Abstract
Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.
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Data availability
The datasets generated during and/or analyzed during the current study are available as Excel spreadsheets in Supplementary Tables 1–10. Source data are provided with this paper. All additional data is available upon request from the corresponding authors.
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Acknowledgements
This work was supported by the Claudia Adams Barr Program (E.T.C.), the Lavine Family Fund (E.T.C.), the Pew Charitable Trust (E.T.C.), National Institutes of Health (NIH) CA259739-01 (E.T.C.), AG071966-01 (E.T.C.), NIH DK123095 (E.T.C.), The Smith Family Foundation (E.T.C.), T32CA236754 (N.D.), the National Cancer Center (NCI) (H.X. and N.D.) NCI R35 CA210030 (K. Stegmaier) and P50 CA206963 (K. Stegmaier), K99AG073461 (H.X.), NCI R35 CA231991 (B.F.C.), the Linde Family Foundation (S.D.-P.), the Doris Duke Charitable Foundation (S.D.-P.), Deerfield 3DC (S.D.-P.), Taiho Pharmaceuticals (S.D.-P.), NCI K99 CA263161 (S.L), and NIH (4R00DK123321-03), Mathers Foundation (MF-2204-02617) and American Cancer Society (DBG-23-983219-01-TBE) (E.L.M). S.L. is a Fellow of the Leukemia & Lymphoma Society. This work was based upon research conducted at the Northeastern Collaborative Access Team beamlines, which were funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). The Eiger 16M detector on the 24-ID-E beamline was funded by a NIH-Office of Research Infrastructure Programs High-End Instrumentation grant (S10OD021527). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.
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Contributions
E.T.C. and N.S.G. conceived of and designed the study. N.D. and S.L. performed cellular experiments and analyzed data. W.J., M.F., J.C. and T.Z. designed and conducted chemical syntheses. N.D. E.M.H, J.L.J. and T.M.Y carried out and analyzed data from mass spectrometry experiments. N.D. assisted with protein expression and purification. H.X. developed the CPT screening platform. E.V.V., K. Senkane and B.F.C. conceived, designed and conducted gel-based screening experiments. E.L.M. performed macrophage experiments and metabolomics experiments. S.D.-P. oversaw CK expression and purification and crystallization. J.C. performed molecular modeling. K. Stegmaier and S.L. contributed to conceptual design and provided cellular reagents. E.T.C. and N.S.G. directed research, oversaw the experiments and wrote the manuscript with assistance from the other authors. L.C.C oversaw kinome profiling experiments.
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N.S.G. is a founder, science advisory board member (SAB) and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK, Shenandoah (board member), Larkspur (board member) and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi. E.T.C. is a founder, board member and equity holder in Matchpoint Therapeutics and Aevum Therapeutics. T.Z. and J.C. are founders, equity holders and consultants in Matchpoint Therapeutics. J.C. is a scientific co-founder M3 Bioinformatics & Technology Inc., and consultant and equity holder for Soltego and Allorion. K. Stegmaier has funding from Novartis Institute of Biomedical Research and Kronos Bio, consults for and has stock options in Auron Therapeutics and previously consulted for Kronos Bio and AstraZeneca. T.M.Y. is a co-founder, stockholder and on the board of directors of DESTROKE, Inc. T.Z., W.J., N.D., N.S.G., J.C. and E.T.C. are inventors on a patent for CK inhibitors WO 2022/087433 A8. All other authors declare no potential conflict of interest. B.F.C. is a founder and scientific advisor to Vividion Therapeutics.
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Extended data
Extended Data Fig. 1 Characterizing CKi interaction with CKB.
(a) Establishing gel-based screening of recombinant CKB in Ramos cell lysates for engagement of cysteine 283 by competition labeling with iodoacetamide-rhodamine. Single experiment shown. (b) Fragment competition labeling of recombinant CKB in Ramos cell lysates identifies small molecules that compete with iodoacetamide-rhodamine labeling. CKi is highlighted in green. Single experiment shown. (c) Structure of CKi-alkyne. (d) Left: Intact protein MS of 800 nM human recombinant CKB incubated for 2 h with DMSO at 37 °C. Right: Intact protein MS of 800 nM human recombinant CKB incubated for 2 h with 1.6 μM CKi-alkyne at 37 °C. (e) UCSD-AML1 cells incubated with alkyne-CKi followed by conjugation to flouorophore for gel based detection. Fluorophore-conjugated iodoacetamide included as a promiscuous labeling control. (f) Crystal structure active site of human CKB bound to CKi highlighting Cys283, ADP, creatine, location of ligands based on PDB: 3B6R.
Extended Data Fig. 2 Effects of CKi on recombinant CKB.
(a) Electron density of CKi in the active site of CKB. A 2FO-FC map of CKi and its attached residue (Cys283) at sigma 1.0. (b) % CKi engagement with human recombinant CKB assessed by monitoring intact mass of CKB over 2 h at 37 °C. (c) Recombinant CKB phosphotransfer activity ± incubation with CKi for indicated times. n = 3 independent experiments. (d) Kinact/Ki determined using % CKi engagement with human recombinant CKB assessed by monitoring intact mass of CKB over 2 h at 37 °C at different concentrations of CKi. n = 3. (e) ATP/ADP ratio determined in UCSD-AML1 after treating with 0, 1.25, 2.5 5, 10, and 20 μM CKi for 16 h using ADP/ATP ratio assay kit. n = 3 biological cell replicates. (f) Structure of select CKi analogs with increasing hydrocarbon lengths. (g) Recombinant CKB phosphotransfer activity ± 2 h preincubation with CKi analogs with increasing hydrocarbon length. (h) Creatine and phosphocreatine abundance in UCSD-AML1 and A549 cells treated with 10 μM CKi. n = 5. (i) HEK293 cells over-expressing (OE) Flag-CKB exhibit significant increase in total cellular CKB content. Representative blot from 2 independent experiments shown.
Extended Data Fig. 3 Phosphoproteome remodeling induced by CKi and MitoCKi.
(a) The effect of CKi on cell viability in a range of cancer cell lines. HEK; HEK293 cells, HEKOECKB are cells generated in Extended Data Fig. 2i. n = 8. (b) Cell type specific phosphoproteomic signature of UCSD-AML1 and A549 cells treated for 16 h with 10 μM CKi or 5 μM MitoCKi. n = 5. (c) Full phosphoproteomic analysis of UCSD-AML1 or A549 cells treated with 10 μM CKi or 5 μM MitoCKi for 16 h. (n = 5). Upregulated and downregulated sites were then subjected to kinase enrichment analysis as describe in Methods. (d) Volcano plots showing phosphoproteomic analysis of UCSD-AML1 or A549 cells treated with 10 μM CKi or 5 μM MitoCKi for 16 h. n = 5. (e) Volcano plots showing phosphoproteomic analysis of UCSD-AML1 or A549 cells treated with 10 μM CKi or 5 μM MitoCKi for 16 h. n = 5.
Extended Data Fig. 4 CKi flow cytometry analysis.
(a) Flow cytometry analysis using Annexin V and 7-AAD staining of UCSD-AML1 or A549 cells treated with DMSO or 10 μM CKi for 16 h. n = 3. (b) Flow cytometry analysis using Click-iT Plus EdU Alexa Fluor 647 and FxCycle Violet staining of UCSD-AML1 or A549 cells treated with DMSO or 10 μM CKi for 16 h. n = 3.
Extended Data Fig. 5 CKi and MitoCKi flow cytometry analyses.
(a) Flow cytometry analysis using Annexin V and 7-AAD staining of UCSD-AML1 or A549 cells treated with DMSO, 2.5 or 5 μM MitoCKi for 16 h. (b) Quantification of fraction of cells that are apoptotic after 16 h of treatment with DMSO, 2.5 or 5 μM MitoCKi. n = 3. (c) Flow cytometry analysis using Click-iT Plus EdU Alexa Fluor 647 and FxCycle Violet staining of UCSD-AML1 or A549 cells treated with DMSO, 2.5 or 5 μM MitoCKi for 16 h. (d) Quantification of fraction of cells that are in S, G0/G1 and G2/M phase of the cell cycle after 16 h of treatment with DMSO, 2.5 or 5 μM MitoCKi. n = 3.
Extended Data Fig. 6 Kinome analysis and TLR pathway analysis of CKi.
(a) Kinome-wide selectivity profile for compound CKi at 10 μM. CKi was tested at 10 μM on a panel of 468 kinases (Creatine Kinases are not in the panel). The results are displayed as red circles with their sizes correlating with the inhibitor’s binding affinity (percent DMSO control). Complete dataset is included in Supplementary Table 7. (b) Immunoblot of IκBα and phosphorylated IκBα in THP1 cells ± CKi and ± LPS. Representative blot from 3 independent experiments shown.
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Supplementary Tables 1–10.
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Darabedian, N., Ji, W., Fan, M. et al. Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor. Nat Chem Biol 19, 815–824 (2023). https://doi.org/10.1038/s41589-023-01273-x
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DOI: https://doi.org/10.1038/s41589-023-01273-x
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