Selective isolation and identification of N‐terminal blocked peptides from tryptic protein digests

Abstract: A method for the easy isolation and direct sequencing of N‐terminally blocked peptide in proteins refractory to N‐terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the N^α‐blocked peptide using ion‐exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N‐terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.     

Transport characteristics of peptides and peptidomimetics: I. N‐methylated peptides as substrates for the oligopeptide transporter and P‐glycoprotein in the intestinal mucosa

Abstract: Peptides and peptidomimetics often exhibit poor oral bioavailability due to their metabolic instability and low permeation across the intestinal mucosa. N‐Methylation has been used successfully in peptide‐based drug design in an attempt to improve the metabolic stability of a peptide‐based lead compound. However, the effect of N‐methylation on the absorption of peptides through the intestinal mucosa is not well understood, particularly when transporters, i.e. the oligopeptide transporter (OPT) and P‐glycoprotein (P‐gp), modulate the passive diffusion of these types of molecules. To examine this, terminally free and terminally modified (N‐acetylated and C‐amidated) analogs of H‐Ala‐Phe‐Ala‐OH with N‐methyl groups on either the Ala‐Phe or Phe‐Ala peptide bond were synthesized. Transport studies using Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa, showed that N‐methylation of the Ala‐Phe peptide bond of H‐Ala‐Phe‐Ala‐OH stabilized the molecule to protease degradation, and the resulting analog exhibited significant substrate activity for OPT. However, N‐methylation of the Phe‐Ala peptide bond of H‐Ala‐Phe‐Ala‐OH did not stabilize the molecule to protease degradation, and the substrate activity of the resulting molecule for OPT could not be determined. Interestingly, N‐methylation of the Phe‐Ala peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ decreased the substrate activity of the molecule for the efflux transporter P‐gp. In contrast, N‐methylation of the Ala‐Phe peptide bond of the terminally modified tripeptide Ac‐Ala‐Phe‐Ala‐NH₂ increased the substrate activity of the molecule for P‐gp.     

13C‐ and 14C‐labelling of N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐methyleneamino] guanidinium acetate

N‐[1‐(4‐chlorophenyl)‐1H‐pyrrol‐2‐yl‐¹³C₄‐methyleneamino]guanidinium acetate has been synthesized by a four‐step procedure. This involved reduction of the Weinreb amide N,N′‐dimethyl‐N,N′‐dimethyloxybutane‐1,4‐diamide‐1,2,3,4‐¹³C₄ by Dibal‐H to give the corresponding unstable dialdehyde which is reacted in situ with 4‐chloroaniline to form 1‐(4‐chlorophenyl)‐1H‐pyrrole‐¹³C₄. This pyrrole analogue underwent a Vilsmeyer acylation with POCl₃/DMF followed by final reaction with aminoguanidine bicarbonate to produce the desired labelled compound with 99% atom ¹³C. By using DMF [α‐¹⁴C] a radio‐labelled analogue was synthesized with a specific activity of 60 mCi/mmol. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of [2‐13C, 4‐13C]‐(2R,3S)‐catechin and [2‐13C, 4‐13C]‐(2R,3R)‐epicatechin

The first synthesis of doubly labeled, [2‐¹³C, 4‐¹³C]‐(2R,3S)‐catechin 15 and [2‐¹³C, 4‐¹³C]‐(2R,3R)‐epicatechin 18 starting from labeled 2‐hydroxy‐4, 6‐bis(benzyloxy)acetophenone 3 and labeled 3, 4‐bis(benzyloxy)‐benzaldehyde 7 are described. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of C‐14 and C‐13, H‐2‐labeled IKK inhibitor: [14C] and [13C4,D3]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido[3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide

[¹⁴C]‐N‐(6‐Chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5B ), an IKK inhibitor, was synthesized from [¹⁴C]‐barium carbonate in two steps in an overall radiochemical yield of 41%. The intermediate, [carboxyl‐¹⁴C]‐2‐methylnicotinic acid, was prepared by the lithiation and carbonation of 3‐bromo‐2‐methylpyridine. [¹³C₄,D₃]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5C ) was synthesized from [1,2,3,4‐¹³C₄]‐ethyl acetoacetate and [D₄]‐methanol in six steps in an overall yield of 2%. [¹³C₄]‐2‐methylnicotic acid, was prepared by condensation of [¹³C₄]‐ethyl 3‐aminocrotonate and acrolein, followed by hydrolysis with lithium hydroxide. Copyright © 2006 John Wiley & Sons, Ltd.     

Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS‐TTEGDA support for the investigation of HCV infection in human blood

Abstract: Four peptides were designed and synthesized on a highly solvating copolymer of tetraethyleneglycol diacrylate cross‐linked polystyrene (PS‐TTEGDA) support with very high purity and yield. The polymer was synthesized in various cross‐linking densities (1, 2, 3, 4, 5 and 10%) using radical aqueous suspension polymerization. Four per cent PS‐TTEGDA resin showed rigidity and mechanical characteristics comparable with those of divinylbenzene cross‐linked polystyrene (PS‐DVB) support. Swelling and solvation characteristics of PS‐TTEGDA were much higher than PS‐DVB support in all solvents used in solid‐phase peptide synthesis. Forty‐eight hour treatment of the support with neat trifluoroacetic acid did not show any change in its infrared spectra. PS‐TTEGDA could be functionalized with chloromethyl, aminomethyl and hydroxymethyl functional groups under various controlled conditions. Synthetic utility of the support was demonstrated by the synthesis of four peptides selected from the envelope and nonstructural protein region of the prototype hepatitis C virus (HCV). These peptides were later used successfully to develop a peptide‐based immunoassay (PBEIA) for the detection of HCV immunity. Peptides designed from the NS1 and NS4 protein regions were found to be very promising for the development of a new diagnostic kit to detect HCV infection in human blood. Peptide purity was tested by RP‐FPLC and the peptide identity was confirmed by amino acid analysis.     

The glycan‐mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation

N‐linked glycans such as 234 and 276 gp₁₂₀ glycans are vital components of HIV evasion from humoral immunity and important for HIV‐1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120‐CD4‐8ANC195 complex with 234 and 276 gp₁₂₀ glycans, 234 gp₁₂₀ glycan, 276 gp₁₂₀ glycan, and without glycan were performed. Our results reveal that 276 gp₁₂₀ glycan can enhance gp120‐CD4 and gp120‐antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp₁₂₀ glycan primarily reinforces gp120‐antibody interactions and weakly affects gp120‐CD4 interactions as it mainly lies between gp120 and antibody. The co‐operating of two glycans can enhance gp120‐CD4 and gp120‐antibody associations. Through the structural analysis, it can be seen that 234 gp₁₂₀ glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors.     

Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches

4‐methyl‐N‐ethcathinone (4‐MEC), the N‐ethyl homologue of mephedrone, is a novel psychoactive substance of the beta‐keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4‐MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography‐mass spectrometry (GC‐MS) and liquid chromatography‐high resolution‐tandem mass spectrometry (LC‐HR‐MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N‐deethylation, hydroxylation of the 4‐methyl group followed by further oxidation to the corresponding 4‐carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N‐methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N‐deethyl‐dihydro isomers and the 4‐carboxy‐dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4‐MEC should be detectable in urine by the GC‐MS and LC‐MSⁿ standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.     

Lipophilic derivatization of synthetic peptides belonging to NS3 and E2 proteins of GB virus‐C (hepatitis G virus) and its effect on the interaction with model lipid membranes

Abstract: The synthesis by solid‐phase methodologies of peptides belonging to structural and non‐structural proteins of GB virus C as well as its N‐α‐acylation with myristate and palmitate fatty acids is described. To explore the peptide–lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non‐structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.     

Effect of hepatic CYP inhibitors on the metabolism of sildenafil and formation of its metabolite,N‐desmethylsildenafil,in rats in vitro and in vivo

Objectives It has been reported that hepatic cytochrome P450 (CYP)2C9 and CYP3A4 are responsible for the metabolism of sildenafil and formation of its metabolite, N‐desmethylsildenafil, in humans. However, in‐vivo studies in rats have not been reported. Methods Sildenafil (20 mg/kg) was administered intravenously to rats pretreated with sulfaphenazole, cimetidine, quinine hydrochloride or troleandomycin, inhibitors of CYP2C6, CYP2C11, CYP2D subfamily and CYP3A1/2, respectively. In‐vitro studies using rat liver microsomes were also performed. Key findings The area under the plasma‐concentration time curve (AUC) was increased and clearance of sildenafil decreased in rats pretreated with cimetidine or troleandomycin. The AUC ratio for N‐desmethylsildenafil (0–4 h): sildenafil (0–∞) was significantly decreased only in rats pretreated with cimetidine. Similar results were obtained in the in‐vitro study using rat liver microsomes. Conclusions Sildenafil is metabolised via hepatic CYP2C11 and 3A1/2, and N‐desmethylsildenafil is mainly formed via hepatic CYP2C11 in rats. Thus, rats could be a good model for pharmacokinetic studies of sildenafil and N‐desmethylsildenafil in humans.     

Cadmium‐induced microsatellite instability in the kidneys and leukocytes of C57BL/6J mice

Cadmium is a cytotoxic, carcinogenic, and mutagenic industrial product or byproduct. The correlation between metal exposure and microsatellite instability (MSI) has been reported by several groups. In the present study, 50 C57BL/6J mice at 6 weeks of age were divided into five groups and intraperitoneally injected with 0, 0.25, 0.5, 1, or 2 mg/kg cadmium chloride quaque die alterna for 4 weeks. Then, the liver, kidney, testis, leukocytes, bone marrow, and small intestine were collected from the treated mice and weighed. Portions of these tissues were fixed for further histological analysis, and the remaining tissues were subjected to genomic DNA extraction for the analysis of a panel of 42 microsatellite markers. The liver and testis weight coefficients were significantly changed in the 1 and 2 mg/kg cadmium chloride‐treated groups compared with the control group. Simultaneously, severe histopathologic changes in the liver and kidneys, along with a complete disorganization of testicular structure and obvious severe necrosis in the testes were observed in the cadmium‐treated group. The cadmium accumulated in the liver and kidneys of the mice in all cadmium‐treated groups; the tissue cadmium concentrations were significantly higher than those in the control group. After STR scanning, MSI was found at three loci (D15Mit5, D10Mit266, and DxMit172) in the kidneys and leukocytes of mice in the lower dose groups (0.25 and 0.5 mg/kg). In summary, we have successfully established a sub‐chronic cadmium exposure model and confirmed that cadmium exposure can induce MSI in mice. We also identified two loci that could be regarded as “hotspots” of microsatellite mutation in mice. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 683–692, 2015.     

Development of the Larval Amphibian Growth and Development Assay: effects of chronic 4‐tert‐octylphenol or 17β‐trenbolone exposure in Xenopus laevis from embryo to juvenile

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized test guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of the Environment. The LAGDA was designed to evaluate apical effects of chronic chemical exposure on growth, thyroid‐mediated amphibian metamorphosis and reproductive development. During the validation phase, two well‐characterized endocrine‐disrupting chemicals were tested to evaluate the performance of the initial assay design: xenoestrogen 4‐tert‐octylphenol (tOP) and xenoandrogen 17β‐trenbolone (TB). Xenopus laevis embryos were exposed, in flow‐through conditions, to tOP (nominal concentrations: 0.0, 6.25, 12.5, 25 and 50 µg l^–1) or TB (nominal concentrations: 0.0, 12.5, 25, 50 and 100 ng l^–1) until 8 weeks post‐metamorphosis, at which time growth measurements were taken, and histopathology assessments were made of the gonads, reproductive ducts, liver and kidneys. There were no effects on growth in either study and no signs of overt toxicity, sex reversal or gonad dysgenesis. Exposure to tOP caused a treatment‐related decrease in circulating thyroxine and an increase in thyroid follicular cell hypertrophy and hyperplasia (25 and 50 µg l^–1) during metamorphosis. Müllerian duct development was affected after exposure to both chemicals; tOP exposure caused dose‐dependent maturation of oviducts in both male and female frogs, whereas TB exposure caused accelerated Müllerian duct regression in males and complete regression in >50% of the females in the 100 ng l^–1 treatment. Based on these results, the LAGDA performed adequately to evaluate apical effects of chronic exposure to two endocrine‐active compounds and is the first standardized amphibian multiple life stage toxicity test to date. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.