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Quantification of Binding of Small Molecules to Native Proteins Overexpressed in Living Cells.
Yin, Yuwen; Zhao, Serena Li; Rane, Digamber; Lin, Zhihong; Wu, Meng; Peterson, Blake R.
Afiliación
  • Yin Y; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 W. 12th Ave., Columbus, Ohio 43210, United States.
  • Zhao SL; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 W. 12th Ave., Columbus, Ohio 43210, United States.
  • Rane D; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 W. 12th Ave., Columbus, Ohio 43210, United States.
  • Lin Z; The Ohio State University Comprehensive Cancer Center─Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, 460 W. 10th Ave., Columbus, Ohio 43210, United States.
  • Wu M; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 W. 12th Ave., Columbus, Ohio 43210, United States.
  • Peterson BR; The Ohio State University Comprehensive Cancer Center─Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, 460 W. 10th Ave., Columbus, Ohio 43210, United States.
J Am Chem Soc ; 146(1): 187-200, 2024 01 10.
Article en En | MEDLINE | ID: mdl-38118119
ABSTRACT
The affinity and selectivity of small molecules for proteins drive drug discovery and development. We report a fluorescent probe cellular binding assay (FPCBA) for determination of these values for native (untagged) proteins overexpressed in living cells. This method uses fluorophores such as Pacific Blue (PB) linked to cell-permeable protein ligands to generate probes that rapidly and reversibly equilibrate with intracellular targets, as established by kinetic assays of cellular uptake and efflux. To analyze binding to untagged proteins, an internal ribosomal entry site (IRES) vector was employed that allows a single mRNA to encode both the protein target and a separate orthogonal fluorescent protein (mVenus). This enabled cellular uptake of the probe to be correlated with protein expression by flow cytometry, allowing measurement of cellular dissociation constants (Kd) of the probe. This approach was validated by studies of the binding of allosteric activators to eight different Protein Kinase C (PKC) isozymes. Full-length PKCs expressed in transiently transfected HEK293T cells were used to measure cellular Kd values of a probe comprising PB linked to the natural product phorbol via a carbamate. These values were further used to determine competitive binding constants (cellular Ki values) of the nonfluorescent phorbol ester PDBu and the anticancer agent bryostatin 1 for each isozyme. For some PKC-small molecule pairs, these cellular Ki values matched known biochemical Ki values, but for others, altered selectivity was observed in cells. This approach can facilitate quantification of interactions of small molecules with physiologically relevant native proteins.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteína Quinasa C / Ésteres del Forbol Límite: Humans Idioma: En Revista: J Am Chem Soc Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteína Quinasa C / Ésteres del Forbol Límite: Humans Idioma: En Revista: J Am Chem Soc Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos