Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) via Knoevenagel Reaction Yields an Effective Mitochondria-Targeted Protonophoric Uncoupler.

Mitochondrial uncouplers are actively sought as potential therapeutics. Here, we report the first successful synthesis of mitochondria-targeted derivatives of the highly potent uncoupler 3,5-ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847), bearing a cationic alkyl(triphenyl)phosphonium (TPP) group. As a key step of the synthesis, we used condensation of a ketophenol with malononitrile via the Knoevenagel reaction. SF-C5-TPP with a pentamethylene linker between SF6847 and TPP, stimulating respiration and collapsing membrane potential of rat liver mitochondria at submicromolar concentrations, proved to be the most effective uncoupler of the series. SF-C5-TPP showed pronounced protonophoric activity on a model planar bilayer lipid membrane. Importantly, SF-C5-TPP exhibited rather low toxicity in fibroblast cell culture, causing mitochondrial depolarization in cells at concentrations that only slightly affected cell viability. SF-C5-TPP was more effective in decreasing the mitochondrial membrane potential in the cell culture than SF6847, in contrast to the case of isolated mitochondria. Like other zwitterionic uncouplers, SF-C5-TPP inhibited the growth of Bacillus subtilis in the micromolar concentration range.

Selectivity of cation transport across lipid membranes by the antibiotic salinomycin.

The ionophoric antibiotic salinomycin is in the phase of preclinical tests against several types of malignant tumors including breast cancer. Notwithstanding, the data on its ion selectivity, although being critical for its therapeutic activity, are rather scarce. In the present work, we studied the ability of salinomycin to exert cation/H+-exchange across artificial bilayer lipid membranes (BLM) by measuring electrical potential on planar BLM in the presence of a protonophore and fluorescence responses of the pH-sensitive dye pyranine entrapped in liposomes. The following order of ion selectivity was obtained by these two methods: K+ > Na+ > Rb+ > Cs+ > Li+. Measurements of the monovalent cation-induced quenching of fluorescence of thallium ions in methanol showed that salinomycin effectively binds potassium and calcium but poorly binds sodium and lithium ions. At high concentrations, salinomycin transports Ca2+ through membranes of liposomes and mitochondria, as measured by using the calcium-sensitive dye Fluo-5 N. The data obtained can be used in the mechanistic studies of the anti-tumor activity of salinomycin and its selective cytotoxicity towards cancer stem cells.

Photodynamic activity rather than drilling causes membrane damage by a light-powered molecular nanomotor.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

Usnic Acid-Mediated Exchange of Protons for Divalent Metal Cations across Lipid Membranes: Relevance to Mitochondrial Uncoupling.

Usnic acid (UA), a unique lichen metabolite, is a protonophoric uncoupler of oxidative phosphorylation, widely known as a weight-loss dietary supplement. In contrast to conventional proton-shuttling mitochondrial uncouplers, UA was found to carry protons across lipid membranes via the induction of an electrogenic proton exchange for calcium or magnesium cations. Here, we evaluated the ability of various divalent metal cations to stimulate a proton transport through both planar and vesicular bilayer lipid membranes by measuring the transmembrane electrical current and fluorescence-detected pH gradient dissipation in pyranine-loaded liposomes, respectively. Thus, we obtained the following selectivity series of calcium, magnesium, zinc, manganese and copper cations: Zn2+ > Mn2+ > Mg2+ > Ca2+ >> Cu2+. Remarkably, Cu2+ appeared to suppress the UA-mediated proton transport in both lipid membrane systems. The data on the divalent metal cation/proton exchange were supported by circular dichroism spectroscopy of UA in the presence of the corresponding cations.

Antibiotic Pyrrolomycin as an Efficient Mitochondrial Uncoupler.

Pyrrolomycins C (Pyr_C) and D (Pyr_D) are antibiotics produced by Actinosporangium and Streptomyces. The mechanism of their antimicrobial activity consists in depolarization of bacterial membrane, leading to the suppression of bacterial bioenergetics through the uncoupling of oxidative phosphorylation, which is based on the protonophore action of these antibiotics [Valderrama et al., Antimicrob. Agents Chemother. (2019) 63, e01450]. Here, we studied the effect of pyrrolomycins on the isolated rat liver mitochondria. Pyr_C was found to be more active than Pyr_D and uncoupled mitochondria in the submicromolar concentration range, which was observed as the mitochondrial membrane depolarization and stimulation of mitochondrial respiration. In the case of mitoplasts (isolated mitochondria with impaired outer membrane integrity), the difference in the action of Pyr_C and Pyr_D was significantly less pronounced. By contrast, in inverted submitochondrial particles (SMPs), Pyr_D was more active as an uncoupler, which caused collapse of the membrane potential even at the nanomolar concentrations. The same ratio of the protonophoric activity of Pyr_D and Pyr_C was obtained by us on liposomes loaded with the pH indicator pyranine. The protonophore activity of Pyr_D in the planar bilayer lipid membranes (BLMs) was maximal at ~pH 9, i.e., at pH values close to pKa of this compound. Pyr_D functions as a typical anionic protonophore; its activity in the BLM could be reduced by the addition of the dipole modifier phloretin. The difference between the protonophore activity of Pyr_C and Pyr_D in the mitochondria and BLMs can be attributed to a higher ability of Pyr_C to penetrate the outer mitochondrial membrane.

Alkyl esters of 7-hydroxycoumarin-3-carboxylic acid as potent tissue-specific uncouplers of oxidative phosphorylation: Involvement of ATP/ADP translocase in mitochondrial uncoupling.

An impressive body of evidence has been accumulated now on sound beneficial effects of mitochondrial uncouplers in struggling with the most dangerous pathologies such as cancer, infective diseases, neurodegeneration and obesity. To increase their efficacy while gaining further insight in the mechanism of the uncoupling action has been remaining a challenge. Encouraged by our previous promising results on lipophilic derivatives of 7-hydroxycoumarin-4-acetic acid (UB-4 esters), here, we use a 7-hydroxycoumarin-3-carboxylic acid scaffold to synthesize a new series of 7-hydroxycoumarin (umbelliferone, UB)-derived uncouplers of oxidative phosphorylation - alkyl esters of umbelliferone-3-carboxylic acid (UB-3 esters) with varying carbon chain length. Compared to the UB-4 derivatives, UB-3 esters proved to be stronger uncouplers: the most effective of them caused a pronounced increase in the respiration rate of isolated rat heart mitochondria (RHM) at submicromolar concentrations. Both of these series of UB derivatives exhibited a striking difference between their uncoupling patterns in mitochondria isolated from liver and heart or kidney, namely: a pronounced but transient decrease in membrane potential, followed by its recovery, was observed after the addition of these compounds to isolated rat liver mitochondria (RLM), while the depolarization of RHM and rat kidney mitochondria (RKM) was rather stable under the same conditions. Interestingly, partial reversal of this depolarization in RHM and RKM was caused by carboxyatractyloside, an inhibitor of ATP/ADP translocase, thereby pointing to the involvement of this mitochondrial membrane protein in the uncoupling activity of both UB-3 and UB-4 esters. The fast membrane potential recovery in RLM uncoupled by the addition of the UB esters was apparently associated with hydrolysis of these compounds, catalyzed by mitochondrial aldehyde dehydrogenase (ALDH2), being in high abundance in liver compared to other tissues. Protonophoric properties of the UB derivatives in isolated mitochondria were confirmed by measurements of RHM swelling in the presence of potassium acetate. In model bilayer lipid membranes (liposomes), proton-carrying activity of UB-3 esters was demonstrated by measuring fluorescence response of the pH-dependent dye pyranine. Electrophysiological experiments on identified neurons from Lymnaea stagnalis demonstrated low neurotoxicity of UB-3 esters. Resazurin-based cell viability assay showed low toxicity of UB-3 esters to HEK293 cells and primary human fibroblasts. Thus, the present results enable us to consider UB-3 esters as effective tissue-specific protonophoric mitochondrial uncouplers.

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Alkyl esters of umbelliferone-4-acetic acid as protonophores in bilayer lipid membranes and ALDH2-dependent soft uncouplers in rat liver mitochondria.

A great variety of coumarin-related compounds, both natural and synthetic, being often brightly fluorescent, have shown themselves beneficial in medicine for both therapeutic and imaging purposes. Here, in search for effective uncouplers of oxidative phosphorylation, we synthesized a series of 7-hydroxycoumarin (umbelliferone, UB) derivatives combining rather high membrane affinity with the presence of a hydroxyl group deprotonable at physiological pH - alkyl esters of umbelliferone-4-acetic acid (UB-4 esters) differing in alkyl chain length. Addition of UB-4 esters to isolated rat liver mitochondria (RLM) resulted in their rapid depolarization, unexpectedly followed by membrane potential recovery on a minute time scale. According to TLC and HPLC data, incubation of RLM with UB-4 esters caused their hydrolysis, which led to disappearance of the uncoupling activity (recoupling). Both mitochondrial recoupling and hydrolysis of UB-4 esters were suppressed by inhibitors of mitochondrial aldehyde dehydrogenase (ALDH2), disulfiram and daidzin, thus pointing to the involvement of this enzyme in the recoupling of RLM incubated with UB-4 esters. The protonophoric mechanism of mitochondrial uncoupling by UB-4 esters was proved in experiments with artificial bilayer lipid membranes (BLM): these compounds induced proton-selective electrical current across planar BLM and caused dissipation of pH gradient on liposomes. UB-4 esters showed antibacterial activity against Bacillus subtilis, Staphylococcus aureus and Mycobacterium smegmatis.

MPC-Based Prediction of Anti-Mutant Effectiveness of Antibiotic Combinations: In Vitro Model Study with Daptomycin and Gentamicin against Staphylococcus aureus.

To explore whether combined treatments with daptomycin and gentamicin can prevent the development of Staphylococcus aureus resistance, and whether the possible restriction is associated with changes in antibiotic mutant prevention concentrations (MPCs), the enrichment of daptomycin- and gentamicin-resistant mutants was studied by simulating 5-day single and combined treatments in an in vitro dynamic model. The MPCs of the antibiotics in the combination were determined at concentration ratios equal to the ratios of 24 h areas, under the concentration-time curve (AUCs) of the antibiotics, as simulated in pharmacodynamic experiments. The MPCs of both daptomycin and gentamicin decreased in the presence of each other; this led to an increase in the time when antibiotic concentrations were above the MPC (T>MPC). The increases in T>MPCs were concurrent with increases of the anti-mutant effects of the combined antibiotics. When anti-mutant effects of the antibiotics in single and combined treatments were plotted against the T>MPCs, significant sigmoid relationships were obtained. These findings suggest that (1) daptomycin-gentamicin combinations prevent the development of S. aureus resistance to each antibiotic; (2) the anti-mutant effects of antibiotic combinations can be predicted using MPCs determined at pharmacokinetic-based antibiotic concentration ratios; (3) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations.

Anti-mutant efficacy of antibiotic combinations: in vitro model studies with linezolid and daptomycin.

OBJECTIVES: To explore whether linezolid/daptomycin combinations can restrict Staphylococcus aureus resistance and if this restriction is associated with changes in the mutant prevention concentrations (MPCs) of the antibiotics in combination, the enrichment of resistant mutants was studied in an in vitro dynamic model. METHODS: Two MRSA strains, vancomycin-intermediate resistant ATCC 700699 and vancomycin-susceptible 2061 (both susceptible to linezolid and daptomycin), and their linezolid-resistant mutants selected by passaging on antibiotic-containing medium were used in the study. MPCs of antibiotics in combination were determined at a linezolid-to-daptomycin concentration ratio (1:2) that corresponds to the ratio of 24 h AUCs (AUC24s) actually used in the pharmacokinetic simulations. Each S. aureus strain was supplemented with respective linezolid-resistant mutants (mutation frequency 10-8) and treated with twice-daily linezolid and once-daily daptomycin, alone and in combination, simulated at therapeutic and sub-therapeutic AUC24s. RESULTS: Numbers of linezolid-resistant mutants increased at therapeutic and sub-therapeutic AUC24s, whereas daptomycin-resistant mutants were enriched only at sub-therapeutic AUC24 in single drug treatments. Linezolid/daptomycin combinations prevented the enrichment of linezolid-resistant S. aureus and restricted the enrichment of daptomycin-resistant mutants. The pronounced anti-mutant effects of the combinations were attributed to lengthening the time above MPC of both linezolid and daptomycin as their MPCs were lowered. CONCLUSIONS: The present study suggests that (i) the inhibition of S. aureus resistant mutants using linezolid/daptomycin combinations can be predicted by MPCs determined at pharmacokinetically derived antibiotic concentration ratios and (ii) T>MPC is a reliable predictor of the anti-mutant efficacy of antibiotic combinations as studied using in vitro dynamic models.

Lysine 72 substitutions differently affect lipid membrane permeabilizing and proapoptotic activities of horse heart cytochrome c.

Peroxidase activity of cytochrome c (cyt c)/cardiolipin (CL) complex is supposed to be involved in the initiation of apoptosis via peroxidative induction of mitochondrial membrane permeabilization. As cyt c binding to CL-containing membranes is at least partially associated with electrostatic protein/lipid interaction, we screened single-point mutants of horse heart cyt c with various substitutions of lysine at position 72, considered to play a significant role in both the binding and peroxidase activity of the protein. Contrary to expectations, K72A, K72R and K72L substitutions exerted slight effects on both the cyt c binding to CL-containing liposomal membranes and the cyt c/H2O2-induced calcein leakage from liposomes, used here as a membrane permeabilization assay. Both the binding and permeabilization were decreased to various extents, but not significantly, in the case of K72E and K72N mutants. A drastic difference was found between the sequence of the permeabilizing activities of the cyt c variants and the previously described order of their proapoptotic activities (Chertkova et al., 2008).

Protonophoric action of BAM15 on planar bilayers, liposomes, mitochondria, bacteria and neurons.

Small molecules capable of uncoupling respiration and ATP synthesis in mitochondria are protective towards various cell malfunctions. Recently (2-fluorophenyl){6-[(2-fluorophenyl)amino](1,2,5-oxadiazolo[3,4-e]pyrazin-5-yl)}amine (BAM15), a new compound of this type, has become popular as a potent mitochondria-selective depolarizing agent producing minimal adverse effects. To validate protonophoric mechanism of BAM15 action, we examined its behavior in bilayer lipid membranes (BLM). BAM15 proved to be a typical anionic protonophore with the activity on planar membranes being suppressed upon decreasing membrane dipole potential. In both planar BLM and liposomes, BAM15 induced proton conductance with the potency close to that of the classical protonophoric uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP). In isolated rat liver mitochondria (RLM), BAM15 caused membrane potential collapse, increased respiration rate and induced Ca2+ efflux at concentrations slightly higher than those for CCCP. Surprisingly, the uncoupling action of BAM15 on isolated RLM, in contrast to that of CCCP, was partially reversed by carboxyatractyloside (CATR), an inhibitor of adenine nucleotide translocase, thereby indicating involvement of this protein in the BAM15-induced uncoupling. BAM15 inhibited growth of Bacillus subtilis at micromolar concentrations. In electrophysiological experiments on molluscan neurons, BAM15 caused plasma membrane depolarization and suppression of electrical activity, but the effect developed more slowly than that of CCCP.

The mitochondria-targeted derivative of the classical uncoupler of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone is an effective mitochondrial recoupler.

The synthesis of a mitochondria-targeted derivative of the classical mitochondrial uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP) by alkoxy substitution of CCCP with n-decyl(triphenyl)phosphonium cation yielded mitoCCCP, which was able to inhibit the uncoupling action of CCCP, tyrphostin A9 and niclosamide on rat liver mitochondria, but not that of 2,4-dinitrophenol, at a concentration of 1-2 µM. MitoCCCP did not uncouple mitochondria by itself at these concentrations, although it exhibited uncoupling action at tens of micromolar concentrations. Thus, mitoCCCP appeared to be a more effective mitochondrial recoupler than 6-ketocholestanol. Both mitoCCCP and 6-ketocholestanol did not inhibit the protonophoric activity of CCCP in artificial bilayer lipid membranes, which might compromise the simple proton-shuttling mechanism of the uncoupling activity on mitochondria.

Verification of a Novel Approach to Predicting Effects of Antibiotic Combinations: In Vitro Dynamic Model Study with Daptomycin and Gentamicin against Staphylococcus aureus.

To explore whether susceptibility testing with antibiotic combinations at pharmacokinetically derived concentration ratios is predictive of the antimicrobial effect, a Staphylococcus aureus strain was exposed to daptomycin and gentamicin alone or in combination in multiple dosing experiments. The susceptibility of the S. aureus strain to daptomycin and gentamicin in combination was tested at concentration ratios equal to the ratios of 24 h areas under the concentration-time curve (AUC24s) of antibiotics simulated in an in vitro dynamic model in five-day treatments. The MICs of daptomycin and gentamicin decreased in the presence of each other; this led to an increase in the antibiotic AUC24/MIC ratios and the antibacterial effects. Effects of single and combined treatments were plotted against the AUC24/MIC ratios of daptomycin or gentamicin, and a significant sigmoid relationship was obtained. Similarly, when the effects of single and combined treatments were related to the total exposure of both drugs (the sum of AUC24/MIC ratios (∑AUC24/MIC)), a significant sigmoid relationship was obtained. These findings suggest that (1) the effects of antibiotic combinations can be predicted by AUC24/MICs using MICs of each antibacterial determined at pharmacokinetically derived concentration ratios; (2) ∑AUC24/MIC is a reliable predictor of the antibacterial effects of antibiotic combinations.

Usnic acid as calcium ionophore and mast cells stimulator.

Usnic acid (UA), a secondary lichen metabolite, has long been popular as one of natural fat-burning dietary supplements. Similar to 2,4-dinitrophenol, the weight-loss effect of UA is assumed to be associated with its protonophoric uncoupling activity. Recently, we have shown that the ability of UA to shuttle protons across both mitochondrial and artificial membranes is strongly modulated by the presence of calcium ions in the medium. Here, by using fluorescent probes, we studied the calcium-transporting capacity of usnic acid in a variety of membrane systems comprising liposomes, isolated rat liver mitochondria, erythrocytes and rat basophilic leukemia cell culture (RBL-2H3). At concentrations of tens of micromoles, UA appeared to be able to carry calcium ions across membranes in all the systems studied. Similar to the calcium ionophore A23187, UA caused degranulation of RBL-2H3 cells. Therefore, UA, being a protonophoric uncoupler of oxidative phosphorylation, at higher concentrations manifests itself as a calcium ionophore, which could be relevant to its overdose toxicity in humans and also its phytotoxicity.

Membrane Binding of Neuronal Calcium Sensor-1: Highly Specific Interaction with Phosphatidylinositol-3-Phosphate.

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Predicting the antistaphylococcal effects of daptomycin-rifampicin combinations in an in vitro dynamic model.

To predict the effects of combined use of antibiotics on their pharmacodynamics, the susceptibility of Staphylococcus aureus to daptomycin-rifampicin combinations was tested at concentration ratios equal to the ratios of daptomycin and rifampicin 24-h areas under the concentration-time curve (AUC24s) simulated in an in vitro dynamic model. In combination with rifampicin, daptomycin MICs decreased 2- to 31-fold, whereas rifampicin MICs were similar with or without daptomycin. The enhanced susceptibility of S. aureus to daptomycin combined with rifampicin resulted in both an increase of the actual AUC24/MIC ratios and also more pronounced antibacterial effects compared with single treatments. The areas between the control growth and time-kill curves (ABBCs) determined in combined and single daptomycin treatments were plotted against AUC24/MIC on the same graph (r2 0.90). These findings suggest that the effects of daptomycin-rifampicin combinations can be predicted by AUC24/MICs of daptomycin using its MIC determined at pharmacokinetically derived daptomycin-to-rifampicin concentration ratios.

Pyrrolomycins Are Potent Natural Protonophores.

The escalating burden of antibiotic drug resistance necessitates research into novel classes of antibiotics and their mechanism of action. Pyrrolomycins are a family of potent natural product antibiotics with nanomolar activity against Gram-positive bacteria, yet with an elusive mechanism of action. In this work, we dissect the apparent Gram-positive specific activity of pyrrolomycins and show that Gram-negative bacteria are equally sensitive to pyrrolomycins when drug efflux transporters are removed and that albumin in medium plays a large role in pyrrolomycin activity. The selection of resistant mutants allowed for the characterization and validation of a number of mechanisms of resistance to pyrrolomycins in both Staphylococcus aureus and an Escherichia coli ΔtolC mutant, all of which appear to affect compound penetration rather than being target associated. Imaging of the impact of pyrrolomycin on the E. coli ΔtolC mutant using scanning electron microscopy showed blebbing of the bacterial cell wall often at the site of bacterial division. Using potentiometric probes and an electrophysiological technique with an artificial bilayer lipid membrane, it was demonstrated that pyrrolomycins C and D are very potent membrane-depolarizing agents, an order of magnitude more active than conventional carbonyl cyanide m-chlorophenylhydrazone (CCCP), specifically disturbing the proton gradient and uncoupling oxidative phosphorylation via protonophoric action. This work clearly unveils the until-now-elusive mechanism of action of pyrrolomycins and explains their antibiotic activity as well as mechanisms of innate and acquired drug resistance in bacteria.

A novel parameter to predict the effects of antibiotic combinations on the development of Staphylococcus aureus resistance: in vitro model studies at subtherapeutic daptomycin and rifampicin exposures.

The search for optimal predictors of anti-mutant effects remains a pressing problem in studies of antibiotic-associated bacterial resistance. To relate the emergence of bacterial resistance with the antibiotic mutant prevention concentration (MPC), a novel integral parameter - the area around the resistance threshold, i.e. MPC level (AAMPC) is proposed. The AAMPC is the algebraic sum of the area under the antibiotic concentration-time curve that is above the MPC (positive area) and the area above the concentration-time curve that is under the MPC (negative area). To assess the predictive performance of AAMPC, the enrichment of resistant Staphylococcus aureus was studied by simulating treatment with daptomycin and rifampicin alone and in combination in an in vitro dynamic model. The enhanced anti-mutant effects of the antibiotic combinations were due to lowering the negative 24-h AAMPCs. These findings suggest that a novel MPC-related parameter is a reliable predictor of mutant enrichment.

Interaction of Potent Mitochondrial Uncouplers with Thiol-Containing Antioxidants.

It is generally considered that reactive oxygen species (ROS) are involved in the development of numerous pathologies. The level of ROS can be altered via the uncoupling of oxidative phosphorylation by using protonophores causing mitochondrial membrane depolarization. Here, we report that the uncoupling activity of potent protonophores, such as carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and fluazinam, can be abrogated by the addition of thiol-containing antioxidants to isolated mitochondria. In particular, N-acetylcysteine, glutathione, cysteine, and dithiothreitol removed both a decrease in the mitochondrial membrane potential and an increase in the respiration rate that is caused by FCCP. The thiols also reduced the electrical current that is induced by FCCP and CCCP across planar bilayer lipid membranes. Thus, when speculating on the mechanistic roles of ROS level modulation by mitochondrial uncoupling based on the antioxidant reversing certain FCCP and CCCP effects on cellular processes, one should take into account the ability of these protonophoric uncouplers to directly interact with the thiol-containing antioxidants.