SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors,Biochemistry

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SMaSh: A Streptavidin Mass Shift Assay for Rapidly Quantifying Target Occupancy by Irreversible Inhibitors
Biochemistry ( IF 2.9 ) Pub Date : 2021-09-23 , DOI: 10.1021/acs.biochem.1c00422
Matthew T Labenski ₁ , Leslie A Bateman ₁ , Lukas T Voortman ₁ , Giulia Giammo ₁ , Susan Cantin ₁ , Lixin Qiao ₁ , Alan F Corin ₁

Affiliation

The streptavidin mass shift (SMaSh) assay is a robust and fast approach for quantifying target protein occupancy by a covalent inhibitor or ligand. It exploits the biotin–streptavidin bond using the Simple Western platform. One measurement on a single sample determines both total and occupied target protein simultaneously and is, therefore, self-normalizing. The approach works in diverse and complex biological matrices and, with no need for matched vehicle-treated controls, readily applies to tissues from animal pharmacology models. Assessing occupancy is critical in the development of targeted covalent drugs. We demonstrate its use by characterizing and validating a variety of chemical probes for Bruton’s tyrosine kinase (BTK, UniprotKB Q10607) and mitogen-activated protein kinase (ERK1/2/MAPK1/2, UniprotKB P28482 and P27361) and determining target engagement of covalent inhibitors for both targets and off-target engagement for ERK. We demonstrated that it works in cell lysates, tissues, and human peripheral blood mononuclear cells. The SMaSh assay is superior to traditional methods and broadly useful as a tool in assessing covalent biological probes or targeted covalent inhibitors.

中文翻译：

SMaSh：通过不可逆抑制剂快速量化目标占有率的链霉亲和素质量转移分析

链霉亲和素质量转移 (SMaSh) 测定是一种可靠且快速的方法，可用于量化共价抑制剂或配体对目标蛋白的占用。它使用 Simple Western 平台利用生物素-链霉亲和素键。对单个样品的一次测量同时确定总目标蛋白和占据的目标蛋白，因此是自归一化的。该方法适用于多种复杂的生物基质，无需匹配的载体处理对照，可轻松应用于动物药理学模型的组织。评估占用率对于开发靶向共价药物至关重要。我们通过表征和验证布鲁顿酪氨酸激酶 (BTK, UniprotKB Q10607) 和丝裂原活化蛋白激酶 (ERK1/2/MAPK1/2, UniprotKB P28482 和 P27361）并确定共价抑制剂的靶标参与和 ERK 的脱靶参与。我们证明它在细胞裂解物、组织和人外周血单核细胞中起作用。SMaSh 检测优于传统方法，可广泛用作评估共价生物探针或靶向共价抑制剂的工具。

更新日期：2021-10-06

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