Previously Published Phosphatase Probes have Limited Utility Due to their Unspecific Reactivity

• Published in: Chembiochem : a European journal of chemical biology 2024-11, Vol.25 (22), p.e202400333-n/a
• Main Authors: ter Brake, F. H. G., Luttikhuizen, S. A. F. M., Wel, T., Gagestein, B., Florea, B. I., Stelt, M., Janssen, A. P. A.
• Format: Article
• Language: English
• Subjects: Activity-Based Protein Profiling; Allosteric properties; Chemical activity; Chemical Proteomics; Development strategies; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Humans; Indolequinones - chemistry; Indolequinones - metabolism; Labeling; Lysates; Molecular Probes - chemistry; Molecular Structure; Phosphatase; Phosphatases; Probes; Protein Tyrosine Phosphatase, Non-Receptor Type 11 - antagonists & inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11 - metabolism; Protein-tyrosine-phosphatase; Proteins; Quinones; SHP2; Sulfones - chemistry; Sulfones - metabolism; Sulfones - pharmacology; Therapeutic targets; Tyrosine; Vinyl sulfone
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.202400333

This study explores the use of activity‐based protein profiling to study protein tyrosine phosphatases. With the discovery of allosteric SHP2 inhibitors, this enzyme family has resurfaced as interesting drug targets. Therefore, we envisioned that previously described direct electrophiles and quinone methide‐based traps targeting phosphatases could be applied in competitive activity‐based protein profiling assays. This study evaluates three direct electrophiles, specifically, a vinyl sulfonate, a vinyl sulfone, and an α‐bromobenzylphosphonate as well as three quinone methide‐based traps as activity‐based probes. For all these moieties it was previously shown that they could selectively engage in assays with purified or overexpressed phosphatases in bacterial lysates. However, this study demonstrates that probes based on these moieties all suffer from unspecific labelling. Direct electrophiles were either unspecific or not activity‐based, while quinone methide‐based traps showed dependence on phosphatase activity but also resulted in unspecific labelling due to diffusion after activation. This phenomenon, termed ′bystander’ labelling, occurred even with catalytically inactive SHP2 mutants. We concluded that alternative strategies or chemistries are needed to apply activity‐based protein profiling in phosphatase research. Moreover, this study shows that quinone methide‐based designs have limited potential in probe and inhibitor development strategies due to their intrinsic reactivity. This study evaluates activity‐based protein profiling (ABPP) probes targeting protein tyrosine phosphatases, including three direct electrophiles and three quinone methide‐based traps. Despite previously reported success in purified enzyme assays, all probes show unspecific labelling or non‐activity‐based behaviour. Quinone methide‐based traps also cause ′bystander’ labelling, limiting their potential for ABPP and inhibitor development in phosphatase research.