Development of potent and selective degraders of PI5P4Kγ

Abstract

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), a family of three members in mammals (α, β and γ), have emerged as potential therapeutic targets due to their role in regulating many important cellular signaling pathways. In comparison to the PI5P4Kα and PI5P4Kβ, which usually have similar expression profiles across cancer cells, PI5P4Kγ exhibits distinct expression patterns, and pathological functions for PI5P4Kγ have been proposed in the context of cancer and neurodegenerative diseases. PI5P4Kγ has very low kinase activity and has been proposed to inhibit the PI4P5Ks through scaffolding function, providing a rationale for developing a selective PI5P4Kγ degrader. Here, we report the development and characterization of JWZ-1-80, a first-in-class PI5P4Kγ degrader. JWZ-1-80 potently degrades PI5P4Kγ via the ubiquitin-proteasome system and exhibits proteome-wide selectivity and is therefore a useful tool compound for further dissecting the biological functions of PI5P4Kγ.

Keywords: PI5P4Kγ; PROTAC; Protein degrader; Selective.

Fig. 1.

(A) PI-4,5-P₂ is generated by phosphorylation of PI-4-P by PI4P5K and PI5P4K-catalyzed phosphorylation of PI-5-P. (B) Chemical structure of pan-PIP4K inhibitor THZ-P1–2 and noncovalent compound Ac-THZ-P1–2. (C) Illustration of potential exit vector from co-crystal structure of THZ-P1–2 and PIP4K2A (PDB-ID: 6OSP).

Fig. 2.

(A) Immunoblot analysis of PI5P4Kγ in Molt4 cells treated with CRBN-based compounds for 6 h. (B) Immunoblot analysis of PI5P4Kγ in Molt4 cells treated with VHL-based compounds for 6 h. Quantitation on the right. Quantified data represents mean ± SEM from two independent biological replicates. * p < 0.05; ** p < 0.01; *** p < 0.005 compared with the DMSO -treated control in a t-test.

Fig. 3.

(A) Chemical structures of compounds 11 and 17. (B) Immunoblot analysis of PI5P4Kγ in Molt4 cells treated with indicated concentrations of selected compounds 11 and 17. Data in the bar graphs are the means ± SEM (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.005 **** p < 0.0001 compared with the DMSO-treated control in a t-test. (C) Immunoblot analysis of PI5P4Kγ in Molt4 treated with 1 μM of compounds 11 and 17 at the indicated time points.

Fig. 4.

(A) Chemical structure of 11-Neg. (B) Immunoblot analysis of PI5P4Kγ in Molt4 cells treated with 11 or 11-Neg for 6 h. (C) Immunoblot analysis of PI5P4Kγ in Molt4 cells pretreated for 2 h with Ac-THZ-P1–2, VHL ligand, bortezomib or MLN4924, and then treated with 17 for 6 h.

Fig. 5.

(A) Immunoblot analysis of PI5P4Ks in Molt4 cells treated with compounds 11, 12, 13, 14, 17 for 6 h. (B) Quantitative proteomics of compound 17 (JWZ-1–80) showing relative abundance of proteins in Molt4 cells treated for 5 h at 1 μM.

Fig. 6.

(A) Immunoblot analysis of PI5P4Kγ in HEK293 cells treated with compound 17 for 24 h. (B) Immunoblot analysis of PI5P4Kγ in Jurkat cells treated with compound 17 for 24 h. (C) Immunoblot analysis of PI5P4Kγ in K562 cells treated with compound 17 for 24 h. (D) Immunoblot analysis of PI5P4Kγ in MCF7 cells treated with compound 17 for 24 h.

Scheme 1.

Synthesis of Compounds 1−9. Reagents and conditions: (a) Pd(PPh₃)₂Cl₂, K₂CO₃, MeCN, 100 °C; (b) m-phenylenediamine, DIPEA, NMP, 150 °C, 4 h; (c) 4-((tert-butoxycarbonyl)amino)benzoic acid, HATU, DIPEA, DMF, rt; (d) TFA/DCM, rt (e) 2 M NaOH, Dioxane, 80 °C; (f) HATU, DIPEA, ligand, DMF, rt.

Scheme 2.

Synthesis of Compounds 10−20. Reagents and conditions: (a) HATU, DIPEA, ligand, DMF, rt; (b) methyl 4-(chlorocarbonyl)benzoate, Et₃N, DCM 0 °C; (c) 2 M NaOH, Dioxane, 80 °C; (d) HATU, DIPEA, DMF, rt.