Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors

Journal Pre-proof Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors Wenchao Lu, Jun Wang, Yong Li, Hongru Tao, Huan Xiong, Fulin Lian, Jing Gao, Hongna Ma, Tian Lu, Dan Zhang, Xiaoqing Ye, Hong Ding, Liyan Yue, Yuanyuan Zhang, Huanyu Tang, Naixia Zhang, Yaxi Yang, Hualiang Jiang, Kaixian Chen, Bing Zhou, Cheng Luo PII:

S0223-5234(19)30919-5

DOI:

https://doi.org/10.1016/j.ejmech.2019.111767

Reference:

EJMECH 111767

To appear in:

European Journal of Medicinal Chemistry

Received Date: 10 August 2019 Revised Date:

24 September 2019

Accepted Date: 6 October 2019

Please cite this article as: W. Lu, J. Wang, Y. Li, H. Tao, H. Xiong, F. Lian, J. Gao, H. Ma, T. Lu, D. Zhang, X. Ye, H. Ding, L. Yue, Y. Zhang, H. Tang, N. Zhang, Y. Yang, H. Jiang, K. Chen, B. Zhou, C. Luo, Discovery and biological evaluation of vinylsulfonamide derivatives as highly potent, covalent TEAD autopalmitoylation inhibitors, European Journal of Medicinal Chemistry (2019), doi: https:// doi.org/10.1016/j.ejmech.2019.111767. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Discovery and Biological Evaluation of Vinylsulfonamide Derivatives as Highly Potent, Covalent TEAD Autopalmitoylation Inhibitors Wenchao Lua,b,l,#, Jun Wanga,d,#, Yong Lib,c,#, Hongru Taoa,e,#, Huan Xiongc, Fulin Lianf, Jing Gaog, Hongna Maa,h, Tian Lua,d, Dan Zhanga,i, Xiaoqing Yea,j, Hong Dinga, Liyan Yuea, Yuanyuan Zhanga, Huanyu Tang

b,c

, Naixia Zhangf, Yaxi Yangb,c, Hualiang

Jianga,b, Kaixian Chena,b,k, Bing Zhou b,c,*, Cheng Luo a,b,k,* a

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China b

University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China

c

Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute

of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China d

Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University

of Chinese Medicine, 138 Xianlin Road, Nanjing 210023, China e

Department of Chemistry, College of Sciences, Shanghai University, 99 Shangda Road,

Shanghai 200444, China f

Department of Analytical Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, 555 Zuchongzhi Road, Shanghai 201203, China g

Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China h

Department of Pharmacy, Guiyang University of Traditional Chinese Medicine, South Dong Qing Road, Guizhou 550025, China

i

Key Laboratory of Guizhou for Fermentation Engineering and Biomedicine, School of Pharmaceutical Sciences, Guizhou University, Guizhou 550025, China

j

College of Life Sciences, Zhejiang Sci-Tech University, 928 No.2 Street, Hangzhou 310018, China

k

Open Studio for Druggability Research of Marine Natural Products, Pilot National Laboratory for Marine Science and Technology (Qingdao), 1 Wenhai Road, Aoshanwei, Jimo, Qingdao 266237, China

l Now in the Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA# These authors contributed equally.

Highlights: 1. DC-TEADin02 is the most potent TEAD autopalmitoylation inhibitor ever reported. 2. DC-TEADin02 inhibits TEADs transcription activity and downstream gene expression. 3. DC-TEADin02 shows minimal reactivity with biologically relevant nucleophiles. 4. DC-TEADin02 is selective over other lipid-binding proteins 5. The study demonstrates how virtual screen cooperating with rational optimization.

ABSTRACT: Transcriptional enhancer associated domain family members (TEADs) are the most important downstream effectors that play the pivotal role in the development, regeneration and tissue homeostasis. Recent biochemical studies have demonstrated that TEADs could undergo autopalmitoylation that is indispensable for its function making the lipid-binding pocket an attractive target for chemical intervention. Herein, through structure-based virtual screen and rational medicinal chemistry optimization, we identified DC-TEADin02 as the most potent, selective, covalent TEAD autopalmitoylation inhibitor with the IC50 value of 197±19 nM while it showed minimal effect on TEAD-YAP interaction. Further biochemical counter-screens demonstrate the specific thiol reactivity and selectivity of DC-TEADin02 over the kinase family, lipid-binding proteins and epigenetic targets. Notably, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression. Taken together, our findings proved the validity of modulating transcriptional output in the Hippo signaling pathway through irreversible chemical interventions of TEADs autopalmitoylation activity, which may serve as a qualified chemical tool for TEADs palmitoylation-related studies in the future. KEYWORDS: Hippo pathway; TEAD transcription factor; Covalent inhibitor; Palmitoylation inhibitor.

1. Introduction The Hippo signaling pathway is an evolutionarily conserved signaling network that plays essential role in cell proliferation, organ size control, stem cell self-renewal and homeostasis maintenance[1, 2]. Transcriptional enhancer associated domain family members (TEADs) are transcriptional factor families that act as downstream effectors in the regulation of transcriptional output of the Hippo signaling pathway[3]. The transcriptional activity of TEADs could be dynamically regulated by well-characterized transcriptional coactivators YAP/TAZ and other indispensable Hippo-independent coactivators like the vestigial-like (VGLL) protein family and the p160 coactivator family in the context of Hippo signaling[4, 5]. In recent years, a plethora of evidence suggests TEADs could undergo S-palmitoylation under physiological conditions, which is required for its stability and function[6]. In 2016, PuiYee Chan and co-workers demonstrated that palmitoylation-deficient TEAD mutants showed impaired YAP-induced transcriptional activities with minimal effect on TEADs localization[7]. Although the study showed far-reaching importance of TEAD palmitoylation and the lipid-interaction proteome was generally considered with broader ligandability compared to the whole fraction of proteome, which showed great opportunities for pharmacological intervention, little progress has been achieved in the development of small molecule inhibitors[8]. Tremendous efforts have been devoted to the drug discovery of TEAD-YAP inhibitors[9-15]. However, only a handful of TEAD autopalmitoylation inhibitors have been reported. The current reported palmitoylation inhibitors including 2-BP, cerulenin lack specificity and potency. In 2015, Pobbati et al. discovered flufenamic acid, a FDA-approved non-steroidal anti-inflammatory drug, as the moderate TEAD autopalmitoylation inhibitor with the KD value of 73 µM, which occupied two distinct sites of

TEAD-YBD with minimal effect on TEAD-YAP interaction[16]. Further chemistry modifications led to the identification of the allosteric inhibitor TED-347 based on the scaffold of flufenamic acid, which could inhibit TEAD-YAP interaction and TEAD autopalmitoylation activity simultaneously[13]. Thus, it still holds enormous potential to develop more potent, selective TEAD autopalmitoylation inhibitors with novel chemotype and well-established mechanisms of action for the illumination of biological processes and future therapeutic innovation[17]. Herein, starting with the non-covalent scaffold identified using computational approaches, we described the discovery, optimization and biological characterization of DC-TEADin02 as the most potent, selective TEAD autopalmitoylation inhibitor targeting the conserved cysteine within the palmitate binding pocket while it showed minimal effect on TEAD-YAP interaction. Further biochemical selectivity profiling demonstrated its specific thiol reactivity and selectivity over epigenetic targets, kinases family and lipid-binding proteins. In cells, DC-TEADin02 inhibited TEADs transcription activity leading to downregulation of YAP-related downstream gene expression, making it a very useful chemical probe for TEAD palmitoylation-related studies in the future.

2. Results and discussion Structure-based virtual screening. Exploitation and characterization of potential drug-actionable pocket is the cornerstone in drug discovery studies[18]. Thus, SiteMap panel integrated in Schrödinger program suite was used to identify the potential drug-binding pocket of TEAD4. The results revealed that the hydrophobic central cavity was more druggable than TEAD-YAP interface with the Dscore value of 1.364, which showed reasonable size and enclosure for small-molecule binding (Table S1). Since there is no high-throughput assay available for the

identification of palmitoylation inhibitors, molecular docking-based virtual screen of in-house diversity-oriented compound collections containing approximately 22,000 compounds was conducted using Standard Precision mode in Glide panel (Figure 1A). The top-ranked 1,000 hits were picked out for further cluster analysis and visual inspection. Considering the diversity of hit scaffolds, 50 candidates were eventually chosen for further bio-orthogonal reaction-based validation.

Figure 1. The discovery of DC-TEADin01 as the moderate TEAD4 autopalmitoylation inhibitor. (A) The flowchart of the structure-based virtual screening campaign. (B-C) DC-TEADin01 and

flufenamic acid dose dependently inhibited TEAD4 autopalmitoylation activity. The quantification results of band density were analyzed in ImageJ (NIH).

Hit Validation. Activity-based protein profiling (ABPP) has been widely applied to characterize palmitoylated proteins by providing quantitative readouts in native biological systems[19-22]. Thus, we sought to use Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reactions to evaluate the inhibitory effects of 50 candidate compounds against TEAD autopalmitoylation activity via the ‘clickable’ palmitoyl alkyne-coenzyme A handle and biotin azide. Among them, DC-TEADin01 showed moderate inhibitory activity with the IC50 value of 32 µM, which was more potent than the previously reported TEAD autopalmitoylation inhibitor flufenamic acid (Figure 1B-C). Upon the preliminary evaluation using enzymatic assays, the direct binding was further investigated via a series of additional biochemical and biophysical assays. As evidenced in Figure 2A, DC-TEADin01 favored protein folding and induced a positive Tm shift for over 4℃ in a dose dependent manner, demonstrating that the observed inhibitory activity against TEAD4 was due to entropically driven binding instead of substantial destabilization of protein structure.[23] Then, ligand-based

NMR

spectroscopy

was

applied

to

detect

the

binding[24].

In

the

Carr-Purcell-Meiboom-Gill (CPMG) experiments, the addition of TEAD4-YBD protein led to a significant decrease of DC-TEADin01 NMR signal (Figure 2B). Moreover, enhanced resonance of the ligand signal was also observed in the STD spectrum (Figure 2C). We also performed competition experiments with palmitoyl CoA, a known binder in the pocket, to exclude the possibility of non-specific binding. As illustrated in Figure 2D, palmitoyl CoA partially displaced DC-TEADin01 validating its specific interaction. Surface plasmon resonance (SPR) experiments

also indicated that DC-TEADin01 bound to TEAD4-YBD with a KD of 41 µM whereas flufenamic acid bound to TEAD4-YBD with a KD of 84 µM under our assay conditions (Figure 2E, Figure S1). Additionally, the molecular docking studies demonstrated that DC-TEADin01 inserted into the deep substrate tunnel of TEAD4-YBD, making extensive hydrophobic contact with the surrounding residues including F393, F415, V389, L377, F373 and M366 (Figure 2F). Taken together, we identified DC-TEADin01 as the bona fide hit, which could be efficiently optimized into more potent ligands[25].

Figure 2. Biochemical and biophysical methods demonstrated the direct binding of DC-TEADin01. (A) The protein thermal shift assays. Synthetic YAP peptide was used as the positive control with the ratio of 1:5. (B-C) CPMG and STD NMR spectrum assays. (D) Competitive NMR binding assay using 1 µM palmitoyl CoA. (E) SPR assays. (F) The predicted binding mode of DC-TEADin01 with TEAD4-YBD in molecular docking studies. Design and SAR Analysis of Covalent Inhibitors. The development of irreversible covalent inhibitors has generated considerable interest and has been encouraged by the clinical

success of BTK inhibitor Ibrutinib and EGFR inhibitor Afatinib for the treatment of chronic lymphocytic leukemia and non-small-cell lung cancer[26]. To further develop more potent small molecular TEAD inhibitors, we try to rationally design the covalent inhibitor dependent on the nucleophilic cysteine adjacent to the pocket, which is 5.4 Å away from the amine group of DC-TEADin01 (Figure 2F). Then a series of compounds featuring a vinylsulfonamide moiety were synthesized and evaluated using in vitro palmitoylation assays (Table 1). Notably, the addition of the covalent moiety yields DC-TEADin03 with significant improvement on its inhibitory activity. The replacement of the oxygen atom with carbon atom in the linker region (DC-TEADin10) was still tolerable, while variation of the length of the linker (DC-TEADin06) led to a moderate decrease on its activity. Considering the hydrophobicity of the pocket, the phenyl group was replaced with more hydrophobic naphthalene moiety to provide DC-TEADin02, which showed significant inhibitory activity at 800 nM and 200 nM. Further introduction of the water-soluble substituents led to the loss of activity (DC-TEADin07-DC-TEADin12). Compared with DC-TEADin02, DC-TEADin04 and DC-TEADin13 showed the minimal effect on TEAD4 palmitoylation. Thus, the more property-like inactive analog DC-TEADin13 were used as a negative control in the following studies.

Table 1. The inhibitory activities of vinylsulfonamide derivatives.

ID

Structure

Inh@800nM

Inh@200nM

DC-TEADin02

91.6%±5.2%

72.5%±7.3%

DC-TEADin03

94.8%±2.9%

62.2%±6.3%

DC-TEADin04

25.2%±6.5%

-4.1%±1.2%

DC-TEADin05

1.1%±2.5%

8.4%±3.3%

DC-TEADin06

52.5%±6.2%

21.0%±2.8%

DC-TEADin07

10.1%±3.6%

0.3%±4.8%

DC-TEADin08

35.0%±3.7%

15.6%±5.2%

DC-TEADin09

25.4%±7.0%

5.4%±1.4%

DC-TEADin10

92.5%±5.6%

59.2%±6.2%

DC-TEADin11

92.1%±3.8%

44.3%±4.7%

DC-TEADin12

82.5%±5.2%

18.9%±1.2%

DC-TEADin13

6.5% ±1.8%

1.2% ±2.1%

Binding mode analysis. To further delineate the binding mode of DC-TEADin02 and elucidate the mode-of-action of the series of compounds, covalent docking was performed using Covalent Docking panel integrated in Maestro 9.2. The results show that DC-TEADin02 adopts the similar conformation with endogenous myristic acid (Figure 3A). Additionally, the sulphamide group adjacent to the opening of the pocket forms the hydrogen bonds with K344 and C367, which shares the similar binding mode to the carboxyl group of flufenamic acid with TEAD2[16] (Figure 3B). The naphthalene moiety inserts into the deep substrate tunnel, making extensive hydrophobic interactions with adjacent residues in the β-sandwich motif including F229, M370, I374, F393, I395, F415 (Figure 3C-D). Consistent with the lack of inhibitory activity, the phenyl and piperidine moiety of DC-TEADin04 and DC-TEADin13 would be expected to clash sterically with F229, which renders it improbable for potent binding (Figure 3E). Taken together, the

docking results rationalized the structure-activity relationship and established a solid binding mode for further structural decoration (Figure 3F).

Figure 3. In silico docking of DC-TEADin02 and its derivatives into the palmitoylation site of TEAD4. (A-B) The alignment of DC-TEADin02 (green) with myristic acid (yellow) (PDB code: 5OAQ)[27] and flufenamic acid (yellow) (PDB code: 5DQ8)[16]. (C) The detailed interactions between DC-TEADin02 and TEAD4. (D) Schematic representation of interactions between DC-TEADin02 and TEAD4 using LigPlot+[28].The hydrophobic interactions and hydrogen bonds were depicted in red arcs and green dotted lines, respectively. (E) The superposition of DC-TEADin02 (white) with the negative compound DC-TEADin13 (green). (F) Local surface view of the binding pocket.

Characterization

of

DC-TEADin02

as

Potent,

Covalent

TEAD4

autopalmitoylation inhibitor. For in vitro palmitoylation assay, DC-TEADin02 inhibited TEAD autopalmitoylation activity with the IC50 value of 197±19 nM whereas DC-TEADin13 showed minimal effect at 50 µM (Figure 4A-B). In order to demonstrate that DC-TEADin02 formed the covalent linkage to TEAD4-YBD, mass spectrometry was conducted to analyze the protein mass change upon compound binding. A higher mass peak was detected with the difference in mass by +323 Da, which was in accordance with the molecular weight of DC-TEADin02 via a direct Michael addition (Figure 4C). Additionally, the HPLC-MS/MS spectrum of a peptide from DC-TEADin02 treated sample [IHRSPLC(C19H17NO2S) EYMINFIHK] demonstrated the specific modification on Cys367 of TEAD (Figure S2). Notably, the interactions of DC-TEADin02 with wild-type TEAD4-YBD resulted in a significant thermal transition with

the △Tm value of ~7 ℃ at the ratio of 1:1 while the reference compound TED-347 showed substantial destabilization of protein structure indicating different mechanism of action (Figure 4D, Figure S3). To further clarify the specific modification of DC-TEADin02 on Cys367, all cysteines of TEAD4-YBD were individually mutated to alanine for thermal shift evaluation. Compared with other TEAD4 mutants, only TEAD4-YBD C367A mutant significantly abolished the compound effect on protein thermal stability (Figure 4E, Figure S4). We also explored the compound effect on TEAD-YAP interaction in the fluorescence polarization (FP) assay using fluorescein isothiocyanate (FITC)-labeled YAP peptide61-100. The results indicated that DC-TEADin02 showed minimal effect on TEAD-YAP interaction making it a very qualified chemical tool for TEAD palmitoylation-related studies, which is in consistent with the mechanism of action of previously reported TEAD autopalmitoylation chemical probe flufenamic acid (Figure S5). Considering palmitoyl CoA used in CuAAC-based click chemistry methods is the biologically active thiol, we then tested DC-TEADin02 for its ability to form covalent adducts with CoA, which may also inhibit TEAD4 autopalmitoylation. As evidenced in Figure 4F, after 8 h incubation, no covalent adducts have been detected by UPLC-MS. Similarly, DC-TEADin02 is unreactive with GSH, which excludes its non-proteinaceous thiol reactivity (Figure 4G). Additionally, the inhibitory activity of DC-TEADin02 was unaffected in the presence of excess bovine serum albumin (BSA), which can mitigate thiol reactivity (Figure 4G). Collectively, these results demonstrated the specific thiol reactivity of DC-TEADin02. It has been well characterized in the chemoproteomics studies that kinases harboring many targetable, active site-proximal cysteines account for the off-target effect of covalent inhibitors owing to its high nucleophilicity[29, 30]. Thus, we conducted kinome profiling to evaluate

compound selectivity. The results showed that it did not inhibit the activity of kinases at 10 µM (Figure 4H, Table S2). Additionally, it was selective over epigenetic targets in the biochemical selectivity profiles (Table S3). Considering the fact that the non-covalent scaffold of

DC-TEADin02 may bind to other lipid binding proteins, we tested the compound effect on the FABP3-5 using thermal shift assays. The results showed that DC-TEADin02 showed minimal effect on FABP3-5 demonstrating the specific interaction with TEAD lipid-binding pocket (Figure S6).

Figure 4. The biophysical and biochemical characterization of DC-TEADin02. (A) The chemical s structures of DC-TEADin02 and its property-like inactive analog DC-TEADin13. (B) The inhibitory activity of DC-TEADin02 and DC-TEADin13 through CuAAC-based click chemistry. The band intensity was quantified in ImageJ (NIH). The experiments were performed in triplicate and the data was shown as mean ± SD. (C) Mass spectrometry demonstrated the covalent modification of DC-TEADin02 with TEAD4-YBD protein. (D-E) DC-TEADin02 significantly improved the thermostability of TEAD and its mutant protein except C367A mutant. Synthetic YAP peptide was used as the positive control with the ratio of 1:5. (F) DC-TEADin02 was incubated up to 8 h with 5-fold excess GSH/CoA for additional adduct analyses in UPLC-MS. (G) DC-TEADin02 inhibited TEAD autopalmitoylation in the presence of excess BSA. (H) Kinase profiling with the compound concentration of 10 µM. Each experiment was performed with one replicate to ensure data quality.

Cellular Activity of DC-TEADin02. In order to further evaluate the inhibitory effect of compounds in cells, we performed TEADs specific luciferase reporter assays with DC-TEADin02 and the negative compound DC-TEADin13. As expected, the treatment of cells transfected with the TEAD luciferase reporter with DC-TEADin02 led to the significant reduction in TEAD reporter activity compared with DC-TEADin13. Additionally, the treatment of DC-TEADin02 showed similar levels versus control cells in TEADs-independent reporter systems demonstrating the specific regulation on Hippo pathway (Figure 5A). We also evaluated TEAD downstream gene expression patterns in colorectal cancer cells HCT 116, which has been evidenced with relative high expression of TEAD4 in the web-accessible GENT database (Figure S5)[31, 32]. Consistent with the inhibition of TEAD transcriptional activity, the treatment of DC-TEADin02 suppressed

downstream gene CTGF and CYR61 expression at a dose-dependent manner while it showed minimal effect on TEAD-YAP interaction, which was in accordance with the findings in vitro (Figure 5B-C). In order to further demonstrate the direct interaction between DC-TEADin02 and TEAD4 and evaluate the cellular uptake of the compound, we performed the cellular thermal shift assay (CETSA) in intact HCT 116 cell. The results showed that the detected soluble fraction of TEAD4 was increased upon the compound treatment, demonstrating its cellular uptake and direct binding on TEAD4 (Figure 5D).

Figure 5. DC-TEADin02 inhibited the TEAD transcriptional activity downregulating the downstream gene expression. (A) The effect of DC-TEADin02 and DC-TEADin13 on TEAD-dependent/independent reporter system. (B) Quantitative RT-PCR analysis demonstrated the downregulation of TEAD-specific genes upon compound treatment. (C) The pull-down assay to detect TEAD-YAP interaction. (D) HCT 116 cells were treated with 50 µM DC-TEADin02 for 4 h in CETSA experiments to demonstrate the direct binding of DC-TEADin01 with TEAD4 in intact cells. The results shown are mean ± SD of three replicates (*P<0.05, **P < 0.01, ***P < 0.001, ****P<0.0001, N.S. P>0.05). (E) The modification of TEAD4 upon 50 µM DC-TEADin02 treatment for 4 hr was detected by MS experiments.

Moreover, HEK293T cells transfected with Flag-TEAD4 was treated with 50 µM DC-TEADin02 for 4 hr. Then Flag-TEAD4 protein was extracted by affinity purification with anti-flag resin from the cell lysate and submitted for MS analysis. As expected, the direct modification on Cys367 of Flag-TEAD4 was detected demonstrating the on-target effect in cells (Figure 5E).

3. Conclusion In eukaryotic organisms, fatty acylation is increasingly recognized as one of the most well-characterized post-translational modifications that could dynamically and spatiotemporally regulate protein stability, localization, trafficking and protein-membrane interactions[33]. Global studies demonstrate that in mammalian cells proteins could be modified by at least six types of lipids [34]. Among them, S-palmitoylation is the uniquely reversible fatty acylation that orchestrates diverse biological processes[35].

Emerging evidence demonstrates that pharmacologically antagonize protein palmitoylation may have great potential for the treatment of a variety of malignant diseases[36]. In 2013, Petrova et al. developed Hhat inhibitors based on high-throughput screen, which could specifically block Shh palmitoylation overcoming drug resistance[37]. In 2018, Hansen and Haag et al. demonstrated that covalent inhibition of STING palmitoylation could significantly inhibit STING signaling cascade and inflammatory cytokine production, which shed light on the treatment of STING-dependent autoinflammatory disease[38-40]. Taken together, these evidences suggest targeting protein palmitoylation could be a valid option for future therapeutic development.

In mammalian cells, a variety of key signaling proteins could be lapidated including RAS, Hedgehog, Shh, Wnt etc[36]. And numerous palmitoyl-proteomic studies have provided many useful resources for related studies in this area[41]. In 2016, Chan and co-workers demonstrated that TEADs transcription family could also undergo autopalmitoylation through a reversible thioester bond, which is sensible to hydrolysis under physiological conditions. The enzymatic S-palmitoylation is indispensable for YAP-induced TEADs transcriptional activities, making it an attractive target for chemical intervention. Although recent years have witnessed massive gain in our understanding of its biochemical function, the development of TEAD autopalmitoylation inhibitors lags behind and is still at its infancy[42]. Herein, based on integrated structure-based virtual screen and CuAAC-based click chemistry biorthogonal evaluation, we identified DC-TEADin01 as the moderate TEAD4 autopalmitoylation inhibitor. A series of biochemical and biophysical methods were adopted to validate the reliability of its scaffold. Further rational chemical optimization led to the identification of DC-TEADin02 as

the most potent, selective, cell active covalent TEAD autopalmitoylation inhibitor bearing a previously unexplored chemotype with the IC50 value of 197±19 nM with minimal effect on TEAD-YAP interaction making it a useful chemical probe for TEAD palmitoylation-related studies. DC-TEADin02 did not interact with GSH and CoA and its inhibitory activity was not comprised in the presence of excessive BSA with specific thiol activity. Additionally, it showed great selectivity against kinase family harboring function cysteines, lipid binding proteins and epigenetic families demonstrating the specificity of both covalent and non-covalent motifs. In cells, it could covalently bind to the Cys367 of TEAD4 protein, leading to the inhibition of TEADs transcriptional activity and downstream gene expression. As the starting point, DC-TEADin02 may serve as a qualified lead compound to develop more potent, subtypeselective irreversible TEAD autopalmitoylation inhibitors, which shed light on future therapeutic innovations. These results provide an efficient paradigm of how virtual screening cooperating with rational optimization could efficiently promote the drug discovery progress. The similar paradigm could be easily and broadly extended to other undruggable targets besides transcription factors. Our results demonstrate the feasibility and the value of using small molecule inhibitors to selectively target protein lipidation in cell signaling, which represent a major advance on the road for functional study in the context of Hippo signaling. 4. Material and methods 4.1 Chemistry Unless otherwise stated, all materials and anhydrous solvents were purchased from commercial suppliers and used without further purification. All products were confirmed by NMR

spectra and high-resolution mass spectra. NMR spectra were obtained on a Bruker Avance 400 MHz or Varian Inova 400 MHz spectrometer using tetramethylsilane as an internal reference. Coupling constants (J values) were reported in Hertz (Hz) and proton coupling patterns were described as broad (b), singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). High-resolution mass spectra were conducted on a triple TOF 5600+ MS/MS system (AB Sciex, Concord, Ontario, Canada) in the negative or positive ESI mode. Reactions were monitored by thin-layer chromatography or UPLC-MS (Waters Acquity UPLC H-class with Waters SQ Detector 2) analysis. Column chromatography was performed on silica gel (200-300 mesh). All tested compounds were purified to ≥ 95% purity

as determined by ultra performance liquid

chromatography.

4.1.1 Synthesis of 4-(p-tolyloxy)aniline (DC-TEADin01)

Scheme1. Synthesis scheme of DC-TEADin01 Reagents and conditions: 1a) K2CO3, DMSO, 90 ℃, 8 h. 1b) H2, Pd/C, MeOH, 6 h. General synthesis procedure 1a： ：p-Cresol (200 mg, 1.85 mmol), K2CO3 (767 mg, 5.55 mmol) and 4-Fluoronitrobenzene (261 mg, 1.85 mmol) were added into DMSO (15 mL). The mixture was stirred at 90 ℃ for 8 h. The reaction mixture was diluted and extracted with EtOAc. The organic layer was washed with water for 5 times then evaporated. The residue was chromatographed

(silicagel,

Petroleum

ether/EtOAc=100:1)

to

give

1-methyl-4-(4-nitrophenoxy)benzene (410 mg, 97%) as a white solid, 1H NMR (400 MHz, Chloroform-d) δ 8.24-8.17 (m, 2H), 7.28-7.22 (m, 2H), 7.04-6.97 (m, 4H), 2.41 (s, 3H). General synthesis procedure 1b： ：1-methyl-4-(4-nitrophenoxy)benzene (DC-TEADin01-1, 100 mg, 0.44 mmol), Pd/C (10 mg) were added in methanol with several drops of HCl and stirred at room temperature under H2 for 6 h. The reaction mixture was filtered using Celite and evaporated. The

residue

was

chromatographed

(silicagel,

Petroleum

ether/EtOAc=20:1)

to

give

4-(p-tolyloxy)aniline (DC-TEADin01, 71 mg, 81%) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 7.09 (d, J = 8.2 Hz, 2H), 6.89-6.81 (m, 4H), 6.70- 6.64 (m, 2H), 3.56 (s,2H), 2.31 (s, 3H). HRMS (ESI): m/z [M + H]+ calculated for C13H14NO – 200.107, found 200.1074.

4.1.2 Synthesis of N-(4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide (DC-TEADin02) Br NO2 1b

2a OH B OH

2b

NO2

O

NH2

DC-TEADin02-1

DC-TEADin02-2

N H

O S

DC-TEADin02

Scheme 2.

Scheme2. Synthesis scheme of DC-TEADin02 Reagents and conditions: 2a) 1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II) , DCE:2M aqueous Na2CO3 (v/v) =3:1, 100 ℃, 2 h. 1b) H2, Pd/C, MeOH, 6 h. 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h General synthesis procedure 2a ： 4-Nitrobenzyl bromide (500 mg, 2.43 mmol), 2-Naphthaleneboronic

acid

(500

mg,

2.91

mmol),

1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II) (0.05 mmol, 34 mg) were dissolved in degassed 1,2-Dichloroethane (15 mL) and 2 M sodium carbonate solution (5 mL). The mixture was stirred at 100 ℃ under argon for 2 h. The reaction mixture was diluted with DCM and filtered

using Celite, then saturated aqueous NH4Cl was added to the filtrate and extracted with EtOAc. The combined organic layers were dried over Na2SO4 and evaporated. The residue was chromatographed (silicagel, Petroleum ether/EtOAc=200:1) to give 2-(4-nitrobenzyl)naphthalene (DC-TEADin02-1，550 mg, 90%) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 8.18-8.13 (m, 2H), 7.86-7.75 (m, 3H), 7.64 (s, 1H), 7.52-7.43 (m, 2H), 7.41-7.35 (m, 2H), 7.27 (dd, J = 8.4, 1.7 Hz, 1H), 4.24 (s, 2H). General synthesis procedure 1b： ：Starting with 2-(4-nitrobenzyl)naphthalene (DC-TEADin02-1), 4-(naphthalen-2-ylmethyl)aniline (DC-TEADin02-2) was obtained as a white solid by using general synthesis procedure 1b (yield 90%). 1H NMR (400 MHz, Methanol-d4) δ 7.85-7.77 (m, 3H), 7.70 (s, 1H), 7.50-7.42 (m, 4H), 7.37-7.31 (m, 3H), 4.22 (s, 2H). Hydrogens of NH2 were not found. General synthesis procedure 2b： ：4-(naphthalen-2-ylmethyl)aniline (DC-TEADin02-2, 80 mg, 0.34 mmol) was stirred with pyridine (0.5 mL) in DCM (10 mL) at 0 ℃, then 2-Chloroethanesulfonyl chloride (56 mg, 0.343 mmol) which was diluted with DCM (2 mL) was slowly added into the mixture. The reaction mixture was stirred for 2 h then evaporated. The residue was diluted and extracted with EtOAc then washed with water for 3 times. The organic layers were dried over Na2SO4 and evaporated. The residue was chromatographed (silicagel, Petroleum ether/EtOAc=5:1) to give N-(4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide (DC-TEADin02) (93 mg, 85%) as a white solid. 1H NMR (400 MHz, Chloroform-d) δ 7.78 (m, 3H), 7.61 (s, 1H), 7.51-7.39 (m, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.4 Hz, 2H), 6.63 (s, 1H), 6.54 (dd, J = 16.5, 9.9 Hz, 1H), 6.26 (d, J = 16.5 Hz, 1H), 5.94 (d, J =

9.9 Hz, 1H), 4.10 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C19H18NO2S 324.1053, found 324.1062.

4.1.3 Synthesis of 4-phenoxyphenyl ethenesulfonate (DC-TEADin03)

Scheme 3. Synthesis scheme of the DC-TEADin03 Reagents and conditions: 3a) 4-phenoxyphenol, TEA, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. General synthesis procedure 3a： ：4-phenoxyphenol (186 mg, 1 mmol) was stirred with TEA (278 µL, 2 mmol) in Anhydrous DCM (10 mL) at 0 ℃, then 2-Chloroethanesulfonyl chloride (105 µL, 1mmol) which was diluted with DCM (2 mL) was slowly added into the mixture. The reaction mixture was stirred for 2h then evaporated. The residue was diluted and extracted with EtOAc then washed with water for 3 times. The organic layers were dried over Na2SO4 and evaporated. The

residue

was

chromatographed

(silicagel,

Petroleum

ether/EtOAc=3:1)

to

give

4-phenoxyphenyl ethenesulfonate (DC-TEADin03) (193 mg, 70%) as a colorless oil. 1H NMR (400 MHz, Chloroform-d) δ 7.36 (t, J = 7.9 Hz, 2H), 7.22-7.10 (m, 3H), 7.05-6.95 (m, 4H), 6.68 (dd, J = 16.6, 10.0 Hz, 1H), 6.39 (d, J = 16.7 Hz, 1H), 6.18 (d, J = 10.0 Hz, 1H).

4.1.4 Synthesis of N-(2-phenoxyphenyl)ethenesulfonamide (DC-TEADin04)

Scheme 4. Synthesis scheme of the DC-TEADin04

General

synthesis

procedure

3a

：

Starting

with

2-phenoxyaniline,

N-(2-phenoxyphenyl)ethenesulfonamide (DC-TEADin04) was obtained as a colorless solid by using general synthesis procedure 3a (yield 76%). 1H NMR (400 MHz, Chloroform-d) δ 7.59 (dd, J = 7.6, 1.9 Hz, 1H), 7.36 (t, J = 7.9 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.08 (pd, J = 7.5, 1.7 Hz, 2H), 6.98 (d, J = 8.2 Hz, 2H), 6.88 (s, 1H), 6.86 (dd, J = 7.7, 1.7 Hz, 1H), 6.51 (dd, J = 16.5, 9.9 Hz, 1H), 6.28 (d, J = 16.5 Hz, 1H), 5.93 (d, J = 9.9 Hz, 1H). HRMS (ESI): m/z [M + H]+ calculated for C19H18NO2S−324.1053, found 324.1062. HRMS (ESI): m/z [M + H]+ calculated for C14H14NO3S 276.0689, found 276.0692.

4.1.5 Synthesis of N-(naphthalen-2-yl)ethenesulfonamide (DC-TEADin05)

Scheme 5. Synthesis scheme of the DC-TEADin05 General

synthesis

procedure

3a

：

Starting

with

naphthalen-2-amine,

N-(naphthalen-2-yl)ethenesulfonamide (DC-TEADin05) was obtained as a white solid by using general synthesis procedure 3a (yield 80%). 1H NMR (400 MHz, Chloroform-d) δ 7.79 (t, J = 9.1 Hz, 3H), 7.65 (d, J = 1.9 Hz, 1H), 7.52-7.41 (m, 2H), 7.31 (dd, J = 8.7, 2.2 Hz, 1H), 6.99 (s, 1H), 6.61 (dd, J = 16.5, 9.9 Hz, 1H), 6.32 (d, J = 16.5 Hz, 1H), 5.95 (d, J = 9.9 Hz, 1H). HRMS (ESI): m/z [M + H]+ calculated for C12H12NO2S 234.0583, found 234.0584.

4.1.6 Synthesis of N-(4-(benzyloxy)phenyl)ethenesulfonamide (DC-TEADin06)

Scheme 6. Synthesis scheme of the DC-TEADin06 General

synthesis

procedure

3a

：

Starting

with

4-(benzyloxy)aniline,

N-(4-(benzyloxy)phenyl)ethenesulfonamide (DC-TEADin06) was obtained as a white solid by using general synthesis procedure 3a (yield 75%). 1H NMR (400 MHz, Chloroform-d) δ 7.37 (dq, J = 21.1, 7.3, 6.5 Hz, 5H), 7.14 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.9 Hz, 2H), 6.54 (dd, J = 16.5, 9.9 Hz, 1H), 6.40 (s, 1H), 6.20 (d, J = 16.6 Hz, 1H), 5.92 (d, J = 9.9 Hz, 1H), 5.04 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C15H16NO3S 290.0845, found 290.0854.

4.1.7

Synthesis

of

N-(1,2,3,4-tetrahydroisoquinolin-6-yl)-4-(vinylsulfonamido)benzamide

(DC-TEADin07)

Scheme7. Synthesis scheme of the DC-TEADin07 Reagents and conditions: 7a) EDCI, 4-DMAP, DCM, overnight. 1b) H2, Pd/C, MeOH, 6 h. 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. 7b) HCl (4 M solution in 1,4-Dioxane), 2 h. General

synthesis

procedure

7a

，

1b

and

2b

：

Tert-butyl

6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (248 mg, 1 mmol), 4-nitrobenzoic acid (167 mg, 1 mmol), EDCI (212 mg, 1.1 mmol), and catalytic quantity of 4-DAMP are added into DCM

(10 mL) and the mixture were stirred for 8 h. The reaction mixture was diluted with DCM and filtered using Celite, then evaporated. Starting with the residue which was not purified, Tert-butyl 6-(4-(vinylsulfonamido)benzamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate (DC-TEADin07-1) was obtained as a white solid by using general synthesis procedure 1b and 2b (yield 45%). 1H NMR (400 MHz, Methanol-d4) δ 7.87 (d, J = 8.9 Hz, 2H), 7.55-7.41 (m, 2H), 7.28 (d, J = 8.9 Hz, 2H), 7.11 (d, J = 8.5 Hz, 1H), 6.71 (dd, J = 16.5, 10.0 Hz, 1H), 6.24 (d, J = 16.5 Hz, 1H), 6.01 (d, J = 9.9 Hz, 1H), 5.48 (s, 2H), 4.53 (s, 2H), 3.63 (t, J = 5.9 Hz, 2H), 2.83 (t, J = 5.9 Hz, 2H), 1.49 (s, 9H). General

synthesis

procedure

7b

：

Tert-butyl

6-(4-(vinylsulfonamido)benzamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate (DC-TEADin07-1, 30 mg, 0.07 mmol) was added into a flask, then HCl (4 M solution in 1,4-Dioxane, 3 mL) was added. The mixture was stirred for 2 h. Then the reaction mixture was evaporated

to

get

N-(1,2,3,4-tetrahydroisoquinolin-6-yl)-4-(vinylsulfonamido)benzamide

(DC-TEADin07) as a HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 10.50 (s, 1H), 10.23 (s, 1H), 9.57 (s, 2H), 7.93 (d, J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 1H), 6.85 (dd, J = 16.4, 9.9 Hz, 1H), 6.21 (d, J = 16.4 Hz, 1H), 6.10 (d, J = 9.9 Hz, 1H), 4.19 (s, 2H), 3.34 (s, 2H), 3.00 (t, J = 5.9 Hz, 2H). (as a HCl salt). HRMS (ESI): m/z [M + H]+ calculated for C18H20N3O3S 358.122, found 358.1211.

4.1.8 Synthesis of N-(4-(vinylsulfonamido)phenyl)-2-naphthamide (DC-TEADin08)

Scheme 8. Synthesis scheme of the DC-TEADin08 General synthesis procedure 7a： ：Starting with 2-naphthoic acid (172 mg, 1 mmol) and (208 mg, 1 mmol), tert-butyl (4-(2-naphthamido)phenyl)carbamate (DC-TEADin08-1) was obtained as a white solid by using general synthesis procedure 7a (yield 85%). 1H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.30 (s, 1H), 8.55 (s, 1H), 8.10 – 7.94 (m, 4H), 7.73 – 7.55 (m, 4H), 7.43 (d, J = 8.6 Hz, 2H), 1.47 (s, 9H). General

synthesis

procedure

(4-(2-naphthamido)phenyl)carbamate

7b

and

2b

：

Starting

with

tert-butyl

(DC-TEADin08-1),

N-(4-(vinylsulfonamido)phenyl)-2-naphthamide (DC-TEADin08) was obtained as a white solid by using general synthesis procedure 7b and 2b (yield 70%). 1H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.55 (s, 1H), 8.09-7.96 (m, 4H), 7.74 (dd, J = 9.0, 2.5 Hz, 2H), 7.66-7.58 (m, 2H), 7.15 (d, J = 8.9 Hz, 2H), 6.76 (dd, J = 16.4, 9.9 Hz, 1H), 6.07 (d, J = 16.4 Hz, 1H), 6.01 (d, J = 10.0 Hz, 1H). One hydrogen of NH was not found. HRMS (ESI): m/z [M + H]+ calculated for C19H17N2O3S 353.0954, found 353.0962.

4.1.9

Synthesis

(DC-TEADin09)

of

N-(2-benzoyl-1,2,3,4-tetrahydroisoquinolin-6-yl)ethenesulfonamide

Scheme 9. Synthesis scheme of the DC-TEADin09 Reagents and conditions: 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. 7b) HCl (4M solution in 1,4-Dioxane), 2h. 3a) Benzoyl chloride, TEA, Anhydrous Dichloromethane, 0 ℃, 2 h. General

synthesis

procedure

2b

：

Starting

with

6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate,

tert-butyl tert-butyl

6-(vinylsulfonamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate

(DC-TEADin09-1)

was

obtained as a white solid by using general synthesis procedure 2b (yield 70%). 1H NMR (400 MHz, Chloroform-d) δ 7.01 (dd, J = 24.2, 9.7 Hz, 3H), 6.70 (s, 1H), 6.55 (dd, J = 16.5, 9.9 Hz, 1H), 6.27 (d, J = 16.5 Hz, 1H), 5.95 (d, J = 9.9 Hz, 1H), 4.52 (s, 2H), 3.61 (t, J = 5.2 Hz, 2H), 2.79 (t, J = 5.7 Hz, 2H), 1.48 (s, 9H). General

synthesis

procedure

7b

and

3a

：

6-(vinylsulfonamido)-3,4-dihydroisoquinoline-2(1H)-carboxylate N-(2-benzoyl-1,2,3,4-tetrahydroisoquinolin-6-yl)ethenesulfonamide

Starting

with

tert-butyl

(DC-TEADin09-1), (DC-TEADin09)

was

obtained as a white solid by using general synthesis procedure 7b and 3a (yield 60%). 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 7.47-7.38 (m, 5H), 7.23-6.86 (m, 3H), 6.74 (dd, J = 16.4, 10.0 Hz, 1H), 6.08 (d, J = 16.4 Hz, 1H), 6.00 (d, J = 9.9 Hz, 1H), 4.58 (d, J = 82.8 Hz, 2H), 3.65

(d, J = 124.2 Hz, 2H), 2.78 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C18H19N2O3S 343.1111, found 343.1118.

4.1.10 Synthesis of N-(4-benzylphenyl)ethenesulfonamide (DC-TEADin10)

Scheme 10. Synthesis scheme of the DC-TEADin10 General

synthesis

procedure

3a

：

Starting

with

4-benzylaniline,

N-(4-benzylphenyl)ethenesulfonamide (DC-TEADin10) was obtained as a pale yellow solid by using general synthesis procedure 3a (yield 70%). 1H NMR (400 MHz, Chloroform-d) δ 7.34-7.09 (m, 9H), 6.57 (dd, J = 16.5, 9.9 Hz, 1H), 6.46 (s, 1H), 6.29 (d, J = 16.5 Hz, 1H), 5.97 (d, J = 9.9 Hz, 1H), 3.97 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C15H16NO2S 274.0896, found 274.0899.

4.1.11 Synthesis of N-(3-hydroxy-4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide (DC-TEADin11)

Scheme 11. Synthesis scheme of the DC-TEADin11 Reagents and conditions:

11a) NBS, Benzoyl peroxide, CCl4, 80 ℃, 8 h. 2a)

1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II), DCE:2 M aqueous Na2CO3 (v/v) =3:1, 100 ℃, 2 h. 11b) 47% aqueous HBr, Methyl trioctyl ammonium chloride, 120 ℃, 12 h. 1b) H2, Pd/C, MeOH, 6 h. 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. General synthesis procedure 11a： ：2-methoxy-1-methyl-4-nitrobenzene (500 mg, 2.99 mmol), NBS (586 mg, 3.29 mmol) and Benzoyl peroxide (72 mg, 0.299 mmol) were added into CCl4 (30 mL) and stirred at 80 ℃ for 8 h. The reaction mixture was evaporated. The residue was chromatographed

(silicagel,

Hexane/EtOAc=100:1)

to

give

1-(bromomethyl)-2-methoxy-4-nitrobenzene (DC-TEADin11-1, 662 mg, 90%) as a white solid. 1

H NMR (400 MHz, Chloroform-d) δ 7.82 (dd, J = 8.3, 2.2 Hz, 1H), 7.73 (d, J = 2.2 Hz, 1H), 7.49

(d, J = 8.3 Hz, 1H), 4.54 (s, 2H), 4.00 (s, 3H). General synthesis procedure 2a： ：Starting with 1-(bromomethyl)-2-methoxy-4-nitrobenzene and 2-Naphthaleneboronic

acid

(DC-TEADin11-1),

2-(2-methoxy-4-nitrobenzyl)naphthalene

(DC-TEADin11-2) was obtained as slight yellow solid by using general procedure 2a (DC-TEADin11-2, yield 90%). 1H NMR (400 MHz, Chloroform-d) δ 7.84-7.75 (m, 4H), 7.73 (d, J = 2.2 Hz, 1H), 7.62 (s, 1H), 7.49-7.41 (m, 2H), 7.32 (dd, J = 8.5, 1.8 Hz, 1H), 7.20 (d, J = 8.2 Hz, 1H), 4.19 (s, 2H), 3.94 (s, 3H). General synthesis procedure 11b： ：2-(2-methoxy-4-nitrobenzyl)naphthalene (DC-TEADin11-2, 500 mg, 1.705 mmol) and methyl trioctyl ammonium chloride (689 mg, 1.705 mmol) were added into 47% aqueous HBr (50 mL) and stirred at 120 ℃ for 12 h. The reaction mixture was extracted

2 times with EtOAc and the oganic layer was washed with H2O for 3 times then evaporated to get 2-(naphthalen-2-ylmethyl)-5-nitrophenol (DC-TEADin11-3, 476 mg, 99%) as a yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 7.85-7.72 (m, 4H), 7.70-7.63 (m, 2H), 7.47 (td, J = 6.6, 5.9, 3.5 Hz, 2H), 7.37-7.30 (m, 1H), 7.27 (d, J = 6.6 Hz, 1H), 5.79 (s, 1H), 4.22 (s, 2H). General synthesis procedure 1b and 2b： ：Starting with 2-(naphthalen-2-ylmethyl)-5-nitrophenol (DC-TEADin11-3),

N-(3-hydroxy-4-(naphthalen-2-ylmethyl)phenyl)ethenesulfonamide

(DC-TEADin11) was obtained as brown oil by using general procedure 1b and 2b (yield 57%). 1

H NMR (400 MHz, Chloroform-d) δ 7.77 (ddd, J = 12.8, 8.6, 5.5 Hz, 3H), 7.63 (s, 1H), 7.49-7.38

(m, 2H), 7.33 (dd, J = 8.3, 1.6 Hz, 1H), 7.04 (d, J = 8.1 Hz, 1H), 6.75 (d, J = 2.1 Hz, 1H), 6.61 (dd, J = 8.1, 2.0 Hz, 2H), 6.54 (dd, J = 16.5, 9.9 Hz, 1H), 6.28 (d, J = 16.6 Hz, 1H), 5.94 (d, J = 9.9 Hz, 1H), 5.48 (s, 1H), 4.08 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C19H18NO3S 340.1002, found 340.1007.

4.1.12

Synthesis

(DC-TEADin12)

of

N-(4-((7-hydroxynaphthalen-2-yl)methyl)phenyl)ethenesulfonamide

Scheme 12. Synthesis scheme of the DC-TEADin12 Reagents

and

conditions:

12a)

TBSCl,

imidazole,

1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium 1,4-Dioxane,

90

℃,

4

h.

2a)

DMF, (II),

2

h.

12b)

AcOK,

Bis(pinacolato)diboron,

1-(bromomethyl)-4-nitrobenzene,

1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II), DCE:2M aqueous Na2CO3 (v/v) =3:1, 100 ℃, 2 h. 1b) H2, Pd/C, MeOH, 6 h. 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. 12c) TBAF(1.0mol/L in THF), 2 h. General synthesis procedure 12a and 12b： ：7-bromonaphthalen-2-ol (500 mg, 2.24 mmol), TBSCl (338 mg, 2.25 mmol) and imidazole (229 mg, 3.36mmol) were added into DMF (20 mL). The reaction mixture was stirred for 2 h. The mixture was diluted with EtOAc and extracted with EtOAc for 3 times then evaporated. Without purification, the residue, AcOK and 1,1-Bis(diphenylphosphino)-ferro-cene]dichloropalladium (II), Bis(pinacolato)diboron was added into 1,4-Dioxane (25 mL) and stirred for 4h at 90 ℃. The reaction mixture was diluted with DCM and filtered using Celite then evaporated. The residue was chromatographed to give tert-butyldimethyl((7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)oxy)silane (DC-TEADin12-1). 1H NMR (400 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.79-7.65 (m, 3H), 7.24 (d, J = 2.2 Hz, 1H), 7.11 (dd, J = 8.8, 2.4 Hz, 1H), 1.39 (s, 12H), 1.01 (s, 9H), 0.24 (s, 6H). General

synthesis

procedure

2a

：

Starting

with

tert-butyldimethyl((7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-yl)oxy)silane (DC-TEADin12-1),

tert-butyldimethyl((7-(4-nitrobenzyl)naphthalen-2-yl)oxy)silane

(DC-TEADin12-2) was obtained as white solid by using general procedure 2a (yield 80%). 1H NMR (400 MHz, Chloroform-d) δ 8.15 (d, J = 8.7 Hz, 2H), 7.70 (dd, J = 8.4, 6.7 Hz, 2H), 7.47 (s,

1H), 7.38 (d, J = 8.7 Hz, 2H), 7.12 (dd, J = 8.5, 1.6 Hz, 2H), 7.06 (dd, J = 8.8, 2.3 Hz, 1H), 4.20 (s, 2H), 1.02 (s, 9H), 0.25 (s, 6H). General

synthesis

procedure

1b,

2b

and

12c

：

Starting

with

tert-butyldimethyl((7-(4-nitrobenzyl)naphthalen-2-yl)oxy)silane (DC-TEADin12-2), the residue of DC-TEADin12-3 was obtained by using general procedure 1b and 2b. Without purification, the residue was stirred with TBAF (1.0 mol/L in THF) for 2 h then evaporated. N-(4-((7-hydroxynaphthalen-2-yl)methyl)phenyl)ethenesulfonamide

(DC-TEADin12)

was

obtained as a slight yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 9.75 (s, 2H), 7.66 (t, J = 8.2 Hz, 2H), 7.48 (s, 1H), 7.18 (d, J = 8.4 Hz, 2H), 7.13- 6.95 (m, 5H), 6.72 (dd, J = 16.5, 9.9 Hz, 1H), 6.05 (d, J = 16.4 Hz, 1H), 5.97 (d, J = 9.9 Hz, 1H), 3.96 (s, 2H). HRMS (ESI): m/z [M + H]+ calculated for C19H16NO3 338.0856, found 338.0856.

4.1.13

Synthesis

N-(4-(naphthalen-2-ylmethyl)-3-(piperidin-4-yloxy)phenyl)ethane-sulfonamide (DC-TEADin13)

Scheme 13. Synthesis scheme of the DC-TEADin13

of

Reagents and conditions: 13a) tert-butyl 4-hydroxypiperidine-1-carboxylate, DIAD, DPPBE, Anhydrous THF, 0 ℃→rt ， 8 h. 1b) H2, Pd/C, MeOH, 6h. 2b) Pyridine, Anhydrous Dichloromethane, 2-Chloroethanesulfonyl chloride, 0 ℃, 2 h. 7b) HCl (4 M solution in 1,4-Dioxane), 2 h. General synthesis procedure 13a, 1b and 2b ： 2-(naphthalen-2-ylmethyl)-5-nitrophenol (DC-TEADin11-3, 100 mg, 0.36 mmol), tert-butyl 4-hydroxypiperidine-1-carboxylate (87 mg, 0.432 mmol) and DPPBE (209 mg, 0.468 mmol) were added into THF (10 mL) at 0 ℃. The mixture was stirred for 10 min then DIAD (95 mg, 0.468 mmol) was added. The reaction proceeds at room temperature for 8 h. The reaction mixture was evaporated and chromatographed to give a crude

product

of

DC-TEADin13-1.

Starting

with

DC-TEADin13-1,

tert-butyl

4-(2-(naphthalen-2-ylmethyl)-5-(vinylsulfonamido)phenoxy)piperidine-1-carboxylate (DC-TEADin13-2) was obtained as a white solid by using general procedure 1b and 2b (yield 50%). 1H NMR (400 MHz, Chloroform-d) δ 7.82-7.68 (m, 3H), 7.59 (s, 1H), 7.47-7.37 (m, 2H), 7.30 (dd, J = 8.4, 1.6 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.81 (d, J = 1.9 Hz, 1H), 6.62-6.58 (m, 2H), 6.54 (dd, J = 16.6, 9.9 Hz, 1H), 6.26 (d, J = 16.5 Hz, 1H), 5.95 (d, J = 9.9 Hz, 1H), 4.48 (dt, J = 6.3, 3.2 Hz, 1H), 4.06 (s, 2H), 3.49 (s, 2H), 3.37 (s, 2H), 1.85 (s, 2H), 1.72 (s, 2H), 1.45 (s, 9H). General

synthesis

procedure

7b

：

Starting

with

tert-butyl

4-(2-(naphthalen-2-ylmethyl)-5-(vinylsulfonamido)phenoxy)piperidine-1-carboxylate (DC-TEADin13-2), N-(4-(naphthalen-2-ylmethyl)-3-(piperidin-4-yloxy)phenyl)ethenesulfonamide (DC-TEADin13) was obtained as a slight yellow solid (HCl salt) by using general procedure 7b. 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 9.09 (s, 1H), 8.97 (s, 1H), 7.87- 7.78 (m, 3H), 7.66 (s, 1H), 7.45

(tt, J = 7.0, 5.2 Hz, 2H), 7.38 (dd, J = 8.5, 1.7 Hz, 1H), 7.15 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 2.1 Hz, 1H), 6.77 (dd, J = 16.4, 9.9 Hz, 1H), 6.70 (dd, J = 8.1, 1.9 Hz, 1H), 6.12 (d, J = 16.4 Hz, 1H), 6.03 (d, J = 9.9 Hz, 1H), 4.55 (t, J = 7.2 Hz, 1H), 4.02 (s, 2H), 3.14 (s, 2H), 3.09 (s, 2H), 2.08 (t, J = 11.4 Hz, 2H), 1.91 – 1.78 (m, 2H) (as HCl salt). HRMS (ESI): m/z [M + H]+ calculated for C24H27N2O3S 423.1737, found 423.1744.

4.2 Structure-based Virtual Screening. Binding site prediction. The crystal structure of human TEAD4 was fetched from the protein data bank (PDB ID: 5OAQ)[43]. Specifically, the covalently bound myristate and unwanted waters were initially deleted. The sitemap panel in the Maestro program (Maestro, version 9.1; Schrödinger, LLC: New York, 2010) was applied to visualize and evaluate potential drug binding sites. Then the optimization of the remaining protein structure was properly performed for docking calculations through the Protein Preparation Wizard panel. Other parameters were set as the default. Docking procedures. The in-house compound database containing ca. 20,000 compounds with diverse scaffolds was prepared through LigPrep panel integrated in the Maestro program to generate all low-energy stereoisomers and possible ionization states of the ligands in the pH range of 7.0 ± 2.0 with Epik. The receptor grids were generated centered at the Cys367 that could undergo autopalmitoylation. Then docking-based virtual screen was conducted with the GLIDE standard precision (SP) mode. Similarly, the covalent docking studies were performed in Covalent Docking panel in the Maestro program. Cys367 was selected as the centroid of the enclosing box with the box size of 20.0 Å. Other parameters were set as the default.

4.3 Protein Expression and Purification

The yap-binding domain (YBD) of human TEAD4 (217-434) was cloned in pET28a vector with the N terminal 6x His tag. The recombinant protein was induced at 16 °C using 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 14-16 h for overexpression in Escherichia coli OverExpress C43 (DE3) chemically competent cells (2nd Lab, EC100). Then cells were harvested followed by sonication and the supernatant was loaded onto the HisTrap FF column (GE Healthcare). The hTEAD4-YBD was eluted with elution buffer (50 mM Hepes 8.0, 300 mM NaCl, 10% Glycerol, 1 mM TCEP, 125 mM imidazole). To remove the endogenous fatty acid, the protein was incubated with 0.2 mM hydroxylamine for 2 h and further purified through gel filtration chromatography in the final buffer (25 mM Hepes pH 8.0, 100 mM NaCl, 1 mM TCEP and 5% glycerol). Collected fractions were concentrated to 5 mg/mL and flash frozen for further use.

4.4 In Vitro Palmitoylation Assay Briefly, different concentrations of synthetic compounds or the positive control flufenamic acid (CSNpharm, CSN12744) were pretreated with 400 nM TEAD4-YBD protein. After 2 h incubation, 2.5 µM palmitoyl alkyne-coenzyme A (Cayman chemical, No. 15968) was added into the centrifugal tubes for another 30 min on ice. The click chemistry reaction was conducted at room temperature as previously described[44]. The reactions were quenched with 6x SDS-sample loading buffer containing 30 mM EDTA and analyzed by immunoblotting. The relative band density was quantified in ImageJ (NIH).

4.5 Thermal Shift Assay Thermal shift assays were performed on Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems). For each reaction, 5 µM purified protein (TEAD4/FABP3-5) mixed with 5× SYPRO

Orange Dye (Sigma-Aldrich, S5692) were prepared in the assay buffer (50 mM Hepes pH 8.0, 100 mM NaCl, 1 mM TCEP). Then 0.5 µL DMSO or different concentrations of compounds were finally added to the assay plate. All samples were tested in triplicate using Quant Studio 6 Flex Real-Time PCR system (Applied Biosystems). Subsequent analysis of fluorescence signal was performed using protein thermal shift software (version 1.2, Applied Biosystems).

4.6 Surface Plasmon Resonance The SPR experiments were performed at 25 °C on Biacore T200 instruments (GE Healthcare) as previously described[45]. hTEAD4-YBD protein (217-434) was directly immobilized onto the CM5 biosensor chip to a level of 3,500 response unit using the standard amine coupling approach. Binding kinetic measurements were conducted in the assay buffer (25 mM Hepes 7.4, 150 mM NaCl, 0.05% P20, 0.2% DMSO). Data was processed using Biacore T200 Evaluation software to calculate the equilibrium dissociation constant.

4.7 Nuclear Magnetic Resonance Experiments The NMR experiments were performed using Bruker AVANCE III 600 MHz spectrometer at 25℃ as previously described[46, 47]. For Carr-Purcell-Meiboom-Gill (CPMG) and saturation transfer difference (STD) experiments, compounds were dissolved to a final concentration of 200 µM in the presence of 5% DMSO-d6 with 5 µM hTEAD4-YBD. For competitive experiments, 1 µM palmitoyl CoA (Sigma, P9716) was used for data acquisition.

4.8 Mass Spectrum Intact protein mass measurement. Protein solution was diluted to a concentration of 1 mg/mL in water with 0.1% formic acid. A total of 2 µg TEAD4 was injected for LC/MS analysis. The intact

protein high resolution mass measurement was analyzed using an Ultimate 3000 LC coupled with a Orbitrap Fusion mass spectrometer equipped with a HESI ion source (Thermo Fischer Scientific, San Jose, CA, USA). Intact protein samples were separated with an Agilent PLRP-S column (1.0 X 50 mm, 5 µm) using a 15 min gradient at a flow rate of 0.300 mL/min. Mobile phase A was made up of water with 0.1% formic acid, while Mobile phase B was made up of acetonitrile with 0.1% formic acid. The LC/MS raw data was processed using BioPharma Finder (Version 2.0， Thermo Fischer Scientific, San Jose, CA, USA) to generate intact protein masses with ReSpectTM (Isotopically Unresolved) deconvolution algorithm. Drug conjugate sites analysis. Sample was precipitated by acetone at −20 °C for 30 min to remove unbound drug compounds. Precipitated proteins were dried in air and resuspended in 100 mM ammonium bicarbonate. Sequencing-grade modified trypsin was added to each sample (enzyme to protein ratio 1:25, w/w) and incubated at 37°C for 16 h. 20 µg of the digested peptide mixture was desalted by C18 tip. Enzyme digests of peptide mixture were analyzed on the easy nano-LC1000 system (Thermo Fischer Scientific, San Jose, CA, USA) using a self-packed column (75 µm × 150 mm; 3 µm ReproSil-Pur C18 beads, 120 Å, Ammerbuch, Germany) at a flow rate of 300 nL/min. The mobile phase A of RP-HPLC was 0.1% formic acid in water, and B was 0.1% formic acid in acetonitrile. The peptides were eluted using a 60 min gradient (2-5% B for 1 min, 5−35% B for 47 min, 35−45% B for 5 min, 45%-100% B for 2min, 100% B hold for 5min) into a nano-ESI orbitrap Fusion mass spectrometer (Thermo Fischer Scientific, San Jose, CA, USA). The mass spectrometer was operated in data-dependent mode with each full MS scan (m/z 350−1500) followed by MS/MS in a duty cycle of 3s with the parameters: for HCD activation: 2-4 precursor ion charge, 35 normalized collision energy; for ETD activation: 3-6 precursor ion charge,

using calibrated charge dependent ETD parameters. Dynamic Exclusion™ was set for 30 s. Precursor ions were isolated by quadrupole with an isolation window of 1.6 Da. The full mass and the subsequent MS/MS analyses were scanned in the Orbitrap analyzer with R = 60,000 and R= 15,000, respectively. The raw data was processed using Proteome Discoverer (Version 2.2). The following criteria were used to identify the conjugated peptides: Trypsin/P was selected as the digestive enzyme with two potential missed cleavages. The search included variable modifications of methionine oxidation and drug conjugation on Cysteine (+323.098Da), both HCD and ETD activation were selected, fixed value PSM validator module was used for PSM confident validation. For the detection of covalent modification of TEAD4 in cells, HEK293T cells transfected with Flag-TEAD4 (FL) in 15cm dish were treated with 50 µM DC-TEADin02 for 4 h. Then cells were harvested and lysed using lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% NP40, 1 mM PMSF and 2.5 mM EDTA). The supernatant of cell lysates was immunoprecipitated with anti-Flag G1 Affinity Resin (GenScript, Cat. No. L00432). The beads were washed with PBS for three times and then sent for MS analysis.

4.9 Fluorescence Polarization Assay Fluorescence polarization assay was performed in 384-well solid black plates (Corning, 3575) using assay buffer (25 mM Hepes pH7.4, 150 mM NaCl, 0.1 mg/ml BSA, 1 mM DTT and 0.01% NP-40). The components for the assay were added as follows: (1) 200 nM TEAD4 protein was pretreated with compounds or DMSO for 1 h. (2) 20 nM FITC-Yap61-100 peptide was added into each well and incubated for another 30 min at room temperature. Fluorescence polarization signal was measured using Envision Multilabel plate reader (PerkinElmer) with excitation wavelength at

480 nm and emission wavelength at 520 nm.

4.10 Evaluation of Off-target Liabilities DC-TEADin02 (1 mM in DMSO, 1 mL) and GSH/CoA (5 mM in PBS buffer, 1 mL) were added together and the mixture was incubated for indicated time at 37 ℃. The mixture was analyzed by UPLC-MS after 8 h incubation. The analysis process lasted for 10 min with a gradient of 10% acetonitrile to 100% acetonitrile in 0.1% formic acid aqueous solution.

4.11 Pull-down Assay 10 µg His-TEAD4-YBD protein was pre-incubated with DMSO or indicated concentrations of DC-TEADin02 for 2 h. Then the protein samples were captured by nickel agarose beads (GE Healthcare). HEK 293T cells were harvested using lysis buffer (Cell Signaling Technology, 9803) and centrifuged at 14,000 g for 20 min to collect the supernatant containing endogenous YAP protein. The captured His-TEAD4-YBD and supernatant were mixed and incubated for 4 h for efficient binding. After several washes with cell lysis buffer, the SDS-sample loading buffer was added to samples for further immunoblotting experiments.

4.12 Selectivity Profiling. The kinase profiling was conducted using pharma discovery services KinaseProfilerTM (Eurofins Scientific, Dundee, UK) with DC-TEADin02 at 10 µM. 190 kinases were tested with one replicate to ensure the data quality. The detailed mean values of inhibitory activities were shown in supporting information. For epigenetic targets, the experimental conditions were performed in Shanghai Chempartner Co., Ltd as previously described[48].

4.13 Database Analysis.

The differential expression patterns of TEAD4 across normal and tumor tissues were analyzed in web-accessible GENT database (http://medical-genome.kribb.re.kr/GENT/)[49]. The Cancer Genome Atlas (https://cancergenome.nih.gov/) was used for TEAD4 expression analysis based on clinical samples.

4.14 Cell Lines. HEK293T and HCT 116 cells were bought from the American Type Culture Collection (ATCC). The cells were tested for mycoplasma and verified by STR Authentication. All cells were grown at 37 °C with 5% CO2 in media as recommended by the supplier.

4.15 Luciferase Assays. HEK293T cells were seeded in 24-well plates (Corning) at 2*105/well density. Cells were transfected with 200 ng 8xGTIIC-Luc TEADs reporter construct (Addgene, #34615) or TOPFlash Beta-catenin reporter (Addgene, #12456) and 20 ng pGL4.75 Renilla construct (Promega, E6931). The transfected cells were treated with various concentrations of compounds or DMSO control. Luciferase signal was measured using dual-luciferase reporter assay kit (Promega, E1980) according to the manufacturer’s guidelines. Data was collected in EnVision Multilabel Plate Reader (PerkinElmer) and plotted in Prism software.

4.16 Quantitative RT-PCR Experiments Total RNA was extracted from cells using FastPure Cell/Tissue Total RNA Isolation kit (Vazyme Biotech, RC101) and was reverse transcripted into cDNA using HiScript Q RT SuperMix (Vazyme Biotech, R122-01). qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Q331-02) and detected by Quant Studio 6 Flex Real-Time PCR system (Applied

Biosystems). All the experiments were performed according to the manufacturer’s instructions (Vazyme Biotech). The primers were listed in Table S4.

4.17 Cellular Thermal Shift Assay (CETSA) Intact cell CETSA was performed on HCT 116 cells according to the protocol previously described[50]. Briefly, the cells were seeded equally in 15 cm dish and treated with 50 µM DC-TEADin02 or an equal volume of DMSO, respectively, at 37°C for 4 h. Then the cells were digested by trypsin and washed with PBS. After several washes, cells were resuspended in PBS containing freshly added protease inhibitors (Roche) and divided equally into six PCR tubes. Cells in each tube were heated at indicated temperatures for 3 min and kept at room temperature for 3 min. Heated cells were lysed by three cycles of freezing (in liquid nitrogen, 1 min) and thawing (in water at room temperature, 1 min). The cell lysates were centrifuged at 20,000 g for 30 min at 4 °C. The soluble fractions were isolated for immunoblotting analysis. Primary antibodies used were as follows: Anti-TEAD4 antibody (Abcam, ab58310) and Anti-GAPDH rabbit polyclonal antibody (BBI life sciences, D110016).

4.18 Statistical Analyses Statistical analyses were conducted in Prism software. Unpaired two-tailed student’s t test was utilized for the analysis of statistical significance. *P<0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001, N.S. P>0.05.

Appendix A. Supplementary data Supplementary data related to the article could be found at the attached file ‘TEADs supporting information’.

Author Contributions Conception and design: B.Z and C.L.; Development of methodology: W.L., J.W., H.T. and Y.L.; Acquisition of data: W.L., J.W., H.T. J.G. F.L. and LY..; Analysis and interpretation of data: W.L., J.W., H.T., Y.L., H.X., H.M. H.D., Y.Z. and Y. Y. Writing, review, and/or revision of the manuscript: W.L., J.W., H.T., Y.L. and Y.Z. Administrative, technical, or material support: X.Y., H.D., Z.C., P.X., H.Z. and L.Y. Study supervision: H.J., K.C., Y.Z., B.Z., and C.L.

Notes The authors declare no competing financial and non-financial interest.

Acknowledgements We appreciate anonymous referees for comments on this work and are extremely grateful to the staff members of the Large-scale Protein Preparation System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Lab, China for providing technical support and assistance in data collection and analysis. We gratefully acknowledge financial support from the Ministry of Science and Technology of China (2015CB910304 to Y.Z.), the National Natural Science Foundation of China (81625022, 91853205, 81821005 to C.L., 81803438 to H.J., 91753207 to B.Z. and 81430084 to K.C); the National Science and Technology Major Project (2018ZX09711002-004-013, 2018ZX09711002-006-001,

2018ZX09711002-007 to H.J.), K. C.

Wong Education Foundation to C.L and B. Z., Chinese Academy of Sciences (XDA12020353 and XDA12050401 to C.L., XDA12020361 to B. Z.), Science and Technology Commission of Shanghai Municipality (18431907100 to H.J.), National Key R&D Program of China (2018YFA0508200 to B. Z.), Shanghai Rising-Star Program (Grant No. 17QA1405000 to B. Z.),

the National Science and Technology Major project (2018ZX09711002 to T.H.) and China Postdoctoral Science Foundation (2017M621571 to L. Y.).

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