RESUMO
Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data.
Assuntos
Produtos Biológicos/metabolismo , Cromatografia Líquida/métodos , Peptídeos/metabolismo , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem/métodos , Bactérias/genética , Bactérias/metabolismo , Contaminação de Medicamentos/prevenção & controle , Humanos , Preparações Farmacêuticas/metabolismo , Proteínas/genéticaRESUMO
The SAR features have been further explored for (2-benzhydryl-4-phenyl-thiazol-5-yl)acetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. The introduction of a nitrogen or a methyl substituent in the benzhydrylic position offer two alternative drugable scaffolds attractive for unsymmetrically substituted derivatives. An imidazole analogue lacks activity due to formation of a favored coplanar intramolecular hydrogen bond. The pyrimidine derivative 18 represents a potent and selective compound that will be subject to continued investigations.
Assuntos
Compostos Benzidrílicos/química , Pirimidinas/química , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Tiazóis/química , Animais , Compostos Benzidrílicos/síntese química , Compostos Benzidrílicos/farmacocinética , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Humanos , Ligação de Hidrogênio , Imidazóis/química , Camundongos , Modelos Moleculares , Nitrogênio/química , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinéticaRESUMO
Structure-activity relationships have been established by exploring the eastern and western side of 5-thiazolyleacetic acids as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Benzhydryl motifs in the 2-position of the thiazole was found to be most advantageous. The 4-thiazole position should either carry 3- or 4-fluorophenyl rings or a 4-pyridyl suitably substituted in the flanking 2-position. Several compounds with single digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained. The compound series display a good PK profile and selectivity over a large number of other targets.
Assuntos
Ácido Acético/química , Biblioteca de Peptídeos , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Tiazóis/química , Ácido Acético/metabolismo , Ácido Acético/farmacologia , Animais , Linhagem Celular , Humanos , Ratos , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Células Th2 , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Structure-activity relationships of three related series of 4-phenylthiazol-5-ylacetic acids, derived from two hits emanating from a focused library obtained by in silico screening, have been explored as CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) antagonists. Several compounds with double digit nanomolar binding affinity and full antagonistic efficacy for human CRTH2 receptor were obtained in all subclasses. The most potent compound was [2-(4-chloro-benzyl)-4-(4-phenoxy-phenyl)-thiazol-5-yl]acetic acid having an binding affinity of 3.7nM and functional antagonistic effect of 66 nM in a BRET and 12 nM in a cAMP assay with no functional activity for the other PGD2 DP receptor (27 microM in cAMP).
Assuntos
Ácido Acético/síntese química , Biblioteca de Peptídeos , Receptores Imunológicos/antagonistas & inibidores , Receptores de Prostaglandina/antagonistas & inibidores , Tiazóis/síntese química , Ácido Acético/metabolismo , Ácido Acético/farmacologia , Animais , Células COS , Chlorocebus aethiops , Humanos , Ligação Proteica/fisiologia , Receptores Imunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacologiaRESUMO
Hits from an in silico derived focused library for CRTH2 were transformed into highly selective antagonists with favorable ADME properties. Oral administration of 4-bromo-2-(1-phenyl-1H-pyrazole-4-carbonyl)phenoxyacetic acid (19) inhibited peribronchial eosinophilia and mucus cell hyperplasia in a mouse model of allergic asthma, supporting the therapeutic potential of this novel compound class. In addition, this selective pharmacological tool compound provides further evidence for CRTH2 as a relevant therapeutic target for treatment of Th2- and eosinophil-related inflammation.