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A selective small-molecule STAT5 PROTAC degrader capable of achieving tumor regression in vivo

Nature Chemical Biology volume 19, pages 703–711 (2023)Cite this article

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Abstract

Signal transducer and activator of transcription 5 (STAT5) is an attractive therapeutic target, but successful targeting of STAT5 has proved to be difficult. Here we report the development of AK-2292 as a first, potent and selective small-molecule degrader of both STAT5A and STAT5B isoforms. AK-2292 induces degradation of STAT5A/B proteins with an outstanding selectivity over all other STAT proteins and more than 6,000 non-STAT proteins, leading to selective inhibition of STAT5 activity in cells. AK-2292 effectively induces STAT5 depletion in normal mouse tissues and human chronic myeloid leukemia (CML) xenograft tissues and achieves tumor regression in two CML xenograft mouse models at well-tolerated dose schedules. AK-2292 is not only a powerful research tool with which to investigate the biology of STAT5 and the therapeutic potential of selective STAT5 protein depletion and inhibition but also a promising lead compound toward ultimate development of a STAT5-targeted therapy.

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Fig. 1: Design of STAT5 SH2 domain ligands and PROTAC degraders.
Fig. 2: AK-2292 selectively depletes STAT5A/B proteins in cells.
Fig. 3: Transcriptomic analysis of AK-2292-treated CML cell lines.
Fig. 4: Evaluation of the effect of AK-2292 on STAT6 activity.
Fig. 5: AK-2292 exerts growth-inhibitory activity in CML cell lines.
Fig. 6: PD effect and efficacy of AK-2292 in mice.

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Data availability

Co-crystal structures are available in the Protein Data Bank (7TVB for AK-305:STAT5A and 7TVA for AK-2292:STAT5A). Proteomics data are deposited and available in the Proteomics Identifications Database (PRIDE) (accession number PXD037895). Transcriptomic profiling data are available at Gene Expression Omnibus with accession GSE217647. Source data are provided with this paper.

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Acknowledgements

We are grateful for financial support from Oncopia Therapeutics, Proteovant Therapeutics and Roivant Sciences. This study is also supported, in part, by the Cancer Center Support Grant (P30CA046592) from the National Cancer Institute, National Institutes of Health, to Rogel Cancer Center of the University of Michigan. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract number DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (grant 085P1000817). Proteomic profiling was assisted by the Proteomics Resource Facility in the Department of Pathology of the University of Michigan. Transcriptomic profiling was assisted by the Advanced Genomics Core at Biomedical Research Core Facilities of the University of Michigan. Animal studies were assisted by the In-Vivo Animal Core of the University of Michigan. Pharmacokinetics studies were performed by the Pharmacokinetics Core at the University of Michigan. 19F and 31P NMR spectra were obtained by the University of Michigan BioNMR Core Facility. We thank G. W. A. Milne for critical proofreading of the manuscript.

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  1. These authors contributed equally: Atsunori Kaneshige, Longchuan Bai.

Authors and Affiliations

  1. Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    Atsunori Kaneshige & Shaomeng Wang

  2. Department of Internal Medicine, University of Michigan, Medical School, Ann Arbor, MI, USA

    Atsunori Kaneshige, Longchuan Bai, Mi Wang, Donna McEachern, Renqi Xu, Yu Wang, Wei Jiang, Hoda Metwally, Paul D. Kirchhoff, Lijie Zhao & Shaomeng Wang

  3. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Jennifer L. Meagher & Jeanne A. Stuckey

  4. Department of Biostatistics, School of Public Health, University of Michigan, Ann Arbor, MI, USA

    Hui Jiang

  5. Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    Meilin Wang, Bo Wen & Duxin Sun

  6. Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA

    Duxin Sun, Jeanne A. Stuckey & Shaomeng Wang

  7. Department of Pharmacology, Medical School, University of Michigan, Ann Arbor, MI, USA

    Shaomeng Wang

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Contributions

A.K., L.B., M.W. and S.W. conceived of the project idea, made intellectual contributions, designed experiments, performed experiments, analyzed and interpreted the data and wrote the manuscript. D.M., J.L.M., P.D.K., L.Z., M.W., B.W., D.S. and J.A.S. made intellectual contributions, designed experiments, performed experiments, analyzed and interpreted the data and wrote the manuscript. R.X., Y.W., W.J., H.M. and H.J. made intellectual contributions, designed experiments, performed experiments and analyzed and interpreted the data. Experimentally, A.K. synthesized all the final compounds. R.X. developed the key tyrosine mimetic group, which was used in all the final compounds. M.W., A.K. and L.B. evaluated the binding affinity of compounds. L.B. and A.K. evaluated the degradation efficiency of synthesized degraders and performed cell viability assay. P.D.K. performed the molecular modeling. L.B. performed the proteomic profiling and transcriptomic profiling. J.L.M. and J.A.S. solved co-crystal structures of the highlighted inhibitor and degrader. L.Z. performed the apoptosis assay. D.M., Y.W., W.J. and H.M. performed all the efficacy studies. D.M., Y.W., W.J. and H.M. dosed compounds to mice and prepared tissue samples for the pharmacodynamics and pharmacokinetics analyses. M.W., B.W. and D.S. performed all the pharmacokinetics studies. S.W. conceived experiments and supervised the work.

Corresponding author

Correspondence to Shaomeng Wang.

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Competing interests

The University of Michigan has filed patent applications on inhibitors and degraders described in this study, which have been licensed to Oncopia Therapeutics, Proteovant Therapeutics and Roivant Sciences. S.W., A.K., L.B. M.W., D.M. and J.S. are co-inventors on one or more of these patent applications and receive royalties from the University of Michigan. The University of Michigan has received a research contract from Oncopia/Proteovant/Roivant, for which S.W. serves as the principal investigator. S.W. is a paid consultant to Proteovant/Roivant and owns equity in Roivant. The remaining authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Binding affinities of STAT5/6 ligands.

Chemical structure and binding data of key representative ligands to STAT5A, STAT5B and STAT6. Data represent n = 3 independent experiments.

Extended Data Fig. 2 Structure and degradation efficiency relationship.

(A). Structure and DC50 relationship of key degraders based on AK-013. (B) Structure and DC50 relationship of key degraders based on AK-305 and structurally similar analogues. Data represent at least n = 2 independent experiments.

Source data

Extended Data Fig. 3 Binding affinity and degradation efficiency evaluation among STAT family proteins.

(A) Summary of Ki values of AK-013, AK-305, AK-2292, and AK- 2292Me with STAT1, STAT3, STAT4, STAT5A, STAT5B, STAT6 and Cereblon. Data represent n = 3 independent experiments. (B)Table of AK-2292’s DC50 values and Dmax values in KU812 cells and PBMCs. Values are calculated from Fig. 2A and Fig. 2B.

Extended Data Fig. 4 Key parameter tables of proteomics analysis.

(A) Protein abundance table of STAT proteins in cells treated with AK-2292 under indicated concentrations and treatment durations, analyzed by multiplexed quantitative proteomics analysis (Fig. 2D). P-value: two-sided Student’s t-test. KU812 (>6,800 quantified proteins), PBMCs (>6,640 quantified proteins), NCO2 (>6,800 quantified proteins). N/D: not detected. Values are derived from three independent replicates. (B) KU812 cells were treated with AK-2292Me under indicated concentrations and treatment durations for multiplexed quantitative proteomics analysis. P-value: two-sided Student’s t-test. KU812 (>6,800 quantified proteins). Values are derived from three independent replicates.

Extended Data Fig. 5 All down- or up-regulated genes in RNAseq analysis.

List of all the down- or up-regulated genes ≥2-fold p-value < 0.05 in KU812 cells, treated with AK-2292 1 µM for 8 h and analyzed by RNA-seq. The experiment was performed in three independent replicates. Statistical testing was performed with two-sided Student’s t-test.

Extended Data Fig. 6 AK-2292 induces STAT5 degradation and exerts growth-inhibitory activity in CML cell lines.

 Growth-inhibitory activities of AK-2292 were determined as IC50 by Cell Titer Glo (CTG) assay (3 days). IC50 data represent n= 3 independent experiments. STAT5 degradation efficiency of AK-2292 was calculated as DC50 by immunoblot analysis. DC50 data represent at least n = 2 independent experiments.    AK-2292 responding cell lines are marked in bold.

Extended Data Fig. 7 PD effect in CD-1 mice.

Female CD-1 mice were treated with AK-2292 at 100 mg/kg or 200 mg/kg i.p. and tissues were analyzed by immunoblotting and a quantification table. Blots represent n = 3 independent experiments.

Source data

Extended Data Fig. 8 Weight change and blood chemistry in Balb/C mice toxicity study.

(A) Balb/C mice were treated with 100 mg/kg of AK-2292 and 200 mg/kg through I.P. 5 days per week for 2 weeks and the body weights of mice were measured every dosing day (n = 5). Values in the plots are shown as mean ± s.d. from n = 5 mice per treatment arm. (B) Balb/C mice were treated with 100 mg/kg of AK-2292 and 200 mg/kg through I.P. 5 days per week for 2 weeks and the blood was analyzed. Values in the table are derived from n = 5 mice per group. Statistical testing was performed with two-sided Student’s t-test.

Extended Data Fig. 9 Cell permeability and plasma protein binding evaluation.

(A) Permeability and Efflux Ratio Determination of AK-2292 & AK-305 in Caco-2 Cells. Papp: apparent permeability coefficient (cm/s). Data shown represent three independent replicates. See also method section. (B) Percentage of protein binding of AK-2292 in Human and Mice Plasma. Data shown represent two independent replicates.

Extended Data Fig. 10 Pharmacokinetics study of AK-2292 and predicted free drug concentration in plasma.

(A) SCID mouse was dosed with AK-2292 10 mg/kg through IP route and Vivo PK parameters were analyzed. Cmax = Maximum observed concentration, Tmax = Time to reach Cmax, AUC(0-tldc) = Area under the concentration-time curve from time zero to time of last detectable concentration, AUC(0-inf) = Area under the concentration-time curve from time zero to infinite, CL = Systemic clearance, CL: Apparent clearance, Vz/F: Volume of distribution associated with the terminal elimination phrase, Terminal elimination half-life (t½) was calculated based on data points (>= 3) in the terminal phase with correlation of coefficient > 0.90. Values in the plot are shown as mean ± s.d. from n = 3 independent experiments. (B) Predicted free drug concentrations in plasma in mice when dosed with AK-2292 at 100 mg/kg, using the data in Extended Data Fig. 9B and 10A. DC50 and DC90 values were calculated using the data in Supplementary Fig. 7. (C) Predicted free drug concentrations in plasma in mice when dosed with AK-2292 at 200 mg/kg, using the data in Extended Data Fig. 9B and 10A. DC50 and DC 90 values were calculated using the data in Supplementary Fig. 7.

Supplementary information

Supplementary Information

Supplementary Figs. 1–18, source data of supplementary figures and chemistry data.

Reporting Summary

Supplementary Data

Structures of the best-ranked homology models.

Source data

Source Data Fig. 2

Unprocessed western blots of Fig. 2 and proteomics data table.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 7

Unprocessed western blots.

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Kaneshige, A., Bai, L., Wang, M. et al. A selective small-molecule STAT5 PROTAC degrader capable of achieving tumor regression in vivo. Nat Chem Biol 19, 703–711 (2023). https://doi.org/10.1038/s41589-022-01248-4

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