Nt 115 instrument

6xHis-GBP and 6xHis-GSAAT was labeled using the Protein Labeling Kit RED-NHS 2nd Generation (NanoTemper; Munich, Germany) according to the manufacturer’s instructions. Briefly, 20 µM of 6× His-GBP recombinant proteins were incubated with NHS labeling buffer and 120 µM of the dye RED-NHS second generation at room temperature for 30 min in dark. The labeled proteins were eluted by loading them onto an equilibrated B column resin by adding 500 µL of PBS buffer. The degree of labeling of the proteins was checked using UV–Vis spectrophotometry at 650 and 280 nm and was approximately 0.5–0.7. Ten to 20 µM of ligand 6xHis-GSAAT were mixed with the labeled target proteins 6×His-GBP with or without GluTR in a series of 1:1 dilutions, producing ligand concentrations ranging from 5 nM to 110 μM. After 5 min of incubation, the samples were loaded into Monolith NT.115 capillaries, and the thermophoresis was recorded in a NT.115 instrument (NanoTemper) at a constant temperature of 30°C, 40% light-emitting diode power and 100% excitation. MST signal from data of three different independent experiments was analyzed using MO.Affinity Analysis software v.2.1.3.

To determine compound-DYRK1A kinase binding affinity, purified His-tagged kinase domain was labeled using RED-Tris-NTA 2nd Generation Labeling Kit (NanoTemper, Munich, Germany). Dissociation constants were determined using a direct binding assay in which 10 µL of the unlabeled compound in a concentration ranging from 250 µM to 8 nM was incubated with 10 µL of 20 nM of labeled DYRK1A. All measurements were performed in triplicates and carried out using Monolith NT. 115 instrument (NanoTemper, Munich, Germany) in PBS-T buffer (137 nM NaCl, 2.5 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄ pH 7.4 with 0.005% Tween-20) at room temperature with 80% excitation and 40% MST power. The Kd values were calculated using MO Affinity Analysis software (NanoTemper, Munich, Germany).

The melting points of the target products were measured using a WRX-4 micro-melting-point apparatus (Shanghai Yice Apparatus & Equipment Co., Ltd., Shanghai, China). ¹H and ¹³C nuclear magnetic resonance (NMR) spectral analyses were performed on a JEOL-ECX 500 NMR spectrometer using CDCl₃ or DMSO-d6 as the solvent and tetramethylsilane (TMS) as an internal standard. High-resolution mass spectrometry (HRMS) was conducted on a LTQ Orbitrap (Thermo Scientific, Missouri, USA). The CD spectrum of helicase was performed through a JASCO J-1500 spectropolarimeter from JASCO (Tokyo, Japan) Co., Ltd. Binding studies were performed on a nanotemper monolith NT.115 instrument (Nanotemper, Munich, Germany) for microscale thermophoresis (MST). The ATPase activity experiment used enzyme assay reagent kits purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All analytical reagents were used in the experiment obtained from Energy Chemical (Shanghai Saen Chemical Technology Co., Ltd., Shanghai, China), without further drying or purification.