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Methods Mol Biol ; 2043: 75-91, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463904


A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.

ChemMedChem ; 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31469937


The growth hormone secretagogue receptor type 1a (GHS-R1a) is a class A rhodopsin-like G protein coupled receptor (GPCR) that is expressed in a variety of human tissues and is differentially expressed in benign and malignant prostate cancer. Previously, the peptidomimetic [1-Nal4 ,Lys5 (4-fluorobenzoyl)]G-7039 was designed as a molecular imaging tool for positron emission tomography (PET). However, this candidate was a poor binder (IC50 =69 nm), required a lengthy four-step radiosynthesis, and had a cLogP above 8. To address these challenges, we now report on changes targeted at the 4th position of G-7039. A 2-fluoropropionic acid (2-FPA) group was added on to Lys5 to determine the potential binding affinity of the [18 F]-2-FP radiolabeled analogue, which could be prepared by simplified radiochemistry. Lead candidate [Tyr4 ,Lys5 (2-fluoropropionyl)]G-7039 exhibited an IC50 of 0.28 nm and low picomolar activity toward GHS-R1a. Molecular docking revealed a molecular basis for this picomolar affinity.

Eur J Med Chem ; 157: 1500-1511, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30282322


The ghrelin receptor is a seven-transmembrane (7-TM) receptor known to have an increased level of expression in human carcinoma and heart failure. Recent work has focused on the synthesis of positron emission tomography (PET) probes designed to target and image this receptor for disease diagnosis and staging. However, these probes have been restricted to small-molecule quinalizonones and peptide derivatives of the endogenous ligand ghrelin. We describe the design, synthesis and biological evaluation of a series of 4-fluorobenzoylated growth hormone secretagogues (GHSs) derived from peptidic (GHRP-1, GHPR-2 and GHRP-6) and peptidomimetic (G-7039, [1-Nal4]G-7039 and ipamorelin) families in order to test locations for the insertion of fluorine-18 for PET imaging. The peptidomimetic G-7039 was found to be the most suitable for 18F-radiolabelling as its non-radioactive 4-fluorobenzoylated analogue ([1-Nal4,Lys5(4-FB)]G-7039), had both a high binding affinity (IC50 = 69 nM) and promising in vitro efficacy (EC50 = 1.1 nM). Prosthetic group radiolabelling of the precursor compound [1-Nal4]G-7039 using N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) delivered the PET probe [1-Nal4,Lys5(4-[18F]-FB)]G-7039 in an average decay-corrected radiochemical yield of 48%, a radio-purity ≥ 99% and an average molar activity of >34 GBq/µmol. This compound could be investigated as a PET probe for the detection of diseases that are characterised by overexpression of the ghrelin receptor.

Hormônio do Crescimento/metabolismo , Peptidomiméticos/farmacologia , Tomografia por Emissão de Pósitrons/métodos , Receptores de Grelina/análise , Relação Dose-Resposta a Droga , Desenho de Drogas , Células HEK293 , Humanos , Estrutura Molecular , Peptidomiméticos/síntese química , Peptidomiméticos/química , Receptores de Grelina/metabolismo , Relação Estrutura-Atividade
Eur J Med Chem ; 123: 822-833, 2016 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-27541265


The ghrelin receptor, also referred to as the growth hormone secretagogue receptor 1a (GHS-R1a), is a G protein-coupled receptor (GPCR) primarily expressed in the brain and pituitary. The wide spectrum of biological functions of GHS-R1a has rendered it a target for therapeutic drugs and for molecular imaging agents, for a variety of diseases. An improved understanding of the binding mechanism of a ligand to GHS-R1a would provide guidance on ligand design. This study investigates the binding of G-7039, a peptidomimetic agonist, to the GHS-R1a. A series of computational studies including homology modeling, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were carried out in conjunction with amino acid replacements on G-7039. The results suggest that the first three residues on the N-terminal segment of the peptidomimetic are bound to three hydrophobic sub-pockets in the receptor binding site, with the driving force for binding mainly from hydrophobic interactions. It has been reported that a charge-charge interaction between the positively charged terminal amine of the agonist and Glu124 on the receptor serves as an anchor point for binding. However, our studies suggest that this interaction is not strong enough to anchor a ligand to the ghrelin receptor in the absence of hydrophobic interactions. The resulting computational model provides insight into structure activity relationship analysis for the ghrelin receptor and will assist in future ligand design.

Substituição de Aminoácidos , Simulação por Computador , Peptidomiméticos/metabolismo , Receptores de Grelina/genética , Receptores de Grelina/metabolismo , Espaço Extracelular/metabolismo , Humanos , Simulação de Acoplamento Molecular , Peptidomiméticos/química , Ligação Proteica , Estrutura Secundária de Proteína , Receptores de Grelina/química , Homologia de Sequência de Aminoácidos , Termodinâmica