ResidenceTimer™

ResidenceTimer-icoonDo you want to know the residence time of your compound on its kinase target?
 Do you want to know the kinetic selectivity of your compound on a panel of kinases?

 At NTRC we developed ResidenceTimer™. This service measures target residence time and kinetic selectivity reliably and with short turn-around time.

Characteristics

Advantages

Drugs that reside on their target for several hours will have the advantage that their pharmacodynamic effect will last even after the drug has been eliminated from systemic circulation. A variety of registered kinase inhibitor drugs exert their pharmacological effect through a prolonged target residence time, a.o., lapatinib (GSK), ponatinib (Ariad), and sorafenib (Bayer).

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BiaCore assays of ponatinib

Drug-target residence time is an important factor in compound optimization and for determining so-called ‘kinetic selectivity’. Especially for compounds where the target residence time exceeds the half-life in circulation, measuring kinetic selectivity is more predictive for in vivo effect than determining selectivity on the basis of IC50 or Kd.

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Residence Time of ponatinib measured on 10 different kinases

ResidenceTimer™ is our service to measure target residence time reliably and with short turn-around time. In ResidenceTimer™ we measure the kinetics of compounds binding to targets by making use of surface plasmon resonance. For protein kinases ResidenceTimer™ makes use of kinases that are specifically biotinylated at the N-terminus, so that immobilization on a streptavidin-coated chip occurs in a uniform way. A panel of more than 40 different kinases has been established (currently being expanded to 60 assays). To accurately measure target residence time, we are use a BiaCore T200 (GE Healthcare). Biacore T200 has been specifically developed for measuring the binding of small molecules to protein targets.

Click here for full list of kinases assays available in our ResidenceTimer™ service.

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Kinases Available in ResidenceTimer™

Method

Surface plasmon resonance allows the direct monitoring of the binding of small molecules to protein targets. In contrast to competitive binding assays, surface plasmon resonance does not require target-specific probes. The immobilization of active protein kinases can be very challenging, however, as most enzymes rapidly lose activity at low pH during normal amine-based immobilization methods. We have used a panel of kinases that have incorporated one biotin label per molecule and that are immobilized at neutral pH. The binding ability of each kinase was validated using selective ligands. All binding experiments were carried out in one uniform assay buffer, making results highly comparable across kinases [1].

All kinases are immobilized to an appropriate resonance level. Binding experiments are based on single cycle kinetics: i.e., compounds are injected consecutively at five concentrations over the surface with immobilized kinases, and, in parallel, over a reference surface. The injections are matched by reference injections, or by buffer containing no ligand. Both buffer injections and the reference flow channel signal are subtracted from the actual signal to ensure that the binding curve is of good quality and only reflects the compound signal (double referencing). By monitoring extended dissociation times we can accurately determine residence times even for compounds with a very long residence time. Compounds are measured in triplo in which we aim for a binding response of more than 10 RU. Binding curves are fitted with a single ligand binding kinetics protocol and parameters reported. Raw data are provided to the client together with a report.

[1] Willemse-Seegers et al. Compound Selectivity and Target Residence Time of Kinase Inhibitors Studied with Surface Plasmon Resonance (2017), J. Mol. Biol. (429), 4, 574–586.

ResidenceTimer™ Kinase Panel

Kinase Domain Pre-profiled therapeutics
ABL (ABL1)
full length ponatinib, bosutinib, dasatinib, imatinib
AKT1 catalytic
pan-Akt inhibitor
AKT2 catalytic
pan-Akt inhibitor
ALK cytoplasmatic 
crizotinib
ALK[L1196M] cytoplasmatic
crizotinib
AurA (AURKA) full length pan-Aurora inhibitors
AurB* (AURKB) full length pan-Aurora inhibitor
AXL cytoplasmatic crizotinib, sunitinib
BMX full length
bosutinib, dasatinib
BRAF catalytic domain
dabrafenib, vemurafenib
BTK* full length ponatinib
BTK full length ponatinib
CHK1 (CHEK1) full length bosutinib, sunitinib
CSF-1R/FMS cytoplasmatic dasatinib
DDR2 cytoplasmatic
ponatinib, dasatinib, imatinib, sorafenib
EGFR cytoplasmatic afatinib, erlotinib, gefitinib
EGFR[T790M/L858R] cytoplasmatic osimertinib
FAK (PTK2) truncated crizotinib
FGFR1* cytoplasmatic ponatinib, sunitinib, pan-FGFR inhibitor
FGFR1 cytoplasmatic ponatinib, sunitinib, pan-FGFR inhibitor
FGFR3* cytoplasmatic sunitinib, pan-FGFR inhibitor
FGFR4 cytoplasmatic  sunitinib, pan-FGFR inhibitor
FLT3 cytoplasmatic
imatinib, dasatinib
FYN[isoform a] full length ponatinib, regorafenib
IGF1R* cytoplasmatic
staurosporine, cabozantinib, IGF-1R inhibitors
IKKα (CHUK) full length IKKβ inhibitor
IKKα (CHUK) inactive mutant full length IKKβ inhibitor
IRAK4 full length crizotinib, sunitinib
ITK* full length crizotinib, sunitinib
JAK2 catalytic domain staurosporine
KDR (VEGFR2) cytoplasmatic
vandetanib
KIT* cytoplasmatic
pazopanib, masitinib, staurosporine
LCK full length ponatinib, regorafenib
LYNa full length ponatinib, regorafenib
MAP2K1 inactive full length crizotinib, dasatinib, MEK inhibitors
MAP3K5 catalytic
 staurosporine,  bosutinib, sunitinib, pan-Akt inhibitor
MET cytoplasmatic
 crizotinib
p38α (MAPK14) inactive truncated
 sorafenib, p38 MAPK inhibitors
p38α (MAPK14) truncated sorafenib, p38 MAPK inhibitors
PAK4 full length staurosporine, bosutinib, pan-Akt inhibitor
PIK3CA/PIK3R1 full length pictilisib, dactolisib
PIK3CB/PIK3R1 full length apitolisib
PIK3CD/PIK3R1 full length idelalisib, duvelisib
PIK3CG/PIK3R1 full length apitolisib, duvelisib
PKN1 full length pan-Akt inhibitor
PLK1 full length PIk1 inhibitor
PYK2 (PTK2B) full length crizotinib
RET cytoplasmatic
ponatinib, regorafenib, vandetanib, dasatinib, sorafenib
ROCK1 catalytic
ROCK, multikinase, angiokinase inhibitors
SRC full length ponatinib, regorafenib
SRPK1 full length staurosporine
SYK* full length staurosporine
SYK full length staurosporine
TIE2* cytoplasmatic
ponatinib, vandetanib
TNIK catalytic
 bosutinib, sunitinib
TRKA (NTRK1)* cytoplasmatic
Trk inhibitor, ponatinib
TRKA (NTRK1) cytoplasmatic
Trk inhibitor, ponatinib
TRKB (NTRK2)* cytoplasmatic
Trk inhibitor, ponatinib
TRKB (NTRK2) cytoplasmatic
Trk inhibitor, ponatinib
TRKC (NTRK3)* catalytic
sorafenib, Trk inhibitor

* pre-activated by treatment with ATP after purification and before immobilization