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

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].

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
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 Name | Entrez Gene Symbol |
ABL1(F317I)-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(F317I)-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(F317L)-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(F317L)-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(H396P)-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(H396P)-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(Q252H)-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(Q252H)-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(T315I)-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1(T315I)-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1-nonphosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
ABL1-phosphorylated | c-abl oncogene 1, receptor tyrosine kinase | ABL1 |
CSF1R | colony stimulating factor 1 receptor | CSF1R |
CSF1R-autoinhibited | colony stimulating factor 1 receptor | CSF1R |
FLT3 | fms-related tyrosine kinase 3 | FLT3 |
FLT3-autoinhibited | fms-related tyrosine kinase 3 | FLT3 |
KIT | v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog | KIT |
KIT-autoinhibited | v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog | KIT |