Research ArticleBIOCHEMISTRY

PHOTACs enable optical control of protein degradation

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Science Advances  21 Feb 2020:
Vol. 6, no. 8, eaay5064
DOI: 10.1126/sciadv.aay5064
  • Fig. 1 PROTACs and PHOTACs.

    (A) Schematic depiction of a PROTAC. Formation of a ternary complex between an E3 ligase, a PROTAC, and a POI leads to degradation of the POI. (B) Chemical structures of PROTACs dBET1 and dFKBP-1. (C) Schematic depiction of a PHOTAC. The molecules toggle between an inactive form (yellow pentagon) and an active form (red star) upon irradiation.

  • Fig. 2 Structure and synthesis of PHOTACs.

    (A) Members of the PHOTAC-I series targeting BRDs. (B) Synthesis of PHOTAC-I-3 starting from lenalidomide. (C) Members of the PHOTAC-II series targeting FKBP12.

  • Fig. 3 Photophysical properties, switching, and bistability of PHOTACs.

    (A) Switching of PHOTAC-I-3 between the trans isomer (left) and the cis isomer (right). (B) Ultraviolet-visible (UV-vis) spectra PHOTAC-I-3 following irradiation with different wavelengths for 5 min. (C) Fraction of trans–PHOTAC-I-3 in the PSS. (D) Thermal relaxation of cis–PHOTAC-I-3 at 37°C in DMSO. (E) Reversible switching and photochemical stability of PHOTAC-I-3. (F) Switching of PHOTAC-II-5 between the trans isomer (left) and the cis isomer (right). (G) UV-vis spectra PHOTAC-II-5 following irradiation with different wavelengths for 5 min. (H) Thermal relaxation of cis–PHOTAC-II-5 at 37°C in DMSO. (I) Switching of PHOTAC-II-6 between the trans isomer (left) and the cis isomer (right). (J) UV-vis spectra PHOTAC-II-6 following irradiation with different wavelengths for 5 min. (K) Thermal relaxation of cis–PHOTAC-II-6 at 37°C in DMSO.

  • Fig. 4 Light-dependent viability of RS4;11 cells.

    (A) Viability of RS4;11 acute lymphoblastic leukemia cells after treatment with PHOTAC-I-3 for 72 hours in the dark or under pulsed (100 ms every 10 s) 390-, 477-, or 545-nm irradiation. (B) Viability of RS4;11 after (+)-JQ1 treatment for 72 hours in the dark or under pulsed (100 ms every 10 s) 390-nm irradiation. (C) Viability of RS4;11 cells after 72 hours in the dark or under pulsed (100 ms every 10 s) 390-nm irradiation.

  • Fig. 5 Optical control of BRD2–4 levels.

    (A) Immunoblot analysis after treatment of RS4;11 cells with PHOTAC-I-3 for 4 hours at different concentrations. Cells were either irradiated with 100-ms pulses of 390-nm light every 10 s (left) or kept in the dark (right). MLN (MLN4924) was added as an additional control. (B) Time course of BRD2–4 degradation, c-MYC levels, and PARP1 cleavage assayed by immunoblotting. RS4;11 cells were treated with PHOTAC-I-3 (1 μM) and collected at the indicated time points. PHOTAC-I-3 has no effect on BRD2–4 levels in the dark over several hours. (C) Immunoblot of a rescue experiment demonstrating the reversibility of degradation promoted by PHOTAC-I-3 through thermal relaxation (left) or optical inactivation by 525-nm pulsed irradiation (right, 100 ms every 10 s). (D) Color dosing: Wavelength dependence of BRD2/4 degradation promoted by 300 nM PHOTAC-I-3. (E) Immunoblot analysis after treatment of RS4;11 cells with PHOTAC-I-3 and combinations with lenalidomide or (+)-JQ1 for 4 hours to confirm a CRBN-based mechanism. Cells were either irradiated with 100-ms pulses of 390-nm light every 10 s (left) or kept in the dark (right). (F) Optical degradation of BRD4 with the thalidomide derivative PHOTAC-I-10. Immunoblot analysis of RS4;11 cells after treatment with PHOTAC-I-10 for 4 hours at different concentrations, which were either irradiated with 100-ms pulses of 390-nm light every 10 s (left) or kept in the dark (right).

  • Fig. 6 Optical control FKBP12 degradation.

    (A) Immunoblot analysis of FKBP12 after treatment of RS4;11 cells with PHOTAC-II-5 for 4 hours at different concentrations. Cells were either irradiated with pulses of 390-nm light (left, 100 ms every 10 s) or kept in the dark (right). (B) Time course of FKBP12 degradation visualized by immunoblotting. RS4;11 cells were treated with PHOTAC-II-5 (100 nM) and collected at the indicated time points. (C) Immunoblot analysis of FKBP12 after treatment of RS4;11 cells with PHOTAC-II-6 for 4 hours at different concentrations. Cells were either irradiated with pulses of 390-nm light (left, 100 ms every 10 s) or kept in the dark (right). (D) Time course of FKBP12 degradation visualized by immunoblotting. RS4;11 cells were treated with PHOTAC-II-6 (100 nM) and collected at the indicated time points.

  • Table 1 Antibodies used in this study.

    PCNA, proliferating cell nuclear antigen; HRP, horseradish peroxidase; IgG, immunoglobulin G.

    AntibodiesSourceIdentifier
    β-ActinCell Signaling
    Technology
    #4970
    BRD2Bethyl LaboratoriesA302-583A
    BRD3Bethyl LaboratoriesA302-368A-1
    BRD4Cell Signaling
    Technology
    #13440
    c-MYCCell Signaling
    Technology
    #5605
    PARP1Cell Signaling
    Technology
    Catalog no. 9542S
    PCNADakoCatalog no. M0879
    αTUBULINSigma-AldrichT6557
    γTUBULINSigma-AldrichT6074
    FKBP12Santa Cruz
    Biotechnology
    Catalog no. sc-133067
    CUL4ABethyl LaboratoriesA300-739A
    MCM2Santa Cruz
    Biotechnology
    Catalog no. sc-9839
    Anti-rabbit IgG,
    peroxidase-linked
    antibody
    Thermo Fisher
    Scientific
    NA934
    Anti-mouse IgG,
    peroxidase-linked
    antibody
    Thermo Fisher
    Scientific
    NA931
    Anti-goat IgG-HRPSanta Cruz
    Biotechnology
    Catalog no. sc-2354

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/8/eaay5064/DC1

    Fig. S1. UV-vis characterization.

    Fig. S2. Thermal relaxation.

    Fig. S3. Viability of RS4;11 acute lymphoblastic leukemia cells after treatment with PHOTAC-I for 72 hours in the dark or under pulsed (100 ms every 10 s) 390-nm irradiation.

    Fig. S4. Immunoblot analysis of PHOTAC-I-3.

    Fig. S5. Immunoblot analysis of BRD4 after treatment of RS4;11 cells with PHOTACs.

    Fig. S6. Model of the photoswitch-cereblon interaction.

    Fig. S7. CRBN knockdown control.

    Fig. S8. Me-PHOTAC-I-3 control.

    Fig. S9. FKBP12 Immunoblots in RS4;11 cells.

    Fig. S10. Immunoblot analysis of FKBP12 after treatment of RS4;11 cells.

    General information

    Synthetic Procedures and Characterization

    References (6064)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. UV-vis characterization.
    • Fig. S2. Thermal relaxation.
    • Fig. S3. Viability of RS4;11 acute lymphoblastic leukemia cells after treatment with PHOTAC-I for 72 hours in the dark or under pulsed (100 ms every 10 s) 390-nm irradiation.
    • Fig. S4. Immunoblot analysis of PHOTAC-I-3.
    • Fig. S5. Immunoblot analysis of BRD4 after treatment of RS4;11 cells with PHOTACs.
    • Fig. S6. Model of the photoswitch-cereblon interaction.
    • Fig. S7. CRBN knockdown control.
    • Fig. S8. Me-PHOTAC-I-3 control.
    • Fig. S9. FKBP12 Immunoblots in RS4;11 cells.
    • Fig. S10. Immunoblot analysis of FKBP12 after treatment of RS4;11 cells.
    • General information
    • Synthetic Procedures and Characterization
    • References (6064)

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