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ClickRNA-PROTAC for Tumor-Selective Protein Degradation and Targeted Cancer Therapy
Xucong Teng,# Xuan Zhao,” Yicong Dai, Xiangdong Zhang, Qiushuang Zhang, Yuncong Wu, Difei Hu, and Jinghong Li*
Cite This: J. Am. Chem. Soc. 2024, 146, 27382-27391
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Supporting Information
ABSTRACT: Proteolysis-targeting chimeras (PROTACs) show 5’ T2A SNAP-E3 3’ promise in tumor treatment. However, the E3 ligases VHL and Tumor Cell AUG UAG CRBN, commonly used in PROTAC, are highly expressed in only Tumor 3º Specific a few tumors, thus limiting the application scope and efficacy of ACC 5 mRNA 5’ UAG PROTAC drugs. Furthermore, the lack of tumor specificity in 3ª Normal Cell ADAR PROTAC drugs can result in toxic side effects. Therefore, there is 5° SNAP-E3 3’ an urgent need to develop tumor-selective PROTAC drugs that do UIG STOP not rely on endogenous E3 ligases. In this study, we introduce the 5 SNAP-E3 1 POI ClickRNA PROTAC ClickRNA-PROTAC system, which involves the expression of a SNAP Click Reaction fusion protein of the E3 ubiquitin ligase SIAH1 and SNAPTag E3 E3 through mRNA transfection and recruits the protein of interest E2 SNAP Ub (POI) using bio-orthogonal click chemistry. ClickRNA-PROTAC can effectively degrade various proteins such as BRD4, KRAS, and NFKB simply by replacing the warhead molecules. By employing a tumor-specific mRNA-responsive translation strategy, ClickRNA- PROTAC can selectively degrade POIs in tumor cells. Furthermore, ClickRNA-PROTAC demonstrated strong efficacy in targeted cancer therapy in a xenograft mouse model of adrenocortical carcinoma. In conclusion, this approach offers several advantages, including independence from endogenous E3 ubiquitin ligases, tumor specificity, and programmability, thereby paving the way for the development of PROTAC drugs.
INTRODUCTION
Proteolysis-targeting chimera (PROTAC) is a novel therapeu- tic strategy based on the ubiquitin-proteasome system (UPS) in living cells.1-3 PROTAC drugs typically consist of two modules: a ligand of the protein of interest (POI) and a ligand of the E3 ubiquitin ligase. By mediating the interaction between the E3 ubiquitin ligase and POI, the POI is ubiquitinated and then degraded by the UPS.4,5 This mechanism provides a new approach for targeting previously undruggable proteins, with many candidate drugs already entering clinical trials.6,7 For example, Arvinas’ ARV-110 and ARV-471 targeting the androgen receptor and estrogen receptor in prostate cancer and breast cancer, respectively, have entered clinical Phase II or III.8,9 Most PROTAC drugs currently in clinical stages utilize the E3 ligases VHL and CRBN to mediate the ubiquitination of target proteins.10 However, the expression levels of E3 ubiquitin ligases (Figure 1A, representing 13 typical E3 ubiquitin ligases for PROTAC) vary in different tumors, limiting the application scope and efficacy of the PROTAC drugs in cancer treatment. Furthermore, the lack of tumor specificity in PROTAC molecules can lead to the degradation of target proteins in normal cells, causing harm to normal cells and resulting in toxic side effects. Therefore, there is an urgent need to
develop tumor-selective PROTAC drugs that do not rely on endogenous E3 ligases.
Covalent self-labeling tags can covalently couple small molecules to target proteins through covalent chemical bonds.11-13 This technique offers advantages such as high connection stability, high specificity, and good bio-orthogon- ality. For instance, SNAPTag can specifically recognize and covalently bind benzylguanine (BG) derivatives,14 while HaloTag can stably covalently bind with chloroalkanes.1 Covalent self-labeling tags can selectively regulate intracellular protein-protein interactions through genetically encoded tagged proteins, aligning well with the principles of PROTACs.16,17 Recently, Crews et al. have reported HaloPROTACs, which utilize the HaloTag and chloroalkanes to mediate the ubiquitination and degradation of targeted proteins.18,19 However, the methods reported so far aim to regulate the expression and degradation of exogenous proteins
Received: May 10, 2024
Revised: September 13, 2024
Accepted: September 16, 2024
Published: September 25, 2024
JACS
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ACS Publications
AXC
A
RNF114
RNF4
DCAF16
DCAF15
BIRC2
MDM2
SIAH1
MARCHF5
CRBN
VHL
PRKN
NEDD4L
CHIP
ACC
BLCA
BRCA
CESC
CHOL
COAD
DLBC
ESCA
GBM
HNSC
KICH
KIRC
KIRP
LAML
LGG
LIHC
LUAD
LUSC
MESO
OV
PAAD
PCPG
PRAD
READ
SARC
SKCM
STAD
TGCT
THCA
THYM
UCEC
UCS
UVM
Z-score
2
-1
0
1
2
3
4
B
mRNA
Sensing Region
T2A
SNAP-E3
5’
Coding Region
3’
BG-DBCO
AUG
UAG
Ligand-N3
Tumor Cell
Normal Cell
3’
Tumor Specific mRNA
ACC
5’
5’
UAG
3’
ADAR
STOP
5’
T2A
SNAP-E3
3’
5’
T2A
SNAP-E3
3’
AUG
UIG
AUG
UAG
POI
SNAP
Click Reaction
E3
Proteasome
Ub
E3
SNAP
U
Ub
E2
Ub
instead of targeted degradation of tumor-related proteins. 17,18
mRNA therapeutics can be used to encode antigen proteins for vaccines or to guide a patient’s cells to produce therapeutic proteins.2º In recent years, significant progress has been made in the development of mRNA therapeutics and vaccines including the optimization of mRNA sequences,21 chemical modification strategies,22,23 and efficient delivery systems.24,25 Recently, several methods have been reported to regulate the translation of mRNA encoded proteins in a cell-type specific manner. For example, the synthetic introns can be activated by splicing factor mutations in tumor cells, resulting in tumor-cell- targeted splicing and expression of mRNA.26 In addition, the mRNA base editing strategy mediated by adenosine deaminase RNA specific (ADAR), named CellREADR (cell access through RNA sensing by endogenous ADAR), can induce A- to-I base editing of the stop codon UAG in a cell-specific manner, thereby activating the translation of downstream proteins.27 However, these methods have not been used in the development of tumor-targeted mRNA therapeutics.
Herein, we developed the ClickRNA-PROTAC system (Figure 1B), which can achieve the efficient degradation of
the target protein selectively in tumor cells. ClickRNA- PROTAC expresses the fusion protein of the covalent tag and E3 ubiquitin ligase in living cells by mRNA transfection and functionalizes the fusion protein by a diphenylcyclooctyne (DBCO) modified ligand. Then, the azide-conjugated POI ligand is ligated to the fusion protein by bio-orthogonal click chemistry and induces the ubiquitination and degradation of POIs. Additionally, utilizing the tumor-specific mRNA- responsive translation regulatory strategy based on A-I base editing of the UAG stop codon, ClickRNA-PROTAC can selectively degrade POIs in tumors cells. To construct the ClickRNA-PROTAC system, we first screened an optimal fusion protein from 52 covalent tag-E3 ubiquitin ligase (Tag- E3) fusion proteins. We found that E3 ubiquitin ligase SIAH1 fused with SNAPTag at the N-terminal (SIAH1-SN) could be highly expressed in cells and mediate targeted protein degradation based on the principle of ClickRNA-PROTAC. Taking advantage of bio-orthogonal click chemistry, we proved that ClickRNA-PROTAC can degrade different proteins such as bromodomain containing 4 (BRD4), KRAS Proto- Oncogene GTPase (KRAS), and NFKB (p65) by replacing
A
E3-SC
E3-HC
B
E3 ligase
SNAPTag
E3 ligase
Halo Tag
N
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A
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BG-DBCO
N
E3-SN
E3-HN
N3
$
3
SNAPTag
E3 ligase
Halo Tag
E3 ligase
CI
JQ1-N3
N
C
N
C
Halo-DBCO
ČI
C
CHIP-SN
NEDD4L-SC
NEDD4L-SN
PARKIN-SC
SIAH1-SN
BIRC2-SN
RNF4-SN
RNF114-SN
D
BIRC2-HN
RNF4-HN
RNF114-HC
SNAP-E3
Control
CHIP-HN
NEDD4L-HN
NEDD4L-HC
PARKIN-HN
Halo-E3
VHL-HC
Control
KDa
KDa
150
BRD4
55.3
62.5
97.6
74.8
36.2
93.3
41.9
100.1
100
150
BRD4
Relative Expression
Relative Expression
36.2
51.5
99.8
36.4
64.8
89.6
101.3
100
43
ß-Actin
43
ß-Actin
warhead molecules. By constructing the mRNA specifically translated in tumor cells to express SIAH1-SN, ClickRNA- PROTAC can selectively work in adrenocortical carcinoma cells but not in normal cells. Furthermore, we validated the performance of ClickRNA-PROTAC for targeted cancer therapy in a xenograft mouse model of adrenocortical carcinoma by tumor-selective degradation of BRD4. ClickR- NA-PROTAC efficiently degrades target proteins and offers advantages such as independence from endogenous E3 ubiquitin ligases, tumor specificity, and programmability, providing a new direction for the development and clinical usage of PROTAC drugs.
RESULTS
Design of ClickRNA-PROTAC. A typical PROTAC system consists of an E3 ubiquitin ligase ligand, a target protein- ligand, and a linker to facilitate the best interaction between the E3 ubiquitin ligase and target protein. The ClickRNA- PROTAC system includes an azide-conjugated ligand of POI, a covalent tag-E3 ubiquitin ligase (Tag-E3) fusion protein, and a DBCO-conjugated ligand of a covalent tag (Figure 1B). Taking the BRD4 ligand JQ1 as an example, the conjugation of the azide group did not affect its ability to capture the target BRD4 protein (Figure S1A). The usage of the Tag-E3 fusion protein eliminates the need for a traditional E3 ubiquitin ligase ligand, and the exogenously transfected E3 ubiquitin ligase can overcome the limitations of various E3 ubiquitin ligase expression levels in different tumors. We also developed an mRNA base editing strategy to achieve selective construction of the ClickRNA-PROTAC system in tumor cells without harming normal cells, thereby preventing side effects in tumor therapy. This synthetic mRNA consists of a 5’ sensing region and a 3’ protein-coding region, with a UAG stop codon in the middle to prevent the translation of the 3’ protein-coding region in its initial state (Figure 1B). In tumor cells, the 5’ sensing region and the tumor-specific mRNA hybridize and form a double-stranded RNA with an A-C mismatch, which activates A-to-I base editing mediated by ADARs, converting
the UAG stop codon to the UIG codon. In this way, the translation of the 3’ protein-coding region is reactivated. Subsequently, the T2A self-cleaving peptide undergoes self- cleavage to release the effector protein. Due to the lack of tumor-specific mRNA in normal cells, double-stranded RNA containing A-C mismatch cannot be formed, and A-I base editing cannot be activated, resulting in an intact UAG stop codon. Therefore, ClickRNA-PROTAC can selectively degrade POIs in only tumor cells.
Construction and Optimization of the Tag-E3 Fusion Proteins and the Azide-Conjugated Ligands. Although it is not difficult to construct the Tag-E3 fusion protein, the covalent tag may be ubiquitinated by the fused E3 ubiquitin ligase, leading to its degradation. Therefore, it is necessary to screen for the best combination of a covalent tag and E3 ubiquitin ligase that can form an efficiently expressed fusion protein in cells. We constructed 52 different Tag-E3 fusion proteins based on 13 representative E3 ubiquitin ligases from different families and 2 widely used covalent tags SNAPTag and HaloTag (Figure 2A). We transfected plasmids of the 52 fusion proteins into HEK293FT cells, respectively, and verified the expression levels of the 52 fusion proteins by Western blotting (WB) analysis. As shown in Figure S2, we identified 16 well-expressed fusion proteins, including NEDD4L-SC, SIAH1-SN, NEDD4L-HN, and others.
Next, we attempted to investigate whether the 16 well- expressed Tag-E3 fusion proteins could degrade the POI under the principle of ClickRNA-PROTAC. As a proof of concept, we selected the BRD4 protein as the POI (Figure 2B), which is widely known for its roles as a transcriptional and epigenetic regulator pivotal in the pathogenesis of diverse cancers, making it a prototypical target for PROTAC technology.28-30 By analyzing the relative expression levels of BRD4 with WB, we observed that some Tag-E3 fusion proteins successfully degraded the BRD4 protein (Figure 2C and D), demonstrating the feasibility of the ClickRNA-PROTAC system. Among them, SIAH1-SN, VHL-HC, and PARKIN-HN exhibited the best degradation capabilities, degrading approximately 74% of
| No. | Name | x | No. | Name | x |
|---|---|---|---|---|---|
| 1 | JQ1-N3-C4 | (CH2)4 | 6 | JQ1-N3-02 | (CH2CH2O)2 |
| 2 | JQ1-N3-C6 | (CH2)6 | 7 | JQ1-N3-O3 | (CH2CH2O)3 |
| 3 | JQ1-N3-C8 | (CH2)8 | 8 | JQ1-N3-04 | (CH2CH2O)4 |
| 4 | JQ1-N3-C10 | (CH2)10 | 9 | JQ1-N3-05 | (CH2CH2O)5 |
| 5 | JQ1-N3-C12 | (CH2)12 | 10 | JQ1-N3-06 | (CH2CH2O)6 |
| 11 | JQ1-N3-07 | (CH2CH2O)7 |
A
N
N
N
N
N3
S
N
O
JQ1-N3-X
CI
B
Control
Control
KDa 1 2 3 4 5 CO
6 7 8 9 10 11
JQ1-N3-X
150
BRD4
Relative Expression
98.9
100.3
54.5
0
99.6
100
100.0
99.8
51.7
100.3
100.7
100.6
100
43
ß-Actin
C
D
SIAH1-SN
SIAH1-SN
+
JQ1-N3-C10
JQ1-N3-C10
MG-132
(-)-JQ1-N3-C10
Bortezomib
KDa 150
BRD4
KDa 150
BRD4
43
ß-Actin
43
ß-Actin
Although SIAH1-SN is effective, it still cannot completely degrade the target protein. In PROTAC molecules, the length of the linker directly impacts the interaction between POI and E3 ubiquitin ligase, which in turn affects the Dmax.31 Therefore, we optimized the chain length between the azide group and the POI ligand molecule to increase the Dmax. As shown in Figure 3A and the Supporting Information, we designed and synthesized 11 JQ1-N3 molecules with different chain lengths. WB results revealed that JQ1-N3-C10 was the optimal azide-conjugated ligand (Figure 3B). (-)-JQ1-N3- C10, an enantiomer of JQ1-N3-C10,32,33 cannot bind to BRD4 and therefore cannot function as a protein-ligand component in PROTAC. Using (-)-JQ1-N3-C10 as a negative control, we demonstrated that the combination of SIAH-SN protein and JQ1-N3-C10 can effectively degrade the BRD4 protein, serving as the optimal ClickRNA-PROTAC elements (Figure 3C). Additionally, the proteasome inhibitor MG-132 or bortezomib can effectively inhibit the degradation of BRD4 protein, thus confirming the proteasome-dependent degradation mechanism of the ClickRNA-PROTAC system (Figure 3D).
Targeted Protein Degradation Ability and Versatility of ClickRNA-PROTAC. After obtaining the optimal fusion protein SIAH1-SN and the optimal azide-conjugated ligand JQ1-N3-C10, we attempted to validate the performance of the ClickRNA-PROTAC system delivered in mRNA form. Prior to this, the biocompatibility of SIAH1-SN mRNA and the BG- DBCO component needed to be assessed. According to Figure S3, transfection of 5-400 ng of SIAH1-SN mRNA in a 24-well
plate, addition of 10-2000 nM BG-DBCO, and simultaneous transfection of 400 ng SIAH1-SN mRNA with 10-2000 nM BG-DBCO all maintained good cell viability. Under conditions of 400 ng of SIAH1-SN mRNA and 1 µM BG-DBCO, the IC50 of JQ1-N3-C10 was determined to be 300 nM (Figure 4A). Furthermore, under the same conditions, the degradation of BRD4 by ClickRNA-PROTAC showed JQ1-N3-C10 dose- dependency, with a DC50 of approximately 77.2 nM (Figure 4B). Flow cytometry analysis indicated significant apoptosis caused by ClickRNA-PROTAC (Figure S4). Moreover, ClickRNA-PROTAC demonstrated efficient degradation of BRD4 protein in various cell lines including human HeLa, HepG2, and mouse N2a, mESC (Figure S5).
The design of the bio-orthogonal click reaction in the ClickRNA-PROTAC system allows for the targeted degrada- tion of different POIs by simply replacing the warhead molecules, offering great versatility and convenience. MRTX849 is a ligand for KRAS protein, and NFKB oligodeoxynucleotide (NFKB-ODN) contains the binding motif of transcription factor NFKB (p65). Both of them are utilized in the construction of PROTAC molecules.34 We conjugated the C10-linker and azide group on MRTX849 and NFKB-ODN to generate two new warheads for ClickRNA- PROTAC (Figure S6A). The interaction capabilities of these two warheads with their target proteins have been validated through affinity enrichment and competitive binding experi- ments (Figure S6B). As shown in Figures 4C and 4D, WB results confirmed that ClickRNA-PROTAC could be used to degrade KRAS and p65 proteins. Furthermore, the proteomic results further elucidated the targeted protein degradation capability of ClickRNA-PROTAC for BRD4, KRAS, and p65
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Cell Viability (%)
100
(+)-JQ1-N3-C10 (nM)
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KDa 150
BRD4
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43
ß-Actin
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(+)-JQ1-N3-C10 (nM)
C
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NFKB-ODN-N3
H
T
GTCCTTTCAGGGGT-C10-N3
MRTX849-N3-C10
CTGGAAAGTCCCCA-3’
SIAH1-SN
+ +
SIAH1-SN
+
MRTX849-N3-C10 + + -
NFKB-ODN-N3
+
MG132
+
MG132
+
KDa
25
KRAS
KDa
72
p65
43
ß-Actin
43
ß-Actin
E
5.5
F
6.5
G
6.0
5.0
6.0
5.5
4.5
5.5
5.0
4.0
5.0
4.5
-log10(pvalue)
3.5
BRD4
-log10(pvalue)
4.5
-log10(pvalue)
4.0
SIAH1
4.0
3.0
SIAH1
3.5
p65
p52
SIAH1
3.5
CDK6
2.5
3.0
KRAS
3.0-
2.0
2.5
2.5
IKBB
1.5
2.0
2.0
1.5
1.5
1.0
1.0
1.0-
0.5
0.5
0.5
0.0
0.0
0.0
-4
-3
-2
log2(FoldChange)
-1
0
1
2
3
4
-4
-3
log2(FoldChange)
-2
-1
0
1
2
3
4
-6
-5
-4
log2(FoldChange)
-3
-2
-1
0
1
2
3
4
5
6
(Figures 4E-G). These results indicated that different small molecule or ODN warheads could be used for ClickRNA- PROTAC with good performance.
In addition, using MRTX849-N3-C10 and NFKB-ODN-N3 as warhead molecules of ClickRNA-PROTAC, we tested the KRAS and p65 degradation ability of all 16 well-expressed fusion proteins (Figure S2), respectively. As shown in Figure
S7, SIAH1-SN still had the best degradation ability for KRAS and p65. Interestingly, with the optimization of linker length in these 2 warhead molecules, RNF4-SN, VHL-HC, CHIP-HN, and PARKIN-HN also showed a good degradation effect and could almost completely degrade POIs. These results expanded the toolbox of E3 ligase fusion proteins for ClickRNA- PROTAC available in different scenarios.
A
5’
mCherry
STOP
T2A
NanoLuc
B
3’
115X
AUG
UAG
Inactivated
6000
5000
Tumor
4000
Normal
3’
3000
119X
Target mRNA
ACC
5’
RPM
2000
967X
1000
50
5’
UAG
3’
25-
0
ADAR
B2M-003
AC132217.4-001
IGF2-003
5’
mCherry
T2A
NanoLuc
3’
AUG
UIG
Activated
C
D
Overall Survival
E
ClickRNA-PROTAC
ClickRNA-PROTAC
100000
156 X
SW-13
0
LOW BRD4 Group
High BRD4 Group
NanoLuc / mCherry
10000
HACC
Logrank p=0.0093
0.8
HR(high)=4.6
Percent survival
Untreated
Untreated
p(HR)=0.016
1000
n(high)=19
0.6
n(low)=19
KDa
100
BRD4
0.4
150
10-
43
ß-Actin
1
0.2
SW-13
HACC
IGF2-1
IGF2-2
IGF2-3
IGF2-4
IGF2-5
IGF2-6
IGF2-7
IGF2-8
IGF2-9
IGF2-10
IGF2-11
IGF2-12
B2M
AC132217.4-1
AC132217.4-2
0.0
0
50
100
150
Months
Construction of the ClickRNA-PROTAC System Tar- geting Adrenocortical Carcinoma. Based on the tumor- specific mRNA-responsive translation regulatory strategy, we designed and screened the sequence of the tumor-specific mRNA sensing region that can specifically activate the translation of effector proteins in adrenocortical carcinoma (ACC). We designed the coding sequence of the mCherry fluorescent protein at the 5’ end of synthetic mRNA, resulting in the constant translation of mCherry for monitoring the transfection efficiency by fluorescence imaging (Figure 5A). Downstream of the tumor mRNA sensing region is the coding sequence of NanoLuc luciferase, resulting in the tumor-specific mRNA-responsive translation of NanoLuc. The ability of the tumor-specific mRNA sensing region to activate tumor-specific protein translation can be evaluated by the ratio of NanoLuc chemiluminescent signal to mCherry fluorescence signal.
By analyzing bulk mRNA-seq data from the TCGA database using the GEPIA2 webtool,35 we selected three ACC-specific highly expressed transcripts, B2M-003, AC132217.4-001, and IGF2-003 (Figure 5B), and designed 15 sequences of tumor- specific mRNA sensing regions recognizing different regions of these transcripts. The results revealed that the sensing sequence IGF2-4 could specifically induce effector protein translation in the adrenocortical carcinoma SW-13 cell line, while the translation level of the same mRNA in normal adrenocortical HACC cells was very low with a signal difference of 156-fold (Figure 5C). Further analysis showed that IGF2 mRNA is only highly expressed in adrenocortical carcinoma, pheochromocytoma, paraganglioma, and uterine leiomyosarcoma, with very low expression in all other normal tissues (Figure S8), making it an ideal tumor-specific mRNA for triggering ClickRNA-PROTAC. Tumor survival analysis of
clinic data and bulk mRNA-seq data from TCGA indicated that ACC patients with high expression of BRD4 have significantly poorer survival, suggesting BRD4 as a potential target for treating ACC (Figure 5D). BRD4 degradation and cell viability experiments demonstrated that ClickRNA- PROTAC could specifically degrade BRD4 in the SW-13 cell line and kill SW-13 cells, while normal HACC cells were unaffected due to the inability to express SIAH1-SN (Figure 5E and Figure S9).
ClickRNA-PROTAC for Targeted Therapy of Adreno- cortical Carcinoma in Vivo. Given the successful protein degradation and ACC tumor-selective cytotoxicity of ClickR- NA-PROTAC in vitro, we further investigated the responsive- ness of ClickRNA-PROTAC in an ACC xenograft mouse model. We injected SW-13 cell suspension into female Balb/c nude mice and initiated drug administration 9 days post- implantation, dosing every 5 days for three cycles, monitoring the mice’s status in a 15-day period (Figure 6A). Observations of tumor volume revealed that ClickRNA-PROTAC signifi- cantly inhibited tumor growth (Figure 6B). Compared to the traditional dBET1 PROTAC molecule,36 ClickRNA-PROTAC exhibited superior antitumor effects (Figure 6C). WB analysis of tumor lysates confirmed the degradation of BRD4 in tumor tissue and the induction of cell apoptosis (Figure 6D). Following the completion of the antitumor study, major organs of the mice (such as heart, liver, lungs, spleen, and kidneys) were subjected to H&E staining examination, showing no significant tissue damage (Figure 6E). The body weights of mice also did not change significantly after administration (Figure S10). Therefore, ClickRNA-PROTAC can selectively kill tumors with no apparent side effects, demonstrating its potential for clinical treatment of ACC.
A
Day -9 Tumor inoculation
Day 0
Day 5
Day 10
Day 15
Monitoring
B
C
1
400
Tumor volume (mm3)
1
2
2
300
3
4
5
3
200
*
4
100
5
0
0
2
4
6
8
10
12
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16
Drug treatment (Days)
1
2 3
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E
1
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1 SIAH1-SN+BG-DBCO+JQ1-C10-N3
Heart
2 dBET1
3 SIAH1-SN+JQ1-N3-C10
4 SIAH1-SN+BG-DBCO+(-)-JQ1-N3-C10
Kidney
5 Vehicle
D
Liver
KDa
1
2
3
4
5
17
Cleaved
caspase-3
Lung
150
BRD4
43
ß-Actin
Spleen
The ClickRNA-PROTAC system requires the sequential administration of SIAH1-SN mRNA, BG-DBCO, and an azide- conjugated ligand of POI. This design is highly convenient for testing new POIs and new ligands. It is also practical and convenient for cellular-level experiments, such as studying the function and regulatory mechanisms of relevant biological processes by targeted degradation of the POI. However, the sequential administration of three components can be cumbersome for applications in antitumor drug development. Alternatively, we can directly use a dual-component ClickRNA-PROTAC system consisting of a small molecule conjugate of BG with the POI ligand (BG-POIL). BG-POIL can be codelivered with SIAH1-SN mRNA, and upon translation of SIAH1-SN protein inside the cell, BG-POIL can immediately assemble with it to form a protein complex that ubiquitinates and degrades the POI.
As a proof of concept, we conjugated BG-DBCO with JQ1- N3-C10 to obtain BG-JQ1 (Figure S11A). We cotransfected
SIAH1-SN mRNA with BG-JQ1 into SW-13 and HACC cell lines simultaneously and observed that this system could degrade BRD4 protein selectively and efficiently (Figure S11B). Subsequently, we validated the BG-JQ1 strategy in a SW-13 tumor-bearing mouse model, using dBET1 and JQ1 as control groups. The results indicate that simultaneous administration of SIAH1-SN mRNA with BG-JQ1 effectively inhibits tumor growth, consistent with the results of the three- component system (Figure S11C-G). However, the structure of BG-JQ1 needs to be further optimized to obtain better pharmacological properties.
DISCUSSION
In this work, we demonstrated the feasibility of ClickRNA- PROTAC that uses Tag-E3 fusion protein to degrade POIs. We designed and screened a fusion protein, SIAH1-SN, which is well expressed and capable of degrading POIs in cells and used it to construct the ClickRNA-PROTAC system. Based on
bio-orthogonal click reaction, ClickRNA-PROTAC can achieve the degradation of different proteins such as BRD4/ KRAS/p65 by replacing warhead molecules. We designed and optimized an IGF2-003 transcript responsive synthetic mRNA encoding SIAH1-SN, resulting in selective activation of ClickRNA-PROTAC in the ACC tumor cell. We also confirmed that ClickRNA-PROTAC can be used for targeted tumor therapy in an ACC xenograft mouse model.
E3 ubiquitin ligases play a central role in the PROTAC technology. The usage of appropriate E3 ubiquitin ligases is a critical factor determining the efficiency and selectivity of PROTACs.37 However, the expression levels of E3 ubiquitin ligases vary in different tumors,10 limiting the application and therapeutic effects of PROTAC. We addressed this issue by genetically encoding and expressing the E3 ubiquitin ligase fusion protein SIAH1-SN. During our experiments, we found that some Tag-E3 fusion proteins may undergo self- degradation (Figure S2). Therefore, we screened multiple fusion proteins to identify those suitable for ClickRNA- PROTAC. Additionally, according to recent research, there may be better E3 ubiquitin ligases that can be applied in the ClickRNA-PROTAC method.38 Similarly, recruiting deubiqui- tinases (DUBs) to POIs to construct deubiquitinase-targeting chimeras (DUBTACs) for stabilizing POIs can also be achieved by our method,38,39 which is worth further exploration in the future. mRNA drugs can be efficiently delivered to disease tissues and produce high expression levels of therapeutic proteins. This advantage is expected to be used to solve the problem of low bioavailability of traditional small molecule PROTAC drugs, but the premise to achieve this goal is to construct protein-based PROTAC elements. Previous methods, such as bioPROTAC, have achieved targeted protein degradation by fusing interacting proteins or antibodies with E3 ubiquitin ligases.40,41 However, this method lacks flexibility in changing POIs, requiring the redevelopment and optimization of new bioPROTAC elements for each new POI. In contrast, our ClickRNA-PROTAC method can degrade different POIs by replacing warhead molecules based on the design of bio-orthogonal click reactions.
Controllable PROTAC technology aims to achieve precise control of PROTAC activity by introducing activatable elements such as photosensitive groups, chemical switches, or bioreactive molecules.42 These strategies increase the temporal and spatial specificity of PROTAC therapy, reducing side effects.43 For example, light-controlled PROTACs can be activated under specific light exposure.44,45 Our group has reported an X-ray radiation-mediated PROTAC molecule (RT-PRO) for controlling the activation of PROTAC prodrugs.46 RT-PRO can be activated by X-ray radiation, effectively inhibiting cell proliferation in vitro and suppressing tumor growth in a xenograft mouse model in vivo. This provides a new approach for precisely activating PROTAC prodrugs to reduce systemic toxicity. Similarly, switches activated by drugs or biomolecules can ensure that PROTACs only take effect under specific physiological conditions, enhancing the safety and efficacy of treatment.47 In this work, we proposed an mRNA-based tumor-selective PROTAC strategy that achieves targeted tumor therapy through tumor- specific mRNA responsive base editing and SIAH1-SN translation. To the best of our knowledge, ClickRNA- PROTAC is the first application of tumor-specific mRNA- mediated selective base editing for tumor targeted therapy.
CONCLUSION
Collectively, ClickRNA-PROTAC has several attractive features, addressing current issues with PROTAC. First, our method overcomes the limitation of differential E3 ubiquitin ligase expression levels between different tumors, expanding the application range of PROTAC technology. Second, the tumor targeting of ClickRNA-PROTAC reduces side effects. Third, our method is programmable, allowing for the degradation of different POIs by replacing warheads. In summary, we proposed a novel ClickRNA-PROTAC strategy that efficiently degrades POIs, with advantages such as independence from endogenous E3 ligases, tumor targeting, and programmability, providing a new avenue for the development and clinical use of PROTAC drugs.
ASSOCIATED CONTENT
SI Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.4c06402.
Methods, compound synthesis, abbreviations and full names of carcinoma, and biochemical evaluation data (PDF)
Plasmid sequences (ZIP)
AUTHOR INFORMATION
Corresponding Author
Jinghong Li - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China; Beijing Life Science Academy, Beijing 102209, China; New Cornerstone Science Laboratory, Shenzhen 518054, China; Center for BioAnalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei 230026, China; @ orcid.org/ 0000-0002-0750-7352; Email: jhli@mail.tsinghua.edu.cn
Authors
Xucong Teng - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China; Beijing Life Science Academy, Beijing 102209, China; New Cornerstone Science Laboratory, Shenzhen 518054, China; Center for BioAnalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei 230026, China; @ orcid.org/ 0000-0002-1514-0562
Xuan Zhao - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Yicong Dai - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Xiangdong Zhang - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China Qiushuang Zhang - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Yuncong Wu - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Difei Hu - Department of Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China
Complete contact information is available at: https://pubs.acs.org/10.1021/jacs.4c06402
Author Contributions
“X.T. and X.Z. contributed equally to this paper.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of China (No. 22034004, No. 22027807), the National Key Research and Development Program of China (No. 2021YFA1200104), the New Cornerstone Science Foundation, China Postdoctoral Science Foundation (No. 2023M731972, No. BX20230179, No. 2023M741974, and GZC20231316) and Beijing Life Science Academy Initiative Scientific Research Program (No. 2023000CA0050, No. 2023100CC0240 and No. 2023100CC0250). We thank Bin Yu (Core Facility, Center of Biomedical Analysis, Tsinghua University) for technical support with Flow cytometry analysis.
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