br Results br Discussion The structures presented

Results
Discussion The structures presented here were solved at high BW 723C86 hydrochloride and show in detail how DDR1 achieves high affinity for imatinib and ponatinib, respectively. Both type II inhibitors bind in their more potent extended conformations to the inactive DFG-out conformation of the kinase domain. Differences to ABL are observed primarily in the P-loop, where DDR1 adopts the active conformation common to the KIT–imatinib complex (KIT, mast/stem cell growth factor receptor) [43]. As a result, residues in the DDR1 P-loop that confer drug resistance when introduced in ABL are solvent exposed and tolerated. DDR1 also assembles a cage-like structure around the inhibitor pocket by tethering the activation segment to the αD helix. This alternative loop arrangement stabilizes the DFG-out conformation of DDR1 and establishes a distinct packing from other structures. This conformation is exploited by the first DDR1-selective type II inhibitors that carry variant head and linker moieties that restrict interaction with the gatekeeper residue [35], [44]. Interestingly, the ether bridge of DDR1-IN-1 is also found in the MET (hepatocyte growth factor receptor) inhibitor LY2801653, which has entered clinical trials for advanced cancer and inhibits DDR1 with IC50 and EC50 values of less than 1nM [45]. Imatinib-mediated inhibition of breakpoint cluster region-ABL has shown remarkable safety and efficacy against CML [26]. Perhaps more significantly, the recognition of imatinib activity against other kinases, notably KIT and PDGFR (platelet-derived growth factor receptor), has led to its effective use in other oncology indications [46], [47] and ongoing clinical trials in fibrosis [48]. Collagen-induced activation of the RTKs DDR1 and DDR2 is similarly observed in fibrotic diseases and neoplastic tissue suggesting that DDR inhibition may be a beneficial off-target effect. Furthermore, ponatinib and dasatinib show potent activity against mutant DDR2 in models of squamous cell lung cancer [20] and indeed dasatinib has entered clinical trials for this indication [49]. DDR kinases share a conserved threonine gatekeeper residue with ABL and are therefore likely to remain susceptible to drug resistance mutations at this site. The aminopyrimidine head group of imatinib is hydrogen bonded to the gatekeeper Thr701 in DDR1 analogous to its interaction with the gatekeeper Thr315 in ABL [27]. In CML, mutation of the gatekeeper Thr315 to Ile confers drug resistance [50], suggesting that an analogous mutation in DDR1 and DDR2 would also confer resistance to imatinib.
Materials and Methods
Acknowledgements
Introduction The discoidin domain receptors, DDR1 and DDR2, are two closely related receptor tyrosine kinases (RTKs) that contain a discoidin (DS) homology domain in their extracellular regions. The DDRs were initially discovered by homology cloning based on their catalytic kinase domains and were orphan receptors until 1997, when two independent groups discovered that several different types of collagen are functional DDR ligands (Shrivastava et al., 1997, Vogel et al., 1997). RTKs are a large family (58 proteins in humans) of single-pass transmembrane receptors, characterized by structurally diverse extracellular ligand-binding regions and conserved cytosolic kinase domains. Based on their extracellular domain architecture, RTKs are divided into 20 subfamilies. RTK-dependent cellular signaling controls critical cellular processes, such as proliferation and differentiation, cell survival, cell migration, and cell cycle control (Lemmon and Schlessinger, 2010). Typical RTKs (exemplified by the most studied receptors, members of the EGF, and insulin receptor subfamilies) are activated by soluble peptide-like growth factors. It was therefore surprising that the DDRs are activated by collagens, major components of all types of extracellular matrix (ECM) (Kadler et al., 2007). Before this discovery, integrins were considered to be the only class of cell surface receptors that could transmit signals into cells by binding ECM components. Integrins are heterodimers of noncovalently associated α and β chains that constitute the main family of ECM receptors for cell adhesion (Hynes, 2002). Of the 24 distinct integrins in higher vertebrates, four serve as collagen-binding receptors (Leitinger, 2011).