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Kinase Activation State Can Affect Inhibitor Binding Affinity

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Kinase activation is often driven by the phosphorylation of residues in key regulatory elements, including, among others, the activation loop (A-loop) and receptor tyrosine kinase (RTK) autoinhibitory juxtamembrane (JM) domains. A-loop phosphorylation stabilizes a catalytically active kinase conformation characterized by a “DFG-in” structure at the loop’s N-terminus, whereas JM domain phosphorylation relieves autoinhibition by dissociating the JM domain from the catalytic domain, where it locks down an inactive “DFG-out” A-loop conformation and sterically occludes the active site. For both of these mechanisms, inhibitor binding affinity can be highly activation state-dependent. For example, the Type II ABL/KIT inhibitor imatinib, which recognizes an inactive “DFG-out” kinase conformation, binds with high affinity to nonphosphorylated ABL, but with reduced affinity (30-fold) to activated ABL, phosphorylated on the A-loop. Imatinib also has a dramatic affinity preference (1,000-fold) for non-autoinhibited KIT (JM domain phosphorylated and dissociated) over the autoinhibited state (JM domain nonphosphorylated and docked) that is explained by steric clashes between the inhibitor and the docked JM domain. The activation state dependence of binding reflects an inhibitor’s binding mode and therefore provides a rapid biochemical method to gain structural insights in the absence of cocrystal data.

Inhibitor Binding Mode Classification

The majority of ATP-competitive kinase inhibitors are classified as having either Type I or Type II binding modes. Although both Type I and II inhibitors generally contact the ATP binding site, only Type II inhibitors access an “allosteric” site unmasked in the inactive DFG-out conformation. Consequently, Type II inhibitor binding can be significantly more sensitive to the phosphorylation state of the A-loop than Type I inhibitor binding. Examples of Type I and Type II inhibitors are listed in Table 1.


Table 1. Select Type I and Type II Kinase Inhibitors



Drug
Inhibitor
Type
Primary
Target

Status
Imatinib II ABL1 FDA-Approved
Nilotinib II ABL1 FDA-Approved
Dasatinib I ABL1 FDA-Approved
Sorafenib II VEGFR2 FDA-Approved
Gefitinib I EGFR FDA-Approved
Erlotinib I EGFR FDA-Approved

Type I and Type II inhibitors are embodiments of related but distinct paradigms for ATP-competitive kinase inhibition. An inhibitor’s binding mode can impact several key parameters in drug discovery, including enzyme inhibition kinetics, offsets between in vitro and cellular potency, nearest neighbor & kinome-wide selectivity, on target residence time & pharmacodynamics, interactions with upstream and downstream signaling molecules, and intellectual property position. Since the optimal binding mode is likely to be target-specific, it is an essential parameter to characterize for multiple leads at program outset and during optimization. When the optimal binding mode is unknown a priori, a strategy to pursue two lead series with distinct binding modes can de-risk early lead selection decision making. However, binding mode determination can be difficult, time consuming, and expensive, often requiring the use of x-ray crystallography or in silico modeling.

scanMODE: classify inhibitors as having Type I or Type II binding modes without a requirement for cocrystal structures

scanMODE includes a panel of ABL assay pairs, phosphorylated or nonphosphorylated on the A-loop, and capitalizes on several key observations that enable the use of these assay pairs to serve as surrogates to classify an inhibitor’s binding mode as Type I or Type II.

  • Type II inhibitors bind preferentially to the nonphosphorylated state of ABL, whereas Type I inhibitor binding is phosphorylation state-independent
  • An inhibitor’s binding mode is generally maintained across kinases (e.g. imatinib is a Type II ABL inhibitor and a Type II LCK inhibitor).
  • Inhibitors that primarily target kinases other than ABL are correctly classified as Type I or Type II when tested against the differentially phosphorylated ABL assay pairs
  • A significant fraction of known kinase inhibitors have sufficient off-target affinity for ABL and/or clinically relevant ABL mutants to qualify for scanMODE analysis


Panel Highlights and Benefits

  • Classify inhibitors as having Type I or Type II binding modes without a requirement for cocrystal structures
  • Further differentiate the detailed binding modes of inhibitors within the Type I and Type II classes
  • Collect activation state-specific biochemical PDGFR family RTK inhibition data that are required to predict & interpret potency in cellular assays

scanMODE classifies inhibitor binding mode by measuring phosphorylation state-dependent affinity changes

Type I Kinase Inhibitor
Affinity Independent of A-loop Phosphorylation

Type II Kinase Inhibitor
Affinity Dependent on A-loop Phosphorylation

Type I Kinase Inhibitor Dasatinib

Binding constant (Kd) determinations were measured for interactions between dasatinib, a known Type I inhibitor and ABL preparations differentially phosphorylated on the A-loop. Dasatinib exhibited no affinity preference for either non-phosphorylated state (Kd = 0.027 nM) or the phosphorylated state (Kd = 0.019 nM)[click graph to enlarge].
Type II Kinase Inhibitor Imatinib

Binding constant (Kd) determinations were measured for interactions between imatinib, a known Type II inhibitor, and ABL preparations differentially phosphorylated on the A-loop.  Imatinib exhibited a 30-fold affinity preference for the non-phosphorylated state (Kd = 1.4 nM) relative to the phosphorylated state (Kd = 56 nM) [click graph to enlarge].

Further differentiate detailed binding modes of inhibitors within the Type I/II classes

scanMODE also includes a panel of PDGFR family RTK assay pairs (CSF1R, FLT3, KIT) in the autoinhibited (JM domain docked) and non-autoinhibited (JM domain not docked) states. Unlike the case for ABL A-loop phosphorylation, both Type I and Type II inhibitor affinities are dependent on the PDGFR family RTK activation state, with large and often dramatic preferences for the non-autoinhibited state observed for all inhibitors tested (Table 2). These binding affinity preferences are inhibitor-specific and report on the compatibility of an inhibitor’s binding mode with the autoinhibited conformation. In the autoinhibited state, the docked JM domain can interfere with inhibitor binding in two ways: first, by sterically clashing with the inhibitor directly, and, second, by stabilizing an enzyme conformation incompatible with inhibitor binding. Whereas inhibitors such as sunitinib (Figure 2) and dasatinib show relatively small affinity preferences and have binding modes compatible with the autoinhibited conformation, imatinib (Figure 2) and nilotinib binding are sterically incompatible with JM domain docking and the affinity preferences are much larger (Table 2).

Thus, structural insights are gained by measuring an inhibitor’s affinity preference for the non-autoinhibited state, the magnitude of which reports on the compatibility of an inhibitor’s binding mode with the autoinhibited conformation. Reference crystal structures of autoinhibited CSF1R (2OGV), FLT3 (1RJB), and KIT (1T45) are publicly available. Since a significant fraction of known kinase inhibitors have off-target affinity for PDGFR family RTKs, these data can provide structural insights for inhibitors targeting kinases outside of the PDGFR family as well.

Table 2. Activation state-dependent KIT inhibitor binding provides structural insights

  KIT Activation State  





Inhibitor




Inhibitor Type



Non-autoinhibited
Kd (nM)




Autoinhibited
Kd (nM)

Fold Affinity Preference for Non-autoinhibited State
Inhibitor Binding Mode Compatibility with Autoinhibited Conformation
Dasatinib I  0.12 1.2 10 High Compatibility
Sunitinib I  0.11 3 30 High Compatibility
CEP-701 I 110 7500 70 Moderate Compatibility
Ki-20227 II 0.48 54 110 Moderate Compatibility
AC220 II 1.6 190 120 Moderate Compatibility
PKC-412 I 210 30000 140 Moderate Compatibility
Sorafenib II 11 2200 200 Moderate Compatibility
Nilotinib II 25 15000 600 Low Compatibility
MLN-518 II 2.4 1500 630 Low Compatibility
Imatinib II 6.5 4400 680 Low Compatibility


Figure 2. Affinity preferences for the non-autoinhibited state report on the compatibility of an inhibitor’s binding mode with the autoinhibited conformation



Sunitinib binding compatible with autoinhibited conformation - small affinity preference for non-autoinhibited state
Imatinib binding incompatible with autoinhibited conformation - clashes with docked JM domain (see arrow) large affinity preference for non-autoinhibited state

scanMODE: collect activation state-specific biochemical PDGFR family RTK inhibition data required to predict & interpret potency in cellular assays

Both Type I and Type II inhibitor affinities are dependent on the PDGFR family RTK activation state, with large and often dramatic preferences for the non-autoinhibited state observed for all inhibitors tested (see Table 2 above). It is therefore critical to know the activation state being queried in biochemical assays when predicting cellular potency and when interpreting cellular data. In Figure 3 we display biochemical and cellular potency data for a panel of KIT inhibitors. Binding affinity (Kd) data were collected for the autoinhibited and non-autoinhibited states of KIT, and enzyme IC50 data for two inhibitors were also measured by three commercial providers using KIT preparations with undefined activation states. Cellular potency was measured for ligand-stimulated wild type KIT. The results show that both the in vitro enzyme activity IC50s and the autoinhibited state Kds can greatly under-predict cellular potency, whereas the non-autoinhibited Kd data are most predictive and give the expected potency offsets (in vitro Kd < cellular IC50).

In conclusion, highly potent PDGFR family RTK inhibitors can be missed in biochemical assays using enzyme preparations for which the activation state is undefined. scanMODE activation state-specific assays provide a unique solution for measuring biochemical potency for inhibitors of this kinase family.

Figure 3. Comparison of biochemical and cellular KIT inhibitor potency data

KIT Inhibitor Potency Data

Key References

  • Liu, Y and Gray, N. S. (2006) Rational design of inhibitors that bind to inactive kinase conformations. Nat. Chem. Biol. 2, 358-364.
  • Wodicka, L. et al., (2010) Activation State-Dependent Binding of Small Molecule Kinase Inhibitors: Structural Insights from Biochemistry. Chem. Biol. 17, 1241-9.


scanMODE Assay Panel

Listed below are the assays currently available for screening and profiling.

 

KGS ▲Kinase NameEntrez Gene Symbol
ABL1(F317I)-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(F317I)-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(F317L)-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(F317L)-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(H396P)-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(H396P)-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(Q252H)-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(Q252H)-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(T315I)-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1(T315I)-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1-nonphosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
ABL1-phosphorylatedc-abl oncogene 1, receptor tyrosine kinaseABL1
CSF1Rcolony stimulating factor 1 receptorCSF1R
CSF1R-autoinhibitedcolony stimulating factor 1 receptorCSF1R
FLT3fms-related tyrosine kinase 3FLT3
FLT3-autoinhibitedfms-related tyrosine kinase 3FLT3
KITv-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologKIT
KIT-autoinhibitedv-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologKIT