Formulation and evaluation of anion transporters in nanostructured lipid carriers.

Six novel click-tambjamines (1-6) bearing an alkyl chain of varying length linked to the imine moiety have been formulated in nanostructured lipid carriers (NLCs) to evaluate their transmembrane anion transport activity both when free (i.e., not encapsulated) and nanoformulated. Nanostructured lipid carriers (NLCs) are an example of drug delivery systems (DDSs) that stand out because of their versatility. In this work we show that NLCs can be used to efficiently formulate highly lipophilic anionophores and experiments conducted in model liposomes reveal that these formulations are adequate to deliver anionophores without compromising their transport activity. This result paves the way to facilitate the study of highly lipophilic anionophores and their potential use as future drugs.

Small Molecule Anion Carriers Correct Abnormal Airway Surface Liquid Properties in Cystic Fibrosis Airway Epithelia.

Cystic fibrosis (CF) is a genetic disease characterized by the lack of cystic fibrosis transmembrane conductance regulator (CFTR) protein expressed in epithelial cells. The resulting defective chloride and bicarbonate secretion and imbalance of the transepithelial homeostasis lead to abnormal airway surface liquid (ASL) composition and properties. The reduced ASL volume impairs ciliary beating with the consequent accumulation of sticky mucus. This situation prevents the normal mucociliary clearance, favouring the survival and proliferation of bacteria and contributing to the genesis of CF lung disease. Here, we have explored the potential of small molecules capable of facilitating the transmembrane transport of chloride and bicarbonate in order to replace the defective transport activity elicited by CFTR in CF airway epithelia. Primary human bronchial epithelial cells obtained from CF and non-CF patients were differentiated into a mucociliated epithelia in order to assess the effects of our compounds on some key properties of ASL. The treatment of these functional models with non-toxic doses of the synthetic anionophores improved the periciliary fluid composition, reducing the fluid re-absorption, correcting the ASL pH and reducing the viscosity of the mucus, thus representing promising drug candidates for CF therapy.

Small molecule inhibitors of bacterial transcription complex formation.

Knoevenagel condensation was employed to generate a set of molecules potentially capable of inhibiting the RNA polymerase-σ70/σA interaction in bacteria. Synthesis was achieved via reactions between a variety of indole-7-carbaldehydes and rhodanine, N-allylrhodanine, barbituric acid or thiobarbituric acid. A library of structurally diverse compounds was examined by enzyme-linked immunosorbent assay (ELISA) to assess the inhibition of the targeted protein-protein interaction. Inhibition of bacterial growth was also evaluated using Bacillus subtilis and Escherichia coli cultures. The structure-activity relationship studies demonstrated the significance of particular structural features of the synthesized molecules for RNA polymerase-σ70/σA interaction inhibition and antibacterial activity. Docking was investigated as an in silico method for the further development of the compounds.

From indole to pyrrole, furan, thiophene and pyridine: Search for novel small molecule inhibitors of bacterial transcription initiation complex formation.

The search for small molecules capable of inhibiting transcription initiation in bacteria has resulted in the synthesis of N,N'-disubstituted hydrazines and imine-carbohydrazides comprised of indole, pyridine, pyrrole, furan and thiophene using the respective trichloroacetyl derivatives, carbohydrazides and aldehydes. Replacement of the indole moiety by smaller heterocycles linked by CONHNC linkers afforded a broad variety of compounds efficiently targeting the RNA polymerase-σ(70)/σ(A) interaction as determined by ELISA and exhibiting increased inhibition of the growth of Escherichia coli compared to Bacillus subtilis in culture. The structural features of the synthesized transcription initiation inhibitors needed for antibacterial activity were identified employing molecular modelling and structure-activity relationship (SAR) studies.

Synthesis and biological activity of novel mono-indole and mono-benzofuran inhibitors of bacterial transcription initiation complex formation.

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and σ(70)/σ(A) factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-σ(70)/σ(A) interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

Synthesis and biological activity of novel bis-indole inhibitors of bacterial transcription initiation complex formation.

The increasing resistance of bacteria against clinically approved antibiotics is resulting in an alarming decrease in therapeutic options for today's clinicians. We have targeted the essential interaction between bacterial RNA polymerase and σ(70)/σ(A) for the development of lead molecules exhibiting a novel mechanism of antibacterial activity. Several classes of structurally related bis-indole inhibitors of bacterial transcription initiation complex formation were synthesized and their antimicrobial activities were evaluated. Condensation of indole-7- and indole-2-carbohydrazides with 7- and 2-trichloroacetylindoles or indole-7- and indole-2-glyoxyloyl chlorides resulted in the successful synthesis of 7,7'-, 2,2'-, 2,7'- and 3,2'-linked bis-indole derivatives with -CO-NH-NH-CO- and -CO-CO-NH-NH-CO- linkers. Indole-7-glyoxyloyl chlorides were reacted with hydrazine hydrate in different ratios to afford respective -CO-CO-NH-NH-CO-CO- bis-indole or hydrazide derivatives. The resulting compounds were found to be active against the ß'-CH-σ(70)/σ interaction in ELISA assays and inhibited the growth of both Gram-positive and Gram-negative bacteria. Structure-activity relationship (SAR) studies were performed in order to identify the structural features of the synthesized inhibitors required for biological activity.

Click-tambjamines as efficient and tunable bioactive anion transporters.

A novel class of transmembrane anion carriers, the click-tambjamines, display remarkable anionophoric activities in model liposomes and living cells. The versatility of this building block for the generation of molecular diversity offers promise to develop future drugs based on this design.

In-stent coronary restenosis, but not the type of stent, is associated with impaired endothelial-dependent vasodilatation.

BACKGROUND: Precise mechanisms leading to restenosis are not fully understood. The type of implanted stent and the intensity of atherogenic processes may affects the restenosis rate. AIM: To compare the long-term effects of the coronary stent implantation - paclitaxel-eluting stent (PES) or bare-metal stents (BMS) - on endothelial-dependent flow-mediated dilation (FMD), platelet-derived growth factor (PDGF) and asymmetric dimethylarginine (ADMA) serum levels and to assess the relationship between FMD, PDGF, ADMA and every-stage in-stent restenosis (eISR). METHODS: The study population included 40 patients with coronary artery disease, who underwent elective percutaneous coronary intervention (PCI) of the left anterior descending artery (LAD) with stent implantation (PES - 21 patients; BMS - 19 patients). Follow-up examination was performed 12 months after PCI. RESULTS: There were no differences between the PES and the BMS patients regarding FMD (PES: 11.8+/-7.8%, BMS: 10.5+/-9.2%), PDGF (PES: 5540+/-2209 pg/ml, BMS: 4923+/-2924 pg/ml) and ADMA (PES: 0.474+/-0.04 micromol/l, BMS: 0.456+/-0.03 micromol/l) serum levels. The follow-up angiography was performed when clinically indicated in 25 patients: in 15 patients with PES and 10 patients with BMS implanted. The eISR was found in 12 subjects: in 7 (47%) with PES and in 5 (50%) with BMS (NS). In all patients with eISR, the FMD values were significantly lower (6.1+/-3.5%, p=0.003) compared to the patients without eISR (14.3+/-7.8%). FMD was the only independent risk factor for eISR (OR=0.631, 95% CI 0.412-0.942, p=0.0003). The cut-off point for FMD < or = 8.4% as a parameter predicting eISR was established (p=0.0001, sensitivity: 83.3%, specificity: 92.3%, PPV: 90.9%, NPV: 85.7%). CONCLUSIONS: The type of stent implanted into LAD does not affect the FMD, PDGF and ADMA serum levels assessed one-year after a PCI procedure. The occurrence of an early in-stent restenosis is associated with impaired FMD at the time of one-year follow-up.

Small molecule-facilitated anion transporters in cells for a novel therapeutic approach to cystic fibrosis.

BACKGROUND AND PURPOSE: Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease that originates from the defective function of the CF transmembrane conductance regulator (CFTR) protein, a cAMP-dependent anion channel involved in fluid transport across epithelium. Because small synthetic transmembrane anion transporters (anionophores) can replace the biological anion transport mechanisms, independent of genetic mutations in the CFTR, such anionophores are candidates as new potential treatments for CF. EXPERIMENTAL APPROACH: In order to assess their effects on cell physiology, we have analysed the transport properties of five anionophore compounds, three prodigiosines and two tambjamines. Chloride efflux was measured in large uni-lamellar vesicles and in HEK293 cells with chloride-sensitive electrodes. Iodide influx was evaluated in FRT cells transfected with iodide-sensitive YFP. Transport of bicarbonate was assessed by changes of pH after a NH4 + pre-pulse using the BCECF fluorescent probe. Assays were also carried out in FRT cells permanently transfected with wild type and mutant human CFTR. KEY RESULTS: All studied compounds are capable of transporting halides and bicarbonate across the cell membrane, with a higher transport capacity at acidic pH. Interestingly, the presence of these anionophores did not interfere with the activation of CFTR and did not modify the action of lumacaftor (a CFTR corrector) or ivacaftor (a CFTR potentiator). CONCLUSION AND IMPLICATIONS: These anionophores, at low concentrations, transported chloride and bicarbonate across cell membranes, without affecting CFTR function. They therefore provide promising starting points for the development of novel treatments for CF.

Small molecule anion transporters display in vitro antimicrobial activity against clinically relevant bacterial strains.

Highly active transmembrane anion transporters have demonstrated their activity against antibiotic-resistant and clinically relevant bacterial strains. This type of compound offers promise as a strategy to develop novel antibacterial agents.

Severe dilated cardiomyopathy as a consequence of Ecstasy intake.

Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure with a prevalence of 1:2500. There are several primary and secondary etiologic factors, including gene mutations, infection agents, particularly viruses, toxins, autoimmune, and systemic disorders, and pheochromocytoma, neuromuscular, metabolic, mitochondrial, and nutritional disorders. However, a precise diagnosis can be reached only in no more than 50% of all cases. Herein, we report a rare case of hepatic damage and severe DCM as a consequence of relatively popular socially used narcotic-Ecstasy (3,4-methylenedioxy-N-methylamphetamine [MDMA]).

Inhibitors of bacterial transcription initiation complex formation.

Antibiotic resistance is a growing global problem, with very few new compounds in development. Bacterial transcription is an underutilized target for antibiotics, which has been attributed to the similarity of the active site of RNA polymerases (RNAPs) across all domains of life and the ease with which resistance can arise through point mutation at multiple sites within this conserved region. In this study we have taken a rational approach to design a novel set of compounds that specifically target the formation of transcription initiation complexes by preventing the unique bacterial σ initiation factor from binding to RNAP. We have identified the region of RNAP to which these compounds bind and demonstrate that one compound, GKL003, has an inhibition constant in the low nanomolar range. This compound has activity against both Gram-positive and -negative organisms, including a community acquired methicillin-resistant strain of the major pathogen Staphylococcus aureus.

Mental stress, heart rate and endothelial function in patients with syndrome X.

BACKGROUND: The aim of the study was to determine whether the baseline heart rate (HR) and changes in HR after mental stress (MS) can influence endothelial function in syndrome X. METHODS: Forty four patients with syndrome X (F/M: 21/23, mean age: 55.4 +/- 10.7 years) were examined. The endothelium-dependent flow-mediated dilation (FMD) was defined as the percentage change in the brachial artery diameter during reactive hyperaemia related to baseline (%FMD). The %FMD was assessed before and after (at 10, 30, and 45 min) standardised three-minute MS. HR and blood pressure were monitored simultaneously. The %FMD values were compared between subgroups characterised by baseline HR, maximum HR and DHR, and HR after MS below and over the median values. RESULTS: The values of %FMD measured at 10, 30 and 45 min after MS (4.39 +/- 5.4%, 4.99 +/- 3.9%, 4.03 +/- 3.5%, respectively; p < 0.001) were significantly lower than baseline values (7.73 +/- 4.9%). Impaired vasodilatation after MS was observed in the following subgroups of patients: those with baseline HR below the median (< 71.5 bpm; baseline: 8.35 +/- 5.8%; 10 min: 2.87 +/- 3.6%, 45 min: 4.56 +/- 3.9%; p < 0.001); those with HR after MS below the median (< 76.5 bpm; baseline: 8.19 +/- 5.5; 10 min: 3.88 +/- 4.3%, 45 min: 4.59 +/- 3.7%; p < 0.01); and those with maximum HR after MS below the median (< 84 bpm; baseline: 8.88 +/- 5.6%; 10 min: 3.88 +/- 3.8%, 30 min: 5.88 +/- 3.9%, 45 min: 4.51 +/- 3.8; p < 0.01). CONCLUSION: The stress-induced endothelial dysfunction syndrome X is related to the baseline HR and the changes in HR after MS, suggesting that the autonomic nervous system plays a part in its pathogenesis. (Cardiol J 2007; 14: 180-185).