Dasatinib-Red is a derivative of Dasatinib, and has been validated as a fluorescent tracer for HTRF Kinase Binding assays.

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Dasatinib-Red is a derivative of Dasatinib, and has been validated as a fluorescent tracer for HTRF Kinase Binding assays.

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Overview

This compound is derived from Dasatinib, which is a well known inhibitor of the BCR-ABL and SRC families of Kinases. Dasatinib, which is an ATP-competitive Kinase inhibitor, is used as a drug to treat chronic myelogenous leukemia and acute lymphoblastic leukemia. It has been shown to bind with an affinity of <100 nM to about 16% of the Kinases and mainly to RTK. It is therefore useful to address multiple Kinases in the HTRF Kinase Binding assay platform. The ‘d2’ fluorescent dye used absorbs with its maximum near 650 nm and fluoresces near 670 nm. It is used as an acceptor molecule in HTRF assays.

Dasatinib-Red is supplied as a 25 µM solution in DMSO.

Benefits

  • EQUILIBRIUM BINDING
  • PHARMACOLOGICAL STUDIES
  • INHIBITOR MODE OF ACTION

Kd determination: assay principal

​The binding of Dasatinib-Red is detected in a sandwich assay format using the Anti Tag labeled with Europium Cryptate (donor) which binds to the tagged Kinase, and a red fluorescent tracer labelled with d2 (acceptor).

The detection principle is based on HTRF® technology.  The HTRF ratio (665/620) will increase upon the addition of more of the tracer, and will saturate depending on the dissociation constant (Kd) of the tracer to the tagged kinase. 

​To know whether Dasatinib-Red is the tracer best suited to your tagged kinase of interest, we advise you to try the Kinase Binding Discovery kits.

Principal of kinase binding saturation assay using dasatnib-red

Kd determination assay protocol

Saturation binding experiments of  Dasatinib-Red can be run in 96- or 384-well plates (20 µL final volume).

First, a dilution series ranging between 0 and 1  µM of Dasatinib-Red in the Kinase Binding Buffer is prepared in a 96-well non-binding plate. Next, 5 µL of Kinase Binding Buffer are dispensed into the final 96- or 384-well plate. Then 5 µL of tagged-Kinase are added, followed by 5 µL of Anti-tag Eu-cryptate. Finally, 5 µL of the red tracer solution are added. 

The HTRF ratio is measured after 1 H of incubation.

Protocol for kinase binding saturation assay using dasatinib-red

IC50/Ki determination assay principle

The binding of Dasatinib-Red is detected in a sandwich assay format using a specific Anti-GST, 6HIS antibody or Streptavidin labeled with Europium Cryptate (donor), which binds to the tagged Kinase, and a red fluorescent Dasatinib labelled with d2 (acceptor).

The detection principle is based on HTRF® technology.  The HTRF ratio (665/620) will increase upon the addition of more Dasatinib-Red, and will saturate depending on the dissociation constant (Kd ) of Staurosporine-Red to the tagged kinase. With the addition of an inhibitor of the kinase, Dasatinib-Red will be displaced and the HTRF signal will disappear, depending on the dose.

Principal of kinase binding inhibitor assay using dasatinib-red

IC50/Ki determination assay protocol

​​Pharmacological evaluation of inhibitors of interest can be run in 96- or 384-well plates.

First, a dilution series of inhibitor ranging between 40 µM and 0.23 nM is prepared, wih 5 µL of each concentration dispensed into the plate. Then 5 µL of tagged-Kinase are added, followed by 5 µL of anti-tag Eu-cryptate. Finally, 5 µL of Dasatinib-Red solution are added, prepared at 4x the final concentration.  The HTRF ratio is measured after 1 H of incubation.

Analyses of the data give typical dose response curves ranging between 10 µM and 56 pM, enabling evaluation of the IC50/Ki values for the inhibitor of interest.

Protocol for kinase binding inhibitor assay using dasatinib-red

Saturation Binding (Kd)

Before perfoming competitive binding studies of inhibitors, the dissociation constant ​(Kd) of Dasatinib-Red has to be determined on the tagged Kinase of interest. This can be done with the help of the Kinase-GST/6HIS/biotin Discovery kits. A typical saturation binding experiment is performed using tracer concentrations between 0 and 250 nM, and measuring total- and non-specific binding signals. Subtracting the non-specific from the total binding signal gives the specific signal, which can be analysed to give the Kd. Here an example is shown on the  determination of the Kd (22 nM) of Dasatinib-Red on 5 nM BRAF-GST. ​

Kd curves of Dasatinib-Red on BRAF-GST

Competitive Binding (IC50/Ki)

Dose response curves of various known kinase inhibitors (Staurosporine, Dasatinib, PP2, Imatinib, Tozasertib, Sunitinib, Gefitinib, and Sorafenib) were measured using Dasatinib-Red at its Kd (22 nM) on 5 nM BRAF-GST.  Dasatinib and Sorafenib showed good potencies, in good correlation with literature values.​​ Staurosporine, Tozasertib, Sunitib and Gefinitib did not compete, as can be expected for BRAF.

Inhibitor effect of various inhibitors on Dasatinib-red /BRAF-GST binding
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