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. 2024 Sep 3;15(9):1606-1614.
doi: 10.1021/acsmedchemlett.4c00319. eCollection 2024 Sep 12.

Discovery of GNE-6893, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of HPK1

John C Tellis  1 , BinQing Wei  1 , Michael Siu  1 , Le An  1 , Grace Kayan Chan  1 , Yong Chen  2 , Xiangnan Du  1 , Lewis Gazzard  1 , Baihua Hu  2 , James Kiefer  1 , Satoko Kakiuchi-Kiyota  1 , Michael Lainchbury  3 , Jonathan L Linehan  1 , Xifeng Luo  2 , Sushant Malhotra  1 , Rohan Mendonca  1 , Jodie Pang  1 , Yinqing Ran  1 , Vijay Sethuraman  1 , Eileen Seward  3 , Chris Sneeringer  1 , Dian Su  1 , Weiru Wang  1 , Ping Wu  1 , John G Moffat  1 , Timothy P Heffron  1 , Edna F Choo  1 , Bryan K Chan  1
Affiliations

Discovery of GNE-6893, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of HPK1

John C Tellis et al. ACS Med Chem Lett. .

Abstract

Hematopoietic progenitor kinase 1 (HPK1) serves a key immunosuppressive role as a negative regulator of T-cell receptor (TCR) signaling. HPK1 loss-of-function is associated with augmentation of immune function and has demonstrated synergy with immune checkpoint inhibitors in syngeneic mouse cancer models. These data offer compelling evidence for the use of selective small molecule inhibitors of HPK1 in cancer immunotherapy. We identified a novel series of isoquinoline HPK1 inhibitors through fragment-based screening that displayed promising levels of biochemical potency and activity in functional cell-based assays. We used structure-based drug design to introduce key selectivity elements while simultaneously addressing pharmacokinetic liabilities. These efforts culminated in a molecule demonstrating subnanomolar biochemical inhibition of HPK1 and strong in vitro augmentation of TCR signaling in primary human T-cells. Further profiling of this molecule revealed excellent kinase selectivity (347/356 kinases <50% inhibition @ 0.1 μM), a favorable in vitro safety profile, and good projected human pharmacokinetics.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Initial lead compound 1: biochemical and cellular potency, including primary human T-cell IL2 assay, and liver microsome stability. aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% of the control compound (red dotted line) and the IL2 EC(0.5×). eLM = liver microsome predicted clearance (mL/min/kg), H = Human, R = Rat, M = Mouse, N = NADPH.
Figure 2
Figure 2
Modeling of a bell-shaped dose–response curve by combination of HPK1 inhibition and antagonistic off-target effect. A/B (left): Predicted dose–response curves for two hypothetical compounds: A – equivalent potencies for inhibition of HPK1 (green curve) and unknown antagonistic off-target (red curve); B – compound with 100× selectivity between HPK1 and off-target. Solid blue line shows combined effect as observed increase in IL2 production above stimulated non-compound-treated cells. C/D (right): Illustration of the use of reference compound normalization to compare IL2 peak width for compounds inducing different maximal response; IL2 level is normalized relative to the maximal level produced by reference compound. Compound doses generating an IL2 response equal to half-maximal control compound response are estimated for the activating (EC(0.5×), green dashed line) and inhibitory (IC(0.5×), red dashed line) components of the dose–response curve. The ratio W(0.5×) (black arrow) indicates the relative selectivity window.
Figure 3
Figure 3
Optimization of the solvent-exposed region. (A) X-ray crystal structure of cyclopropyl amide 1 in HPK1 kinase domain with the targeted Gly24 interaction highlighted. (B) Pyrazole analog 2 establishing the key H-bond. (C) Pyrazole-lactam analog 3, using conformational constraint to reinforce the Gly24 H-bond. aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% of the control compound (red dotted line) and the IL2 EC(0.5×). eLM = liver microsome predicted clearance (mL/min/kg), H = Human, R = Rat, M = Mouse, N = NADPH.
Figure 4
Figure 4
(A) X-ray crystal structure of aminopyridine 4 in the HPK1 kinase domain. (B) Biochemical and cellular potency of 4, including primary human T-cell IL2 assay, permeability, hepatocyte stability, and in vivo PK (rat and mouse). aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% of the control compound (red dotted line) and the IL2 EC(0.5×). eMDCK permeability assay (apical-to-basolateral, cm/s × 10–6). fHep stability = hepatocyte predicted clearance (mL/min/kg), H = human, R = Rat, M = Mouse. gRat PK: Sprague–Dawley, 0.5 mg/kg i.v. dose, plasma clearance measured in mL/min/kg, Vss = volume of distribution (L/kg), t1/2 = i.v. half-life (measured in h). hMouse PK: C57BL/6, 1 mg/kg i.v. dose or 25 mg/kg p.o. dose, blood clearance measured in mL/min/kg.
Figure 5
Figure 5
Seminal carbamate 5: biochemical and cellular potency, including primary human T-cell IL2 assay, permeability, hepatocyte stability, and in vivo PK (rat and mouse). aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% that of the control compound (red dotted line) and the IL2 EC(0.5×). eMDCK permeability assay (apical-to-basolateral, cm/s × 10–6). fHep stability = hepatocyte-predicted clearance (mL/min/kg), H = human, R = Rat, M = Mouse. gRat PK: Sprague–Dawley, 0.5 mg/kg i.v. dose, plasma clearance measured in mL/min/kg, Vss = volume of distribution (L/kg), t1/2 = i.v. half-life (measured in h). hMouse PK: C57BL/6, 1 mg/kg i.v. dose or 25 mg/kg p.o. dose, blood clearance measured in mL/min/kg.
Figure 6
Figure 6
Morpholinopyridine analog 6: biochemical and cellular potency, including primary human T-cell IL2 assay, permeability, hepatocyte stability, and in vivo PK (rat and mouse). aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% that of the control compound (red dotted line) and the IL2 EC(0.5×). eMDCK permeability assay (apical-to-basolateral, cm/s × 10–6). fHep stability = hepatocyte predicted clearance (mL/min/kg), H = human, R = Rat, M = Mouse. gRat PK: Sprague–Dawley, 0.5 mg/kg i.v. dose, plasma clearance measured in mL/min/kg, Vss = volume of distribution (L/kg), t1/2 = i.v. half-life (measured in h). hMouse PK: C57BL/6, 1 mg/kg i.v. dose or 25 mg/kg p.o. dose, blood clearance measured in mL/min/kg.
Figure 7
Figure 7
Tool compound GNE-6893. (A) Cocrystal structure of GNE-6893 bound to HPK1 kinase domain. (B) Biochemical and cellular potency, including primary human T-cell IL2 assay, permeability, and hepatocyte stability. aHTRF enzymatic assay (n > 2). bJurkat cell assay measuring phosphorylation of SLP76. cPrimary human T-cell assay measuring IL2 secretion after CD3/CD28 stimulation and compound treatment; IL2 EC(0.5×) = the concentration at which the response surpasses 50% of the response of a control compound (green dotted line). dIL2 W(0.5×) = the ratio of the concentration at which the response passes below 50% that of the control compound (red dotted line) and the IL2 EC(0.5×). eMDCK permeability assay (apical-to-basolateral, cm/s × 10–6). fHep stability = hepatocyte predicted clearance (mL/min/kg), H = human, R = Rat, M = Mouse.
Figure 8
Figure 8
GNE-6893 kinase selectivity panel measured at 100 nM.
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