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1.
Annu Rev Biochem ; 89: 557-581, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32208767

RESUMO

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.


Assuntos
Química Farmacêutica/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Técnicas de Sonda Molecular , Terapia de Alvo Molecular/métodos , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Genes Reporter , Humanos , Cinética , Imagem Óptica/métodos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade
2.
SLAS Discov ; 25(2): 176-185, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31709883

RESUMO

Protein kinases are intensely studied mediators of cellular signaling. While traditional biochemical screens are capable of identifying compounds that modulate kinase activity, these assays are limited in their capability of predicting compound behavior in a cellular environment. Here, we aim to bridge target engagement and compound-cellular phenotypic behavior by utilizing a bioluminescence resonance energy transfer (BRET) assay to characterize target occupancy within living cells for Bruton's tyrosine kinase (BTK). Using a diverse chemical set of BTK inhibitors, we determine intracellular engagement affinity profiles and successfully correlate these measurements with BTK cellular functional readouts. In addition, we leveraged the kinetic capability of this technology to gain insight into in-cell target residence time and the duration of target engagement, and to explore a structural hypothesis.


Assuntos
Tirosina Quinase da Agamaglobulinemia/isolamento & purificação , Transferência Ressonante de Energia de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/química , Tirosina Quinase da Agamaglobulinemia/genética , Humanos , Cinética , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química
3.
Medchemcomm ; 8(4): 700-712, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30108788

RESUMO

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

4.
Proc Natl Acad Sci U S A ; 94(26): 14306-13, 1997 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-9405608

RESUMO

Enveloped viruses enter cells by protein-mediated membrane fusion. For influenza virus, membrane fusion is regulated by the conformational state of the hemagglutinin (HA) protein, which switches from a native (nonfusogenic) structure to a fusion-active (fusogenic) conformation when exposed to the acidic environment of the cellular endosome. Here we demonstrate that destabilization of HA at neutral pH, with either heat or the denaturant urea, triggers a conformational change that is biochemically indistinguishable from the change triggered by low pH. In each case, the conformational change is coincident with induction of membrane-fusion activity, providing strong evidence that the fusogenic structure is formed. These results indicate that the native structure of HA is trapped in a metastable state and that the fusogenic conformation is released by destabilization of native structure. This strategy may be shared by other enveloped viruses, including those that enter the cell at neutral pH, and could have implications for understanding the membrane-fusion step of HIV infection.


Assuntos
Hemaglutininas/fisiologia , Orthomyxoviridae/fisiologia , Replicação Viral , Hemaglutininas/química , Fusão de Membrana , Conformação Proteica
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