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Antibiotics: The Need for Innovation
Published in Nathan Keighley, Miraculous Medicines and the Chemistry of Drug Design, 2020
Other antibacterial agents are designed to act on the plasma membrane. The polypeptides valinomycin and gramicidin A cause the uncontrolled movement of ions across the cell membrane. Valinomycin acts as an inverted detergent and complexes a ‘naked’ potassium ion. The hydrophobic outer of the complex can pass the cell membrane and deposit the potassium ion outside the cell, resulting in fatality. Typically, K+ is in high concentration inside the cell and can only pass the membrane via specialised transport proteins and this equilibrium is disrupted by valinomycin. Valinomycin is selective towards K+, having the correct spatial arrangement of donor atoms to displace water ligands and form bonds to K+. Other ions, such as Na+, are not the correct size to fit and displacing water ligands is too difficult. Unfortunately, the toxicity is not selective to bacteria; affecting mammal cells as well. Gramicidin A is a peptide consisting of 15 amino acids, which coil into a helix where hydrophobic side chains point outwards and interact with the membrane. Two helices of gramicidin A must combine to span the membrane and the hydrophobic interior of the helix serves as a channel for the passage of ions. However, gramicidin A is also toxic to humans. Producing compounds to serve as drugs which employ the ‘magic bullet’ approach is a challenge for medicinal chemists. Compounds must be toxic to bacteria, but safe to use and to achieve this, target specificity is essential when developing effective antibiotics in the future.
Ultraviolet and Light Absorption Spectrometry
Published in Adorjan Aszalos, Modern Analysis of Antibiotics, 2020
Zoltan M. Dinya, Ferenc J. Sztaricskai
The UV spectra of depsipeptide antibiotics are usually not specific and show end absorption. Valinomycin exhibits weak absorption at 281 nm in hexane [118,119]. Staphylomycin MI(41), containing α, β-unsaturated carbonyl groups and an oxazole-type chromophoric unit, has the UV parameters λmax (nm) and ε(m2 /mol) in parentheses: 216 (3230) and 270 sh (1110) [120].
Restoration of Membrane Environments for Membrane Proteins for Structural and Functional Studies
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Empty liposomes or proteoliposome were 100-fold diluted into a buffer containing 150 mM NaCl, 20 mM Tris-HCl, pH 7.4, and 2 μM 9-amino-2-methoxy-6-chloroacridine (ACMA, Sigma-Aldrich). This yielded a final K+ concentration of 1.5 mM outside liposomes. Fluorescence intensity was measured every 1 second using a SpectraMax fluorometer (Molecular Devices) for a total of 180 seconds with excitation at 395 nm and emission at 490 nm. The proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP, Sigma-Aldrich) was added to a final concentration of 1 μM after 60 seconds, and the sample was gently mixed with a pipette. Valinomycin (Sigma-Aldrich) was added to a final concentration of 20 nM at 120 seconds.
Why do platelets express K+ channels?
Published in Platelets, 2021
Joy R Wright, Martyn P. Mahaut-Smith
Full platelet activation requires a sustained elevation of intracellular calcium, resulting in the externalization of the negatively charged membrane phospholipid component phosphatidylserine (PS) from the inner leaflet of the platelet membrane. Scott syndrome patients have a mild bleeding phenotype and have been noted to be deficient in the scramblase mechanism that facilitates PS exposure in erythrocytes and platelets, and also in the ability to produce platelet microparticles from the platelet surface membrane [28]. This defect in platelet procoagulant response may be due in part to reduced Gardos channel function, since the impaired procoagulant response in Scott patients following platelet activation with combined collagen and thrombin application was almost completely restored to normal levels by the K+ ionophore valinomycin [13]. Interestingly, valinomycin will insert into both intracellular and surface membranes [29] and could exert its observed effect at least in part by affecting mitochondrial membrane potential which has a key influence on procoagulant activity. Whether KCa3.1 is also present in platelet organellar membranes is unknown. Experiments in SK4-/- transgenic mice suggest that the Gardos channel also plays a role in stromal cell-derived factor 1 (SDF-1)-dependent platelet migration [30]. A further potential role for KCa3.1 in platelets and megakaryocytes is the regulation of cell volume, as proposed in the human megakaryocytic cell line DAMI [31].
Efflux pump inhibitors as a promising adjunct therapy against drug resistant tuberculosis: a new strategy to revisit mycobacterial targets and repurpose old drugs
Published in Expert Review of Anti-infective Therapy, 2020
Liliana Rodrigues, Pedro Cravo, Miguel Viveiros
These compounds, also known as proton translocators or uncouplers, dissipate the PMF and electrochemical gradient, preventing further synthesis of ATP and, consequently, the normal function of cell processes that depend on this energy gradient such as efflux pumps [4,56,86]. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrofenol (DNP) and valinomycin are examples of such compounds that have been widely used to study the role of efflux pumps in mycobacteria. For example, CCCP decreased the MIC of tetracycline in a M. smegmatis strain expressing Rv1258c and decreased the activity of this efflux pump in a M. bovis BCG overexpressing strain [19,30]. Moreover, CCCP was one of the efflux inhibitors that abolished the decreased susceptibility to several compounds, such as ethidium bromide, acriflavine and safranin O in M. tuberculosis strains overexpressing jefA and mmr (Rv3065) (Rv2459) [28,87]. CCCP and valinomycin reduced novobiocin and rifampicin resistance mediated by P55 [18]. It was also demonstrated in several studies that CCCP and DNP reduce efflux pump activity associated with fluoroquinolone resistance [39,68,87]. However, due to their mechanism of action, these compounds are highly cytotoxic and, thus, further studies are needed to determine the concentrations that are simultaneously the safest and most effective.
Discovery of a new mitochondria permeability transition pore (mPTP) inhibitor based on gallic acid
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
José Teixeira, Catarina Oliveira, Fernando Cagide, Ricardo Amorim, Jorge Garrido, Fernanda Borges, Paulo J. Oliveira
Evaluation of AntiOxBEN3 effect on mitochondrial transmembrane electric potential (ΔΨ). Approximate values for mitochondrial transmembrane electric potential (ΔΨ) was estimated through the evaluation of fluorescence changes of safranine O (5 µM) and was recorded on a spectrofluorometer operating at excitation and emission wavelengths of 495 and 586 nm, with a slit width of 5 nm. Increasing concentrations of AntiOxBEN3 (2.5–10 µM) were added to the reaction medium (200 mM sucrose, 1 mM KH2PO4, 10 mM Tris (pH 7.4), and 10 µM EGTA) containing respiratory substrates glutamate/malate (5 and 2.5 mM, respectively) or succinate (5 mM) and RLM (0.5 mg in 2 ml final volume) and allowed to incubate for a 5 min period prior to recording, at 25 °C. In this assay, safranine (5 µM) and ADP (25 nmol) were used to initiate the assay and to induce depolarisation, respectively. Moreover, 1 µM FCCP was added at the end of all experiments to cause complete mitochondrial depolarisation. ΔΨ was calculated using a calibration curve obtained when RLM were incubated in a reaction medium mostly devoid of K+, containing 200 mM sucrose, 1 mM NaH2PO4, 10 mM Tris (pH 7.4), and 10 µM EGTA, supplemented with 0.4 µ.g valinomycin, as previous described22,23. The extension of fluorescence changes of safranine induced by ΔΨ was found to be similar in the standard and K+-free medium. “Repolarisation” corresponds to the recovery of apparent ΔΨ after the complete phosphorylation of ADP added. Lag phase reflects the time required to phosphorylate the added ADP. Values are means ± SEM of five independent experiments.