Fig 1.
Design strategy and synthesis of KHK-6.
(A) Design strategy for KHK-6. (B) Synthesis of KHK-6. Reagents and conditions: (a) 3 M CH3MgBr in Et2O, Et2O, 0 °C to rt, 3 h, 100%; (b) MnO2, THF, 70 °C, 16 h, 82%; (c) ter-butyl carbamate, Pd2(dba)3, Xantphos, Cs2CO3, 1,4-dioxane, 100 °C, 2 h, 44%; (d) 40% TFA in DCM, DCM, rt, 1 h, 81%; (e) PTSA·H2O, IPA, 90 °C, 16 h, 90%.
Fig 2.
KHK-6 is a selective ATP-competitive HPK1 inhibitor.
(A) Proposed binding mode of KHK-6 compound (green sticks) in ATP-binding site of HPK1 (gray ribbons) superimposed with the crystal structure of HPK1-IN-3 (orange sticks; in HPK1 crystal structure, 7M0M) and ceritinib (magenta sticks; in ALK crystal structure, 4MKC). Hydrogen bonding interactions are depicted in red dots, water molecules in red spheres, and flap regions of HPK1 are made transparent for clarity. Water positions were obtained by averaging the position of interacting water molecules in the trajectory of 250-ns molecular dynamics simulation of HPK1 and KHK-6 complex structure. (B) IC50 value was determined at a 1/3 dilution from 1 μM through KinaseProfiler™ by Eurofins. The data represent the mean values of two independent experiments. (C), (D) Cell viability was determined at a 1/3 dilution from 1 μM in Jurkat cells and PBMCs by the Cell Counting Kit-8 (CCK-8) assay. The data represent the mean values of three independent experiments. The data are presented as means ± standard error of the mean. * P < 0.05 (vs. negative control).
Fig 3.
KHK-6 block serine376 phosphorylation of SLP-76 by TCR signaling.
(A) Western blotting of Jurkat cells treated with each concentration of KHK-6 before stimulation for 1 h and stimulated with 2 μg/mL anti-CD3 (immobilized) and 4 μg/mL (soluble) anti-CD28 antibody for 4 h is shown. Relative blot intensity of p-SLP-76 (S376)/SLP-76 protein levels was analyzed. (B) Relative quantification of SLP76 protein phosphorylation on PBMCs treated with a specific concentration of KHK-6 before stimulation and stimulated with Dynabeads Human T-Activator CD3/CD28 at a ratio of 1:1 (Dynabeads: T cells) for 25 min was analyzed. The data are presented as means ± standard error of the mean. # P < 0.05; ## P < 0.01 (vs. negative control); * P < 0.05); ** P < 0.01 (vs. Dynabeads-stimulated control).
Fig 4.
KHK-6 effects on cytokine release in activated T cells.
(A) IL-2 production in Jurkat cells treated with a specific concentration of KHK-6 before stimulation and stimulated with 2 μg/mL (immobilized) anti-CD3 antibody and 4 μg/mL (soluble) anti-CD28 antibody for 2 day was analyzed using a human IL-2 ELISA. The data shown represent the results of three independent experiments. (B), (C) Cytokine production (IL-2 and GM-CSF) in PBMCs treated with specific concentrations of KHK-6 before stimulation and stimulated with Dynabeads Human T-Activator CD3/CD28 at a ratio of 1:1 (Dynabeads:T cells) at the indicated stimulation time. All data were obtained from at least three independent experiments. The data are presented as means ± standard error of the mean. ### P < 0.001 (vs. negative control); * P < 0.05; ** P < 0.01 (vs. Dynabeads-stimulated control).
Fig 5.
Effect of KHK-6 on CD4+ and CD8+ T-cell phenotypic marker expression.
PBMCs were treated with KHK-6 for 1 h in the presence or absence of Dynabeads Human T-Activator CD3/CD28 stimulation. Different T-cell phenotypes of cells positive for early (CD69+) and late markers (CD25+ and HLA-DR+) among human CD4+T cells (A, B, and C) and CD8+ T cells (D, E, and F) were assessed using the iQue® platform. All data were obtained from at least three independent experiments. The data are presented as means ± standard error of the mean. # P < 0.05 (vs. negative control); * P < 0.05; ** P < 0.01 (vs. Dynabeads-stimulated control).
Fig 6.
KHK-6 enhanced PBMC-mediated tumor-killing effect.
Labeled SKOV3 (A) and A549 cells (B) co-cultured with PBMCs. Target cells (2 × 103/well) were treated with KHK-6 before stimulation for 1 h and co-cultured with Dynabeads Human T-Activator CD3/CD28 stimulated effect cell. The ratio of effector cells (PBMCs) to the target cells (SKOV3 or A549) was 1:5. All data were obtained from at least three independent experiments. The data are presented as means ± standard error of the mean. ## P < 0.01; ### P < 0.001 (vs. negative control); * P < 0.05; *** P < 0.001 (vs. Dynabeads-stimulated control).