle spindles during WBV may result in altered synaptic excitation of motoneurons. WBV was performed on adult male Wistar rats, 5 days/wk, for 5 wk, and each daily session consisted of four 30-s runs of vibration at 50 Hz. Fast-type medial gastrocnemius motoneurons were investigated intracellularly in deeply anesthetized animals in the experimental ( = 7, 34 motoneurons) and control ( = 7, 32 motoneurons) groups. Monosynaptic Ia excitatory postsynaptic potentials (EPSPs) were evoked by electrical stimulation of afferent fibers from the synergistic lateral gastrocnemius and soleus muscles. Data were analyzed using a mixed linear model. The central latencies of EPSPs were 0.45-0.9 ms with no differences in the mean values between the analyzed groups ( = 0.291). WBV induced an increase of the mean EPSP amplitude by 28% ( = 0.025), correlated with the resting membrane potential and input resistance, and a shortening of the mean EPSP rise time by 11% ( = 0.012). The potentiation of synaptic excitation of motoneurons was not accompanied by changes of passive membrane properties, pointing to synaptic plasticity. This indicates that WBV may support rehabilitation or training processes aimed at increasing muscle strength on the basis of increased motoneuronal drive. The study provides new information on neuronal plasticity following repeatedly exerted mechanical loading. We demonstrate in electrophysiological experiments on rat lumbar motoneurons that low-volume whole body vibration applied systematically for 5 wk potentiates synaptic excitation from primary muscle afferents. The adaptive changes are expressed by higher amplitudes and shorter rise times of monosynaptic EPSPs evoked in motoneurons of the vibrated group compared with the control.
Topics: Animals; Excitatory Postsynaptic Potentials; Male; Motor Neurons; Muscle, Skeletal; Rats; Rats, Wistar; Spinal Cord; Synapses; Vibration
PubMed: 34855524
DOI: 10.1152/japplphysiol.00519.2021 -
ACS Infectious Diseases Oct 2022Gram-negative bacteria are intrinsically resistant to a plethora of antibiotics that effectively inhibit the growth of Gram-positive bacteria. The intrinsic resistance...
Gram-negative bacteria are intrinsically resistant to a plethora of antibiotics that effectively inhibit the growth of Gram-positive bacteria. The intrinsic resistance of Gram-negative bacteria to classes of antibiotics, including rifamycins, aminocoumarins, macrolides, glycopeptides, and oxazolidinones, has largely been attributed to their lack of accumulation within cells due to poor permeability across the outer membrane, susceptibility to efflux pumps, or a combination of these factors. Due to the difficulty in discovering antibiotics that can bypass these barriers, finding targets and compounds that increase the activity of these ineffective antibiotics against Gram-negative bacteria has the potential to expand the antibiotic spectrum. In this study, we investigated the genetic determinants for resistance to rifampicin, novobiocin, erythromycin, vancomycin, and linezolid to determine potential targets of antibiotic-potentiating compounds. We subsequently performed a high-throughput screen of ∼50,000 diverse, synthetic compounds to uncover molecules that potentiate the activity of at least one of the five Gram-positive-targeting antibiotics. This led to the discovery of two membrane active compounds capable of potentiating linezolid and an inhibitor of lipid A biosynthesis capable of potentiating rifampicin and vancomycin. Furthermore, we characterized the ability of known inhibitors of lipid A biosynthesis to potentiate the activity of rifampicin against Gram-negative pathogens.
Topics: Anti-Bacterial Agents; Erythromycin; Gram-Negative Bacteria; Linezolid; Lipid A; Novobiocin; Oxazolidinones; Rifampin; Vancomycin
PubMed: 36098580
DOI: 10.1021/acsinfecdis.2c00357 -
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