Beyond IC50: Binding kinetics in drug discovery

Drugs with similar affinity can exhibit very different kinetic and binding mechanism profiles, which may contribute to efficacy, safety, duration of action and differentiation from other similar therapies.

Early evaluation of the kinetics of drug-target interactions helps to improve decision-making, to focus on the most promising compounds.

Binding kinetics define efficacy of target occupancy

  • Onset of the drug action is influenced by the association rate (kon): the faster a drug binds to the target, the faster the occupation will occur.
  • Duration of drug action is dependent on the dissociation rate (koff): the longer the duration of binding, the longer the pharmacodynamic effect.
  • Differences in association and dissociation rates are not identified by affinity measurement alone.

Applications

Predict drug clinical efficacy: By using binding kinetics, we help our clients find the most effective compounds early.

In biochemical and cellular assays performed at equilibrium, the drug and target are present at constant concentrations and thus the IC50 accurately reflects the concentration of the drug-target complex. However, in the body the concentration of a drug constantly changes due to several physiological processes. Hence, affinity is not the only relevant parameter that predicts target occupancy and ultimately efficacy: clear understanding of binding kinetics provides a more complete picture of what drives target occupancy in
vivo1-6.

The correlation of in vivo efficacy with residence time has been demonstrated across a spectrum of target types.

Target classTargetReferences
GPCRsA3RXia L. et al. (2017) J Med Chem. 14;60(17):7555-7568.
GPCRsA2ARHothersall J.D. et al. (2017) Mol Pharmacol. 91(1):25-38.
GPCRsβ2ARRosethorne E.M. et al (2016) Mol Pharmacol. 89(4):467-75.
GPCRsCB1Louvel J. et al (2015) Eur J Med Chem. 28;101:681-91.
GPCRsCCR2Vilums M. et al (2015) ChemMedChem. 10(7):1249-58.
GPCRsCCR5Swinney D.C. et al. (2014) Br J Pharmacol. 171(14):3364-75.
GPCRsHRH1Bosma R. et al. (2017) Front Pharmacol. 25;8:667.
GPCRsGIPRNordskov M.B.G et al. (2020) Basic Clin Pharmacol Toxicol. 126 Suppl 6:122-132.
GPCRsNK1RNederpelt I. et al. (2017) Sci Rep. 26;7(1):14169.
GPCRsmGluR2Doornbos M.L.J. et al. (2017) J Med Chem. 10;60(15):6704-6720.
GPCRsM3 mAChRGuo D. et al. (2018) Methods Mol Biol. 1705:197-206.
Nuclear receptorsERCopeland R.A. (2016) Nat Rev Drug Discov. 15(2):87–95.
KinasesABLPuttini M. et al. (2008) Haematologica 93, 653–661.
KinasesAURK B, AURK CAnderson K. et al. (2009) Biochem J. 420(2):259-65.
KinasesBTKBradshaw J.M. et al. (2015) Nat Chem Biol. 11:525–531.
KinasesCDK2, CDK9Ayaz P. et al. (2016) ACS Chem Biol 11(6):1710-9.
KinasesCDK7Marineau J.J. et al. (2021). doi: 10.1021/acs.jmedchem.1c01171.
KinasesEGFRWood E.R. et al. (2004) Cancer Res. 64, 6652–6659.
Kinasesp38α MAPKWalter N.M. et al. (2017) J Med Chem. 12;60(19):8027-8054.
KinasesTTKUitdehaag J.C.M. et al. (2017) J Mol Biol. 7;429(14):2211-2230.
KinasesRIP1KYoshikawa M. et al (2018) J Med Chem. 61:2384–2409.
ProteasesAChEKharlamova A.D. et al (2016) Biochem J. 473(9):1225-36.
ProteasesBACE1Eketjäll S. et al. (2016) J Alzheimers Dis.  50(4): 1109–1123.
Epigenetic enzymesDOT1LDaigle S.R. et al (2013) Blood. 122(6): 1017–1025.
Epigenetic enzymesHDAC1, HDAC2Wagner F.F. et al. (2016) Bioorg Med Chem. 15;24(18):4008-4015.
Epigenetic enzymesEZH2Van Aller G.S. et al. (2014) ACS Chem Biol. 21;9(3):622-9.
SynthasesCOX-2Tian G. et al. (2021) Biochemistry 60(31):2407-2418.
Ion channelsL-type calciumCopeland R.A. (2016) Nat Rev Drug Discov. 15(2):87–95.
Ion channelsM2 protonDrakopoulos A. et al. (2018) ACS Med Chem Lett. 9(3):198-203.

Comprehensive reviews of the importance of drug-target kinetics

  1. Copeland R.A. (2016) Nat Rev Drug Discov. 15(2):87–95.
  2. Doris A Schuetz D.A. (2017) Drug Discov Today. 22(6):896-911.
  3. Swinney D.C. (2004) Nat Rev Drug Discov. 3(9):27–41.
  4. Zhang R. et al. (2010) Expert Opin Drug Discov. 5(11):1023-9.
  5. Tummino P.J. et al. (2008) Biochemistry. 47(20):5481–5492.
  6. Lu H. at al. (2010) Curr Opin Chem Biol. 14(4):467–474.

Extend the duration of action: The longer a drug remains bound to its target, the longer it remains active, meaning less frequent doses are needed. 

There are many medicines for which binding kinetics contribute to efficacy and duration of action. One example is Candesartan, an antihypertensive drug that lowers blood pressure more efficiently than any other member of the same class, such as Losartan.

While Candesartan and Losartan share the same binding site, similar pharmacokinetics and receptor occupancy profiles, their binding kinetics are completely different1-4. In a clinical trial study, the long-lasting pharmacodynamic effect of Candesartan provided greater efficacy at a lower dose (16 mg) than Losartan (100 mg) and maintained efficacy in the event of a missed dose5.

Left: Dose–response curves for the effect of Candesartan (orange) and Losartan (blue) on blood pressure in hypertensive patients. Right: Pharmacodynamic, pharmacokinetic and clinical features of Candesartan and Losartan. Adapted from Elmfeldt D. et al (2002).

References

  1. Vauquelin G. et al (2010) Br J Pharmacol. 161(3): 488–508.
  2. Tummino P.J. et al. (2008) Biochemistry. 47(20):5481–5492.
  3. Copeland R.A. et al. (2006) Nat Rev Drug Discov 5(9):730-739.
  4. Swinney D.C. (2004) Nat Rev Drug Discov. 3(9):27–41.
  5. Elmfeldt D. et al (2002) Blood Press. 11(5):293-301.

Compound differentiation: Kinetic profiling in the early stages of drug discovery enables better identification of compounds with therapeutic potential that merit further progression.

Simple affinity measurements may mask the true impact of structural changes made in the same chemical series.

Ayaz and colleagues show this with a series of Roniciclib analogs of CDK21. They found that Roniciclib, the trifluoromethyl derivatives (CF3) and bromo analogues (Br) showed similar kinase and cell proliferation inhibition along with comparable pharmacokinetic profiles. However, Roniciclib and the CF3 derivatives had longer residence times and superior efficacy in tumor growth inhibition relative to the corresponding bromo analogues.

Left: Comparison of the residence times of compounds 1–5 and Roniciclib. The CF3 derivatives (orange) have up to 15 times longer residence times than their Br analogues (blue). Right: Comparison of cell proliferation and antitumor efficacy. The CF3-substituted compounds show superior efficacy in tumor growth inhibition relative to the Br analogs despite their similar EC50 values. Adapted from Ayaz, P. et al. (2016).

References

  1. Ayaz, P. et al. (2016) ACS Chem. Biol. 11, 1710–1719.

Maximize therapeutic windows: Early modulation of on- and off-target kinetics can help build safety margins and reduce adverse events.

It is now understood that drug safety is difficult to achieve by solely considering affinity. Indeed, many reports link selectivity to the kinetic profile of the drugs.

Find out more by reading the appropriate case study:

Affinity doesn’t tell the whole story of MET inhibitor Foretinib

Understand PK/PD disconnects: Kinetic profiling often bridges the gap between PK/PD prediction and observation.


Systemic exposure as a driver of pharmacologic effects provides limited value in guiding compound optimization, selection, and advancement when PK/PD disconnects are observed within a chemical series.  Binding kinetics can help in this scenario not only guiding compound design but also predicting target occupancy and drug dose.

Build better models: PK/PD models that integrate kinetics better define therapeutic windows and therefore dose regimens.

Client feedback and several studies1-8 suggest that the pharmacokinetic/pharmacodynamic models that integrate binding kinetics, instead of just affinity, better predict:

  • Target engagement.
  • Drug dose.
  • Treatment schedule.
  • Real selectivity and potential toxicities.

Simulations of the time course of in vivo target occupancy (TO) by Dasatinib show that conventional PK/PD approaches predict lower efficacy and a higher dose than is actually needed. Affinity based models also fail to predict the real selectivity and consequently, potential toxicities.

Simulations of Dasatinib TO with ABL and off-targets SRC and FYN using binding kinetics (PK-BK) and affinity (PK-Kd) models. Left: Current clinical treatment schedules do not show differences in modelling results. Right: When a longer administration schedule is considered, PK-BK models predict different TO with an increase of the AUC. Adapted from Georgi V. et al (2018).

This effect of binding kinetics on drug PK has been demonstrated in mice. The comparison of the PK profile of inhibitors with similar Kd but different residence time between the WT and epoxide hydrolase (EH) knockout mice, reveals that drugs with longer residence time bind to EH longer, protecting them from being metabolized or eliminated from the body.

Left: Long residence time drug (20 min) was administrated at 0.3 mg/Kg. The PK profiles are very different after 48 h. The drug is fully eliminated in KO mice within 72 h, while in WT mice the drug blood level stayed above the Kd at postdosing day 14. Right: Short residence time drug (6 min) was administrated at 1 mg/Kg. The PK profiles are similar. The drug is fully eliminated in WT mice within 168 h and in KO mice within 144 h. Adapted from Lee K.S.S. et al. (2019).

References

  1. Georgi V. et al (2018) J Am Chem Soc. 140(46):15774–15782.
  2. Daryaee F. et al (2017) Chem Sci. 8(5): 3434–3443.
  3. Walkup G.K. et.al (2015) Nat Chem Biol. 11(6):416–423.
  4. Lee K.S.S. et al. (2019) ACS Central Sci. 5(9):1614–1624.
  5. De Witte W.E.A. (2018) J Pharmacokinet Pharmacodyn. 45(4):621-635.
  6. Ramsey J.S. et al (2011) Br J Pharmacol. 164(3):992-1007.
  7. Hong Y. et al. (2008) Clin Pharmacokinet. 47(2):129-37.
  8. Yassen A. et al (2005) J Pharmacol Exp Ther. 313(3):1136-49.

Strengthen intellectual property: By using binding kinetics, our clients enrich the structural diversity of their pipeline

Understanding binding kinetics early in drug discovery gives access to a more diverse chemical space, better defined biology and therefore more scope for intellectual property.

Technology

KINETICfinder®

KINETICfinder® is Enzymlogic’s patented high-throughput screening platform that delivers all key binding kinetic (kon / koff / residence time) and affinity (kd) parameters for reversible binders quickly and at scale.

Our highly robust platform provides accurate and reproducible compound–target interaction data that enables improved screening of compounds in early discovery: to iterate medicinal chemistry, understand PK/PD disconnects and build better models to define therapeutic windows.

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
ACK (TNK2) ACK, ACK-1, ACK1, p21cdc42Hs Clear line of sight
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

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