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1.
J Pharm Sci ; 112(8): 2285-2291, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37062414

RESUMO

Many biotherapeutics such as monoclonal antibodies (mAbs) consist of various glycoforms, which can have different PK properties upon administration to animals and human. As a result, it is necessary to monitor the abundance of glycoforms and limit lot-to-lot variability during the manufacturing process. However, limited information is known about the clearance of mAb glycoforms from ocular space upon intravitreal injection. We present here an assessment of glycoform clearance of a biotherapeutic mAb (IgG1) from rabbit vitreous humor, aqueous humor and retina tissue using LC/MS. The results show that G0, G0F and G1F have similar T1/2, while mannose-5 has a longer T1/2 and is cleared slower in rabbit ocular space, which contradicted with what has been reported in the literature in which Mann5 was cleared faster systematically.


Assuntos
Anticorpos Monoclonais , Retina , Animais , Coelhos , Humanos , Cromatografia Líquida , Cinética , Espectrometria de Massas
2.
ACS Chem Biol ; 14(3): 342-347, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30807093

RESUMO

The impact of covalent binding on PROTAC-mediated degradation of BTK was investigated through the preparation of both covalent binding and reversible binding PROTACs derived from the covalent BTK inhibitor ibrutinib. It was determined that a covalent binding PROTAC inhibited BTK degradation despite evidence of target engagement, while BTK degradation was observed with a reversible binding PROTAC. These observations were consistently found when PROTACs that were able to recruit either IAP or cereblon E3 ligases were employed. Proteomics analysis determined that the use of a covalently bound PROTAC did not result in the degradation of covalently bound targets, while degradation was observed for some reversibly bound targets. This observation highlights the importance of catalysis for successful PROTAC-mediated degradation and highlights a potential caveat for the use of covalent target binders in PROTAC design.


Assuntos
Tirosina Quinase da Agamaglobulinemia , Inibidores de Proteínas Quinases , Pirazóis , Pirimidinas , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Piperidinas , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteólise , Pirazóis/química , Pirazóis/metabolismo , Pirimidinas/química , Pirimidinas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
3.
J Am Chem Soc ; 140(3): 932-939, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29232121

RESUMO

Selective covalent inhibition of kinases by targeting poorly conserved cysteines has proven highly fruitful to date in the development of chemical probes and approved drugs. However, this approach is limited to ∼200 kinases possessing such a cysteine near the ATP-binding pocket. Herein, we report a novel approach to achieve selective, irreversible kinase inhibition, by targeting the conserved catalytic lysine residue. We have illustrated our approach by developing selective, covalent PI3Kδ inhibitors that exhibit nanomolar potency in cellular assays, and a duration of action >48 h in CD4+ T cells. Despite conservation of the lysine residue throughout the kinome, the lead compound shows high levels of selectivity over a selection of lipid and protein kinases in biochemical assays, as well as covalent binding to very few off-target proteins in live-cell proteomic studies. We anticipate this approach could offer a general strategy, as an alternative to targeting non-conserved cysteines, for the development of selective covalent kinase inhibitors.


Assuntos
Lisina/química , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular , Classe I de Fosfatidilinositol 3-Quinases , Descoberta de Drogas , Humanos , Lisina/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica
4.
ACS Chem Biol ; 12(10): 2515-2521, 2017 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-28876896

RESUMO

Chemoproteomics profiling of kinase inhibitors with kinobeads enables the assessment of inhibitor potency and selectivity for endogenously expressed protein kinases in cell lines and tissues. Using a small panel of targeted covalent inhibitors, we demonstrate the importance of measuring covalent target binding in live cells. We present a differential kinobeads profiling strategy for covalent kinase inhibitors where a compound is added either to live cells or to a cell extract that enables the comprehensive assessment of inhibitor selectivity for covalent and noncovalent targets. We found that Acalabrutinib, CC-292, and Ibrutinib potently and covalently bind TEC family kinases, but only Ibrutinib also potently binds to BLK. ZAK was identified as a submicromolar affinity Ibrutinib off-target due to covalent modification of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150 next to the DFG loop, demonstrating an alternative route to covalent inactivation of this kinase, e.g., to inhibit canonical TGF-ß dependent processes.


Assuntos
Acrilamidas/farmacologia , Benzamidas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirazinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Acrilamidas/química , Adenina/análogos & derivados , Linfócitos B , Benzamidas/química , Linhagem Celular , Humanos , Piperidinas , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Pirazinas/química , Pirazóis/química , Pirimidinas/química
5.
J Med Chem ; 60(13): 5455-5471, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28591512

RESUMO

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Quinoxalinas/farmacologia , Tanquirases/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Quinoxalinas/síntese química , Quinoxalinas/química , Relação Estrutura-Atividade , Tanquirases/metabolismo
6.
ACS Chem Biol ; 11(9): 2541-50, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27384741

RESUMO

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.


Assuntos
Sondas Moleculares/química , Análise de Célula Única , Células HeLa , Humanos
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