Combinatorial design, selection and synthesis of peptide inhibitors against human glutathione transferase p1-1

Certain glutathione S-transferase (GST) isoenzymes detoxify the cell from xenobiotics, thus becoming inhibition targets when overexpressed in various tumours leading to MDR. We developed a combinatorial strategy aiming at designing peptide inhibitors against the hGSTP1-1 isoenzyme involved in MDR. We employed a combinatorial peptide library featuring engineered E. coli cells harboring a plasmid able to express a fusion protein containing random 12 peptides which were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. After five selection rounds, clones were screened for hGSTP1-1 binding and those with the strongest signal were selected and sequenced. Sequence alignments showed a core binding sequence which, along with selected peptide fragments, were synthesized using the solid phase methodology and Fmoc/tBu chemistry on 2-chlorotrityl chloride solid support. The four peptides were studied for their inhibition potency against hGSTP1-1 allozymes A, B and C

Peptide cell-display for selection of inhibitors against human glutathione transferase P1-1 (hGSTP1-1) allozymes

We developed a combinatorial strategy aiming at designing peptide inhibitors against the hGSTP1-1 isoenzyme involved in MDR. We developed a combinatorial strategy aiming at designing peptide inhibitors against the hGSTP1-1 isoenzyme. We employed a combinatorial peptide library featuring engineered E. coli cells harboring a plasmid able to express a fusion protein containing random 12peptides. After five selection rounds, clones were screened for hGSTP1-1 binding (dot blot hybridization) and those with the strongest signal were selected and sequenced. Sequence alignments showed a core binding sequence which, along with selected peptide fragments, were synthesized using the solid phase methodology . The synthetic peptides were studied for their inhibition potency against three human GSTP1-1 allozymes, A, B and

Synthesis of glutathione analogues and screening as substrates & inhibitors for human glutathione transferase p1‐1

A major detoxification mechanism of the cell involves the glutathione transferase (GST)‐catalyzed formation of glutathione (GSH) conjugates with various xenobiotics Based on the same mechanism, GST overexpression may lead to multidrug resistant phenotypes Therefore, several compounds with inhibitory potency against GSTs have been developed as potential tools fortackling GST-­‐attributed MDR. Several individual compounds and prodrugs have been proposed as GST‐inhibiting substances. In addition, GSH analogues have been considered as specific GST inhibitors, with particular attention been directed towards the synthesis of GSH analogues stable against γ‐glutamyltranspeptidase (γGT) and peptidases, as GST inhibitors

Reliable Characterization of Organic & Pharmaceutical Compounds with High Resolution Monochromated EEL Spectroscopy

Organic and biological compounds (especially those related to the pharmaceutical industry) have always been of great interest for researchers due to their importance for the development of new drugs to diagnose, cure, treat or prevent disease. As many new API (active pharmaceutical ingredients) and their polymorphs are in nanocrystalline or in amorphous form blended with amorphous polymeric matrix (known as amorphous solid dispersion-ASD), their structural identification and characterization at nm scale with conventional X-Ray/Raman/IR techniques becomes difficult. During any API synthesis/production or in the formulated drug product, impurities must be identified and characterized. Electron energy loss spectroscopy (EELS) at high energy resolution by transmission electron microscope (TEM) is expected to be a promising technique to screen and identify the different (organic) compounds used in a typical pharmaceutical or biological system and to detect any impurities present, if any, during the synthesis or formulation process. In this work, we propose the use of monochromated TEM-EELS, to analyze selected peptides and organic compounds and their polymorphs. In order to validate EELS for fingerprinting (in low loss/optical region) and by further correlation with advanced DFT, simulations were utilized

Marine-Derived Surface Active Agents: Health-Promoting Properties and Blue Biotechnology-Based Applications

Surface active agents are characterized for their capacity to adsorb to fluid and solid-water interfaces. They can be classified as surfactants and emulsifiers based on their molecular weight (MW) and properties. Over the years, the chemical surfactant industry has been rapidly increasing to meet consumer demands. Consequently, such a boost has led to the search for more sustainable and biodegradable alternatives, as chemical surfactants are non-biodegradable, thus causing an adverse effect on the environment. To these ends, many microbial and/or marine-derived molecules have been shown to possess various biological properties that could allow manufacturers to make additional health-promoting claims for their products. Our aim, in this review article, is to provide up to date information of critical health-promoting properties of these molecules and their use in blue-based biotechnology (i.e., biotechnology using aquatic organisms) with a focus on food, cosmetic and pharmaceutical/biomedical applications

Design and development of new synthetic analogues of the active pharmaceutical ingredient leuprolide

Leuprolide is a synthetic nonapeptide, analogue of Luteinizing Hormone Releasing Hormone (LHRH or GnRH). Is a super agonist of LHRH and as an active pharmaceutical ingredient has been used in the treatment of many sex hormone-related disorders. Regulation of hypothalamic-gonadal-axis by LHRH analogues is used for the treatment of hormone-dependent diseases, such as endometriosis, benign prostate hyperplasia, fertility disorders, as well as prostate, ovarian and breast cancer to induce reversible chemical castration. The currently available LHRH analogues as drugs and their applications in the treatment of the above diseases lead the scientists to the development of new more potent LHRH analogues with better pharmacological profile. In recent years the biology of GnRH has been revised, due to accumulating evidence that peripheral, extrapituitary, normal and malignant tissues locally produce GnRH and express GnRH binding sites. An autocrine/paracrine role of GnRH at these normal and malignant tissues, including the mammary gland, has been suggested. The direct antiproliferative effect of the LHRH analogues in many malignant tissues inaugurates the field and leads to the design of effective LHRH analogues with new perspectives. To-date, only a few studies have investigated the effects of LHRH analogues in pituitary and cancer cells in parallel. Information concerning the impact of systematic single amino acid modifications of LHRH on pituitary and malignant cells is still lacking. The structural investigation of this field seems to be a very important issue. The aim of this study was the contribution in the field of the structure-activity relationships based in the rational design (bibliographic data, conformational analysis), development (modern synthesis) and biological characterization of the new analogues of Leuprolide. Particularly, with the biological characterization, we tried to compare the binding affinities of the new Leuprolide analogues in the LHRH hypophysial receptor with their direct antiproliferative activity on breast cancer cells. Furthermore, the development of a new synthetic protocol which leads to high yield and purities of the synthesized products was a requisition. Important structural data obtained by conformational analysis of LHRH and Leuprolide using NMR spectroscopy. This conformational study in combination with the bibliographic data led us to design and synthesize new analogues of Leuprolide with main modifications in positions 3 and 6. Trp3 of peptide sequence of Leuprolide is substituted by non natural aromatic amino/imino acids. D-Leu6 is substituted by α,α-dialkyl amino acids L, D-Gly(tBu), D-Lys and D-Dab. In the ε- and γ-amino group of Lysine and diaminoisobutyric acid in position 6 several modifications have been performed by conjugation of natural amino acids (Gly, Ala, Sar) and 2-amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC). ATPC was also incorporated in the N-terminal segment replacing the final dipeptide of LHRH and Leuprolide. Like in the peptide sequence of Leuprolide, most of the new LHRH analogues lack the carboxy-terminal Gly10-amide of LHRH and an ethylamide residue is added to Pro9 (Fujino modification). The new analogues of LHRH were assembled on a [3-((Ethyl-Fmoc-amino)-methyl)-1-indol-1-yl]-acetyl AM resin to provide the peptide amide using Fmoc/tBu methodology. The affinities for the LHRH receptor of the new analogues were determined by competition binding experiments in mouse anterior pituitary αT3-1 cells while their antiproliferative activity was performed with human breast cancer cell lines (MCF-7). Modern peptide synthesis afforded us peptides in high yields which could be easily purified. This study provides binding requirements which can be used in the design of new Leuprolide analogues with high pituitary binding affinity and high antiproliferative activity in breast cancer cells (MCF-7). Results show in many cases differential impact on pituitary binding affinity and direct antiproliferative effect on breast cancer cells which is in accordance with bibliographic reports that mainly support this dissimilarity to different signal transduction mechanisms in hypothalamic and extra hypothalamic malignant tissues. Final this study provides new Leuprolide analogues with higher binding affinity and antiproliferative activity than it has exhibited and they deserve further pharmaceutical/biological investigation. This structure-activity relationship study for LHRH analogues at pituitary and breast cancer cell lines contributes to the on-going research of LHRH binding requirements of breast cancer cells and provides new perspectives for the design of effective GnRH analogues