Furthermore the inhibitor binding can also be altered by

Furthermore, the inhibitor binding can also be altered by various mutations. Prime examples are acquired intrinsic resistance mutations that have marred the success of tyrosine kinase inhibitors (TKIs) such as gefitinib, erlotinib and imanitib, prompting efforts for second- and third-line treatments (Daub et al., 2004; Azam and Daley, 2006; Gibbons et al., 2012). One resistance mechanism common to many kinase inhibitors is the mutation of the so-called “gatekeeper” residue that remains the most frequently detected drug-resistance mutation in the clinic. Examples include resistance to TKIs targeting breakpoint cluster region-abelson tyrosine kinase (BCR-ABL) fusion in chronic myelogenous leukaemia (Gorre et al., 2001; Shah et al., 2002), EGFR in nonsmall cell lung cancer (Kobayashi et al., 2005; Pao et al., 2005), platelet-derived growth factor receptor (PDGFR) in hypereosinophilic syndrome (Cools et al., 2003), KIT in gastrointestinal stromal tumours (Tamborini et al., 2006) and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion in lung cancer (Choi et al., 2010b). Modelling in cell culture has also been successfully used to discover clinically relevant acquired resistance and the application of this approach to FGFR driven-cancer Wnt agonist 1 identified a gatekeeper substitution (Chell et al., 2013). Gatekeeper substitutions in FGFR have been also identified in clinical samples, however as primary cancer mutations rather than secondary mutations (Taylor et al., 2009; Shukla et al., 2012; Ang et al., 2015). Taking into account the widespread occurrence of acquired gatekeeper resistance in many kinases and initial laboratory and clinical observations for FGFR, occurrence of this phenomenon in FGFR is widely anticipated. Further factors that can influence drug binding include, pre-existing mutations in the targeted kinase or subtle differences between closely related family members. This has also been documented for the FGFR family members (Brooks et al., 2012; Dieci et al., 2013; Byron et al., 2013) emphasizing the need for further characterisation that would inform treatment.
Methods
Results
Discussion The activating effect of gatekeeper substitutions on kinase activity has been documented for several kinases including SRC, ABL and EGFR using structural and computational methodologies. There are only a few crystal structures available where the most common threonine gatekeeper residue is substituted by methionine (SRC T338M, EGFR T790M and ABL T315I) or isoleucine (SRC T341I) (Getlik et al., 2009; Zhou et al., 2009; O\'Hare et al., 2009; Azam et al., 2008). MD simulations have been performed for EGFR T790M and together with experimental evidence show the activating effect on the EGFR kinase (Sutto and Gervasio, 2013). Furthermore, these previous findings and our data for FGFR1 V651M variant, including measurements of kinases activity, crystallography and MD stimulations (Figs. 1, 2 and 3) reinforce a similar underlying mechanism. As first suggested for SRC T341I (Azam et al., 2008), the valine to methionine substitution in FGFR1 leads to exclusion of water molecules and a re-arrangement of K514 side chain potentiating the hydrophobic spine (that now includes M651) and facilitates coordination of ATP by K514 (Fig. 2B and C). MD simulations suggest a set of further interactions in the active basin of FGFR1V561M and outline allosteric changes that result in a shift towards the active form, including an impact on “molecular brake”, a feature not present in most other kinases (Supplemental Fig. S4). Furthermore, resistance mutations can have an effect on drug binding not only by direct steric hindrance but also by allosterically modulating kinase dynamics as shown for EGFR T790M (Yun et al., 2008; Wan and Coveney, 2011; Sutto and Gervasio, 2013); this may also be a contributory factor in FGFR resistance.