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  • br Results br Discussion The sequence homology based cluster


    Discussion The sequence homology-based clustering of the human kinome by Manning et al. (2002) has been extremely useful to organize kinases into c-Myc Peptide by sequence relationship. Since kinases are such prominent drug targets, we expect that clustering of the kinases by their inhibition phenotype could allow medicinal chemists to quickly visualize what kinases and signaling cascades are often co-inhibited. This classification could reveal interactions relevant to polypharmacology or negative side effects. Importantly, promiscuity is not only a feature of kinase but may also be affected by the specificity of inhibitors tested to identify promiscuous kinases. The inhibitors studied here were chosen to be selective probes, and their selectivity scores are superior to those of the average clinically approved kinase inhibitor. However, when we repeat this clustering analysis with independent inhibitor sets including clinically approved drugs, we identify essentially the same promiscuous kinases (Figure S2B). Since the DFG-Asp-out conformation appears to facilitate the promiscuity of DDR1, we wanted to understand whether other promiscuous kinases are also stable in the DFG-Asp-out conformation. Therefore, we compared the kinase domain sequences of the eight promiscuous kinases with the rest of the human kinome using the Two Sample Logo server (Manning et al., 2002, Vacic et al., 2006). This analysis revealed that residues stabilizing the DFG-Asp-out conformation in DDR1 (Asp671 and Arg752) are enriched among the promiscuous tyrosine kinases (Figures 4A and S5), predicting that this salt bridge could stabilize the DFG-Asp-out conformation also in the other promiscuous kinases. In fact, we found that 25 of the 27 available crystal structures of promiscuous kinases in the PDB are in the DFG-Asp-out conformation and form this salt bridge (Figure 4B and Table S2) (Illig et al., 2011, Mol et al., 2004, Murray et al., 2015, Walter et al., 2007, Wang et al., 2016). This includes the apo structures of CSF1R, KIT, and PGDFRA showing that the DFG-Asp-out conformation is energetically accessible in the absence of ligands (Liang et al., 2016, Mol et al., 2004, Walter et al., 2007). It will be interesting to determine the structural basis for the two promiscuous non-tyrosine kinases MEK5 and YSK4. They both lack the equivalent to the Asp671-Arg752 (DDR1 numbering) salt bridge, and currently no structure is available for these kinases. While we identify DDR1 here as a promiscuous kinase, DDR1 can still be inhibited with selective inhibitors. The salt bridge formed between the activation loop and the C lobe of DDR1 forms a hydrophobic cage that facilitates the binding of many chemically different inhibitors. However, this unique hydrophobic cage has been exploited by highly selective inhibitors for DDR1, such as DDR1-IN-1 (Canning et al., 2014). Interestingly, DDR1-IN-1 bypasses interactions with the threonine gatekeeper residue in DDR1 and instead forms a hydrogen bond with the αD helix in the C lobe of DDR1. This interaction is only possible due to anchoring of the activation loop to the C lobe by the Asp671-Arg752 salt bridge in the DFG-Asp-out conformation adapted by DDR1. In addition to providing insights about promiscuous kinases in general, the results presented here suggest a molecular mechanism for the unusually low kinase activity of DDR1 (Leitinger, 2014). Among promiscuous kinases, Tyr755 is only present in DDR1 and DDR2. This tyrosine is part of DDR1/2\'s YxxxYY motif, which is an autoinhibitory motif conserved between DDR1/2 and the insulin receptor kinase (IRK) family (Artim et al., 2012, Leitinger, 2014). This autoinhibition is released upon tyrosine phosphorylation at this motif. Unlike the promiscuous tyrosine kinases, the IRK family kinases do not contain the Asp-Arg salt bridge. Consequently, they are catalytically more active but less promiscuous than DDR1. The presence of both the Tyr755 and the Asp-Arg salt bridge explains why, despite other sequence similarities, DDR1 is less active than both other promiscuous kinases and the kinases of the IRK family (Wei et al., 1995).