Mandel Sebastian, Hanke Thomas, Prendiville Niall, Baena-Nuevo María, Berger Lena Marie, Farges Frederic, Schwalm Martin Peter, Berger Benedict-Tilman, Kraemer Andreas, Elson Lewis, Saraswati Hayuningbudi, Abdul Azeez Kamal R, Dederer Verena, Mathea Sebastian, Corrionero Ana, Alfonso Patricia, Keller Sabrina, Gstaiger Matthias, Krause Daniela S, Müller Susanne, Röhm Sandra, Knapp Stefan
Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany.
Structure Genomics Consortium Buchmann Institute for Molecular Life Sciences, Johann Wolfgang Goethe-University, Max-von-Laue-Str. 15, D-60438 Frankfurt am Main, Germany.
J Med Chem. 2025 Jul 24;68(14):15026-15049. doi: 10.1021/acs.jmedchem.5c01204. Epub 2025 Jul 2.
Selectivity for closely related isoforms of protein kinases is a major challenge in the design of drugs and chemical probes. Covalent targeting of unique cysteines is a potential strategy to achieve selectivity for highly conserved binding sites. Here, we used a pan-LIMK inhibitor to selectively probe LIMK1 over LIMK2 by targeting the LIMK1-specific cysteine C349 located in the glycine-rich loop region. Binding kinetics of both noncovalent and covalent LIMK inhibitors were investigated, and the fast on-rate and small size of type-I inhibitors were used in the design of a covalent LIMK1 inhibitor. The developed cell-active, isoform-selective LIMK1 inhibitor showed excellent proteome-wide selectivity in pull-down assays, enabling studies of LIMK1 isoform-selective functions in cellular model systems and providing a versatile chemical tool for studies of the LIMK signaling pathway.
对密切相关的蛋白激酶亚型具有选择性是药物和化学探针设计中的一项重大挑战。共价靶向独特的半胱氨酸是实现对高度保守结合位点选择性的一种潜在策略。在此，我们使用一种泛LIMK抑制剂，通过靶向位于富含甘氨酸环区域的LIMK1特异性半胱氨酸C349，选择性地检测LIMK1与LIMK2。研究了非共价和共价LIMK抑制剂的结合动力学，并将I型抑制剂的快速结合速率和小尺寸用于共价LIMK1抑制剂的设计。所开发的具有细胞活性、亚型选择性的LIMK1抑制剂在下拉实验中表现出优异的全蛋白质组选择性，能够在细胞模型系统中研究LIMK1亚型选择性功能，并为LIMK信号通路研究提供了一种通用的化学工具。
