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Cited by 121Protein Degradation and InhibitorsChemical Synthesis and AnalysisCRISPR and Genetic Engineering

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A chemical approach for global protein knockdown from mice to non-human primates

Open access
Sun, Xiuyun, Wang, Jun, Yao, Xia, Zheng, Wen, Mao, Yang, Lan, Tianlong, Wang, Liguo, Sun, Yonghui, Zhang, Xinyi, Zhao, Qiuye, Zhao, Jianguo, Xiao, Rui-Ping, Zhang, Xiuqin, Ji, Guangju, Rao, Yu·2019·Cell Discovery
DOI 10.1038/s41421-018-0079-1PMC PMC6361926
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Abstract

Although conventional genetic modification approaches for protein knockdown work very successfully due to the increasing use of CRISPR/Cas9, effective techniques for achieving protein depletion in adult animals, especially in large animals such as non-human primates, are lacking. Here, we report a chemical approach based on PROTACs technology that efficiently and quickly knocks down FKBP12 (12-kDa FK506-binding) protein globally in vivo. Both intraperitoneal and oral administration led to rapid, robust, and reversible FKBP12 degradation in mice. The efficiency and practicality of this method were successfully demonstrated in both large and small animals (mice, rats, Bama pigs, and rhesus monkeys). Furthermore, we showed this approach can also be applied to effectively knockdown other target proteins such as Bruton's tyrosine kinase (BTK). This chemical protein knockdown strategy provides a powerful research tool for gene function studies in animals, particularly in large animals, for which gene-targeted knockout strategies may remain unfeasible.

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Figures

Fig. 1 RC32-induced degradation of FKBP12 in cell cultures. a Mechanism of action of PROTAC. b Docking mode of RC32 binding to FKBP12 and recruiting CRBN. Moiety in red, linker; moiety in blue, rapamycin; moiety in green, pomalimide. c Chemical structure of RC32. d Immunoblots showing degradation of FKBP12 in Jurkat cells treated with RC32 at the indicated concentrations for 12 h. ß-Actin served as a loading control. e Immunoblots for FKBP12 protein. Jurkat cells were first treated for 3 h with bortezomib (Bortez), carfilzomib (Carf), rapamycin (RAP), or pomalimide (Poma) followed by treatment with RC32 (10 nM) for 2 h. ß-Actin served as a loading control. f DC50 with 12 h of RC32 treatment in Jurkat cells. g Recovery of FKBP12 level after washout of RC32. After treatment for 12 h with 1 μM RC32, the Jurkat cells were washed with fresh culture medium and further cultured in fresh medium for the indicated times. h Selectivity of RC32 against FKBPs in Jurkat cells. i Degradation efficiency of RC32 in different cell lines (100 nM) and primary cardiac myocytes (1 µM) treated for 12 h. Data in (f) and (i) are presented as mean ± SEM of 3 independent experiments
Fig. 1 RC32-induced degradation of FKBP12 in cell cultures. a Mechanism of action of PROTAC. b Docking mode of RC32 binding to FKBP12 and recruiting CRBN. Moiety in red, linker; moiety in blue, rapamycin; moiety in green, pomalimide. c Chemical structure of RC32. d Immunoblots showing degradation of FKBP12 in Jurkat cells treated with RC32 at the indicated concentrations for 12 h. ß-Actin served as a loading control. e Immunoblots for FKBP12 protein. Jurkat cells were first treated for 3 h with bortezomib (Bortez), carfilzomib (Carf), rapamycin (RAP), or pomalimide (Poma) followed by treatment with RC32 (10 nM) for 2 h. ß-Actin served as a loading control. f DC50 with 12 h of RC32 treatment in Jurkat cells. g Recovery of FKBP12 level after washout of RC32. After treatment for 12 h with 1 μM RC32, the Jurkat cells were washed with fresh culture medium and further cultured in fresh medium for the indicated times. h Selectivity of RC32 against FKBPs in Jurkat cells. i Degradation efficiency of RC32 in different cell lines (100 nM) and primary cardiac myocytes (1 µM) treated for 12 h. Data in (f) and (i) are presented as mean ± SEM of 3 independent experimentsCC BY · PubMed Central
Fig. 2 Global knockdown of FKBP12 in mice by i.p. RC32 and recovery of FKBP12 protein levels. a Immunoblotting for FKBP12 in various organs/tissues of mice given RC32 (30 mg/kg, i.p., twice a day for 1 day). b FKBP12 protein levels in different organs/tissues of mice (RC32 group, n = 6; vehicle group, n = 4). c Immunoblots showing recovery of the FKBP12 level in the pancreas after withdrawal of RC32 at day 2. d Recovery of the FKBP12 level in representative organs/tissues after withdrawal of RC32 (n = 6). Data in (b) and (d) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01; n.s., not significant)
Fig. 2 Global knockdown of FKBP12 in mice by i.p. RC32 and recovery of FKBP12 protein levels. a Immunoblotting for FKBP12 in various organs/tissues of mice given RC32 (30 mg/kg, i.p., twice a day for 1 day). b FKBP12 protein levels in different organs/tissues of mice (RC32 group, n = 6; vehicle group, n = 4). c Immunoblots showing recovery of the FKBP12 level in the pancreas after withdrawal of RC32 at day 2. d Recovery of the FKBP12 level in representative organs/tissues after withdrawal of RC32 (n = 6). Data in (b) and (d) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01; n.s., not significant)CC BY · PubMed Central
Fig. 3 FKBP12 protein knockdown by RC32 after oral administration and i.c.v. in mice and i.p. in rats. a Upper panel, immunoblot of FKBP12 degradation in the brain of mice with a single i.c.v. injection (0.2 mg in 2 µL). Lower panel, schematic of i.c.v. injection of RC32. b Statistical analysis of the FKBP12 degradation as in (a) (RC32 group, n = 6; vehicle group, n = 3). c Immunoblots of FKBP12 in tissues/organs from mice given RC32 orally (60 mg/kg, twice a day for 1 day). d Statistical analysis of the FKBP12 degradation as in (c) (n = 3). e Immunoblots of FKBP12 degradation in rats after 1 day of RC32 administration (20 mg/kg, i.p., twice per day). f Statistical analysis of the FKBP12 degradation as in (e) (n = 5). Data in (b), (d), and (f) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01)
Fig. 3 FKBP12 protein knockdown by RC32 after oral administration and i.c.v. in mice and i.p. in rats. a Upper panel, immunoblot of FKBP12 degradation in the brain of mice with a single i.c.v. injection (0.2 mg in 2 µL). Lower panel, schematic of i.c.v. injection of RC32. b Statistical analysis of the FKBP12 degradation as in (a) (RC32 group, n = 6; vehicle group, n = 3). c Immunoblots of FKBP12 in tissues/organs from mice given RC32 orally (60 mg/kg, twice a day for 1 day). d Statistical analysis of the FKBP12 degradation as in (c) (n = 3). e Immunoblots of FKBP12 degradation in rats after 1 day of RC32 administration (20 mg/kg, i.p., twice per day). f Statistical analysis of the FKBP12 degradation as in (e) (n = 5). Data in (b), (d), and (f) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01)CC BY · PubMed Central
Fig. 4 Influence of RC32 administration (30 mg/kg, i.p., twice a day for 2 days and once a day for 28 days) on mouse cardiomyocytes and cardiac function. a Representative confocal line-scan images showing Ca2+ sparks. b Peak of the Ca2+ spark. c Increased full width at half-maximum (FWHM). d Prolonged full-duration at half-maximum (FDHM), rise time, and half-time decay (Tau) (RC32 group, n = 39; vehicle group, n = 44). e Decreased ejection fraction (EF) and f fractional shortening (FS) of the mice in the RC32-treated group. g Increased left ventricular mass (LV mass). h Increased left ventricular posterior wall thickness diastolic (LVPW;d) (RC32 group, n = 17; vehicle group, n = 11). Data in (c–h) are presented as mean ± SEM. (c) and (d) were assessed with two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01) and (e–h) with two-way ANOVA and the Sidak and Tukey tests (*p < 0.05, **p < 0.01)
Fig. 4 Influence of RC32 administration (30 mg/kg, i.p., twice a day for 2 days and once a day for 28 days) on mouse cardiomyocytes and cardiac function. a Representative confocal line-scan images showing Ca2+ sparks. b Peak of the Ca2+ spark. c Increased full width at half-maximum (FWHM). d Prolonged full-duration at half-maximum (FDHM), rise time, and half-time decay (Tau) (RC32 group, n = 39; vehicle group, n = 44). e Decreased ejection fraction (EF) and f fractional shortening (FS) of the mice in the RC32-treated group. g Increased left ventricular mass (LV mass). h Increased left ventricular posterior wall thickness diastolic (LVPW;d) (RC32 group, n = 17; vehicle group, n = 11). Data in (c–h) are presented as mean ± SEM. (c) and (d) were assessed with two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01) and (e–h) with two-way ANOVA and the Sidak and Tukey tests (*p < 0.05, **p < 0.01)CC BY · PubMed Central
Fig. 5 Global knockdown of FKBP12 in Bama pigs by RC32. a Efficiency of RC32 (8 mg/kg, i.p., twice a day for 2 days) in pigs. b Global knockdown of FKBP12 in different organs/tissues from pigs (RC32 group, n = 5; vehicle group, n = 3). Data in (b) are presented as mean ± SEM and were assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01; n.s., not significant)
Fig. 5 Global knockdown of FKBP12 in Bama pigs by RC32. a Efficiency of RC32 (8 mg/kg, i.p., twice a day for 2 days) in pigs. b Global knockdown of FKBP12 in different organs/tissues from pigs (RC32 group, n = 5; vehicle group, n = 3). Data in (b) are presented as mean ± SEM and were assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01; n.s., not significant)CC BY · PubMed Central
Fig. 6 Global knockdown of FKBP12 in rhesus monkeys by RC32 and its functional effects. a Efficiency of RC32 knockdown in monkeys (8 mg/kg, i.p., twice a day for 3 days). b Decreased EF in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. c FS in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. d Decreased systolic blood pressure (SBP) in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. e FKBP12 protein levels in skin and muscle from the RC32-treated group and recovery of the protein levels after withdrawal of the PROTAC. Data in (b–d) are presented as mean ± SEM of 3 independent experiments and were assessed with two-way ANOVA with Sidak and Tukey tests (*p < 0.05, **p < 0.01)
Fig. 6 Global knockdown of FKBP12 in rhesus monkeys by RC32 and its functional effects. a Efficiency of RC32 knockdown in monkeys (8 mg/kg, i.p., twice a day for 3 days). b Decreased EF in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. c FS in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. d Decreased systolic blood pressure (SBP) in the RC32-treated group compared with the vehicle group and recovery after withdrawal of the PROTAC. e FKBP12 protein levels in skin and muscle from the RC32-treated group and recovery of the protein levels after withdrawal of the PROTAC. Data in (b–d) are presented as mean ± SEM of 3 independent experiments and were assessed with two-way ANOVA with Sidak and Tukey tests (*p < 0.05, **p < 0.01)CC BY · PubMed Central
Fig. 7 Global knockdown of BTK in mice by P13IS. a The chemical structure of P13IS. b Efficiency of P13IS (33.3 mg/kg, i.p., three times a day for 11 days) in mice. c Global knockdown of BTK in different organs/tissues from mice (n = 3). d Recovery of the BTK level in representative organs/tissues after withdrawal of P13IS (n = 3). Data in (c) and (d) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01)
Fig. 7 Global knockdown of BTK in mice by P13IS. a The chemical structure of P13IS. b Efficiency of P13IS (33.3 mg/kg, i.p., three times a day for 11 days) in mice. c Global knockdown of BTK in different organs/tissues from mice (n = 3). d Recovery of the BTK level in representative organs/tissues after withdrawal of P13IS (n = 3). Data in (c) and (d) are presented as mean ± SEM and assessed with the two-tailed unpaired Student’s t-test (*p < 0.05, **p < 0.01)CC BY · PubMed Central

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Authors
Sun, X., Wang, J., Yao, X., Zheng, W., Mao, Y., Lan, T., Wang, L., Sun, Y., Zhang, X., Zhao, Q., Zhao, J., Xiao, R.-P., Zhang, X., Ji, G., & Rao, Y.
Affiliations
  • Center for Life Sciences (CN)
  • Chinese Academy of Sciences (CN)
  • Tsinghua University (CN)
  • Peking University (CN)
Corresponding author
  • Zhang, Xinyi
  • Zhang, Xiuqin
  • Ji, Guangju
  • Rao, Yu
ORCID
Venue
Cell Discovery
Publisher
Springer Science and Business Media LLC
Published
2019-02-05
Volume
5
Issue
1
Pages
10-10
ISSN
2056-5968 (ISSN-L)
DOI
10.1038/s41421-018-0079-1
PMC
PMC6361926
OpenAlex
W2914693193
Cited by
118
References
33
Citations by year
2026: 8 · 2025: 13 · 2024: 12 · 2023: 11 · 2022: 12 · 2021: 21 · 2020: 30 · 2019: 11
FWCI
6.12
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Discovery (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
National Natural Science Foundation of China (81573327)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
  • Physical Sciences › Chemistry › Organic Chemistry › Chemical Synthesis and Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
Keywords
PROTACs, Bruton's tyrosine kinase, non-human primates, rhesus monkeys, Bama pig, oral administration, intraperitoneal administration, large animal model, gene function analysis

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