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scanKINETIC - Association/Dissociation Kinetics

Reversibility and Dissociation Kinetics Studies

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Classify inhibitors as irreversible, reversible-slow dissociation or reversible-rapid dissociation

Irreversible, covalent inhibitors and reversible inhibitors that dissociate slowly from a kinase target can have unusual properties in both cellular and in vivo pharmacology models. For example, target inhibition can be maintained for several hours or more, even after the inhibitor has been “washed out” or cleared. In the absence of target dissociation data, these pharmacology results can be difficult to interpret, particularly when multiple inhibitors are being compared. Furthermore, while irreversible/slowly dissociating inhibitors may be desirable for some drug discovery programs, these properties may not be ideal in all cases. KINOMEscan offers a dissociation kinetics service that classifies inhibitors as irreversible, reversible-slow dissociation, or reversible-rapid dissociation.

Experimental Design for Reversibility and Dissociation Kinetics Studies

For each inhibitor (Samples A & B) two parallel dose response curves are prepared and equilibrated with the kinase of interest either continuously (Sample A) or with an intervening reaction dilution step (Sample B). Samples are then read out and the data are fit to the Hill binding equation to calculate Kd values.

Model Data for Reversibility and Dissociation Kinetics Studies

For reversible, rapidly dissociating inhibitors (left panel), the apparent Kd value for Sample B is higher than the Kd value for Sample A by a multiple equal to the Sample B reaction dilution factor (10-fold in this example). For reversible, slowly dissociating inhibitors (center panel), the apparent Kd value for Sample B is higher than the Kd value for Sample A by a multiple less than the Sample B reaction dilution factor, since for Sample B, the inhibitor only partially dissociates after the reaction dilution step. For irreversible inhibitors (right panel), the Kd values for Samples A&B are equivalent, since for Sample B, the inhibitor fails to dissociate after the reaction dilution step [click graph to enlarge].

Association Kinetics Studies

Identify slowly associating inhibitors early in the drug discovery process

The association rates for kinase-inhibitor complexes are variable and can be both inhibitor and kinase-dependent. While many kinase-inhibitor complexes form rapidly (< minutes), others can have extraordinarily slow association rates, requiring several hours to reach equilibrium. Slowly associating inhibitors can yield conflicting potency data in various in vitro, cellular, and in vivo experiments, where the inhibitor-target equilibration time is often assay specific. High affinity, slowly associating inhibitors also have extremely slow dissociation kinetics; the desirability of these kinetic properties ultimately depends on other inhibitor characteristics (e.g. pharmacokinetics) and upon the goals of a specific drug discovery program. In addition, slow association kinetics can provide insight into inhibitor binding mode, as it is well documented that Type II inhibitors, which bind a catalytically inactive “DFG-out” enzyme conformation, often bind more slowly than Type I inhibitors, which are less conformation selective. The KINOMEscan association kinetics service enables the identification of slowly associating inhibitors early in the drug discovery process.

Slow Association Kinetics: p38-alpha/BIRB-796

Association kinetics analysis for the Type II p38-alpha inhibitor BIRB-796. BIRB-796 is a highly potent Type II inhibitor reported to have ultra-slow association kinetics for its target, p38-alpha. In this KINOMEscan association kinetics study, Kd measurements for this interaction were made as a function of co-incubation time (1 or 24 hr). The data reveal a dramatic reduction (30-fold) in the apparent Kd value for the 24 hr time point relative to the 1 hr time point, which is consistent with published studies demonstrating ultra-slow association kinetics for this interaction [click graph to enlarge].
AAK1 ABL1(E255K)-phosphorylated ABL1(F317L)-phosphorylated ABL1(H396P)-nonphosphorylated ABL1(H396P)-phosphorylated
ABL1(Q252H)-nonphosphorylated ABL1(Q252H)-phosphorylated ABL1(T315I)-nonphosphorylated ABL1(T315I)-phosphorylated ABL1(Y253F)-phosphorylated
ABL1-nonphosphorylated ABL1-phosphorylated ALK ALK(C1156Y) ALK(L1196M)
ANKK1 ASK2 AURKA AURKB BMPR1B
BMPR2 BRAF BRAF(V600E) BTK BUB1
CAMK1G CAMK2D CDC2L5 CDK4-cyclinD3 CDK7
CDKL1 CDKL5 CHEK2 CLK2 CSF1R
CSK CSNK2A1 CSNK2A2 DAPK1 DYRK1A
DYRK2 EGFR EGFR(L858R) EGFR(L858R,T790M) EGFR(T790M)
EIF2AK1 EPHA3 EPHB6 FLT3 FLT3(D835V)
FLT3(ITD,D835V) FLT3(ITD,F691L) FLT3(R834Q) FLT3-autoinhibited GRK1
GRK7 GSK3B HASPIN HIPK1 HIPK2
HIPK3 ICK INSR IRAK1 JAK1(JH2domain-pseudokinase)
JAK2(JH1domain-catalytic) JNK1 JNK2 JNK3 KIT
KIT(D816H) KIT-autoinhibited LATS2 LOK LRRK2
LZK MAP3K2 MAP3K3 MAP4K2 MAPKAPK5
MEK1 MEK2 MEK3 MEK4 MKK7
MKNK1 MKNK2 MST4 MYLK NDR1
NEK11 NEK3 NIM1 OSR1 p38-alpha
PAK4 PCTK1 PDGFRA PFPK5(P.falciparum) PHKG2
PIK3C2B PIK3CA PIK3CA(C420R) PIK3CA(E542K) PIK3CA(E545A)
PIK3CA(E545K) PIK3CA(Q546K) PIK3CG PIK4CB PIM1
PLK1 PLK2 PLK3 PLK4 PRKCI
PRKG2 RIOK2 RIPK5 ROCK2 RPS6KA4(Kin.Dom.2-C-terminal)
RSK1(Kin.Dom.2-C-terminal) RSK2(Kin.Dom.1-N-terminal) RSK4(Kin.Dom.1-N-terminal) SBK1 SGK
SNARK SNRK SRMS STK16 TAK1
TAOK1 TAOK2 TAOK3 TBK1 TNIK
TRKA TRKB TYK2(JH1domain-catalytic) TYK2(JH2domain-pseudokinase) ULK1
ULK2 ULK3 VEGFR2 WNK3 YSK1