Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Cyst hatching in Anostraca accelerated by retinoic acid,amplified by Calcium Ionophore A23187, and inhibited by Calcium-channel blockers

Cyst hatching, under standardized conditions, of the Anostracan species Thamnocephalus platyurus and Streptocephalus dichotomus was significantly accelerated but not increased by applying the morphogen retinoic acid (RA). Cyst hatching was enhanced but not accelerated by artificially increasing the inflow of Ca²⁺ to the embryonic cells, using Calcium Ionophore A 23 187. Cyst hatching was accelerated and amplified, to a level in excess of the summed effects of each treatment, by a combined application of RA and ionophore. It was inhibited almost quantitatively by the Calcium-channel blockers Nifedipin and Verapamil. The significance of these findings is discussed.     

One-pot synthesis and cytotoxicity studies of new Mannich base derivatives of polyether antibiotic—Lasalocid acid

Seven Mannich base derivatives of polyether antibiotic Lasalocid acid (2a–2g) were synthesized and screened for their antiproliferative activity against various human cancer cell lines. A novel chemoselective one-pot synthesis of these Mannich bases was developed. Compounds 2a–2c and 2g with sterically smaller dialkylamine substituent, displayed potent antiproliferative activity (IC₅₀: 3.2–7.3 μM), and demonstrated higher than twofold selectivity for specific type of cancer. The nature of Mannich base substituent on C-2 atom at the aromatic ring may be critical in the search for selectivity towards a particular cancer cell.     

Antibacterial activity of singly and doubly modified salinomycin derivatives

The increasing challenge of antibiotic resistance stimulates the search for novel antibacterial agents, especially such that would be effective against multi-drug resistant bacterial strains. Fortunately, natural compounds are excellent sources of potentially new drug leads. Particularly interesting in this context are polyether antibiotic salinomycin (SAL) and its semi-synthetic derivatives, as they exhibit large spectrum of bioactivity. We synthesized and evaluated the antibacterial activity of a series of SAL analogs; four singly (2–3, 15, 17) and two doubly modified (16, 18) derivatives were found to show excellent inhibitory activity not only against planktonic Gram(+) bacterial cells, but also towards select strains of methicillin-resistant staphylococci with the MIC values of 1–4 µg mL⁻¹. Of note, the most promising candidates were more effective in preventing bacterial biofilm formation than unmodified SAL and a commonly used antibiotic – ciprofloxacin. Furthermore, we proved that rational modification of C20 hydroxyl of SAL may reduce genotoxic properties of the obtained analogs. Mechanistically, the structure-activity relationship studies suggested that electroneutral transport mechanism could be beneficial in terms of ensuring high antibacterial activity of SAL derivatives.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Synthesis,antiproliferative and antibacterial activity of new amides of salinomycin

A series of 11 novel amides of salinomycin were synthesized for the first time. All the obtained compounds were found to show potent antiproliferative activity against human cancer cell lines including the drug-resistant cancer cells. Four new salinomycin derivatives revealed good antibacterial activity against clinical isolates of methicillin-resistant Staphylococcus epidermidis (MRSE).     

Halting ionic shuttle to disrupt the synthetic machinery—Structural and molecular insights into the inhibitory roles of Bedaquiline towards Mycobacterium tuberculosis ATP synthase in the treatment of tuberculosis

Therapeutic targeting of the adenosine triphosphate (ATP) machinery of Mycobacterium tuberculosis (Mtb) has recently presented a potent and alternative measure to halt the pathogenesis of tuberculosis. This has been potentiated by the development of bedaquiline (BDQ), a novel small molecule inhibitor that selectively inhibits mycobacterial F₁F_o-ATP synthase by targeting its rotor c-ring, resulting in the disruption of ATP synthesis and consequential cell death. Although the structural resolution of the mycobacterial C₉ ring in co`mplex with BDQ provided the first-hand detail of BDQ interaction at the c-ring region of the ATP synthase, there still remains a need to obtain essential and dynamic insights into the mechanistic activity of this drug molecule towards crucial survival machinery of Mtb. As such, for the first time, we report an atomistic model to describe the structural dynamics that explicate the experimentally reported antagonistic features of BDQ in halting ion shuttling by the mycobacterial c-ring, using molecular dynamics simulation and the Molecular Mechanics/Poisson-Boltzmann Surface Area methods. Results showed that BDQ exhibited a considerably high ΔG while it specifically maintained high-affinity interactions with Glu65_B and Asp32_B, blocking their crucial roles in proton binding and shuttling, which is required for ATP synthesis. Moreover, the bulky nature of BDQ induced a rigid and compact conformation of the rotor c-ring, which impedes the essential rotatory motion that drives ion exchange and shuttling. In addition, the binding affinity of a BDQ molecule was considerably increased by the complementary binding of another BDQ molecule, which indicates that an increase in BDQ molecule enhances inhibitory potency against Mtb ATP synthase. Taken together, findings provide atomistic perspectives into the inhibitory mechanisms of BDQ coupled with insights that could enhance the structure-based design of novel ATP synthase inhibitors towards the treatment of tuberculosis.     

Formation of ion-translocating oligomers by nigericin

Summary At pH 4.0, >10^–7 m nigericin was found capable of conducting net charge transfer across bimolecular lecithin membranes, with a stoichiometry of three uncharged ionophore moieties per cation. At neutral or alkaline pH, nigericin catalyzed the transfer of net charge through dimer forms. In agreement with these results, quantitative analysis of nigericin-potassium complexes formed at pH 4.0 showed a 31 ratio, and a 21 ratio at neutral or alkaline pH. A 11 stoichiometry was observed when the ionophore complex was not transferred from methanol-water to chloroform. Moreover,¹H-NMR spectra of nigericin-cation complexes formed at pH 4.0, displayed clear-cut chemical shift variations different to those observed at neutral or alkaline pH. Thus, it is apparent that acid pH causes a transition from dimeric to trimeric forms of nigericin-cation complexes. The membrane conductance increased up to ten times when negatively charged phosphatidyl glycerol was used, while the conductance decreased in positively charged cetylpyridinium containing membranes at pH 4.0. These results suggest that the nigericin-K⁺ oligomeric complex is positively charged. In this respect, pK _a values around 8.0 were obtained for the nigericin carboxylate group in media of different dielectric constant, indicating that this chemical group is undissociated under these conditions. Moreover, the values for the complex formation constants as well as the G values calculated for the dimers and trimers indicated that such ionophore cation oligomeric complexes are thermodynamically stable.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.