How KINETICfinder® assays work

KINETICfinder® is a highly tuned and optimized TR-FRET Binding Kinetic Assay based on the binding and displacement of an active-site directed fluorescent probe.

Real-time binding of the fluorescent probe is detected using a labeled anti-tag antibody, which binds to the target of interest. The binding of the fluorescent probe and labeled antibody to the target increases the TR-FRET signal, whereas the displacement of the fluorescent probe with a compound decreases the TR-FRET signal.

To ensure that our kinetic assays consistently deliver the highest quality data we always introduce up to 32 controls and a reference compound in each analysis.

Our services

Binding kinetics

KINETICfinder® is a high throughput kinetic platform that delivers all key binding kinetics (kon, koff, residence time) and affinity (Kd) parameters for reversible binders quickly and at scale.

Kinetic selectivity

KINETICfinder® can help you to modulate the therapeutic index and safety profile of your drugs through modification of on- and off-target kinetics (kon, koff, residence time and Kd).

Applications

  • Easier decision-making: Ideal hit-to-lead and lead optimization tool to facilitate parallel SAR and SKR iteration.
  • Kinetic selectivity: Modulate earlier and with confidence the safety profile of your drugs through modification of the on- and off-target kinetics.
  • Understand PK/PD disconnects: Kinetic profiling often bridges the gap between PK/PD prediction and observation
  • Build better models: PK/PD models that integrate kinetics better predict target engagement, drug dose and treatment schedule.

Features and benefits

Reliable: In depth studies demonstrate concordance with literature values.

There is a very high correlation between the Kd, kon and koff values obtained from our assays and the IC50 and kinetics values obtained from activity assays and biophysical methods such as Surface Plasmon Resonance (SPR). Moreover, our assays provide reliable results regardless of the magnitude of the value measured.

Figure 1. KINETICfinder® is an accurate and reliable method. Correlation between the affinity and kinetic constants of 22 inhibitors and 22 different kinases obtained with KINETICfinder® and those obtained from end-point activity assays and biophysical assays (SPR) reported in the literature. The plots display the Pearson correlation coefficient (r) and p-value.

We are experts in kinetic profiling

Enzymlogic is a world leading analytical CRO, expert in generation and interpretation of kinetic data. We are proud to serve the smallest biotech innovators through to 8 of the world’s top 20 Pharma organizations, supporting drug hunters in their search for the most promising compounds in early discovery.

References

  1. Ayaz P. et al. (2016) ACS Chem Biol. 11(6):1710-9.
  2. Conroy A. et al (2009). Cancer Chemother Pharmacol. 64(4):723-32. 
  3. Deininger M. et al (2005) Blood. 105(7):2640-53.
  4. Ferguson FM. et al. (2016) ACS Med Chem Lett. 7(10):908-912.
  5. Folkes AJ. et al. (2008) J Med Chem. 51(18):5522-32.
  6. Iwata H. et al. (2011) Biochemistry. 50(5):738-51.
  7. Gozgit. et al. (2015) 25th Meeting of the European School of Hematology A 1075.
  8. Limvorasak S. et al. (2009) Expert Opin Pharmacother. 10(18):3091-102.
  9. Lannutti BJ. et al. (2011) Blood 117:591-594.
  10. Lin YL. et al. (2013) Proc Natl Acad Sci U S A. 110(5):1664-9.
  11. Manley PW. et al. (2011) Blood 118 (21):1674.
  12. Morando MA. et al. (2015) Sci Rep. 6:24439.
  13. O’Hare T. et al. (2013) Cancer Res. 73(11):3356-70.
  14. O’Hare T. et al. (2009) Cancer Cell. 16(5):401-12.
  15. Pargellis C. et al. (2002) Nat Struct Biol. 9(4):268-72.
  16. Parry D. et al. (2010) Mol Cancer Ther. 9(8):2344-53.
  17. Redaelli S. et al. (2009) J Clin Oncol. 27(3):469-71.
  18. Wedge SR. et al. (2002) Cancer Res. 62(16):4645-55. 
  19. Willemsen-Seegers N. et al. (2017) J Mol Biol. 429(4):574-586.
  20. Winkler DG. et al. (2013) Chem Biol. 20(11):1364-74.

Accurate: Kd, kon and koff values are precise regardless of magnitude across a broad dynamic range.

All our assays go through a rigorous establishment and validation process prior to use and ongoing assay performance is closely monitored to ensure they meet the highest of standards. KINETICfinder® provides precise and reproducible Kd, kon and koff readouts, regardless of the magnitude of the values, in just a single assay.

Figure 2. KineticFinder® provides accurate and reproducible results with a wide dynamic range. Kd, kon and koff values of 85 inhibitors and 70 different kinases from two independent experiments are shown on scatter plots with the axes in logarithmic scale. Each point represents an inhibitor-kinase interaction (470 interactions total). The plots display the Pearson correlation coefficient (r) and p-value.

Sensitive: Our flexible and highly sensitive assays enable the identification and discrimination of a wide spectrum of compounds.

KINETICfinder® is a flexible and highly sensitive platform designed for the identification and discrimination of a wide spectrum of compounds:

  • Slow to rapid binders
  • Weak to potent binders.

Our assays use low protein concentrations (from picomolar to low nanomolar), precise TR-FRET readouts and continuous measurements that afford a broad dynamic range:

  • Kd: µM-pM (6 log)
  • kon: 101-5×107 M-1s-1 (6 log)
  • koff: 10-6-1 s-1 (6 log)

Robust and reproducible: Our assays show outstanding robustness and reproducibility between plates and days.

The high and sustained stability of our assays guarantees superior quality results over time.

  • Outstanding reproducibility between plates and days.
  • Enables data to be interpreted with confidence.
  • High Z-values ensure low false positive/negative rates.

Our assays ensure robust measurement over time

Figure 3. KINETICfinder® enables data to be interpreted with confidence. Typical signal-to-background and Z-values obtained with KINETICfinder® assays. S/B (blue) and Z-values (purple) were calculated based on 32 control wells over 10 hours.
Figure 4. KINETICfinder® provides highly robust measurements over time.
Z-values from 140 independent assays spanning a period of 16 months. Z-values were calculated for each time point in a kinetic assay and averaged over the 10-hour duration.

Our assays are highly reproducible between plates and days

Figure 5. KINETICfinder® provides outstanding reproducibility between plates and days. Analysis of kon, koff and Kd values for a single compound-kinase interaction over a sixteen-month period were performed. Data taken from 3 different compound, enzyme, probe and antibody lots demonstrate less than 5-fold variance.

Without complications: As our assays are performed close to native conditions, they do not encounter the challenges associated with SPR biosensor chips.

Enzymlogic’s kinetic profiling assays are performed in solution, benefiting clients who have experienced limitations when using biosensing technologies like SPR, stemming from complications due to target immobilization on the sensor chip.

FeaturesSPRKINETICfinder®
Target activityPossible lossMaintained. All active sites available
Target conformationPossible changeMaintained
Multiprotein complexesPotential limitationNo issues
Heterogeneity in surface binding sitesComplications in the interpretation of dataNo issues
Nonspecific bindingCan have major influenceLow
Mass transportPotential limitationNo issues
Sensitivity dependent on molecular weightPotential limitationNo issues
Dynamic range konup to 105-106 M-1s-1up to 5×107 M-1s-1
Dynamic range kofffrom 10-5 to 1 s-1from 10-6 to 1 s-1
ThroughputLowHigh

Enzymlogic’s kinetic profiling assays are performed in solution, benefiting clients who have experienced limitations when using biosensing technologies like SPR, stemming from complications due to target immobilization on the sensor chip.

In addition, Enzymlogic assays exhibit higher reproducibility vs SPR given that, in each run we:

  • Measure multiple concentrations of each compound.
  • Measure several compounds (up to 80).
  • Use internal reference compounds.

Also, as chips have a finite lifetime, the use of different SPR chips when building a dataset is also a source of variability.

Talk to us about your kinetic profiling roadblocks. We love a challenge and have successfully supported clients where other approaches have not worked out.

Rapid turnaround and cost effective: We are committed to delivering rapid and reliable data turnaround, on time and to budget, helping you keep your programs on track.

Quality data delivered on schedule

We are laser focused on rapid and reliable data delivery, with a keen eye on timelines and budgets.

  • Standard data turnaround for study requests is 10 business days from compound receipt.
  • Expedited turnaround is available for regularly scheduled ongoing screening programs.

We offer flexible solutions

Together we will define the best way to collaborate through:

  • Ready-to-use or user-defined assays.
  • User-defined screening concentrations.
  • Bespoke assay development.
  • Flexible service models to support your discovery goals and maximize program success rates.
  • Direct contract, sub-contract via your principal CRO service provider or using outsourcing services (including Scientist and Science Exchange).

List of targets ready to use

Our expertise extends beyond kinases to GPCR and other target types.

Search our database to discover whether your target is:
  • May be possible: We have a strong track record of achievement where others fail! Contact us to find out more.
  • Clear line of sight: Establishment requires finalization / validation, which typically takes 2-4 weeks to complete.
  • Good to go: Data within 2 weeks of compound receipt.

* Full length and partial length forms are available.

Target name Alternative names KINETICfinder COVALfinder
AAK1 KIAA1048, DKFZp686K16132 Clear line of sight
ABL1 ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Good to go
ABL1 E255K ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL1 G250E ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL1 H396P ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL1 M351T ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL1 Q252H ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL1 Y253F ABL, c-ABL1, BCR-ABL, CHDSKM, JTK7, bcr/abl, c-ABL, p150, v-abl Clear line of sight
ABL2 (ARG) ARG, ABL isoform b, ABLL Good to go
ACVR2A ACTRII, ACTR2, ACVR2 Clear line of sight
ACVR2B ACTRIIB, ActR-IIB, HTX4 Clear line of sight
AKT1 AKT1, PKB, PKB-ALPHA, PRKBA, RAC, RAC-ALPHA, Good to go
ALK ALK, CD246, NBLST3 Good to go May be possible
ALK C1156Y ALK, CD246, NBLST3 Clear line of sight
ALK F1174L ALK, CD246, NBLST3 Clear line of sight
ALK L1196M ALK, CD246, NBLST3 Clear line of sight
ALK R1275Q ALK, CD246, NBLST3 Clear line of sight
ALK T1151_L1152insT ALK, CD246, NBLST3 Clear line of sight
ALK1 (ACVRL1) ALK1, ALK-1, ACVRLK1, HHT, HHT2, ORW2, SKR3, TSR-I Good to go May be possible
ALK2 (ACVR1) ALK2, FOP, SKR1, TSRI, ACTRI, ACVR1A, ACVRLK2 Clear line of sight

How can we help?

Our experienced project manager will work with you to identify the best way forward for your project.