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  • br Results br Discussion The structures presented here were

    2020-10-15


    Results
    Discussion The structures presented here were solved at high Cy3 azide 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 Cy3 azide 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 Collagen is the most abundant extracellular matrix (ECM) protein of vertebrates (Pérez-Tamayo, 1978). The remodeling of collagen to maintain tissue homeostasis is a critical metabolic process in the health of mammals (Cox and Erler, 2011). Collagen remodeling comprises distinct, well-orchestrated processes, including pericellular proteolysis by membrane-bound or secreted metalloproteinases (Kirmse et al., 2011, Sternlicht and Werb, 2001) and internalization and intracellular degradation of collagen fibrils (Arora et al., 2000, Cullen, 1972, Groves et al., 2008, Madsen et al., 2013, Neurath, 1993, Soames and Davies, 1977). Cell-induced mechanical compaction and reorganization of collagen in the ECM by cell-generated tractional forces (Wolf et al., 2013) contribute to local tissue strengthening and optimize fiber orientation, properties that ultimately affect the survival, growth, and migration of cells (Cox and Erler, 2011, Fang et al., 2014, Paszek et al., 2005). Localized increases of deposition, alignment, and cross-linking of collagen fibrils contribute to ECM stiffening, a process seen in tissue fibrosis and in the stroma associated with epithelial tumors (Levental et al., 2009, Malik et al., 2015, Paszek et al., 2005, Wynn, 2008). Although the mechanisms by which ECM adhesion receptors contribute to ECM stiffening are not defined, it is known that the rate-limiting steps of collagen remodeling by traction are the binding of specific adhesion receptors to collagen fibrils and the generation and application of actomyosin-dependent contractile forces (Kirmse et al., 2011, Kural and Billiar, 2013, Wolf et al., 2013, Wyckoff et al., 2006).