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

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.

Crystal Structure-Guided Design of Bisubstrate Inhibitors and Photoluminescent Probes for Protein Kinases of the PIM Family

“…In contrast, ARC-3126 utilized a different exit vector based on its diverse ATP-competitive moiety BPTP ( Figure 3 C,D). Remarkably, ARC-3126 did not form canonical hydrogen bonds with the hinge backbone but was anchored to the backpocket via a polar interaction with the conserved VAIK lysine (K67) similar to other kinases that harbor a large hydrophobic residue immediately N-terminal to the DFG motif [ 29 ], which is a binding mode that has been associated with favorable selectivity profiles ( Figure 3 D). The non-classical hinge-binding mode of BPTP in the ARC-3126/PIM-1 complex, similar to those of pyrimidinones and pyridazines [ 25 , 30 ], is a distinctive element for its selectivity, and thus for the selectivity of ARC-3126 over the promiscuous inhibitor ARC-3125.…”

Development of Cdc2-like Kinase 2 Inhibitors: Achievements and Future Directions

“…The differential scanning fluorimetry (DSF) assay can directly witness the thermal stability of CLK2 by detecting the quantity of fluorescence dye bound to unfolded CLK2 using real-time reverse transcription polymerase chain reaction (RT-PCR) in Table , entry 5 . It is suitable for HTS and offers advantages such as low loss of protein and high temperature ranges .…”

“…Recently, compound 15 (Figure C), which was previously reported as a CDK inhibitor, exhibited a significant CLK2 inhibitory activity (CLK2 Δ T m = 12 °C) . In addition, the Structural Genomics Consortium (SGC) cooperating with scientists at Luceome Biotechnologies developed the pyrazolopyridazine derivative 16 (Figure C, IC 50 = 4 nM) as a chemical probe for CLK1/2/4.…”

“…In addition, the Structural Genomics Consortium (SGC) cooperating with scientists at Luceome Biotechnologies developed the pyrazolopyridazine derivative 16 (Figure C, IC 50 = 4 nM) as a chemical probe for CLK1/2/4. Compound 16 exhibited significant binding affinity in a cell-based assay (cell CLK2 IC 50 = 70 nM) and interacted with potential off-targets in only STK16 in the NanoBRET target engagement assay conducted with 403 kinases . Compound 17 exhibited weak CLK2 inhibitory activity (IC 50 = 130 nM, Figure C).…”

“…In 2019, Nemec et al reported the discovery of furo­[3,2- b ]­pyridine derivative 24 (MU1210, Figure A) as a nonselective CLK2 inhibitor (IC 50 = 20 nM) that regulated Mdm4 alternative splicing . Compound 24 did not exhibit a strong interaction with the hinge backbone but rather interacted with the back pocket particularly with the tripeptide DFG-1 residue . It has been reported that compound 24 showed favorable pharmacokinetics and high cell activity in MCF7.…”