Abstract
Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.
Highlights
IntroductionMany human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) [3,4,5,6]
The STEP46 major splice variant was chosen over STEP61 since the latter, containing two transmembrane motifs, was not straightforward enough to express and purify at the scale required for high-throughput screening (HTS)
The fluorescence emission readout was highly linear, with STEP46 levels tested over a wide range of concentrations
Summary
Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) [3,4,5,6]. Targeted therapies that inhibit PTK function have been very successful with >30 tyrosine kinase inhibitors (TKIs) in clinical use [7,8]. PTPs are the largest class of protein phosphatases with over 100 members in humans [9,10]. They are challenging targets because they have highly conserved active sites, and small molecules that bind the catalytic site often exhibit poor selectivity. As a result of these challen lack of effective PTP probes has impeded progress in the field for years
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