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    • To test our hypothesis that the relative stability of

    2020-07-28

    To test our lxr agonists that the relative stability of the DFG-Asp-out conformation is related to promiscuity, we looked at data available in the PKIS2 set for a kinase that was tested in a state stabilized in DFG-Asp-in and the DFG-Asp-out conformation. We found data for activation loop phosphorylated and non-phosphorylated Abl kinase. Phosphorylated Abl is stabilized in the DFG-Asp-in active conformation (Hari et al., 2013). The number of compounds Abl binds with high affinity increases in the non-phosphorylated state, in which the DFG-Asp-out conformation is favored (Figure S2C). This implies that also for Abl kinase, the relative stability of the DFG-Asp-out conformation correlates with promiscuity. How could disease-related mutations affect the stability of the DFG-Asp-out conformation and the ability of kinases to bind ligands with high affinity? The Two Sample Logo analysis identified residues across the entire kinase domain that are specific to the group of promiscuous kinases. These residues could affect the overall stability of the DFG-Asp-out conformation, and in turn affect the ability of the kinase to bind ligands (Figure 4A). We found that seven of them correspond to sites of clinical mutations that confer imatinib resistance in Abl (Azam et al., 2003) (Figure S5B). Two of these residues (Abl resistance mutants M370T/I and M491I) are distant from the imatinib binding site and the mechanism of their resistance is unclear. Our model suggests that mutations at these sites destabilize the DFG-Asp-out conformation and thereby weaken the affinity for imatinib. In addition, the D681N/Y/G mutations in PDGFRA (Asp671 of the salt bridge in DDR1) are activating mutations that confer resistance to imatinib and sunitinib (COSMIC Study: COSU419, COSU375) (Zehir et al., 2017). This is consistent with our previous finding that a distributed network of residues stabilizes the DFG-Asp-out conformation in kinases (Seeliger et al., 2007). Mutating these residues reduces the stability of the DFG-Asp-out conformation, conferring resistance to ligands such as imatinib that favor this conformation. Similarly, when we compare the sequences of DDR1 and the less promiscuous DDR2, we find that no amino acids within 5 Å of the inhibitors differ between DDR1 and DDR2. This indicates that in fact differences in secondary shell or even more remote residues underlie the difference in promiscuity between DDR1 and DDR2 (Figures S5C–S5E). Our data indicate that the ability of these promiscuous kinases to bind chemically diverse inhibitors is defined by the hydrophobic pocket formed by the activation loop, which is only accessible in the DFG-Asp-out conformation. Inhibitors do not artificially induce the DFG-Asp-out conformation as was once surmised, but it is a stable, accessible conformation of kinases. Analysis of the available apo structures of the promiscuous kinases PDGFRA, c-Kit, and CSF1R and our MD analysis of DDR1 show that the DFG-Asp-out inactive conformation is the preferred conformation of this unusual set of kinases (Table S2). This analysis of a large chemical genomic dataset identified a phenotypically distinct class of medically important signaling enzymes. Further analysis revealed a shared structural mechanism underlying this characteristic that would not have been obtained by sequence comparison alone. We speculate that future analysis of similar datasets will yield further insight into the function, regulation, and druggability of enzymes.