Light-Induced Protein Degradation with Photocaged PROTACs

Article abstract of DOI:10.1021/jacs.9b06422

Induction of protein degradation is emerging as a powerful strategy to modulate protein functions and alter cellular signaling pathways. Proteolysis-targeting chimeras (PROTACs) have been used to degrade a range of diverse proteins in vitro and in vivo. H

Full text of DOI:10.1021/jacs.9b06422

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Communication
Light-induced protein degradation with photo-caged PROTACs
Gang Xue, Kun Wang, Danli Zhou, Hanbing Zhong, and Zhengying Pan
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 30 Sep 2019
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Light-induced protein degradation with photo-caged PROTACs
Gang Xue†, §, Kun Wang‡, §, Danli Zhou†, Hanbing Zhong*, ‡, and Zhengying Pan*, †
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†State Key Laboratory of Chemical Oncogenomics, Engineering Laboratory for Chiral Drug Synthesis, School of Chemical
Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, 518055, China.
‡Department of Biology, Southern University of Science and Technology, Shenzhen, 518055, China.
Supporting Information Placeholder
A PROTAC molecule is composed of three basic components:
(1) a ligand to recruit E3 ubiquitin ligase, (2) a ligand to bind a
target protein, and (3) a linker to connect the two recruiting ligands.
We selected bromodomain and extraterminal (BET) protein
bromodomain-containing protein 4 (Brd4) as the first target protein
due to its importance as a potential therapeutic target and an elegant
previous work in the literature1,12. The template of pc-PROTACs is
based on the Bradner group’s dBET11, where thalidomide, a ligand
of E3 ligase cereblon (CRBN)13, is linked to JQ1, a ligand of Brd414
(Figure 1b). dBET1 selectively and highly efficiently degrades
BET proteins. The photo-control group has to serve two purposes:
one is to block the formation of binding complexes between pc-
PROTACs and its target protein or E3 ligase, and the other is to be
cleaved upon light irradiation. 4,5-Dimethoxy-2-nitrobenzyl
(DMNB) group can be efficiently cleaved upon irradiation at 365
nm and has been used in numerous cellular studies8. Based on the
CRBN-thalidomide (PDB ID: 4CI1)13 and Brd4-JQ1 (PDB ID:
3MXF)14 crystal structures (Figure S1), we hypothesized that
incorporation of the bulky DMNB group through the amide
nitrogen of the JQ1 moiety (pc-PROTAC1) or the imide nitrogen
of the thalidomide moiety (pc-PROTAC2) (Figure 1c) may hamper
the binding and degradation capability of pc-PROTACs.
ABSTRACT: Induction of protein degradation is emerging as a
powerful strategy to modulate protein functions and alter cellular
signaling pathways. Proteolysis-targeting chimeras (PROTACs)
have been used to degrade a range of diverse proteins in vitro and
in vivo. Here we present a type of photo-caged PROTACs (pc-
PROTACs) to induce degradation activity with light. Photo-
removable blocking groups were added to a degrader of Brd4, and
the resulting molecule pc-PROTAC1 showed potent degradation
activity in live cells only after light irradiation. Furthermore, this
molecule efficiently degraded Brd4 and induced expected
phenotypic changes in zebrafish. Additionally, this approach was
successfully applied to construct pc-PROTAC3 of BTK. Thus a
general strategy to induce protein degradation with light was
established to augment the chemists’ toolbox to study disease-
relevant protein targets.
Selective degradation of proteins rather than direct inhibition of
protein activity emerges as a novel and powerful strategy to
modulate and study the functions of proteins in vitro1 and in vivo2,
and this approach has shown great potential to treat various
diseases in patients3. Small molecule degraders, such as PROTACs,
coined by Sakamoto, Crews and Deshaies in 20014, degrade
proteins of interest via the ubiquitin-proteasome system. A wide
range of PROTACs have been developed to degrade important
proteins, such as Brd41,5, FKBP121,2b, and BTK6. The first oral
PROTAC (ARV-110) targeting androgen receptor has recently
progressed into a phase I clinical study7.
Control of small molecule probe’s activity with higher precision
has been the Holy Grail of chemical biology and drug discovery.
Light with high spatiotemporal resolution has been widely used in
neurobiology, chemical biology8 and disease treatment9 through
the light-controlled release of active small molecule modulators.
We envisioned to incorporate photo-control groups into PROTACs
and developed a general strategy to switch on the degradation
activity of PROTACs by light (Figure 1a). Herein, we report the
discovery of photo-caged PROTACs (pc-PROTACs), which are
inactive without irradiation with light, and upon irradiation, can
quickly release active PROTACs to efficiently degrade the target
proteins and to induce desired phenotypes in both live cells and
zebrafish. Around the submission of our manuscript ， three
preprints of photoactivatable PROTACs were reported online,
utilizing photoswitchable diazo compounds10 or a photocleavable
coumarin group11.
Figure 1. Release of an active PROTAC from pc-PROTAC upon
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light irradiation. (a) Schematic representation of the pc-PROTAC
effects of 0.1  dBET1, and 1  pc-PROTAC1 almost
completely degraded Brd4 with maximum degradation efficacy
(Dmax) of 93% (Figures S5b, 2b). The same degradation experiment
was conducted at various irradiation times (Figure 2c). Reduction
in the Brd4 protein levels was clearly observed after a brief
exposure to the light at 365 nm for only 0.3 min, while longer
exposure time for 3 min led to complete degradation of Brd4. The
kinetics of Brd4 degradation by pc-PROTAC1 upon irradiation for
3 min was examined (Figure S6), and the data showed that Brd4
was almost completely degraded after 4 h, similar to the effect of
dBET1. When cells were incubated with pc-PROTAC1 in the dark
for 2 h and then briefly washed with fresh medium to remove free
pc-PROTAC1, the Brd4 protein levels were still significantly
degraded after the cells were irradiated (Figures S7a, S7b).
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strategy. Under light irradiation, a photo-removable group (purple
circle) is cleaved from pc-PROTAC and an active PROTAC is
released to degrade the protein of interest (POI). (b) Uncaging
reaction of pc-PROTAC1. (c) Chemical structure of pc-PROTAC2.
(d) Photo-induced release efficiency of pc-PROTAC1 to generate
dBET1.
These two pc-PROTACs were synthesized through modified
literature routes1 (Schemes S1, S2). Ultraviolet (UV) spectra of
both compounds showed significantly enhanced absorption in the
region around 365 nm (Figure S2a). The photolysis of pc-
PROTACs and generation of dBET1 upon irradiation (365 nm, 3
mW/cm2) were monitored by high-performance liquid
chromatography. Both pc-PROTAC1 and pc-PROTAC2
disappeared quickly with half-times (t1/2) of 60 s and 105 s,
respectively (Figures 1d, S3b). However, pc-PROTAC1 generated
about 50% of the desired product dBET1 (Figures 1d, S2b), but pc-
PROTAC2 failed to produce appreciable amount of any major
product (Figures S3c). The stability of pc-PROTAC1 in the dark
was also examined in the PBS buffer solution for 24 h.
Approximately 88% of pc-PROTAC1 remained intact and no
dBET1 was released (Figure S4). Therefore, pc-PROTAC1 was
selected for subsequent studies.
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Burkitt's lymphoma cells are sensitive to Brd4 degradation5a,
hence we treated Namalwa cells with a range of concentrations of
dBET1 and pc-PROTAC1 with or without irradiation to
characterize the anti-proliferative activity of these compounds.
Without irradiation, the anti-proliferative activity of pc-PROTAC1
(GI50 = 3.1 ) was substantially weaker than that of dBET1 (GI50
= 0.34 ), and at the highest concentration of 50 , pc-
PROTAC1 was unable to completely inhibit cell proliferation
(Figures 2d). However, upon irradiation, pc-PROTAC1 had a
significant inhibitory effect on cell growth (GI50 = 0.4 ) similar
to that of dBET1. Next the 10-day colony-forming assay was
performed in Brd4-sensitive hepatocellular carcinoma HUH7
cells15 to evaluate the long-term anti-proliferative effects of pc-
PROTAC1. With irradiation pc-PROTAC1 (5 ) showed almost
complete inhibition while pc-PROTAC1 without irradiation
showed no effect to reduce the colony density (Figures 2e). Thus,
these results demonstrated that UV light can induce pc-PROTAC1
to generate significant degradation of target protein Brd4 in the live
cells and inhibit the proliferation of the tumor cells.
Figure 2. In vitro experiments of pc-PROTAC1 with or without
irradiation. (a) Binding affinity of compounds to Brd4
bromodomain 1. (b) Brd4 levels were reduced in a dose-dependent
manner upon irradiation. (c) Results of western blot assay of Brd4
levels in Ramos cells at various irradiation times. (d) Results of
cell viability assay in Namalwa cells. (e) Results of colony-forming
assay in HUH7 Cells with pc-PROTAC1 (5 ).
As expected, the binding affinity of pc-PROTAC1 to Brd4 was
greatly reduced with an IC50 value of 7.6 , while the IC50 values
of JQ1 and dBET1 were 71 nM and 22 nM, respectively (Figure
2a), demonstrating that introduction of the bulky DMNB group at
the JQ1 moiety efficiently reduced the binding ability of pc-
PROTAC1 to Brd4 by more than 100-fold. To determine whether
pc-PROTAC1 can degrade Brd4 under various conditions, Ramos
cells were incubated with various concentrations of the compound
for 1 h and the quantity of Brd4 was measured 12 h later. Consistent
with a substantially weaker binding affinity, 3  pc-PROTAC1
did not show much appreciable degradation of Brd4 in the dark
(Figure S5a). However, upon irradiation with UV light at 365 nm
for 3 min, Brd4 was dose dependently degraded. 0.3  pc-
PROTAC1 significantly reduced the Brd4 levels similar to the
Figure 3. Degradation of endogenous Brd4 protein in zebrafish
embryos. (a) Phenotype of zebrafish embryos treated by dBET1
and pc-PROTAC1 at 24 hpf and 36 hpf. (b) Expression of Brd4
protein in treated embryos examined by western blot.
Then we used zebrafish as a model organism to evaluate the in
vivo activity of pc-PROTAC1. The biological advantages of the
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zebrafish embryo model, such as ex vivo development and optical
to detect Brd4 degradation in vitro5b. Here, an expression plasmid
with a cytomegalovirus (CMV) promoter encoding an enhanced
green fluorescent protein (EGFP) tagged Brd4 protein was injected
into the zebrafish embryos at the single cell stage (Figures 4a, 4b),
and the injected embryos were incubated at 28.5 °C until 24 hpf.
The cells with the EGFP signals were distributed randomly
throughout the embryos. Then, the embryos were treated with
DMSO, dBET1 and pc-PROTAC1 with or without irradiation. The
degradation of EGFP-Brd4 in the embryos was initially observed
as early as 8-10 h after treatment with pc-PROTAC1 with
irradiation or dBET1. After 12 to 24 h, most of the EGFP-Brd4
expressing cells showed substantial degradation of fluorescent
protein (Figure 4c). In contrast, EGFP in embryos treated by
DMSO or pc-PROTAC1 without irradiation were remained (Figure
4c). As a control, embryos injected with a plasmid encoding only
EGFP were also treated as described above, and no degradation of
EGFP was observed (Figure S11). Collectively, these results
demonstrate that UV light can induce pc-PROTAC1 to degrade
Brd4 in the zebrafish embryos.
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transparency, make it an ideal system for developmental biology
studies16 and drug discovery17. Brd4 protein contains three
domains, BD1, BD2 and ET (Figure S8a), which are conserved
among various species18. Specifically, amino acid sequences of the
BD1 and BD2 domains, which contain binding sites of JQ114, are
highly conserved between zebrafish and human (Figure S8b).
Moreover, zebrafish CRBN also showed high sequence similarity
with the human orthologue (Figure S9). In zebrafish, brd4 is widely
expressed at relatively high levels during early embryogenesis18.
Thus, embryos at 12 hours postfertilization (hpf) were treated with
pc-PROTAC1 (100  in embryo medium). After exposure to
light at 365 nm for 10 min, embryos were incubated at 28.5 °C.
Embryos treated with DMSO (1% in embryo medium) served as a
blank control, embryos treated with dBET1 (50  in embryo
medium) were used as a positive control, and embryos treated with
pc-PROTAC1 (100  in embryo medium, but without
irradiation) were used as a negative control. Embryos treated with
pc-PROTAC1 with irradiation or dBET1 showed thinner yolk
extension compared to that in the controls (DMSO and pc-
PROTAC1 without irradiation) at 24 hpf (Figure 3a). The yolk
extension of the embryos treated with pc-PROTAC1 with
irradiation or dBET1 became barely observable at 36 hpf (Figure
3a), suggesting that treatment with dBET1 and uncaging of pc-
PROTAC1 by light disturbed early development of the zebrafish
embryos. To detect the degradation activity of pc-PROTAC1,
western blot was used to assay Brd4 protein levels in the whole cell
extracts of the treated embryos at 36 hpf. The results showed that
Brd4 protein was significantly degraded in the embryos treated
with dBET1 (Figure S10) and pc-PROTAC1 (50 or 100 ) with
irradiation (Figure 3b). These results confirmed the light induced
degradation activity of pc-PROTAC1 in zebrafish.
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Figure 5. BTK degradation by pc-PROTAC3. (a) The chemical
structure of pc-PROTAC3. (b) Upon irradiation, BTK levels were
reduced by pc-PROTAC3 in a dose-dependent manner (mean ±
SD). (c) Results of western blot assay of the BTK levels in Ramos
cells at various irradiation times.
This light-induced degradation strategy is not restricted to Brd4
protein. pc-PROTAC3 (Figures 5a, S12a) was designed and
synthesized (Scheme S3) with the bulky DMNB group connected
to the imide nitrogen of MT-802 developed by the Crews’ group to
degrade BTK protein6a, an important drug target19. pc-PROTAC3
had absorption spectrum, stability and photo-induced release
efficiency similar to those of pc-PROTAC1 (Figures S12b, S12c,
S12d, S12e). Inhibition potency of pc-PROTAC3 against BTK is
close to that of MT-802 since both compounds share the same
binding region to BTK (Figure S12f). Ramos cells were treated
with various concentrations of pc-PROTAC3 and only upon light
irradiation at 365 nm for 3.5 min, BTK levels were significantly
reduced in a dose-dependent manner after 18h (Figures 5b, S13).
Similar to pc-PROTAC1 in degradation of Brd4, the light-induced
degradation of BTK by pc-PROTAC3 was influenced by
irradiation time (Figure 5c).
In conclusion, we have presented a general strategy to develop
photo-caged PROTACs inducible by light to degrade proteins of
interest. The degradation activity of pc-PROTACs upon irradiation
is comparable to that of the corresponding PROTACs in the live
cells. Furthermore, light-induced protein degradation mediated by
pc-PROTACs is effective in zebrafish, a simple yet powerful
animal model for developmental biology and early drug screen
studies. Because light can function at millisecond and submicron
resolutions, we envision that this strategy will show great promise
Figure 4. Degradation of EGFP-tagged Brd4 protein in zebrafish
embryos. (a) Expression plasmid encoding EGFP-tagged Brd4 or
EGFP only. (b) Scheme of visualizing Brd4 protein degradation in
living embryos. (c) Degradation of EGFP-tagged Brd4 protein in
treated embryos at 0, 12 and 24 h after treatment.
Green fluorescent protein provides a powerful means to directly
visualize proteins in the biological systems, and has been utilized
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ASSOCIATED CONTENT
Supporting Information
Compound synthesis, experimental details and supporting figures
are presented in the Supporting Information. The Supporting
Information is available free of charge on the ACS Publications
website.
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: panzy@pkusz.edu.cn; zhonghb@sustech.edu.cn
Author Contributions
§These authors contributed equally.
(7)
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The authors declare no competing financial interests.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China
(81872749), the Shenzhen Science and Technology Innovation
Commission (JCYJ20160226105227446) and the Shenzhen
Municipal Development and Reform Commission for their funding
support. We also thank Zhixue XU and Dr. Chenzhou Hao for their
assistances in the NMR experiments and image generation,
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