Monolith nt automated

To evaluate the binding affinity of JX57 and GRP75, an MST assay was conducted using Monolith NT Automated (NanoTemper Technologies, München, Germany). Purified GRP75 protein was obtained by Zoonbio Biotechnology Co., Ltd. (Nanjing, China). Recombinant His‐tagged GRP75 protein was labeled with RED‐tris‐NTA second generation dye solution (MO‐L018, NanoTemper Technologies), according to the manufacturer's instructions. The concentration of the final labeled GRP75 concentration was 50 nmol L⁻¹; GRP75 was mixed with different concentrations of JX57 and JX66 by pipetting multiple times. All samples were diluted in 1× PBST and contained the same amount of DMSO. The K_d was determined in MO. Control using the K_d fit.

The experiments were performed on Monolith NT Automated (NanoTemper, Munich, Germany) machine with the following parameters:

MST power = 40%

Excitation power = 15%

Temperature = 20 °C

Acquisition mode = Pico-Red

The data was processed using MO.Affinity analysis v2.2.4 (NanoTemper). The change in fluorescence was used to fit the data to either 1:1 K_D model or Hills equation.
For protein–ligand interaction measurements using dye labeling, 6xHis-tagged protein was labeled with RED-Tris dye following manufacturer’s protocol. Following this, an appropriate number of the labeled protein samples with the ligand were prepared by mixing 1 µL of ligand in 100% DMSO with 24 µL of labeled protein in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20. Final DMSO concentration = 4%. The samples were incubated for 30 min, loaded into premium-coated capillaries and measured. The ligand concentrations used were based on solubility studies by NMR and literature data
For non-labeled approach, 24 samples of tau K18^M were prepared from 5.9 to 1000 nM as 1.25× dilutions in the presence of 100 nM of compound 1 (Methylene Blue) in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween 20, 4% DMSO.

During the MST experiments, the concentration of the protein in solution was kept constant while the compounds were titrated. The dilution series of the compounds were prepared by applying a 3:1 ratio with initial concentrations of the compounds: Rivaroxaban: 17.5 μM, Apixaban: 250 μM, and Betrixaban: 100 μM. HEPES buffer supplemented with 0.01% of Pluronic F-127 was used to dilute the stock solutions of the compounds to the initial concentrations (mentioned above). The same buffer with 1.25% of DMSO (v/v) was used as the dilution buffer in the dilution series. Next, a constant amount of protein was added in 1:1 volume ratio to the respective diluted compounds resulting in final concentration of the protein of 4 μM and final concentration of the compounds starting from: Rivaroxaban: 8.75 μM, Apixaban: 125 μM and Betrixaban: 50 μM.
The experiments were performed on LabelFree Capillary Chips in 3 independent runs with one technical repetition using the Monolith NT.Automated (NanoTemper, Munich, Germany) with the following parameters: excitation power: 10% LabelFree, MST Power: medium, Before MST: 3s, MST-On Time 10s and After MST: 1s.

A Monolith NT Automated from NanoTemper Technologies was used for MST assays. FRB, and HDAC1 proteins were fluorescently labeled with the RED-tris-NTA (MO-L008) according to the manufacturer’s instructions. SIRT2 was carried out by the LabelFree method as previously described (Soares da Costa et al., 2016 (link)). All affinity measurements were performed in PBS buffer mixed with 0.05% Pluronic F-127 (Invitrogen, P6866). Rapamycin, arrayed at different concentrations, was incubated with proteins for 30 min before application to Monolith NT standard treated capillaries. Thermophoresis was then determined at 25°C with 15-20% excitation power and middle MST power.

The microscale thermophoresis (MST) assays were carried out according to the method previously reported (41 (link)). Binding affinities of peptidyl substrates with SARS-CoV-2 M^pro (H41A) were measured using Monolith NT.Automated (NanoTemper Technologies). H41A was fluorescently labeled according to the manufacturer’s procedure. Protein was kept in MST Buffer (20 mM Hepes pH 7.0, 300 mM NaCl, 5% glycerol), and the concentration was adjusted to 4 μM. Next, RED-Tris-NTA second-generation dye was added, mixed, and incubated for 30 min at 25 °C in the dark. The final molar ratio of protein to dye was 80:1. Each of the unlabeled peptidyl substrates at 12 different serially diluted concentrations was then mixed with the same volume of labeled protein at room temperature. The samples were then loaded into standard treated capillaries (NanoTemper Technologies) and measured at 25 °C, 40% LED power, and medium MST power. Each assay was repeated three times. Data analyses were performed using MO.Affinity Analysis v.2.2.4 software (NanoTemper Technologies). All of the final plots were made using GraphPad Prism 8.0.