Structural mechanisms determining inhibition of the collagen receptor DDR1 by selective and multi-targeted type II kinase inhibitors

Abstract

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC50 of 9nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.

Keywords: crystallography; drug design; gleevec; oncology; phosphorylation.

Graphical abstract

Fig. 1

Overview of the DDR1 structure. (a) Domain organization of DDR1. (b) Crystal structure of the DDR1 kinase domain in complex with the inhibitor ponatinib. (c) Chemical structures of imatinib and ponatinib, highlighting their “head”, “linker” and “tail” regions. (d) Sequence alignment of the kinase domains of DDR1, DDR2 and ABL. Secondary structure elements are displayed for the DDR1 kinase. Residues labeled with an asterisk (*) form hydrogen bonds with both imatinib and ponatinib. Residues labeled with a double dagger (^‡) form hydrogen bonds with imatinib only.

Fig. 2

Crystal packing in the DDR1–ponatinib complex. (a) The asymmetric unit of the DDR1–imatinib structure contains two protein monomers. Asterisks mark the disordered regions of the activation segments corresponding to strands β8 and β9. (b) The asymmetric unit of the DDR1–ponatinib structure contains a crystallographic dimer held by the self-association of the β8-β9 hairpin (colored blue and orange in chains A and B, respectively). (c) The dimer interface is stabilized by the binding of an additional ponatinib molecule. The linker and tail regions of the interfacial ponatinib molecule contact hydrophobic residues on the lower face, where they also hydrogen bond to the carbonyl of Ile815 (αEF helix) in each protein chain. The opposite face of the β-sheet is stabilized by contacts with the αC helix and phosphate-binding loop (P-loop), including a salt bridge between Arg798 (β8) and Asp668 (αC) (data not shown). (d) Perhaps as a result of these different packing interactions, DDR1 monomers from the imatinib and ponatinib co-structures show a subtle shift in the relative positions of the N-terminal kinase lobes.

Fig. 3

Structural comparison of DDR1 and ABL. (a) Superposition of the structures of DDR1 and ABL–imatinib (PDB ID: 2HYY) reveals a number of structural changes that are highlighted by boxed regions. (b) Sequence alignment of the activation segment of selected kinases. The three potential sites of phosphorylation in DDR1 are highlighted by asterisks. The same region is highlighted on the DDR1 structure (right) showing the same tyrosine side chains in DDR1 (red with residue numbers displayed), IGF1R (PDB ID: 1P4O; green) and ABL (blue) .

Fig. 4

Divergent P-loop structures in DDR1 and ABL lead to changes in ATP pocket shape. Superposition of the DDR1–imatinib (white) and ABL–imatinib (PDB ID: 2HYY; magenta) complexes. DDR1 Glu618 and Phe621 are equivalent to the drug resistance mutations Gly250Glu and Tyr253Phe in ABL. Different conformations are also observed for the A-loop where DDR1 Arg789 corresponds to ABL Arg386.

Fig. 5

DDR1 binding and inhibition by imatinib. (a) Interactions of imatinib in the ATP pocket of DDR1. Colors correspond to the structural features indicated in Fig. 1. Hydrogen bond interactions are shown as dotted lines. (b) ITC measurements of the binding show a K_D value of 1.9 nM. (c) Imatinib efficacy on blocking collagen-induced DDR1 Y513 autophosphorylation in U2OS cells.

Fig. 6

DDR1 binding and inhibition by ponatinib. (a) Interactions of ponatinib in the ATP pocket of DDR1. Colors correspond to the structural features indicated in Fig. 1. Hydrogen bond interactions are shown as dotted lines. (b) ITC measurements of the binding show a K_D value of 1.3 nM. (c) Ponatinib efficacy on blocking collagen-induced DDR1 Y513 autophosphorylation in U2OS cells.

Fig. 7

Structural basis for DDR1-IN-1 selectivity. (a) Chemical structure of DDR1-IN-1. (b) Superposition of the ABL–imatinib complex (red; PDB ID: 2HYY) and the DDR1-IN-1 complex with DDR1 (green; PDB ID: 4CKR) . A green surface representation defines the shape and extent of the ATP pocket in DDR1. The alternative hinge interactions of the two inhibitors are highlighted. (c) Superposition as in (b) highlighting the interactions of the inhibitor “tail” regions. (d) The ether bridge of DDR1-IN-1 orientates the indolin-2-one “head” group away from the ABL P-loop disrupting critical hydrophobic interactions with Tyr253.