DFG-1 Residue Controls Inhibitor Binding Mode and Affinity, Providing a Basis for Rational Design of Kinase Inhibitor Selectivity.

Martin Schröder,Oleg Fedorov,Franz Bracher,Stefan Knapp,Apirat Chaikuad,Alex N Bullock

doi:10.1021/acs.jmedchem.0c00898

Abstract

Selectivity remains a challenge for ATP-mimetic kinase inhibitors, an issue that may be overcome by targeting unique residues or binding pockets. However, to date only few strategies have been developed. Here we identify that bulky residues located N-terminal to the DFG motif (DFG-1) represent an opportunity for designing highly selective inhibitors with unexpected binding modes. We demonstrate that several diverse inhibitors exerted selective, noncanonical binding modes that exclusively target large hydrophobic DFG-1 residues present in many kinases including PIM, CK1, DAPK, and CLK. By use of the CLK family as a model, structural and biochemical data revealed that the DFG-1 valine controlled a noncanonical binding mode in CLK1, providing a rationale for selectivity over the closely related CLK3 which harbors a smaller DFG-1 alanine. Our data suggest that targeting the restricted back pocket in the small fraction of kinases that harbor bulky DFG-1 residues offers a versatile selectivity filter for inhibitor design.

Highlights

Protein kinases constitute one of the largest protein families in the human genome consisting of more than 500 members.[1]
The loss of inhibitor binding affinities against the CLK1 mutant was apparent for other inhibitors tested, except staurosporine (6), with more profound changes in Ki of approximately 9- to 109-fold observed for KH-CB19 (7)[37] and GW807982X (8)[38,39] (Figure 3D and Supplementary Table 4). These results suggested that the smaller DFG-1 alanine is likely responsible for the loss of inhibitor binding in CLK3
The larger V324 potentially restricted the binding cavity and assisted stabilization of the β-carboline in CLK1 through van der Waals interactions, a scenario that was likely absent in the larger pocket of CLK3 with its smaller DFG-1 A319. Such a potential role for the DFG-1 residue was further supported by the CLK3 mutant structure, in which the narrowing of the back pocket by the A319V substitution led to an inverted binding mode which once again resembled that observed in wild-type CLK1 (Figure 5C)

Summary

■ INTRODUCTION

Protein kinases constitute one of the largest protein families in the human genome consisting of more than 500 members.[1]. The larger V324 potentially restricted the binding cavity and assisted stabilization of the β-carboline in CLK1 through van der Waals interactions, a scenario that was likely absent in the larger pocket of CLK3 with its smaller DFG-1 A319 Such a potential role for the DFG-1 residue was further supported by the CLK3 mutant structure, in which the narrowing of the back pocket by the A319V substitution led to an inverted binding mode which once again resembled that observed in wild-type CLK1 (Figure 5C). The potential DFG-1-assisted stabilization of the βcarboline in CLK1 and A319V CLK3, as well as PIM1 and DAPK3, enabled positioning of the nitrile group in the vicinity to the catalytic lysine (K191 and K186 in CLK1 and CLK3, respectively), which may lead to polar contacts explaining more favorable binding and the affinity gains in these kinases Overall, these results established an important role for the DFG-1 residue in determining the nature of the pockets controlling kinase sensitivity. These results demonstrate that many diverse scaffolds can exhibit such atypical binding mechanisms by targeting the bulkier DFG-1 valine residue and achieve high potency for CLK1/2/4 with selectivity over CLK3

■ DISCUSSION AND CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES