Abstract
PTPσ is a dual-domain receptor type protein tyrosine phosphatase (PTP) with physiologically important functions which render this enzyme an attractive biological target. Specifically, loss of PTPσ has been shown to elicit a number of cellular phenotypes including enhanced nerve regeneration following spinal cord injury (SCI), chemoresistance in cultured cancer cells, and hyperactive autophagy, a process critical to cell survival and the clearance of pathological aggregates in neurodegenerative diseases. Owing to these functions, modulation of PTPσ may provide therapeutic value in a variety of contexts. Furthermore, a small molecule inhibitor would provide utility in discerning the cellular functions and substrates of PTPσ. To develop such molecules, we combined in silico modeling with in vitro phosphatase assays to identify compounds which effectively inhibit the enzymatic activity of PTPσ. Importantly, we observed that PTPσ inhibition was frequently mediated by oxidative species generated by compounds in solution, and we further optimized screening conditions to eliminate this effect. We identified a compound that inhibits PTPσ with an IC50 of 10 µM in a manner that is primarily oxidation-independent. This compound favorably binds the D1 active site of PTPσ in silico, suggesting it functions as a competitive inhibitor. This compound will serve as a scaffold structure for future studies designed to build selectivity for PTPσ over related PTPs.
Highlights
Tyrosine phosphorylation is a critical mechanism by which cells exert control over signaling processes
We hypothesized that the active site could be exploited in the development of competitive inhibitors targeted to protein tyrosine phosphatase (PTP)
The tyrosine-like moiety of compound 36 binds in the domain of PTPs anticipated to bind the phosphotyrosine side chain of the known substrate. This integrative approach of computational and biochemical methods led to the identification of several small molecule inhibitors of PTPs
Summary
Tyrosine phosphorylation is a critical mechanism by which cells exert control over signaling processes. Protein tyrosine kinases (PTKs) and phosphatases (PTPs) work in concert to control these signaling cascades, and alterations in the expression or activity of these enzymes hallmark many human diseases [1,2]. The critical role of PTPs in cell function and their role in disease etiology highlight the importance of developing phosphatase agonists and inhibitors. Most importantly, phosphatase active sites display high conservation which hinders the ability to develop catalysis-directed inhibitors with any degree of selectivity [4]. Despite these pitfalls, the emerging role of PTPs in human disease etiology has necessitated a solution. Bidentate inhibitors of PTP1B, implicated in type II diabetes and obesity, have been developed which span both the catalytic pocket and a second substrate binding pocket discovered adjacent to the active site [5,6,7]
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