A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-kinase Degrader

Abstract

Heterobifunctional molecules that recruit E3 ubiquitin ligases, such as cereblon, for targeted protein degradation represent an emerging pharmacological strategy. A major unanswered question is how generally applicable this strategy is to all protein targets. In this study, we designed a multi-kinase degrader by conjugating a highly promiscuous kinase inhibitor with a cereblon-binding ligand, and used quantitative proteomics to discover 28 kinases, including BTK, PTK2, PTK2B, FLT3, AURKA, AURKB, TEC, ULK1, ITK, and nine members of the CDK family, as degradable. This set of kinases is only a fraction of the intracellular targets bound by the degrader, demonstrating that successful degradation requires more than target engagement. The results guided us to develop selective degraders for FLT3 and BTK, with potentials to improve disease treatment. Together, this study demonstrates an efficient approach to triage a gene family of interest to identify readily degradable targets for further studies and pre-clinical developments.

Keywords: BTK; FLT3; chemoproteomics; drug design; kinase; protein degradation.

Figure 1.. Rationally designed multi-kinase degrader TL12-186 presents CRBN-dependent pharmacology.

(A) Chemical structures of the multi-kinase degrader TL12-186, its parental kinase inhibitor TL13-87, and its negative control analog TL13-27. (B) TREEspot visualization of the kinome selectivity profile of TL12-186 (1 μM). See also Table S1. (C) CRBN-DDB1 engagement assay by AlphaScreen, testing the ability of thalidomide, pomalidomide, TL12-186 and TL13-27 at displacing biotinylated thalidomide for CRBN-DDB1 binding at indicated concentrations. Values represent quadruplicate means ± SD. (D) Cellular CRBN engagement assay, testing the ability of thalidomide, pomalidomide, TL12-186 and TL13-27 at preventing CRBN-mediated degradation of FKBP12^F36V-Nluc luciferase at indicated concentrations. Values represent triplicate means ± SD. (E) 2-day proliferation assays of WT and CRBN−/− MOLM-14 cells treated with TL13-87, TL12-186 or TL13-27. Values represent duplicate means ± SD. (F) 2-day proliferation assays of WT and CRBN−/− MOLT-4 cells treated with TL13-87, TL12-186 or TL13-27. Values represent duplicate means ± SD. For all plots within this figure, error bars shorter than the height of the symbols are not drawn.

Figure 2.. Quantitative multiplexed proteomics revealed 28 kinases readily degradable by TL12-186 in two leukemic cell lines.

(A) Relative abundance of 7559 proteins in MOLM-14 cells when treated with TL12-186 (100 nM) for 4 hours, versus adjusted P Values (limma, duplicates, <5% FDR). Relative abundance was calculated using linear models for cells treated with TL12-186 compared to DMSO and TL13-27 controls. Red and blue circles represent significantly downregulated kinases and non-kinases, respectively. See also Tables S2 and S3. (B) Relative abundance of 6609 proteins in MOLT-4 cells when treated with TL12-186 (100 nM) for 4 hours, versus adjusted P Values (limma, triplicates, <5% FDR). Otherwise, as in (A). See also Tables S4 and S5. (C) Relative abundance, as in (A), of 208 kinases in MOLM-14 cells identified by mass spectrometry, versus their KINOMEscan scores. See also Tables S6 and S7. (D) Relative abundance, as in (B), of 176 kinases in MOLT-4 identified by mass spectrometry, versus their KINOMEscan scores. See also Figure S1 and Tables S6.

Figure 3.. Western blotting validation of multi-kinase degradation induced by TL12-186, a CRBN-mediated proteosome-dependent process.

(A) Immunoblots for PTK2B, AURKA, BTK, FLT3 and GAPDH in MOLM-14 cells after 4-hour treatment of DMSO, pomalidomide, TL13-87, TL12-186 or TL13-27 at indicated concentrations. See also Figure S2A. (B) Immunoblots for PTK2B, AURKA, BTK, FLT3 and GAPDH in MOLM-14 cells pre-treated with DMSO, carfilzomib, MLN4924 or pomalidomide for 4 hours, followed by 4-hour co-treatment with DMSO or TL12-186. See also Figure S2B. (C) Immunoblots for PTK2B, AURKA, BTK, FLT3, GAPDH and CRBN in WT and CRBN−/− MOLM-14 cells after 4-hour treatment with DMSO or TL12-186. See also Figure S2C. (D) Immunoblots for PTK2B, AURKA, ITK, WEE1 and GAPDH in MOLT-4 cells after the same treatment regimen described in (A). See also Figure S2D. (E) Immunoblots for PTK2B, AURKA, ITK, WEE1 and GAPDH in MOLT-4 cells after the same treatment regimen described in (B). See also Figure S2E. (F) Immunoblots for PTK2B, AURKA, ITK, WEE1, GAPDH and CRBN in WT and CRBN−/− MOLT-4 cells after 4-hour treatment with DMSO or TL12-186. See also Figure S2F.

Figure 4.. Design and characterization of AC220-based FLT3 degraders.

(A) Immunoblots for FLT3, AURKA and VINC in MOLM-14 cells treated with DMSO, TL12-186 (100 nM) or TL13-27 (100 nM) for the indicated amount of time. (B) Immunoblots for FLT3, BTK, AURKA, LC3 and GAPDH in MOLM-14 cells pre-treated with DMSO, carfilzomib, MLN4924, pomalidomide, bafilomycin A1 or chloroquine for 4 hours, followed by 4-hour co-treatment with DMSO or TL12-186. (C) Chemical structures of the two best AC220-based FLT3 degraders developed, TL13-117 and TL13-149. See also Figure S3A. (D) Immunoblots for FLT3, AURKA, and GAPDH in MOLM-14 cells pre-treated with DMSO or pomalidomide for 4 hours, followed by 12-hour co-treatment with DMSO, TL12-186, AC220, TL13-117, TL13-148 or TL13-149 (all at 100 nM). See also Figures S3B and S3D. (E) Immunoblots for FLT3 and α-tubulin in MOLM-14 cells treated with DMSO, AC220, TL13-117 or TL13-149 at indicated concentrations for 12 hours. See also Figure S3C and S3E. (F) 2-day proliferation assays of WT and CRBN−/− MOLM-14 cells treated with AC220, TL13-117 or TL13-149. Values represent duplicate means ± SD. Error bars shorter than the height of the symbol are not drawn. See also Figure S3F.

Figure 5.. Design and characterization of bosutinib and RN486-based BTK degraders.

(A) Chemical structures of a bosutinib-based and an RN486-based BTK degrader, CJH-005-067 and DD-04-015, respectively. (B) Immunoblots for BTK, PTK2B, AURKA, CDK7 and GAPDH in MOLM-14 cells treated with DMSO, RN486 or DD-04-015 at indicated concentrations for 4 hours. See also Figure S4. (C) 3-day proliferation assays of TMD8 cells treated with RN486 or DD-04-015, with or without washout following 6 hours of treatment. Values represent triplicate means ± SD from one representative experiment. Error bars shorter than the height of the symbol are not drawn.