Ronald Gust

Small cationic antimicrobial peptides delocalize peripheral membrane proteins

Significance Multidrug-resistant bacteria present an acute problem to medicine, generating interest in novel antimicrobial strategies. Antimicrobial peptides currently are being investigated, both as antibiotics and as immunomodulatory agents. Many antimicrobial peptides interact with the bacterial membrane, a previously underexplored antibiotic target. We present a system-based study of the mode of action of small cationic peptides and the mechanisms that bacteria use to defend against them. We show that peptide integration into the membrane causes delocalization of essential peripheral membrane proteins. This delocalization impacts on two cellular processes, namely respiration and cell-wall biosynthesis. We describe a bacterial survival strategy in which mechanosensitive channels in the bacterial membrane establish osmoprotection against membrane-targeting bacteriolytic peptides. Understanding the peptides mode of action and bacterial survival strategies opens up new avenues for devising peptide-based antibacterial strategies. Short antimicrobial peptides rich in arginine (R) and tryptophan (W) interact with membranes. To learn how this interaction leads to bacterial death, we characterized the effects of the minimal pharmacophore RWRWRW-NH2. A ruthenium-substituted derivative of this peptide localized to the membrane in vivo, and the peptide also integrated readily into mixed phospholipid bilayers that resemble Gram-positive membranes. Proteome and Western blot analyses showed that integration of the peptide caused delocalization of peripheral membrane proteins essential for respiration and cell-wall biosynthesis, limiting cellular energy and undermining cell-wall integrity. This delocalization phenomenon also was observed with the cyclic peptide gramicidin S, indicating the generality of the mechanism. Exogenous glutamate increases tolerance to the peptide, indicating that osmotic destabilization also contributes to antibacterial efficacy. Bacillus subtilis responds to peptide stress by releasing osmoprotective amino acids, in part via mechanosensitive channels. This response is triggered by membrane-targeting bacteriolytic peptides of different structural classes as well as by hypoosmotic conditions.

Synthesis, characterization and in vitro antitumour activity of a series of novel platinum(II) complexes bearing Schiff base ligands.

A series was neutral platinum(II) complexes bearing OCH(3)- or F-substituted 3,4-bis(4-fluorophenyl)-1,6-bis(2-hydroxyphenyl)-2,5-diazahexa-1,5-dienes (diarylsalenes) were synthesized and tested for in vitro antitumour activity. The growth inhibitory effects depended on the configuration and the substitution pattern of the salicylidene moiety. The lead compound [meso-3,4-bis(4-fluorophenyl)-1,6-bis(2-hydroxyphenyl)-2,5-diazahexa-1,5-diene]platinum(II) (1-Pt) reduced the cell growth of MCF-7 (IC(50) = 7.6 μM) and MDA-MB 231 cells (IC(50) = 10.0 μM), but was inactive against HT-29 cells at the used concentration range (IC(50) > 20 μM). The change of the configuration (meso → d,l) at the 1,2-diimino-1,2-diarylethane bridge and methoxy substitution led to completely inactive compounds, while fluorine substituents increased the antiproliferative effects depending on their position (3-F < 5-F < 4-F < 6-F). Complex 10-Pt (6-F: IC(50)(MCF-7) = 1.5 μM, IC(50)(MDA-MB 231) = 1.3 μM, IC(50) (HT-29) = 2.6 μM) was as active as cisplatin (IC(50)(MCF-7) = 1.6 μM, IC(50)(MDA-MB 231) = 1.5 μM, IC(50)(HT-29) = 4.1 μM).

Pyrimidine-2,4,6-triones are a new class of voltage-gated L-type Ca2+ channel activators

Cav1.2 and Cav1.3 are the main L-type Ca2+ channel subtypes in the brain. Cav1.3 channels have recently been implicated in the pathogenesis of Parkinson’s disease. Therefore, Cav1.3-selective blockers are developed as promising neuroprotective drugs. We studied the pharmacological properties of a pyrimidine-2,4,6-trione derivative (1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, Cp8) recently reported as the first highly selective Cav1.3 blocker. Here we show, in contrast to this previous study, that Cp8 reproducibly increases inward Ca2+ currents of Cav1.3 and Cav1.2 channels expressed in tsA-201 cells by slowing activation, inactivation and enhancement of tail currents. Similar effects are also observed for native Cav1.3 and Cav1.2 channels in mouse chromaffin cells, while non-L-type currents are unaffected. Evidence for a weak and non-selective inhibition of Cav1.3 and Cav1.2 currents is only observed in a minority of cells using Ba2+ as charge carrier. Therefore, our data identify pyrimidine-2,4,6-triones as Ca2+ channel activators.

Recent methodological and instrumental development in MEKC

The review gives an update about the methodological and instrumental developments in micellar electrokinetic capillary chromatography as a type of CE analytical technique. Here, the last two years development of the technique are particularly presented. Recent approaches to improve sensitivity are discussed. Newly introduced concentration techniques and experimental methods for verification of the different mechanisms and processes of micellar electrokinetic chromatography analysis are highlighted. A theoretical model to explain changes in separation and electrophoretic mobility order of fully charged analytes are demonstrated. Modern approaches for improving compatibility of micellar electrokinetic capillary chromatography to mass spectrometry are also reported.

Thiopyrazole preactivated chitosan: combining mucoadhesion and drug delivery.

The objective of this study was to develop a preactivated chitosan derivative by the introduction of thioglycolic acid followed by 3-methyl-1-phenylpyrazole-5-thiol (MPPT) coupling via disulfide bond formation. The newly synthesized conjugate was characterized in terms of water-absorbing capacity, cohesive properties, mucoadhesion and drug release kinetics. Further in vitro characterization was conducted regarding permeation enhancement of the model compound fluorescein isothiocyanate dextran (FD4) and cytotoxic effects on Caco-2 cells. Based on the attachment of the hydrophobic residue, chitosan-S-S-MPPT test discs showed increased stability of the polymer matrix as well as improved water uptake and liberation of fluorescein isothiocyanate dextran (FD4) compared to chitosan only. The mucoadhesive qualities on porcine intestinal mucosa could be improved 38-fold based on the enhanced bonding between chitosan-S-S-MPPT and mucus through the thiol/disulfide exchange reaction of polymer and mucosal cysteine-rich domains supported by MPPT as the leaving group. This novel biomaterial presents a disulfide conjugation-based delivery system that releases the antibacterial thiopyrazole when the polymer comes into contact with the intestinal mucosa. These properties, together with the safe toxicological profile, make chitosan-S-S-MPPT a valuable carrier for mucoadhesive drug delivery systems and a promising matrix for the development of antimicrobial excipients.

Conformational analysis of bivalent estrogen receptor ligands: from intramolecular to intermolecular binding.

The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra‐ and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.

Evaluation of selected 3D virtual screening tools for the prospective identification of peroxisome proliferator-activated receptor (PPAR) γ partial agonists

The peroxisome proliferator-activated receptor (PPAR) γ regulates the expression of genes involved in adipogenesis, lipid homeostasis, and glucose metabolism, making it a valuable drug target. However, full activation of the nuclear receptor is associated with unwanted side effects. Research therefore focuses on the discovery of novel partial agonists, which show a distinct protein-ligand interaction pattern compared to full agonists. Within this study, we employed pharmacophore- and shape-based virtual screening and docking independently and in parallel for the identification of novel PPARγ ligands. The ten top-ranked hits retrieved with every method were further investigated with external in silico bioactivity profiling tools. Subsequent biological testing not only confirmed the binding of nine out of the 29 selected test compounds, but enabled the direct comparison of the method performances in a prospective manner. Although all three methods successfully identified novel ligands, they varied in the numbers of active compounds ranked among the top-ten in the virtual hit list. In addition, these compounds were in most cases exclusively predicted as active by the method which initially identified them. This suggests, that the applied programs and methods are highly complementary and cover a distinct chemical space of PPARγ ligands. Further analyses revealed that eight out of the nine active molecules represent novel chemical scaffolds for PPARγ, which can serve as promising starting points for further chemical optimization. In addition, two novel compounds, identified with docking, proved to be partial agonists in the experimental testing.

The Biological Activity of Zeise’s Salt and its Derivatives†

With the aim to design new biologically active bioinorganic drugs of aspirin, whose mode of action is based on the inhibition of the cyclooxygenase(COX) enzymes, derivatives of Zeises salt were synthesized in this structure-activity relationship study. Surprisingly, not only these Zeise-aspirin compounds but also Zeises salt itself showed high inhibitory potency against COX enzymes in inu2005vitro assays. In contrast, potassium tetrachloroplatinate and cisplatin did not influence the enzyme activity at equimolar concentrations. It was demonstrated by LC-ESI tandem-mass spectrometry that Zeises salt platinates the essential amino acids Tyr385 (active site of the enzyme) and Ser516 (will be acetylated by aspirin) of COX-1, thereby strongly impairing the function of the enzyme. This finding demonstrates for the first time that Zeises salt is pharmacologically active and is a potent enzyme inhibitor.

A highly sensitive method for in vitro testing of fluorinated drug candidates using high-resolution continuum source molecular absorption spectrometry (HR-CS MAS)

AbstractWe report here the development, optimization, and evaluation of a highly sensitive method for the determination of fluorine in biological matrices employing highresolution continuum source molecular absorption spectrometry (HR-CS MAS), suitable for pharmacological testing of fluorine-containing drug candidates. For this purpose, the most important parameters were studied in detail and subsequently optimized using a multivariate approach based on experimental design methodology. We developed a new approach employing a graphite tube lined with tantalum foil, thereby significantly enhancing sensitivity, while interferences from phosphorus monoxide (PO) molecular absorption due to the complex phosphate-rich matrix were completely eliminated. The limit of detection and the characteristic mass were 5.79 and 6.08xa0pg F, respectively. In order to evaluate the accuracy of the procedure, a recovery test was performed using spiked samples from three bioassays (i.e., DNA binding, protein binding, and cellular uptake) and the recovery rates ranged from 97.4 to 106.4xa0%. The proposed method is applicable for preclinical in vitro testing of fluorinated drug molecules and thereby establishes HR-CS atomic absorption spectrometry instrumentation as a universal tool in medicinal chemistry.n FigureDetermination of fluorine in biological matrices for pharmacological evaluation of fluorinated drug molecules

Characterization of telmisartan-derived PPARγ agonists: importance of moiety shift from position 6 to 5 on potency, efficacy and cofactor recruitment.

Selective modulation of the peroxisome proliferator‐activated receptor gamma (PPARγ) by direct binding of small molecules demonstrates a promising tool for treatment of insulin resistance and typeu20052 diabetes mellitus. Besides its blood pressure‐lowering properties, the AT1‐receptor blocker telmisartan has been shown to be a partial agonist of PPARγ with beneficial metabolic effects in vitro and in mice. In our previous work, comprehensive structure–activity relationship (SAR) studies discussed the different parts of the telmisartan structure and various moieties. Based on these findings, we designed and synthesized new PPARγ ligands with a benzimidazole (agonists 4‐5 and 4‐6), benzothiophene (agonists 5‐5 and 5‐6) or benzofuran (agonists 6‐5 and 6‐6) moiety either at positionu20055 oru20056 of the benzimidazole core structure. Lipophilicity and EC50 values were improved for all new compounds compared with telmisartan. Regarding PPARγ activation, the compounds were characterized by a differentiation assay using 3T3‐L1 cells and a luciferase assay with COS‐7 cells transiently transfected with pGal4‐hPPARgDEF, pGal5‐TK‐pGL3 and pRL‐CMV. A decrease in both potency and efficacy was observed after the shift of either the benzothiophene or the benzofuran moiety from positionu20056 to positionu20055. Selective recruitment of the coactivators TRAP220, SRC‐1 and PGC‐1α, and release of corepressor NCoR1 determined by time‐resolved fluorescence resonance energy transfer (TR‐FRET) was detected depending on residues in positionu20055 oru20056.