Identification of Tau-Tubulin Kinase 1 Inhibitors by Microfluidics-based Mobility Shift Assay from a Kinase Inhibitor Library

by Selleckchem | Oct 29 2023

Tau tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that phosphorylates multiple residues in tau protein. Hyperphosphorylated tau is the main cause of tauopathy, such as Alzheimer's disease (AD). Therefore, preventing tau phosphorylation by inhibiting TTBK1 has been proposed as a therapeutic strategy for AD. However, few substrates of TTBK1 are reported for a biochemical assay and few inhibitors targeting TTBK1 have been reported so far. In this study, we identified a fluorescein amidite (FAM)-labeled peptide 15 from a small peptide library as the optimal peptide substrate for human TTBK1 (hTTBK1). We then developed and validated a microfluidics-based mobility shift assay (MMSA) with peptide 15. We further confirmed that peptide 15 could also be used in the ADP-Glo kinase assay. The established MMSA was applied for screening of a 427-compound kinase inhibitor library, yielding five compounds with IC50s of several micro molars against hTTBK1. Among them, three compounds, AZD5363, A-674563 and GSK690693 inhibited hTTBK1 in an ATP competitive manner and molecular docking simulations revealed that they enter the ATP pocket and form one or two hydrogen bonds to the hinge region with hTTBK1. Another hit compound, piceatannol, showed non-ATP competitive inhibitory effect on hTTBK1 and may serve as a starting point to develop highly selective hTTBK1 inhibitors. Altogether, this study provided a new in vitro platform for the development of novel hTTBK1 inhibitors that might have potential applications in AD prevention.

Comments:

The study you described focuses on Tau tubulin kinase 1 (TTBK1), a kinase involved in phosphorylating tau protein, and its potential as a therapeutic target for Alzheimer's disease (AD). The researchers aimed to identify peptide substrates for TTBK1 and develop assays to screen for TTBK1 inhibitors.

First, the researchers identified a peptide substrate, labeled with fluorescein amidite (FAM), from a small peptide library that was optimal for human TTBK1 (hTTBK1). They then developed a microfluidics-based mobility shift assay (MMSA) using this peptide substrate, which allows for the detection of TTBK1 activity by measuring changes in peptide mobility.

The researchers also validated the use of the identified peptide substrate in the ADP-Glo kinase assay, another method to assess kinase activity. This confirms that the substrate can be used in different assay formats for TTBK1 activity measurement.

Next, the researchers screened a library of 427 kinase inhibitors using the established MMSA. They identified five compounds that showed inhibitory activity against hTTBK1, with IC50 values in the micromolar range. Three of these compounds (AZD5363, A-674563, and GSK690693) were found to competitively inhibit hTTBK1 by binding to the ATP pocket. Molecular docking simulations revealed that these compounds form hydrogen bonds with the hinge region of hTTBK1.

The fourth compound, piceatannol, showed non-ATP competitive inhibition of hTTBK1. This compound may have the potential to serve as a starting point for developing highly selective hTTBK1 inhibitors.

Overall, this study provides a platform for the development of novel hTTBK1 inhibitors using the identified peptide substrate and the MMSA assay. These inhibitors have the potential to be further explored for their therapeutic applications in the prevention of Alzheimer's disease.