Alkenyl Oxindole Is A Novel PROTAC Moiety That Recruits The CRL4DCAF11 E3 Ubiquitin Ligase Complex For Targeted Protein Degradation
Alkenyl Oxindole Is A Novel PROTAC Moiety That Recruits The CRL4DCAF11 E3 Ubiquitin Ligase Complex For Targeted Protein Degradation
Alkenyl Oxindole Is A Novel PROTAC Moiety That Recruits The CRL4DCAF11 E3 Ubiquitin Ligase Complex For Targeted Protein Degradation
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82171416 to R.T.), the Medical Innovation and facilitate the degradation of target proteins. This dual functionality empowers TPD drugs to
Development Project of Lanzhou University elicit stronger therapeutic effects and holds promise for targeting proteins that were previously
(lzuyxcx-2022-156 to R.W.), the CAMS Innovation
deemed “undruggable” [2–4]. Currently, TPD strategies primarily utilize 2 major degradation
Fund for Medical Sciences (CIFMS) (2019-I2M-5-
074, 2021-I2M-1-026, 2021-I2M-3-001 and 2022- pathways: the ubiquitin-proteasome system and the lysosomal degradation pathway [5–7].
I2M-2-002 to R.W.), Guangdong Basic and Applied According to mechanism of action, the major TPD strategies include proteolysis targeting chi-
Basic Research Foundation (2023B1515020075 to meras (PROTACs), lysosome targeting chimeras (LYTACs), and autophagy targeting chime-
R.T.), the Science, Technology and Innovation ras (AUTACs) [8–11]. Among them, PROTAC technology is the most extensively studied and
Commission of Shenzhen Municipality
has achieved significant breakthroughs. It has been successfully applied to degrade more than
(RCBS20210609103800006,
JCYJ20220530112602006 and
100 target proteins, including those previously considered “undruggable” [12]. Moreover,
RCYX20221008092845052 to R.T.), the Lingang more than 20 PROTACs are currently undergoing clinical trials since 2019 [13–16], indicating
Laboratory Grant (LG-QS-202203-11 to R.T.) and that PROTAC technology is a promising strategy for drug discovery.
the China Postdoctoral Science Foundation PROTACs are heterobifunctional molecules consisting of 2 ligand domains joined by a
(2023M731523 to T.W.). The funders had no role chemical linker. One ligand domain binds to the protein target of interest, and the other ligand
in study design, data collection and analysis,
recruits an E3 ubiquitin ligase. By engaging with both the target protein and E3 ligase simulta-
decision to publish, or preparation of the
manuscript. neously, PROTACs facilitate the polyubiquitination and proteasomal degradation of the target
protein [17]. While over 600 E3 ligases have been identified in the human genome [18], only a
Competing interests: The authors have declared
small fraction (<3%) have been successfully recruited by PROTACs [19]. The most commonly
that no competing interests exist.
utilized E3 ligases include CRBN, VHL, MDM2, and IAPs. More recently, KEAP1 [20,21],
Abbreviations: AUATTEC,
: Theabbreviationlisthasbeenupdated:Pleasecheckandconfirmthattheaddedentriesarecorrectlyabbreviated:
autophagosome-tethering RNF114 [22,23], DCAF15 [24], and DCAF16 [25] have expanded the toolbox of accessible E3
compound; AUTAC, autophagy targeting chimera;
ligases. However, the vast majority (>90%) of reported PROTAC molecules continue to rely
BRD4, bromodomain-containing protein 4; BD1,
bromodomain 1; CRISPRi, CRISPR interference; on just 2 ligases: CRBN and VHL [12]. This narrow E3 diversity poses a major challenge, as
CRL, Cullin-RING E3 ligase; CUL4B, cullin-4B; the development and targeting potential of PROTAC degraders is constrained by the limited
DDB1, damage-specific DNA binding protein 1; pool of recruited E3s. Therefore, broadening the range of E3 ligases that can be engaged by
DCAF11, DDB1 and CUL4 associated factor 11; small molecule ligands could unlock new avenues to potentially degrade a wider range of pro-
EGFP, enhanced green fluorescent protein; FACS,
tein targets, as well as circumvent the acquired drug resistance that caused by mutations in cer-
fluorescence activated cell sorting; FBS, fetal
bovine serum; HDR, homology-directed repair;
tain E3 ligases [26,27].
HPLC, high-performance liquid chromatography; The alkenyl oxindole framework is commonly found in synthetic or natural compounds
HRMS, high-resolution mass spectra; LYTAC, that exhibit a wide range of biological activities and have attracted research interests from
lysosome targeting chimera; mHTT, mutant pharmacologists and chemists [28]. Many alkenyl oxindoles have been developed as lead com-
huntingtin protein; NAE1, NEDD8 activating E1 pounds or marketed drugs against tumors, such as sunitinib [29–31]. Recently, 2 alkenyl oxi-
enzyme; NMR, nuclear magnetic resonance; OD,
ndoles (10O5 and AN1) were found to act as molecular glues that tether mutant huntingtin
optical density; PI, propidium iodide; PROTAC,
proteolysis targeting chimera; RBX1, RING-finger protein (mHTT) to autophagy-related protein LC3, leading to the autophagy-lysosomal degra-
protein RING-box1; RT, room temperature; RT- dation of mHTT [32]. This prompted us to test whether this strategy can be expanded to
qPCR, quantitative reverse transcription PCR; TGI, degrade other substrates. To this end, we synthesized a series of heterobifunctional molecules
tumor growth inhibition rate; TLC, thin-layer by linking BRD4 inhibitor JQ1 with different alkenyl oxindoles, followed by assessing their tar-
chromatography; TPD, targeted protein
geted degradation activity. This led to the identification of HL435, a highly potent alkenyl oxi-
degradation; UBA3, ubiquitin like modifier
activating enzyme 3.
ndole-based BRD4 degrader. However, when we investigated the protein degradation
mechanism of HL435, we found that it degraded BRD4 through the ubiquitin-proteasome sys-
tem rather than the autophagy-lysosomal pathway. Based on this unexpected finding, we
hypothesized that alkenyl oxindoles may act as novel E3 ligase ligands. To verify our hypothe-
sis, we performed a pooled CRISPR interference (CRISPRi) screening, from which we revealed
that the E3 ligase complex CRL4DCAF11 is in charge of HL435-induced proteasomal degrada-
tion of BRD4. We further validated the antitumor efficacy of HL435 both in vitro and in vivo.
Overall, we discovered that alkenyl oxindoles can act as recruitment moiety for CRL4DCAF11
and developed alkenyl oxindole-based PROTAC molecules with high degradation efficiency
and antitumor effects, expanding the toolbox of E3 ligases available for PROTAC drug
development.
Results
Compounds development and structure-activity relationship studies on
alkenyl oxindole-based heterobifunctional degraders
To explore the potential of alkenyl oxindole for target protein degradation, we designed and
synthesized a series of heterobifunctional molecules by connecting JQ1 with different alkenyl
oxindoles using various linkers (S1 Data), followed by examining their ability to degrade BRD4
(Figs 1 and S1). Firstly, JQ1 and the reported alkenyl oxindole (10O5) were connected directly
with saturated or unsaturated alkane chains of different lengths to afford compounds H1-H4.
However, they had little ability to degrade BRD4. When PEG linker was used to replace the
alkane chain (H5), the degradation of BRD4 was observed at a concentration of 1.0 μM. Fur-
thermore, the direct connection of linker and 10O5 through amide bond (H6) significantly
improved the degradation ability. Therefore, we conducted a subsequent structural-activity
study on the alkenyl oxindole moiety using the linker of compound H6. Subsequently, we
examined the degradation activity of JQ1-alkenyl oxindole conjugates constructed from differ-
ent alkenyl oxindole derivatives, including the addition of electron-poor substituents on the
phenyl group or the benzo moiety of oxindole core (Fig 1 and H7-H28). The results showed
that enhanced degradation activity can be achieved by substituting the alkenyl oxindole with tri-
fluoromethyl group (Fig 1, R = 6-CF3). These modifications led to the development of HL435
(H27), an excellent BRD4 degrader with a degradation efficiency >99% at 1.0 μM.
The NMR Spectra showed that there are (E) and (Z) isomers in all JQ1-alkenyl oxindole
conjugates we synthesized. In order to clarify whether the isomeric configuration affects its
efficacy in degrading BRD4, we separated and purified the 2 isomeric forms of compound H6
through high-performance liquid chromatography (HPLC), and the western blot (WBAU ) results
: Pleasenoteth
demonstrated that there was no notable disparity in BRD4 degradation activity between the 2
configurations (S2 Fig). We subsequently explored the stability of the isomeric configuration
and found that both configurations in methanol would undergo isomerization over time at dif-
ferent temperatures, with the isomerization rate noticeably decelerated at lower temperatures
(S3 Data). This observation might partially account for the absence of significant differences in
degradation activity between the isomeric configurations.
Fig 1. The structure and target degradation activity of JQ1-alkenyl oxindole conjugates. aBRD4 degradation rate
was relative quantification result of S1 Fig (WB). bThe Ar motif of H4 was 4-iodobenzaldehyde.
https://doi.org/10.1371/journal.pbio.3002550.g001
bafilomycin failed to rescue the degradation of BRD4 induced by HL435, H1, H6, or H7 in dif-
ferent cell lines (Figs 2G, 2H, S5B and S5D). We next treated WT-, ATG5KO-, and ATG4BKO-
Hela cells with HL435 and found that the absence of LC3 or autophagosomes did not affect the
efficiency of HL435 in degrading BRD4 (Fig 2I). Similar results were obtained for H1 (S5C
Fig). These results suggested that the degradation of BRD4 by JQ1-alkenyl oxindole-conju-
gated compounds was independent of the autophagy-lysosomal pathway; thus, we suspected
Fig 2. HL435 potently degrades BRD4 through the ubiquitin-proteasome system. (A) Design and development of HL435 as a potent BRD4 degrader. (B)
Representative WB results; cells were treated with gradient concentrations of HL435 for 12 hours. (C) The DC50 values of HL435 to degrade BRD4 in MDA-MB-231
and MCF-7 cells, from relative quantitative analysis. (D) Representative WB results; MDA-MB-231cells were treated with HL435 at 0.5 μM for gradient time. (E)
Relative quantitative analysis for the time-dependent degradation of BRD4. (F) The relative mRNA levels of BRD4; cells were treated with HL435 at 1.0 μM for 12 hours,
GAPDH used as internal reference. (G) Representative WB results; cells were cotreated with HL435 and chloroquine (CQ) or bafilomycin (Baf) for 6 hours; CQ or Baf
was pretreated for 2 hours. (H) Relative quantitative analysis of BRD4 from G. (I) Representative WB results (n = 3). WT-Hela, ATG5KO-Hela, or ATG4BKO-Hela cells
were treated with indicated concentration of HL435 for 6 hours. (J) WB results for BRD4 degradation; cells were pretreated with PYR-41, MG132, or PS-341 for 2
hours, followed by HL435 cotreatment for 6 hours. (K) Relative quantitative analysis of BRD4 from J. (L) WB results; HL435 and MLN4924 were cotreated at indicated
concentration for 6 hours; MLN4924 was pretreated for 2 hours. (M) Representative WB results. HEK293T cells were cotransfected with Flag-BRD4 and HA-Ub
plasmids for 32 hours, followed by treatment with MG132 for 8 hours and HL435 for 6 hours. Cell lysates were immunoprecipitated with anti-Flag magnetic beads and
immunoblotted for ubiquitination level of BRD4. (N) Representative WB results; JQ1, HL389, or HL435 was treated for 6 hours. (O) Relative quantitative analysis BRD4
from N. The data underlying the graphs in the figure can be found in S5 Data; the raw images for WB in the figure can be found in S1 Raw Images. Data were presented
as mean ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) or Mann–Whitney test (for F). *p < 0.05, **P < 0.01, ***P < 0.001,
****P < 0.0001; ns, no statistical significance.
https://doi.org/10.1371/journal.pbio.3002550.g002
Fig 3. CRISPRi screening identified the CRL4DCAF11 complex as a potential target mediating HL435-induced
proteasomal degradation of BRD4. (A) Design of BRD4 BD1 dual fluorescent reporter. BRD4 BD1 domain was fused
with mScarlet, followed by a P2A self-cleaving fragment and an EGFP. (B) Validation of BD1 reporter response to
HL435 treatment. Representative fluorescent microscope fields for BD1 reporter levels in HEK29T cells treated with
DMSO, HL435 (50 nM), or HL435 (50 nM) and MG132 (5 μM) for 24 hours. Bar = 200 μm. (C) Validation of BD1
reporter response to HL435 treatment. Relative BD1-mScarlet signal in HEK293T cells was determined by the ratio of
mScarlet and EGFP as measured by flow cytometry. The cells were treated with DMSO, HL435 (50 nM), or HL435 (50
nM) and MG132 (5 μM) for 24 hours. (D) Quantification of the relative BD1-mScarlet signal in the indicated groups
was shown in the bar graph (mean ± SD, n = 3 biological replicates). The data underlying this graph can be found in S5
Data. (E) CRISPRi screening strategy. CRISPRi HEK293T cells harboring BD1 reporter were transduced with an
sgRNA library targeting all human E1, E2, and E3 enzymes. Cells were treated with DMSO or HL435 (50 nM, 24
hours) and 5 million cells were taken as “input.” Cells with top 25% relative BD1-mScarlet signal (BD1high) were sorted
via FACS. The frequencies of BD1high and input cells expressing each sgRNA were determined by next-generation
sequencing and were compared to determine sgRNAs enriched or depleted in the BD1high population. The screening
was performed in duplicates. (F) Screening results analyzed by the MAGeCK-iNC pipeline were shown for
HL435-treated and DMSO-treated groups. A positive phenotype indicates the corresponding sgRNA was enriched in
the BD1high population and vice versa. Dots in red, blue, grey, and orange represent positive hits, negative hits,
negative control, and other genes, respectively. Genes encoding components of the CRL4DCAF11 complex and the
NEDD8-activating enzyme were highlighted. The data underlying the graphs can be found in S4 Data. (G) Scatter plot
comparing gene scores for the screens under HL435 and DMSO treatment. Genes encoding components of the
CRL4DCAF11 complex and the NEDD8-activating enzyme were highlighted. The data underlying this graph can be
found in S4 Data. (H) Predicted structure of CRL4DCAF11 complex using Alphafold. Statistical significance was
determined by one-way ANOVA. ****P < 0.0001.
https://doi.org/10.1371/journal.pbio.3002550.g003
Fig 4. HL435 recruits CRL4DCAF11 complex to induce proteasomal degradation of BRD4. (A) Validation of
knockdown efficiency of different hit genes in CRISPRi HEK293T reporter cells by RT-qPCR (mean ± SD, n = 3
technical replicates). (B) Representative fluorescent microscope fields for BD1 reporter levels in the reporter cells
expressing sgRNAs targeting individual hit genes after DMSO or HL435 treatment (50 nM, 24 hours). Bar = 200 μm.
(C) Relative BD1-mScarlet signal was quantified by flow cytometry for hit gene knockdown in BD1 reporter cells after
DMSO or HL435 treatment (50 nM, 24 hours). (D) Quantification of the relative BD1 intensity in the indicated groups
was shown in the bar graph (mean ± SD, n = 3 biological replicates). (E) Validation of knockdown efficiency of 2
sgRNAs targeting DCAF11 in CRISPRi HEK293T reporter cells by RT-qPCR (mean ± SD, n = 3 technical replicates).
(F) WB showing endogenous protein levels of BRD4 and α-tubulin in CRISPRi HEK293T cells expressing sgRNAs
targeting DCAF11 after DMSO or HL435 treatment (50 nM, 24 hours). (G) Co-immunoprecipitation analysis of
HA-DCAF11 with Flag-BD1 in the absence or presence of HL435 (5 μM, 6 hours) in HEK293T cells. The data
underlying the graphs in the figure can be found in S5 Data; the raw images for WB in the figure can be found in S1
Raw Images.
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https://doi.org/10.1371/journal.pbio.3002550.t001
Fig 5. HL435 exhibited excellent antitumor efficacy in vitro and in vivo. (A) IC50 values of HL435 against multiple tumor cell
lines, determinated by the CCK8 assay after 48-hour treatment. (B) Representative flow cytometry analysis results of the cell cycle in
MCF-7 cells, treated with indicated compounds for 24 hours before stained with PI. (C) Quantitative statistical analysis of cell cycle
for B. (D) Representative flow cytometry analysis results and quantitative statistical analysis of apoptosis; MDA-MB-231 cells were
treated with indicated compounds for 36 hours before stained with a 7AAD/APC Apoptosis Detection kit. (E) Representative WB
results of c-Myc and cycle relevant proteins in MCF-7 cells (n = 3). (F) Representative WB results of apoptosis relevant proteins in
MDA-MB-231 cells (n = 3). (G) Picture of stripped xenograft tumors at the end of experiment (day 45). NOD-SICD mice bearing
the MDA-MB-231 xenograft were daily administered with vehicle (10% DMSO + 90% corn oil, IP) or HL435 (20 mg/kg, IP) 6 days
per week for 27 days. (H) Growth curve of xenograft tumors after treatment. (I) Weights of stripped xenograft tumors at day 45. (J)
Body weight curves during treatment. The data underlying the graphs in the figure can be found in S5 Data; the raw images for WB
in the figure can be found in S1 Raw Images. Data were presented as mean ± SEM (n � 3). One-way ANOVA or unpaired t test was
employed to determine statistical significance. *p < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no statistical significance.
https://doi.org/10.1371/journal.pbio.3002550.g005
compounds conjugated with alkenyl oxindoles, we evaluated their antitumor abilities both in
vitro and in vivo. Most of heterobifunctional compounds exhibited more excellent antiproli-
feration abilities against breast cancer cells than JQ1 or alkenyl oxindoles, supporting the more
excellent therapeutic potential of degraders compared to inhibitors. Consistent with the degra-
dation efficiency of BRD4, HL435 showed the best antiproliferative activity overall, with an
IC50 as low as 8.7 nM against prostate cancer cells 22RV1. In addition to outstanding antiproli-
ferative abilities against multiple tumor cells, HL435 can effectively arrest the cell cycle and
induce apoptosis in breast cancer cells by blocking BRD4 downstream signaling pathway in a
concentration-dependent manner. Finally, the antitumor efficacy of HL435 in vivo was vali-
dated in a mouse MDA-MB-231 xenograft model, with good tolerability. These data suggested
that HL435, a compound composed of structure modified alkenyl oxindole and BRD4 inhibi-
tor JQ1, was a promising drug-like lead compound for anticancer drug development.
Although there are more than 600 E3 ubiquitin ligases in human cells, the ligand molecules
currently available to recruit E3 ubiquitin ligases only cover less than 3% [18,19]. In addition,
with the emergence of E3 ubiquitin ligase resistance, PROTACs based on the same E3 ubiquitin
ligase ligand may be ineffective [41–43]. Therefore, the development of new E3 ubiquitin ligase
ligands can not only solve the limitations of existing ligands, but also be a major way to expand
the scope of PROTACs therapeutic targets and provide better treatment opportunities [26].
Moreover, as DCAF11 is localized in the nucleus, the identification of ligands capable of recruit-
ing DCAF11 provides new possibilities for targeting the degradation of nuclear proteins.
In summary, we discovered alkenyl oxindole as a novel PROTAC moiety for targeted pro-
tein degradation via CRL4DCAF11 recruitment. We also developed JQ1-alkenyl oxindole-conju-
gated bifunctional molecules with high BRD4 degradation efficiencies in multiple cell lines
and proved their anticancer effect both in vitro and in vivo. Our study expands the E3
toolbox available for PROTACs, which will potentially broaden the spectrum of degradable
proteins and improve the efficiency of target degradation, as well as circumvent the acquired
drug resistance caused by mutations in certain E3 ligases, providing new possibilities for drug
discovery.
BRD4 plasmids for 32 hours (Flag-BD1 and HA-DCAF11 plasmids for 48 hours) by Hieff
Trans Liposomal Transfection Reagent (Yeasen, China).
DNA constructs
The coding sequence of BRD4 BD1 domain (amino acid N44-E168) was amplified from full-
length of BRD4 cDNA in a pcDNA3.1-BRD4-3Flag with the forward primer (50 -ATGACGAT-
GACAAGACTAGTaaccccccgcccccagagacctcca-30 ) and reverse primer (50 -ccgccttcttctgtgggtag
ctcatttatt-30 ), and it was fused with mScarlet-P2A-EGFP from pRT117 vector into the pLVX-
3Flag-Hygro vector with the forward primer (50 -ctacccacagaagaaGGCGGTGGCTCGGTGAG-
CAA-30 ) and reverse primer (50 -AGGGGCGGGATCCGCGGCCGCttactagtcggttcaactctaggtg-
30 ) by Hieff Clone Universal One Step Cloning Kit (YEASEN, 10922ES20). The coding
sequence of DCAF11 (Youbio, L11006) was inserted into a pcDNA3.1-3HA vector with the
forward primer (50 - TACCTGACTACGCTGGTACCatgggatcgcggaacagcagcag-30 ) and reverse
primer (50 - GATATCTGCAGAATTCctactggggtgaggaaaaggg-30 ) by Hieff Clone Universal
One Step Cloning Kit (YEASEN, 10922ES20).
CRISPRi screening
The overall CRIPSRi screening process was performed as described previously [33,44,45]. In
brief, HEK293T cells harboring the BRD4 BD1 domain dual fluorescence reporter were
infected with the sgRNA library targeting all human E1, E2, and E3 enzymes as described pre-
viously. The MOI value was controlled under 0.3 when library was transduced to the cells. The
cells were selected by 2 μg/mL puromycin for 2 days. After expansion and puromycin selec-
tion, the cells were treated with DMSO and HL435, respectively. FiveAU million
: PleasenotethatasperPLOSstyl
cells were taken
as “input” 24 hours later, and the remaining cells were subsequently collected for FACS, where
the cells were sorted into the top 25% based on the ratio of mScarlet and EGFP signal. For each
sample, cells corresponding to at least 4,000-fold over the library coverage were sorted per rep-
licate. Sorted populations were collected and genomic DNA was isolated using DNAiso
Reagent (Takara, 9770A). sgRNA cassettes were amplified by PCR and subjected to next-gen-
eration sequencing (NGS) by NovaSeq 6000 PE150. Sequencing results were analyzed using
MAGeCK-iNC as previously described [33].
After washed away nonspecifically bound proteins, anti-Flag magnetic beads were boiled with
1× LB for transsexual washout. ToAUseparate
: PleasenotethatasperPLOSstyle;
protein samples, 8%, 10%, and 12% SDS-PAGE
numeralsarenotallowedatthebeg
gels or 3% Tris-Acetate Polyacrylamide Gradient Gels were used and then transferred to a
PVDF membrane (Millipore; Merck KGaA). AboutAU 5% skim
: PleasenotethatasperPLOSstyle;
milk was used to block mem- numeralsareno
branes at room temperature (RT) for 1 hour. Primary antibodies were blotted at 4˚C over-
night. The next day, the membranes were slowly flipped in secondary antibodies conjugated
with horseradish peroxidase for 1 hour at RT. Images were captured by Tanon 5200 (Shanghai,
China). Image J was used to quantify the intensities of bands. The antibodies used in this paper
were as below: Anti-α-Tubulin (T6047) , anti-LC3B (L7543) and anti-β-Actin were purchased
from Sigma (St. Louis, MO, USA); Anti-BRD4 (13440), anti-PARP1 (9542), anti-Caspase 9
(9505), anti-HA (3724), and anti-Cyclin D1 (2922) were purchased from Cell Signaling Tech-
nology (Danvers, MA); Anti-Ubiquitin (sc-8017) was from Santa Cruz (Dallas, TX, USA).
Anti-ATG4B (M134), anti-ATG5 (M153), and anti-Flag were from MBL (Tokyo, Japan); anti-
Cyclin B1(55004-1-AP), anti-p53 (10442-1-AP), anti-p21 (10355-1-AP), anti-c-Myc (10828-
1-AP), and anti-GAPDH (60004-1-Ig), anti-Vinculin (66305-1-Ig), goat anti-mouse IgG (H
+L) (SA00001-1), and goat anti-rabbit IgG (H+L) (SA00001-2) were purchased from
Proteintech.
Animal experiment
Animal experiment was performed at the Experimental Animal Center of Sun Yat-sen Univer-
sity (East Campus), and female NOD-SCID mice were purchased from Guangdong Yaokang
BiotechnologyAU . Inject
: PleasenotethatasperPLOSstyle;
subcutaneously 5 million MDA-MB-231 cellsLtd:;
donotuseInc:; on the right dorsal side of
etc:exceptasappropriateintheaffi
each mouse at the age of 6 to 7 weeks. When tumors size reached 60 to 70 mm3 about half a
month later, mice were randomly divided into 2 groups. Mice were daily injected with vehicle
(10% DMSO + 90% corn oil, IP) or HL435 (20 mg/kg, IP) 6 days per week for 27 days. Volume
of xenograft tumor and body weight of each mouse were measured every 2 to 4 days. Volume
of xenograft tumor = length × width2/2. KillAU all of: PleasenotethatasperPLOSstyle;
mice at day 27 posttreatment and xenograft
donotusesacrificeinref
tumors were excised for weight measurement. TGI (%) = [1 − (TVTreatment/Dx − TVTreatment/
D1) / (TVVehicle/Dx − TVVehicle/D1)] × 100%, X = days posttreament.
Chemistry
Unless otherwise stated, all solvents and the compounds without provided synthesis routes
were commercially purchased. All solvents were purified and dried according to standard
methods before use. The spectra of 1H nuclear magnetic resonance (NMR) was recorded on a
Varian instrument (500 MHz or 400 MHz), and the tetramethylsilane signal or residual protio
solvent signals was used as the internal standard. 13C NMR was recorded on a Varian instru-
ment (125 MHz or 100 MHz). Data for 1H NMR were recorded as follows: chemical shift
(δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, q = quartet or unre-
solved, coupling constant (s) in Hz, integration). Data for 13C NMR were reported in terms of
chemical shift (δ, ppm). The progress of the reaction was monitored by thin-layer
chromatography (TLC) on glass plates coated with a fluorescent indicator (GF254). Flash col-
umn chromatography was performed on silica gel (200 to 300 mesh). The ESI ionization
sources were employed to obtain high-resolution mass spectra (HRMS). The purity of final
key products was confirmed by a Waters e2695 HPLC system equipped with an XBridge C18
(5 um, 4.6 × 250 mm) and eluted with methanol/water (97.5:2.5) at a flow rate of 1.0 mL/min-
ute. The yields indicated were from single step reactions. All compounds used in biological
tests have been further purified by preparative liquid chromatography, and all of them showed
>95% purity using the HPLC methods described above.
Supporting information
S1 Fig. All compounds we developed could degrade BRD4 in a concentration-dependent
manner. Western blotting results for BRD4 degradation. Image J was employed for relative
quantitative analysis. The raw images for WB in the figure can be found in S1 Raw Images.
(TIF)
S2 Fig. BRD4 degradation activity between different configurations of H6. The raw images
for WB in the figure can be found in S1 Raw Images.
(TIF)
S3 Fig. HL435 degraded BRD4 in concentration- and time-dependent manner in MCF-7
cells. (A) Representative WB results for concentration-dependent studies of BRD4 degrada-
tion. (B) Representative WB results for kinetics studies of BRD4 degradation. Image J was
employed for relative quantitative analysis. The raw images for WB in the figure can be found
in S1 Raw Images.
(TIF)
S4 Fig. HL435 potently degraded BRD4 in multiple cell lines. WB results for BRD4 degrada-
tion. Image J was employed for relative quantitative analysis. The raw images for WB in the fig-
ure can be found in S1 Raw Images.
(TIF)
S5 Fig. Compounds conjugated alkenyl oxindoles degraded BRD4 via ubiquitin-protea-
some system. (A) Structure of compounds conjugated alkenyl oxindoles. (B) Autophagy-lyso-
some inhibitor CQ or Baf cannot rescue the degradation of BRD4 by H1. (C) H1 degraded
BRD4 independent of LC3 and autophagosomes. (D) Proteasome inhibitor MG132 but not
autophagy inhibitor CQ or Baf could rescue the degradation of BRD4 by H6 or H7. Cells were
pretreated with MG132, CQ, or Baf for 2 hours, followed by H6 or H7 treatment for 6 hours.
(E) Inhibitors of the ubiquitin-protease system could rescue the degradation of BRD4 by H6.
Cells were pretreated with PYR-41 or MG132 for 2 hours, followed by H6 treatment for 6
hours. The raw images for WB in the figure can be found in S1 Raw Images.
(TIF)
S6 Fig. HL435 arrested cell cycle in MDA-MB-231 cells. (A) Representative flow cytometry
analysis results of the cell cycle. MDA-MB-231 cells were treated with indicated compounds
for 24 hours before stained with PI. (B) Quantitative statistical analysis of cell cycle for A. (C)
Representative WB results of cycle relevant proteins in MDA-MB-231 cells. The data underly-
ing the graphs in the figure can be found in S5 Data; the raw images for WB in the figure can
be found in S1 Raw Images. Data were presented as mean ± SEM (n = 3). Statistical signifi-
cance was determined by one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, no
statistical significance.
(TIF)
S7 Fig. HL435 induced cell apoptosis in MCF-7 cells. (A) Representative flow cytometry
analysis results and quantitative statistical analysis of apoptosis; MCF-7 cells were treated with
indicated compounds for 36 hours before stained with an 7AAD/APC Apoptosis Detection
kit. (B) Representative WB results of apoptosis-relevant proteins. The data underlying the
graphs in the figure can be found in S5 Data; the raw images for WB in the figure can be found
in S1 Raw Images. Data were presented as mean ± SEM (n = 3). Statistical significance was
determined by one-way ANOVA. ***P < 0.001, ****P < 0.0001; ns, no statistical significance.
(TIF)
S8 Fig. Effects of HL435 on the transcript levels of c-MYC (A) and P21 (B) in breast cancer
cell lines. Cells were treated with indicated compounds for 12 hours; GAPDH was used as
internal reference. The data underlying the graphs in the figure can be found in S5 Data. Data
were presented as mean ± SEM. Statistical significance was determined by one-way ANOVA.
*P < 0.05, ***P < 0.001, ****P < 0.0001; ns, no statistical significance.
(TIF)
S1 Data. The synthetic methods of compounds H1-H28.
(PDF)
S2 Data. 1H and 13C NMR Spectra of compounds H1-H28 and D27.
(PDF)
S3 Data. The HPLC data for different configurations of H6 under different preservation
conditions.
(PDF)
S4 Data. CRISPRi screening results and sgRNA sequence of CRL4DCAF11 complex.
(XLSX)
S5 Data. Underlying numerical data for Figs 2C, 2E, 2F, 2H, 2K, 2O, 3D, 4A, 4D, 4E, 5A,
5C, 5D, 5H, 5I, 5J, S6B, S7A and S8.
(XLSX)
S1 Raw Images. Raw images for all WB.
(PDF)
Acknowledgments
We thank Professor Min Li (Sun Yat-sen University) for his kindly gifts (ATG4BKO-Hela,
ATG5KO-Hela, ATG4BKO-HCT116 cell lines). We thank Dr. Xibin Lu for his guidance of
FACS and SUSTech Core Research Facilities for their support on our project.
Author Contributions
Conceptualization: Liang Hong, Rui Wang, Guofeng Li.
Data curation: Ying Wang, Man Zhao, Aima Huang, Fan Sun, Lu Chen, Risheng Lin,
Ming Zhang, Shiyu Xu, Zhihui Sun, Liang Hong, Rui Wang, Ruilin Tian, Guofeng Li.
Formal analysis: Ying Wang, Liang Hong, Rui Wang, Ruilin Tian.
Funding acquisition: Tianzi Wei, Ming Zhang, Liang Hong, Rui Wang, Ruilin Tian.
Investigation: Ying Wang, Tianzi Wei, Man Zhao, Rui Wang, Guofeng Li.
Methodology: Ying Wang, Tianzi Wei, Man Zhao, Aima Huang, Fan Sun, Lu Chen,
Risheng Lin, Yubao Xie, Ming Zhang.
Project administration: Ying Wang, Tianzi Wei, Man Zhao, Aima Huang, Fan Sun, Lu Chen,
Risheng Lin, Yubao Xie, Ming Zhang, Shiyu Xu, Zhihui Sun.
Resources: Ming Zhang, Liang Hong, Rui Wang, Ruilin Tian, Guofeng Li.
Software: Ying Wang, Liang Hong, Rui Wang, Ruilin Tian.
Supervision: Ying Wang, Tianzi Wei, Ming Zhang, Liang Hong, Rui Wang, Ruilin Tian,
Guofeng Li.
Validation: Ying Wang, Tianzi Wei, Fan Sun, Lu Chen, Ming Zhang, Ruilin Tian.
Visualization: Ying Wang, Tianzi Wei, Man Zhao, Aima Huang, Fan Sun, Lu Chen,
Risheng Lin, Yubao Xie, Ming Zhang, Shiyu Xu, Zhihui Sun.
Writing – original draft: Ying Wang, Tianzi Wei, Man Zhao.
Writing – review & editing: Ying Wang, Tianzi Wei, Man Zhao, Liang Hong, Rui Wang,
Ruilin Tian, Guofeng Li.
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