Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer (NSCLC)

Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer (NSCLC) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer (NSCLC) Anqi Li, Ting Ma, Shuai Wang, Yueyang Guo, Qianqian Song, Bin Yu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2593334/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Apr, 2024 Read the published version in Biomedicine & Pharmacotherapy → Version 1 posted You are reading this latest preprint version Abstract Abnormally high expression of lysine-specific demethylase 1A (LSD1) and DCN1 plays a vital role in the occurrence, development, and poor prognosis of non-small cell lung cancer (NSCLC). Accumulating evidence has shown that the development of small-molecule inhibitors dually targeting LSD1 and the UBC12-DCN1 interaction probably have therapeutic promise for cancer therapy. This work reported that WS-384 dually targeted LSD1 and UBC12-DCN1 interactions and evaluated its antitumor effects in vitro and in vivo . Specifically, WS-384 inhibited A549 and H1975 cells viability and decreased colony formation and EdU incorporation. WS-384 could also trigger cell cycle arrest, DNA damage, and apoptosis. Moreover, WS-384 significantly decreased tumor weight and volume in A549 xenograft mice. Mechanistically, WS-384 increased the gene and protein level of p21 by suppressing the neddylation of cullin 1 and decreasing H3K4 demethylation at the CDKN1A promoter. The synergetic upregulation of p21 contributed to cell cycle arrest and the proapoptotic effect of WS-384 in NSCLC cells. Taken together, our proof of concept studies demonstrated the therapeutic potential of dual inhibition of LSD1 and the UBC12-DCN1 interaction for the treatment of NSCLC. WS-384 could be used as a lead compound to develop new dual LSD1/DCN1 inhibitors for the treatment of human diseases in which LSD1 and DCN1 are dysregulated. LSD1 DCN1 Non-small cell lung cancer cancer therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1 Introduction Lung cancer is the most common primary malignant tumor in the world (Ferlay, et al.2015). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of the incidence of lung cancer (Arbour, et al.2019,Chicas-Sett, et al.2020,Choi, et al.2019). Protein neddylation is the process of adding NEDD8, a ubiquitin-like molecule, to target substrate proteins. The neddylation modification is catalyzed by E1 NEDD8-activating enzyme (NAE), E2 NEDD8-conjugating enzymes (UBE2M/UBC12, UBE2F), and E3 NEDD8 ligase (Huang, et al.2004,Zhou, et al.2018). Overexpression of NAE1 and UBC12 has been found in human lung cancers, and their inhibition could suppress tumor growth (Li, et al.2014). The NEDD8-activating enzyme E1 inhibitor MLN4924 completely inhibits the activation of all cullin-RING ligases (CRLs), leading to the accumulation of CRL substrates (Petroski, et al.2005,Zhao, et al.2014). Because each CRL regulates the degradation of specific substrate proteins, it is valuable to develop small molecules that selectively inhibit individual CRL members in biological processes and human diseases (Soucy, et al.2009,Soucy, et al.2009). The crystal structure of the cullin1-RBX1-UBC12-NEDD8-DCN1 complex provides a basis for the design of small-molecule inhibitors suppressing the DCN1-UBC12 protein-protein interaction (Scott, et al.2014). DCN1 is highly expressed in several human tumors, such as primary lung, head, neck, and cervical carcinomas, as an oncogene. DI-591 (Zhou, et al.2017), a triazolo[1,5-a] pyrimidine-based inhibitor WS-383 (Wang, et al.2019), 5-cyano-6-phenyl pyrimidine-based inhibitor DC-2 ( WS-383 and DC-2 were developed by our group) (Zhou, et al.2020), and compound 67 (Hammill, et al.2018) are small molecule inhibitors targeting the DCN1-UBC12 interaction with high-affinity. However, the cellular potencies of these DCN1 inhibitors are still moderate (Zhou, et al.2017,Zhou, et al.2019). Lysine-specific demethylase 1A (LSD1) acts as an enzyme removing mono- and di-methylation from histones or nonhistones, which leads to the repression or activation of gene transcription (Shi, et al.2004,Shi, et al.2005). High levels of LSD1 have been found in diverse malignancies and are closely related to many cellular processes (Ambrosio, et al.2017,Cho, et al.2011,Egolf, et al.2019,Fang, et al.2019,Karakaidos, et al.2019,Wojtala, et al.2019). Numerous LSD1 inhibitors have been reported and underwent clinical trials for cancer therapy, especially for small lung cancer cells (SCLC) and acute myeloid leukemia (AML) (Fang, et al.2019,Li, et al.2019). Our group has focused on LSD1 for a long time and designed a series of LSD1 inhibitors that showed sound antitumor effects (Liu, et al.2020,Wang, et al.2019,Zheng, et al.2013). However, most LSD1 inhibitors have weaker inhibitory effects on solid tumors than other cytotoxic drugs (Maiques-Diaz, et al.2016). Recently, it has been reported that the irreversible LSD1 inhibitor T-3775440 combined with the NAE inhibitor MLN4924 showed powerful synergistic effects in the treatment of AML (Ishikawa, et al.2017). Therefore, designing compounds that could dually inhibit both DCN1 and LSD1 is an effective strategy for treating NSCLC. In a previous study, our group synthesized the compound WS-384 , which suppressed the UBC12-DCN1 interaction and verified its inhibitory effect on cullin1 and cullin3 neddylation (Wang, et al.2019). In this research, we firstly found that WS-384 also had a potential inhibitory effect on LSD1 activity. Therefore, we further investigated the anti-tumor effect of the dual inhibitor WS-384 on NSCLC in vitro and in vivo and explored the underlying mechanisms. Since p21 is the substrate of cullin1, and LSD1 inhibition can increase p21 gene expression by decreasing H3K4 demethylation at the CDKN1A promoter (Liu, et al.2017,Liu, et al.2017,Zhu, et al.2019), we speculate that p21 is the critical regulating factor in the synergistic effect of dual inhibition of DCN1 and LSD1. The results found that WS-384 induced cell cycle arrest and DNA damage by increasing the gene and protein expression of p21 dependent on the inhibition of LSD1 and UBC12-DNC1 interation, respectively, which eventually led to cell apoptosis. Collectively, our work demonstrated that WS-384 could serve as a hit compound for the development of dual LSD1/DCN1 inhibitors for cancer therapy. 2. Materials And Methods 2.1. Chemicals and Reagents WS-384 was synthesized by our group and stored at 4°C. 2.2. Cell lines and cell culture A549 and H1975 cell lines were maintained in RPMI 1640 medium (Biological Industries, Shanghai, China) containing 10% fetal bovine serum (Biological Industries, Shanghai, China). Cell growth was observed under an inverted microscope. All cells were maintained at 37°C and 5% CO 2 in a humidified environment. 2.3. MTT assay The MTT assay determined cytotoxicity. A549 and H1975 cells were seeded in 96-well plates at a density of 3 × 10 3 cells/well and incubated overnight. Then, all cells were changed to a fresh medium containing various concentrations of WS-384 for 24 h, 48 h, and 72 h of treatment. After incubation, MTT was added to each well and incubated for another 4 h. Next, the precipitate was dissolved entirely in 150 \({\mu }\) L DMSO and then shaken on a shaker for 10 min to dissolve and the absorbance was measured at 570 nm. All experiments were repeated in triplicate. 2.4. Clone formation assay H1975 cells and A549 cells were plated in 6-well culture plates at a density of 1000 cells/well and placed in an incubator overnight. Then, the media was replaced with fresh media containing different concentrations of WS-384 for 9 days. After drug administration, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. Subsequently, the cells were washed with ultrapure water three times until the colonies were clear enough. Finally, the cells were stained with a crystal violet solution, photographed, and counted by ImageJ. 2.5. Flow cytometry analysis of cell apoptosis The Annexin V-EGFP apoptosis detection kit (KeyGEN BioTECH, Jiangsu, China). A549 and H1975 cells that underwent apoptosis were evaluated via flow cytometry. A549 and H1975 cells were seeded in 6-well culture plates at a density of 1.8 \(\times\) 10 6 and 2.5 \(\times\) 10 6 , respectively. After incubation overnight, cells were treated with various concentrations of WS-384 . For flow cytometry, 2 \(\times\) 10 6 cells in 200 \({\mu }\) L binding buffer were stained with 2 \({\mu }\) L Annexin V-FITC and 2 \({\mu }\) L PI at 37°C in the dark for 20 min. All cells were analyzed via flow cytometry (488 nm excitation and 530 nm emission filters). 2.6. Flow cytometric analysis of cell cycle A549 and H1975 cells were seeded in 6-well culture plates at a density of 1.8 \(\times\) 10 6 and 2.5 \(\times\) 10 6 , respectively. After incubation overnight, cells were treated with various concentrations of WS-384 . Cells were collected and fixed in 70% prechilled ethanol at -20°C overnight and then stained with propidium iodide (PI) and RNase A (KeyGEN BioTECH, Jiangsu, China). All cells were analyzed via BD Accuri C6 flow cytometry. 2.7. 5-Ethynyl-20-deoxyuridine (EdU) incorporation assay A549 and H1975 cells were seeded in glass-bottom dishes at a density of 5 \(\times\) 10 4 cells/well. After 24 h of treatment with WS-384 , a fresh medium containing EdU labeling agent (RiboBio, Guangzhou, China) was added to the cell culture and incubated for another 4 h. Then, the cells were fixed with 4% paraformaldehyde and incubated with glycine. The cells were stained with an anti-EdU working solution at room temperature, washed with 0.5% Triton X-100 PBS, and then incubated with Hoechst 33342. Finally, cells were observed under a laser confocal scanning microscope (Ma, et al.2017). 2.8. siRNA transfection LSD1, DCN1 and negative control small interfering RNAs (siNC) were transfected into A549 cells with Lipofectamine RNAi max reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Cells were collected and analyzed by flow cytometry or Western blotting after transfection for 72 h. The sequences were as follows: siLSD1 #1, 5′-CUACAUCUUACCUUAGUCATT-3′ (sense), 5′-UGACUAAGGUAAGAUGUAGTT-3′ (antisense); siLSD1#2, 5′-CAGCUGACAUUUGAGGCUATT-3′ (sense), 5′-UAGCCUCAAAUGUCAGCUGTT-3′ (antisense); siNC#, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense), 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense); siDCN1 #1 5′-GGAUAAAGUUCGUCAGUUUTT-3′ (sense), 5′-AAACUGACGAACUUUAUCCTT-3′ (antisense); siDCN1 #2 5 ′-GGACAGGAAGAAGUUAGAATT-3 (sense), 5′-UUCUAACUUCUUCCUGUCCTT-3′ (antisense); siDCN1 #3 5′-GCCAUUGCCUACUGGAACUTT-3′ (sense), 5′-AGUUCCAGUAGGCAAUGGCTT-3′(antisense). 2.9. Cellular thermal shift assay A549 and H1975 cells were collected and then washed twice with PBS. Then, 850 \({\mu }\) L of PBS was added to the cells, and the cells were frozen and thawed repeatedly from liquid nitrogen in a constant temperature water bath 3–5 times. The supernatant was placed into new EP tubes after centrifugation for 20 min at low temperature. The supernatant was added to 10 \({\mu }\) M WS-384 and incubated for 30 min at room temperature. The samples were placed into a PCR instrument, and a loading buffer was added; samples were denatured and used for western blotting. 2.10. Western blot analysis A549 and H975 cells were treated with different concentrations of WS-384 and incubated in an incubator for 48 h. Then, the cells were digested, centrifuged and washed with PBS. The total protein was extracted with RIPA. All samples were kept on ice for 30 min followed by centrifugation at 12000 rpm at 4℃ for 10 min. The supernatant was absorbed and quantified by a BCA protein assay kit (Beyoyime, Hainan, China). Next, the proteins of A549 and H975 cells were separated by sodium dodecyl sulfate 10% SDS-PAGE and transferred to NC membranes by the wet transfer method. After that, the protein in the membranes was incubated with primary antibodies and secondary antibodies, and finally, the protein expression was detected by ECL photoluminescence solution. 2.11. Comet assay The prepared suspension of cells in 0.6% low melting point agarose (LMA) dissolved in PBS was cast onto microscope slides with 0.6% average melting point agarose and laid LMA. The cells were lysed overnight at 4℃ in lysis buffer. DNA was allowed to unwind for 40 min in an electrophoretic solution. Electrophoresis was conducted for 30 min at 25 V and 300 mA. The slides were neutralized with Tris, pH 7.5. The slides were stained with ethidium bromide for 20 min and immediately examined under an Eclipse fluorescence microscope. 2.12. RNA extraction and quantitative RT-PCR A549 and H1975 cells were spread in 12-well plates and treated with WS-384 for 48 h. Next, 500 \({\mu }\) L TRIzol was added to each well. Then, 100 \({\mu }\) L of chloroform was added to each EP tube and centrifuged by cryogenic centrifugation at 12000 rpm for 10 min to absorb the water phase. Isopropanol was added to the EP tube, placed at room temperature for 10 min, and centrifuged at 12000 rpm for 10 min at 4℃; the supernatant was discarded. The RNA precipitate was washed with 75% ethanol and centrifuged at 12000 rpm for 3 min at 4℃, and the supernatant was discarded to obtain RNA precipitate. RNA was dissolved with DEPC water. Then the concentration of nucleic acids was quantified. According to the steps of HiScript Ⅱ Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, China), cDNA was obtained for PCR experiments. The cDNA was diluted according to the expression of the target gene, and PCR was carried out by quantitative fluorescence PCR. 2.13. Animal study The SPF animal lab approved this study of Zhengzhou University. Female BALB/c nude mice (6–8 weeks, 16–18 g) were obtained from Hunan Shrek Jingda Experimental Animal Co. Ltd. Nude mice were inoculated with A549 cells at a density of approximately 1.5 \(\times\) 10 7 cells/200 \({\mu }\) L. Then the nude mice were regrouped after 6 days. WS-384 was administered to nude mice by oral administration at 0, 25 mg/kg, and 50 mg/kg every day. When the average tumor volume reached approximately 900 mm 3 , the mice were euthanized with CO 2 , measured the tumor volume and removed the corresponding organs. During the experiments, nude mice were cared for and handled strictly according to the guidelines of the Animal Ethics Committee of the School of Pharmacy, Zhengzhou University. All experimental protocols were approved by the Animal Ethics Committee of School of Pharmacy, Zhengzhou University. 2.14. Haematoxylin and eosin (H&E) staining The transplanted tumors and central organs of nude mice were fixed in paraformaldehyde for one week and then embedded in paraffin (Feldman, et al.2014). After paraffin slicing, dewaxing, dyeing, dehydration, sealing and other steps were carried out, a microscope was used to take pictures. 2.15. Molecular docking Chem3D software was used to build groups and optimize compound configurations through MM2 molecular mechanics. Then, Mgltools1.5.6 and AutodockTools1.5.6 were used to open the small ligand molecule in PMV, and hydrogen and charge were added to detect the root of the ligand. The 3D structure of the protein was downloaded from the RCSB Protein Data Bank and opened in Mgltools1.5.6 PMV. The original ligands and solvents were removed, all hydrogen atoms were added, the Gasteiger charge was calculated, and nonpolar hydrogen was merged. According to the coordinate file of the original ligand, we determined the grid box coordinates and box size of the docking site, and the Mark genetic algorithm generated the docking structure. The related energy in the grid was calculated by AutoGrid, and semiflexible docking was performed in Autodock. The composition with the lowest point was selected for the analysis of the docking structure pattern and the diagram was made using PyMOL. 2.16. Statistical analysis All experiments were conducted more than three times. The results were analyzed using GraphPad Prism version 8.0.1 to perform one-way ANOVAs and t-tests (GraphPad Software, San Diego, CA, USA). The results are given as the mean ± SD. A p-value less than 0.05 was considered statistically significant. 3 Results 3.1 WS-384 inactivated cullin1 and cullin3 neddylation and accumulated CRL substrates in A549 and H1975 cells. According to our previous reports, WS-384 is an inhibitor of the UBC12-DCN1 protein-protein interaction (Wang, et al.2019). The chemical formula of WS-384 is shown in Fig. 1 A (Wang, et al.2019). To gain further insights into the interaction of WS-384 with target proteins, we conducted molecular modeling studies by docking the compound WS-384 into DCN1. The internal reorganization of binding is shown in Fig. 1 B, and WS-384 could bind well to the binding site of DCN1. WS-384 formed a hydrogen bond interaction with the amino acid residue PRO97 of DCN1 and fit relatively well into the hydrophobic cleft formed by DCN1. WS-384 inhibited the UBC12-DCN1 interaction with an IC 50 value of 14.81 \(\pm\) 0.95 nM. As reported, UBC12-DCN1 mainly mediates cullin1 and cullin3 neddylation (Zhou, et al.2017). The results found that WS-384 blocked the neddylation of cullin1 and cullin3 but had no effect on the neddylation of other cullin members in NSCLC cells (Figs. 1 C and D ). Additionally, WS-384 increased the levels of p21 and Bim (substrates of cullin 1) and NRF-2 (substrate of cullin 3) (Fig. 1 E and F ). In summary, these results suggested that WS-384 selectively inhibited the neddylation of cullin1 and cullin3 by binding to DCN1 and blocking the interaction between UBC12-DCN1 proteins but had no effect on the neddylation of other cullin family members in NSCLC. 3.2 Effects of WS-384 on LSD1 activity in A549 and H1975 cells Next, we explored whether WS-384 also had an inhibitory effect on LSD1. Therefore, we first conducted molecular modeling studies by docking the compound WS-384 into LSD1. The internal reorganization of the binding assay is shown in Fig. 2 A. WS-384 could dock into the active binding pocket of LSD1 and form a hydrogen bond interaction with the amino acid residue HIS564 of LSD1. WS-384 fit relatively well into the hydrophobic cleft formed by LSD1. WS-384 was docked into the active binding pocket of LSD1 and WS-384 inhibited the activity of LSD1 with an IC 50 value of 338.79 \(\pm\) 2.53 nM. Second, we employed a cellular thermal shift assay (CETSA) (Martinez Molina, et al.2013) to assess the target engagement of WS-384 in A549 and H1975 cells. As shown in Fig. 2 B, the thermal stability of LSD1 protein was enhanced by WS-384 at 56°C in H1975 cells and 52°C in A549 cells. Furthermore, WS-384 enhanced the stability of LSD1 protein in a dose-dependent manner, which indicated the cellular target engagement of WS-384 in NSCLC cells. LSD1 can catalytically remove methyl groups from LSD1 substrates H3K4me1/2 (Fu, et al.2017). Herein, we investigated the effects of WS-384 on the methylation of histone H3K4 in A549 and H1975 cells. As demonstrated in Figs. 2 C and D , WS-384 induced the accumulation of H3K4me1/2 in A549 and H1975 cells in a concentration-dependent manner, which was consistent with GSK-LSD1 (an LSD1 inhibitor). These results indicated that WS-384 could also effectively inhibit the activity of LSD1. 3.3 WS-384 inhibited cell proliferation and induced cell apoptosis in a concentration-dependent manner in A549 and H1975 cells. To test whether WS-384 had cytotoxic effects on A549 and H1975 cells, we first conducted an MTT assay. The results showed that WS-384 significantly inhibited A549 and H1975 cell growth dose- and time-dependent (Fig. 3 A and B ). The IC 50 values of WS-384 in A549 and H1975 cells are shown in Table 1 . In the clone formation assays, we observed a significant decrease in the colorogenic ability of A549 and H1975 cells with increased WS-384 concentrations and even complete disappearance of colony formation (Figs. 3 C and D ). To further confirm the effect of WS-384 on cell proliferation, and EdU incorporation assay was performed. The data showed that WS-384 significantly reduced the number of EdU cells compared to the control group's dose-dependent manner (Fig. 3 E and F ). These data revealed that WS-384 inhibited the proliferation of NSCLC cells. Table 1 Cytotoxicity of WS-384 in A549 and H1975 cells. Data are shown as the mean ± SD (n = 3). IC 50 ± SD (µM) A549 H1975 24 h 6.67 ± 0.71 4.23 ± 0.74 48 h 5.87 ± 1.29 2.43 ± 0.41 72 h 3.01 ± 0.44 2.15 ± 0.26 Apoptosis is the leading cause of cell death, so we next examined the effect of WS-384 on cell apoptosis by flow cytometry analysis. As shown in Fig. 4 A, when A549 and H1975 cells were treated with WS-384 , cell apoptosis occurred obviously. At the same time, WS-384 treatment increased the expression of caspase family proteins such as cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, and cleaved PARP (Figs. 4 B and C ). The results suggested that WS-384 could significantly promote NSCLC cell apoptosis. 3.4 P21 acted as an important intermediate regulator responsible for the anticancer effect of WS-384 . WS-384 selectively inhibited neddylation of cullin1/3 over other cullin members and induced the accumulation of downstream proteins p21, Bim and NRF-2 in both A549 and H1975 cells. NRF-2, as a transcription factor, plays a vital role in the regulation of the antioxidant response and lowers ROS levels (Basak, et al.2017,Zhou, et al.2017). The ROS content is vital for cellular function and survival signaling. Therefore, we investigated the cellular effect of WS-384 on ROS generation in NSCLC cells. However, as shown in Fig. 5 A, WS-384 had no significant effect on ROS levels in A549 and H1975 cells, which suggested that the proapoptotic effect of WS-384 was not caused by ROS and NRF-2 might not be an important regulator for the anticancer effect of WS-384 . It has been reported that LSD1 inhibition increases H3K4 methylation on the CDKN1A promoter, which leads to the transcriptional activation of p21 and p21, the typical substrate protein of cullin1 (Liu, et al.2017,Zhu, et al.2019). Therefore, we further investigated the expression of p21, and the results showed that the gene and protein expression of p21 were both significantly increased after WS-384 treatment in A549 and H1975 cells (Fig. 5 B, C, D, and E ). Taken together, these results indicate that p21, not NRF-2, acts as an important intermediate regulator to inhibit A549 and H1975 cell proliferation. 3.5 WS-384 induced cell cycle arrest and DNA damage in A549 and H1975 cells. p21 is an important protein that regulates the cell cycle, and dysregulation is a vital reason for cell proliferation inhibition. Therefore, we stained NSCLC cells with PI and analyzed the cell cycle using flow cytometry. As shown in Fig. 6 A, WS-384 significantly induced G2/M phase arrest in both A549 and H1975 cells. It is generally acknowledged that p21 can bind and inhibit the activity of cyclin CDK4/6 complexes and then play a checkpoint regulator role in cell cycle arrest (Dongoran, et al.2020,Han, et al.2019,Liu, et al.2017,Vilgelm, et al.2019,Xie, et al.2018,Zhu, et al.2019). Therefore, we detected the levels of cell cycle regulatory proteins including cyclin B1, p-CDC2, CDK4 and CDK6. Figures 6 B and C demonstrated that these proteins were downregulated in the WS-384 treated group. To further explore how WS-384 promoted cell cycle arrest and apoptosis, we next investigated the effect of WS-384 on DNA damage. The accumulation of \({\gamma }\) -H2AX (a marker protein of DNA damage) in A549 and H1975 cells was increased after treatment with WS-384 (Fig. 7 A and B ), and this was further confirmed by the comet assay, which revealed an increased percentage of DNA damage (Fig. 7 C and D ). In summary, all these data suggested that WS-384 could induce cell cycle arrest and DNA damage in NSCLC cells. 3.6 Dual inhibition of LSD1 and DCN1-UBC12 interaction led to proliferation inhibition and proapoptotic effects. To further verify the proliferation inhibition and proapoptosis effects induced by the dual inhibition of LSD1 and DCN1, we performed a small interfering RNA (siRNA) transfection experiment (Zhou, et al.2019,Zhu, et al.2019). Quantitative analysis showed that the silencing efficiencies of siLSD1 #2 and siDCN1 #3 were 72.65% and 57.07%, respectively (Figs. 8 A and B ). Moreover, silencing DCN1 by three siRNAs in both A549 cells decreased the DCN1 and neddylation of cullin1 and cullin3. Silencing LSD1 with two siRNAs in A549 cells decreased the LSD1 level and caused the accumulation of H3K4me2 (Figs. 8 A and B ). Therefore, siLSD1 #2 and siDCN1 #3 were used for subsequent experiments. Cell proliferation was also more pronounced after inhibition by siLSD1 and siDCN1, as measured by MTT and colony-forming assays (Fig. 8 C and D ). As shown in Fig. 8 E, the protein level of p21 was significantly increased by LSD1 or DCN1 siRNA, and the relative level of p21 was higher in the combination LSD1 siRNA and DCN1 siRNA treatment group than in the LSD1 siRNA or DCN1 siRNA alone group, which was consistent with the increased expression induced by WS-384 . As indicated in Fig. 8 F, the levels of celaved caspase family proteins (cleaved caspase 3, cleaved caspase 9, and cleaved PARP) were significantly increased by pronounced LSD1 and DCN1 silencing, and proapoptotic, proliferation inhibition and DNA damage inducing effects were simultaneously verified by flow cytometry (Fig. 8 G). Moreover, the expression level of \({\gamma }\) -H2AX and the percentage of DNA damage were significantly increased in cells treated with siLSD1 and siDCN1 compared with cells treated with siLSD1 or siDCN1 alone (Fig. 8 H and I ). Taken together, these data indicated that the pro-apoptosis, proliferation inhibition and DNA damage inducing effect of WS-384 on NSCLC cells was caused by dual inhibition of the LSD1 and DCN1-UBC12 interaction. 3.7 Anti-tumor effect of WS-384 in A549 xenograft mice. To further evaluate the anti-tumor activity of WS-384 in vivo , xenograft models with subcutaneously implanted A549 cells were established. Tumor-bearing mice were treated with vehicle or WS-384 25 mg/kg and 50 mg/kg by oral administration once a day for 39 consecutive days (Fig. 9 A). After drug administration for 39 days, mice were killed, and tumors were harvested and weighed (Figs. 9 B and C ). From the results, tumor growth was significantly suppressed after 15 days in the 50 mg/kg group compared to the control. In addition, tumor weight in the 50 mg/kg group was significantly lower than that of the control group, and there were no significant changes in body weight during administration (Figs. 9 D and E ). We next homogenized the tumor tissue and extracted the tissue protein to further explore the dual-target inhibition effect of WS-384 in vivo . We found that WS-384 suppressed the neddylation of cullin1 and cullin3 and increased the methylation of histone H3K4 by inhibiting DCN1 and LSD1, respectively (Fig. 9 F). The expression level of p21 and γ-H2AX, as well as cleaved caspase family proteins, were all increased (Fig. 9 G). Finally, we investigated the potential side effects of WS-384 on the main organs. As shown in Fig. 9 H, H&E staining results demonstrated no apparent toxicity in the heart, liver, spleen, lung, or kidney after treatment with WS-384 . Additionally, there was no significant difference in ALT, AST, uric acid, or creatinine, consistent with the histopathological results (Fig. 9 I). Taken together, these results suggested that WS-384 could effectively inhibit the growth of NSCLC tumors with low toxicity. 4. Discussion NSCLC is one of the most common human cancers with high morbidity and mortality. Novel therapeutic strategies and compounds with high efficacy and low toxicity are urgently needed. As reported, DCN1 is abnormally highly expressed in many squamous cell carcinomas and plays an essential role in the modulation of the neddylation pathway and tumorigenesis (Broderick, et al.2010,Sarkaria, et al.2006,Yoo, et al.2012). DCN1 could interact with both cullin1/3 and UBC12 and further facilitate cullin neddylation. Several studies have reported some inhibitors targeting the DCN1-UBC12 protein-protein interaction, and unlike MLN4924, these inhibitors showed no significant cytotoxicity (Wang, et al.2019,Zhou, et al.2017). Recently, our group also developed a potent and selective DCN1-UBC12 interaction inhibitor WS-383 (Wang, et al.2019). In this study, we further evaluated the inhibitory effect of WS-384 , an analog of WS-383 , on the DCN1-UBC12 protein-protein interaction and found that WS-384 could effectively inhibit cullin 1 and cullin 3 neddylation in NSCLC cells (Fig. 3 ). Several studies have revealed that LSD1 is an important oncogene inducing tumorigenesis. However, the cytotoxicity of a single LSD1 inhibitor is usually limited in solid tumors, so it is necessary to enhance the anti-tumor effect of the small molecule inhibitor. Based on the structure of LSD1 and DCN1-UBC12, we speculate that it might be possible to design an inhibitor targeting LSD1 and DCN1. We found that WS-384 occupied the FAD active pocket in LSD1 and the DCN1 active pocket, so we inferred that WS-384 had a similar binding pattern at the active sites of LSD1 and DCN1 from the docking model. Moreover, molecular docking analysis and cellular activity experiment results showed that WS-384 could adapt to the ligand-binding pocket of LSD1 and inhibit the activity of LSD1. WS-384 could block the activity of the LSD1 and DCN1-UBC12 interaction at the same time. Interestingly, this finding suggested that WS-384 was a dual inhibitor targeting the LSD1 and DCN1-UBC12 interaction. Next, we tested whether WS-384 had a potential anti-proliferative effect on lung carcinoma cells. First, we used the MTT assay to detect whether WS-384 had cytotoxic effect, and the results showed that WS-384 could significantly decrease cell viability. Second, the cell colony formation and EdU incorporation experiments showed that WS-384 significantly reduced the number of colon and EdU-positive cells compared to the control group. These data indicated that WS-384 obviously inhibited NSCLC cell proliferation. For most compounds that have cytotoxic effects, apoptosis is the main cause of cell death; thus, in our study, we detected cell apoptosis by flow cytometry analysis. When A549 and H1975 cells were treated with WS-384 , cell apoptosis was obvious. Taken together, WS-384 could significantly inhibit cell proliferation and induce NSCLC cell apoptosis. In addition, WS-384 also inhibited tumor growth and induced cell apoptosis in vivo . Our findings also showed that the anti-tumor activity of simultaneous knockdown of LSD1 and DCN1 was the same as that of WS-384 treatment, which further confirmed that the anti-tumor effect of WS-384 was due to dual inhibition of the LSD1 and DCN1-UBC12 interaction. The cyclin-dependent kinase inhibitor p21 plays a vital role in cell cycle regulation, apoptosis and DNA damage. P21, as a downstream protein of cullin1 was significantly increased by WS-384 . We also found that WS-384 could elevate the gene expression of p21 in NSCLC cells, which is consistent with previous studies (Wang, et al.2019). Liangsong Zhu et al reported that LSD1 could directly bind to the p21 promoter, induce H3K4me2 demethylation, and further regulate p21 transcription (Zhu, et al.2019). Therefore, it was suggested that elevated p21 is an important regulatory factor in the synergistic anti-tumor effect of WS-384 . As p21 is an important protein that regulates the cell cycle, we next detected the cell cycle by flow cytometry. The data showed that WS-384 induced G2/M phase arrest and affected cell cycles regulatory proteins such as cyclin B1, p-CDC2, CDK4, and CDK6. From the results, we concluded that p21 was a critical intermediate regulator mediating the anticancer effect of WS-384 . In conclusion, we proposed for the first time the practical anti-tumor effect of WS-384 targeting LSD1 and the DCN1-UBC12 interaction. Assays of cullin protein neddylation, histone H3K4 methylation and knockdown of LSD1 or DCN1 demonstrated the highly active and selective inhibition of the LSD1 and DCN1-UBC12 interaction by WS-384 . Additionally, WS-384 could induce cell cycle arrest and apoptosis in vitro by modulating the p21 signaling pathway. In vivo experiments further verified the anti-tumor effect of WS-384 in A549 bearing xenograft mouse models. Our results lay a theoretical foundation for the clinical application of LSD1 and DCN1-UBC12 protein-protein interaction inhibitors in the treatment of NSCLC. Declarations Funding This work was supported by the National Natural Science Foundation of China (No. 82020108030, U21A20416, 82104279, 81903770, 81973177 and 22277110), Program for Science & Technology Innovation Talents in Universities of Henan Province (No. 21HASTIT045), Key scientific and technological projects of Henan Province (No. 202102310162, 222102310125), China Postdoctoral Science Foundation (Nos. 2019M662550, 2019M662556) and the Natural Science Foundation of Henan Province (no. 222300420069). Author contributions Conception and design: Siqi Feng, Hongmin Liu, Ting Ma, Bin Yu and Anqi Li designed the research. Anqi Li, Ting Ma, Yueyang Guo and Qianqian Song performed the experiments and Anqi Li carried out data analysis. Anqi Li and Ting Ma wrote the manuscript and Siqi Feng revised the manuscript. All of the authors have read and approved the final manuscript. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Code availability Not applicable. Ethical statement All animal experiments and experimental procedures were approved by the Research Ethics Committee of the Drug Research Institute of Zhengzhou University and were in strict compliance with the Guide for the Care and Use of Laboratory Animals of the Drug Research Institute of Zhengzhou University. Consent to participate All participants signed a document of informed consent. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2593334","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":176740887,"identity":"4cdb9b3e-279c-4146-a3b9-79518aa566ee","order_by":0,"name":"Anqi Li","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anqi","middleName":"","lastName":"Li","suffix":""},{"id":176740888,"identity":"0e2c84a9-c65e-4d29-b95b-4a60ff6e18f2","order_by":1,"name":"Ting Ma","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Ma","suffix":""},{"id":176740889,"identity":"bf11c290-2207-4682-8ef1-be58556710f5","order_by":2,"name":"Shuai Wang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shuai","middleName":"","lastName":"Wang","suffix":""},{"id":176740890,"identity":"2f35393e-42ea-4ef4-ad45-2057fc79c6a8","order_by":3,"name":"Yueyang Guo","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yueyang","middleName":"","lastName":"Guo","suffix":""},{"id":176740891,"identity":"45624d8d-6207-47ea-9258-c8004e80ed1c","order_by":4,"name":"Qianqian Song","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qianqian","middleName":"","lastName":"Song","suffix":""},{"id":176740892,"identity":"c3b8a1fb-b6ed-469e-a27a-44300e83a73c","order_by":5,"name":"Bin Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArklEQVRIiWNgGAWjYFACHoYDDBUQpgQJWs6QqoWBsY0ULQY3cg8eLpxXZ29wgPngbR4GuzwitOQlHJ65jY3Z4ABbsjUPQ3IxEVpyDA7zbuNhMzjAYyYN9FdiA3Fa5kjwGBzg/0aKlgYDCaAtbMRpkTzzLuEwz7EEA8nDbMaWcwySCWvhO557+DNPTZ093/HmhzfeVNgR1qJwAMZiBruTkHogkCdo6CgYBaNgFIwCABKBOJFnQiaIAAAAAElFTkSuQmCC","orcid":"","institution":"Zhengzhou University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yu","suffix":""},{"id":176740893,"identity":"f9e89f3d-0bb9-48b7-b2e4-3b0478250604","order_by":6,"name":"Siqi Feng","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siqi","middleName":"","lastName":"Feng","suffix":""},{"id":176740894,"identity":"aa9b66f4-4f43-40f3-b644-ebcb0b52d402","order_by":7,"name":"Hongmin Liu","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongmin","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2023-02-16 06:29:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2593334/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2593334/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1016/j.biopha.2024.116240","type":"published","date":"2024-04-01T15:13:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":33181338,"identity":"ad8b0fd6-4f61-4002-8f52-4f0952fdf6e3","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1849164,"visible":true,"origin":"","legend":"\u003cp\u003eCellular effects of \u003cem\u003eWS-384\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eon cullin neddylation and CRL substrates in A549 and H1975 cells. (A) The chemical formula of \u003cem\u003eWS-384\u003c/em\u003e. (B) Docking results of \u003cem\u003eWS-384\u003c/em\u003e by Autodock. (C, D) The expression of cullin1, cullin2, cullin3, cullin4A, and cullin5 after treatment with \u003cem\u003eWS-384\u003c/em\u003e for 48 h in A549 and H1975 cells. (E, F) The expression of Bim, p21, and NRF-2 after treatment with compounds \u003cem\u003eWS-384\u003c/em\u003efor 48 h in A549 and H1975 cells. GAPDH was used as the loading control. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001; **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/4203c5ed8e12c944fa5171b4.png"},{"id":33181341,"identity":"b06974ec-0208-4ff8-a5f7-7f0d7c23a935","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1074092,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e targeted LSD1 and inhibited the migration of NSCLC cells. (A) Docking results of \u003cem\u003eWS-384\u003c/em\u003e by Autodock. (B) Enhancement of DCN1 protein thermal stability by \u003cstrong\u003eWS-384\u003c/strong\u003e in A549 and H1975 cells. (C, D) Histone methylation in A549 and H1975 cells after treatment with \u003cstrong\u003eWS-384\u003c/strong\u003e for 48 h. The expression of H3K4me1/2 were determined by Western blot. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/89d8393727bdacfa456a5144.png"},{"id":33184612,"identity":"30ee0f99-5cb9-4d88-830c-8b7f86ce16fe","added_by":"auto","created_at":"2023-02-20 15:53:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":869348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e inhibited the proliferation of A549 and H1975 cells. (A, B) A549 and H1975 cells were treated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003e, and cell viability was measured via MTT assay at different time points. (C, D) The effect of \u003cem\u003eWS-384\u003c/em\u003e on the clonogenic ability of A549 and H1975 cells. (E, F) EdU staining of A549 and H1975 cells. Cells were incubated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003efor 48 h and then observed using confocal laser scanning microscopy. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001 compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/f0a96cd96280afcb364f47d8.png"},{"id":33182899,"identity":"b69cdb06-6cb6-4f3f-b8df-50f651305c97","added_by":"auto","created_at":"2023-02-20 15:45:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1207643,"visible":true,"origin":"","legend":"\u003cp\u003eCompound \u003cem\u003eWS-384\u003c/em\u003e induced apoptosis in A549 and H1975 cells. (A) A549 and H1975 cells were incubated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003e for 48 h, and apoptosis was detected by flow cytometry with Annexin V-FITC and PI staining. (B, C) Western blot analysis with antibodies specific for cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved PARP and β-actin was used as the loading control. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/8f3ded5583e4b8e6e7eb7c57.png"},{"id":33181343,"identity":"5a434284-35b4-4ac4-a0bd-36336f1bfb79","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":485995,"visible":true,"origin":"","legend":"\u003cp\u003eP21 acts as an intermediate regulator in A549 and H1975 cells. (A) A549 and H1975 cells were treated with the indicated concentrations of \u003cem\u003eWS-384\u003c/em\u003e for 24 h. The levels of ROS were detected by flow cytometry. (B, C) The expression of p21 after treatment with compound \u003cem\u003eWS-384\u003c/em\u003e for 48 h in A549 and H1975 cells. β-actin was used as a loading control. (D, E) qRT-PCR was used to detect p21 mRNA expression in A549 and H1975 cells. Values are presented as the mean ± SD of three independent experiments * \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, ** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/2997ba4f681d68f68d8598c0.png"},{"id":33181346,"identity":"d20bde59-45a0-4e7d-a835-d46971659083","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":750168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e induced cell cycle arrest in A549 and H1975 cells. (A) Cell flow cytometry was used to detect changes in the cell cycle in A549 and H1975 cells treated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003efor 48 h. (B, C) CDK4, CDK6, p-CDC2, and cyclin B1 protein expression in A549 and H1975 cells after \u003cem\u003eWS-384\u003c/em\u003e treatment for 48 h was determined by Western blotting; β-actin was used as the reference protein. Each value is represented as the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/adf8f2217446192a981d81e4.png"},{"id":33184613,"identity":"a445a5e4-e6f5-454f-bee2-50b5ea297bb8","added_by":"auto","created_at":"2023-02-20 15:53:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1428079,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e induced DNA damage in A549 and H1975 cells. (A, B) γ-H2AX protein expression in A549 and H1975 cells after \u003cem\u003eWS-384\u003c/em\u003e treatment for 48 h was determined by Western blotting; β-actin was used as the reference protein. (C, D) A549 and H1975 cells were treated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003e, and then DNA damage was examined by comet assay. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the control by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/023ece1e03a8ccc5e07fee70.png"},{"id":33182901,"identity":"b874b222-e3e4-4d7d-a53f-66784d20e4a3","added_by":"auto","created_at":"2023-02-20 15:45:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1309538,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of inhibition of LSD1 and DCN1-UBC12 interaction on NSCLC cells. (A) LSD1, H3K4me2 and DCN1 protein levels were detected by Western blot after A549 cells were transfected with small interfering RNAs against LSD1 for 48 h. (B) DCN1, cullin1, cullin3 and LSD1 protein expression was examined in A549 cells transfected with DCN1 siRNA. GAPDH was used as a loading control. (C) The viability of A549 cells transfected with LSD1 and DCN1 siRNA was examined using the MTT assay for 48 h. (D) The effect of LSD1 and DCN1 siRNA on the clonogenic ability of A549 cells. (E) p21 protein levels in A549 cells after LSD1 and DCN1 silencing treatment for 48 h, as determined by Western blotting; GAPDH was used as the reference protein. (F) Cleaved caspase 3, cleaved caspase 9, and cleaved PARP protein expression in A549 cells after LSD1 and DCN1 siRNA treatment for 48 h, as determined by Western blotting; GAPDH was used as the reference protein. (G) Apoptosis of A549 cells induced by LSD1 and DCN1 siRNA treatment for 48 h was detected by Annexin-V FITC and PI staining. (H) -H2AX protein expression in A549 cells after LSD1 and DCN1 siRNA treatment for 48 h, as determined by Western blotting; GAPDH was used as the reference protein. (I) A549 cells were treated with LSD1 and DCN1 siRNA for 48 h, and then DNA damage was examined by comet assay. Each value represents the mean ± SD of at least three independent experiments. * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the control by one-way ANOVA. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05;\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003e \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 compared to the siLSD1 #2 or siDCN1 #3 by one-way ANOVA.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/7fa15aac1de54f55367d128b.png"},{"id":33181348,"identity":"68d93617-4a3c-45c5-ae81-063328b30cb4","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":14966333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e suppresses tumor growth in A549 xenografts. (A) Tumor sizes over time in mice treated with control and \u003cem\u003eWS-384\u003c/em\u003e. (B) The tumor weight in each group (n = 6). (C) The nude mice weight in each group. (D) Tumor volume was measured by calipers once every three days when the tumor reached approximately 120 mm\u003csup\u003e3\u003c/sup\u003e. (F, G) Cullin 1, cullin 3, p21, H3K4me2, and -H2AX protein expression in the tumor tissue after \u003cem\u003eWS-384\u003c/em\u003e treatment for 36 days, as determined by Western blotting; GAPDH was used as the reference protein (n = 3). (H) H\u0026amp;E staining of heart, liver, spleen, lung and kidney sections of nude mice. (I) AST, ALT, uric acid and creatinine were determined after mice were killed. The results are expressed as the mean ± SD. (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/197d591952057d7762cada33.png"},{"id":33181344,"identity":"536b11f5-ec95-408d-9b17-d5ed73622852","added_by":"auto","created_at":"2023-02-20 15:37:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":672385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e, the first-in-class LSD1/DCN1 dual inhibitor, shows therapeutic promise for treating non-small cell lung cancer by increasing the levels of p21.\u003c/p\u003e","description":"","filename":"figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/e5906317dc2dcd234d7f147f.png"},{"id":52842827,"identity":"762d97b5-8e94-4bbe-989d-c587417a6b76","added_by":"auto","created_at":"2024-03-17 15:13:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4571104,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2593334/v1/2d7a366e-2fbe-4437-a9ec-66044354c8f7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer (NSCLC)","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLung cancer is the most common primary malignant tumor in the world (Ferlay, et al.2015). Non-small cell lung cancer (NSCLC) accounts for approximately 85% of the incidence of lung cancer (Arbour, et al.2019,Chicas-Sett, et al.2020,Choi, et al.2019). Protein neddylation is the process of adding NEDD8, a ubiquitin-like molecule, to target substrate proteins. The neddylation modification is catalyzed by E1 NEDD8-activating enzyme (NAE), E2 NEDD8-conjugating enzymes (UBE2M/UBC12, UBE2F), and E3 NEDD8 ligase (Huang, et al.2004,Zhou, et al.2018). Overexpression of NAE1 and UBC12 has been found in human lung cancers, and their inhibition could suppress tumor growth (Li, et al.2014). The NEDD8-activating enzyme E1 inhibitor MLN4924 completely inhibits the activation of all cullin-RING ligases (CRLs), leading to the accumulation of CRL substrates (Petroski, et al.2005,Zhao, et al.2014). Because each CRL regulates the degradation of specific substrate proteins, it is valuable to develop small molecules that selectively inhibit individual CRL members in biological processes and human diseases (Soucy, et al.2009,Soucy, et al.2009).\u003c/p\u003e \u003cp\u003eThe crystal structure of the cullin1-RBX1-UBC12-NEDD8-DCN1 complex provides a basis for the design of small-molecule inhibitors suppressing the DCN1-UBC12 protein-protein interaction (Scott, et al.2014). DCN1 is highly expressed in several human tumors, such as primary lung, head, neck, and cervical carcinomas, as an oncogene. \u003cem\u003eDI-591\u003c/em\u003e (Zhou, et al.2017), a triazolo[1,5-a] pyrimidine-based inhibitor \u003cem\u003eWS-383\u003c/em\u003e (Wang, et al.2019), 5-cyano-6-phenyl pyrimidine-based inhibitor \u003cem\u003eDC-2\u003c/em\u003e (\u003cem\u003eWS-383\u003c/em\u003e and \u003cem\u003eDC-2\u003c/em\u003e were developed by our group) (Zhou, et al.2020), and compound \u003cb\u003e67\u003c/b\u003e (Hammill, et al.2018) are small molecule inhibitors targeting the DCN1-UBC12 interaction with high-affinity. However, the cellular potencies of these DCN1 inhibitors are still moderate (Zhou, et al.2017,Zhou, et al.2019).\u003c/p\u003e \u003cp\u003eLysine-specific demethylase 1A (LSD1) acts as an enzyme removing mono- and di-methylation from histones or nonhistones, which leads to the repression or activation of gene transcription (Shi, et al.2004,Shi, et al.2005). High levels of LSD1 have been found in diverse malignancies and are closely related to many cellular processes (Ambrosio, et al.2017,Cho, et al.2011,Egolf, et al.2019,Fang, et al.2019,Karakaidos, et al.2019,Wojtala, et al.2019). Numerous LSD1 inhibitors have been reported and underwent clinical trials for cancer therapy, especially for small lung cancer cells (SCLC) and acute myeloid leukemia (AML) (Fang, et al.2019,Li, et al.2019). Our group has focused on LSD1 for a long time and designed a series of LSD1 inhibitors that showed sound antitumor effects (Liu, et al.2020,Wang, et al.2019,Zheng, et al.2013). However, most LSD1 inhibitors have weaker inhibitory effects on solid tumors than other cytotoxic drugs (Maiques-Diaz, et al.2016). Recently, it has been reported that the irreversible LSD1 inhibitor T-3775440 combined with the NAE inhibitor MLN4924 showed powerful synergistic effects in the treatment of AML (Ishikawa, et al.2017). Therefore, designing compounds that could dually inhibit both DCN1 and LSD1 is an effective strategy for treating NSCLC.\u003c/p\u003e \u003cp\u003eIn a previous study, our group synthesized the compound \u003cem\u003eWS-384\u003c/em\u003e, which suppressed the UBC12-DCN1 interaction and verified its inhibitory effect on cullin1 and cullin3 neddylation (Wang, et al.2019). In this research, we firstly found that \u003cem\u003eWS-384\u003c/em\u003e also had a potential inhibitory effect on LSD1 activity. Therefore, we further investigated the anti-tumor effect of the dual inhibitor \u003cem\u003eWS-384\u003c/em\u003e on NSCLC \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e and explored the underlying mechanisms. Since p21 is the substrate of cullin1, and LSD1 inhibition can increase p21 gene expression by decreasing H3K4 demethylation at the CDKN1A promoter (Liu, et al.2017,Liu, et al.2017,Zhu, et al.2019), we speculate that p21 is the critical regulating factor in the synergistic effect of dual inhibition of DCN1 and LSD1. The results found that \u003cem\u003eWS-384\u003c/em\u003e induced cell cycle arrest and DNA damage by increasing the gene and protein expression of p21 dependent on the inhibition of LSD1 and UBC12-DNC1 interation, respectively, which eventually led to cell apoptosis. Collectively, our work demonstrated that \u003cem\u003eWS-384\u003c/em\u003e could serve as a hit compound for the development of dual LSD1/DCN1 inhibitors for cancer therapy.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and Reagents\u003c/h2\u003e \u003cp\u003e \u003cem\u003eWS-384\u003c/em\u003e was synthesized by our group and stored at 4\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell lines and cell culture\u003c/h2\u003e \u003cp\u003eA549 and H1975 cell lines were maintained in RPMI 1640 medium (Biological Industries, Shanghai, China) containing 10% fetal bovine serum (Biological Industries, Shanghai, China). Cell growth was observed under an inverted microscope. All cells were maintained at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. MTT assay\u003c/h2\u003e \u003cp\u003eThe MTT assay determined cytotoxicity. A549 and H1975 cells were seeded in 96-well plates at a density of 3 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well and incubated overnight. Then, all cells were changed to a fresh medium containing various concentrations of \u003cem\u003eWS-384\u003c/em\u003e for 24 h, 48 h, and 72 h of treatment. After incubation, MTT was added to each well and incubated for another 4 h. Next, the precipitate was dissolved entirely in 150 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL DMSO and then shaken on a shaker for 10 min to dissolve and the absorbance was measured at 570 nm. All experiments were repeated in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Clone formation assay\u003c/h2\u003e \u003cp\u003eH1975 cells and A549 cells were plated in 6-well culture plates at a density of 1000 cells/well and placed in an incubator overnight. Then, the media was replaced with fresh media containing different concentrations of \u003cem\u003eWS-384\u003c/em\u003e for 9 days. After drug administration, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 20 min. Subsequently, the cells were washed with ultrapure water three times until the colonies were clear enough. Finally, the cells were stained with a crystal violet solution, photographed, and counted by ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Flow cytometry analysis of cell apoptosis\u003c/h2\u003e \u003cp\u003eThe Annexin V-EGFP apoptosis detection kit (KeyGEN BioTECH, Jiangsu, China). A549 and H1975 cells that underwent apoptosis were evaluated via flow cytometry. A549 and H1975 cells were seeded in 6-well culture plates at a density of 1.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e6\u003c/sup\u003e and 2.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e6\u003c/sup\u003e, respectively. After incubation overnight, cells were treated with various concentrations of \u003cem\u003eWS-384\u003c/em\u003e. For flow cytometry, 2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e6\u003c/sup\u003e cells in 200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL binding buffer were stained with 2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL Annexin V-FITC and 2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL PI at 37\u0026deg;C in the dark for 20 min. All cells were analyzed via flow cytometry (488 nm excitation and 530 nm emission filters).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Flow cytometric analysis of cell cycle\u003c/h2\u003e \u003cp\u003eA549 and H1975 cells were seeded in 6-well culture plates at a density of 1.8 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e6\u003c/sup\u003e and 2.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e6\u003c/sup\u003e, respectively. After incubation overnight, cells were treated with various concentrations of \u003cem\u003eWS-384\u003c/em\u003e. Cells were collected and fixed in 70% prechilled ethanol at -20\u0026deg;C overnight and then stained with propidium iodide (PI) and RNase A (KeyGEN BioTECH, Jiangsu, China). All cells were analyzed via BD Accuri C6 flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. 5-Ethynyl-20-deoxyuridine (EdU) incorporation assay\u003c/h2\u003e \u003cp\u003eA549 and H1975 cells were seeded in glass-bottom dishes at a density of 5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e4\u003c/sup\u003e cells/well. After 24 h of treatment with \u003cem\u003eWS-384\u003c/em\u003e, a fresh medium containing EdU labeling agent (RiboBio, Guangzhou, China) was added to the cell culture and incubated for another 4 h. Then, the cells were fixed with 4% paraformaldehyde and incubated with glycine. The cells were stained with an anti-EdU working solution at room temperature, washed with 0.5% Triton X-100 PBS, and then incubated with Hoechst 33342. Finally, cells were observed under a laser confocal scanning microscope (Ma, et al.2017).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. siRNA transfection\u003c/h2\u003e \u003cp\u003eLSD1, DCN1 and negative control small interfering RNAs (siNC) were transfected into A549 cells with Lipofectamine RNAi max reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer\u0026rsquo;s instructions. Cells were collected and analyzed by flow cytometry or Western blotting after transfection for 72 h. The sequences were as follows: siLSD1 #1, 5\u0026prime;-CUACAUCUUACCUUAGUCATT-3\u0026prime; (sense), 5\u0026prime;-UGACUAAGGUAAGAUGUAGTT-3\u0026prime; (antisense); siLSD1#2, 5\u0026prime;-CAGCUGACAUUUGAGGCUATT-3\u0026prime; (sense), 5\u0026prime;-UAGCCUCAAAUGUCAGCUGTT-3\u0026prime; (antisense); siNC#, 5\u0026prime;-UUCUCCGAACGUGUCACGUTT-3\u0026prime; (sense), 5\u0026prime;-ACGUGACACGUUCGGAGAATT-3\u0026prime; (antisense); siDCN1 #1 5\u0026prime;-GGAUAAAGUUCGUCAGUUUTT-3\u0026prime; (sense), 5\u0026prime;-AAACUGACGAACUUUAUCCTT-3\u0026prime; (antisense); siDCN1 #2 5 \u0026prime;-GGACAGGAAGAAGUUAGAATT-3 (sense), 5\u0026prime;-UUCUAACUUCUUCCUGUCCTT-3\u0026prime; (antisense); siDCN1 #3 5\u0026prime;-GCCAUUGCCUACUGGAACUTT-3\u0026prime; (sense), 5\u0026prime;-AGUUCCAGUAGGCAAUGGCTT-3\u0026prime;(antisense).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Cellular thermal shift assay\u003c/h2\u003e \u003cp\u003eA549 and H1975 cells were collected and then washed twice with PBS. Then, 850\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL of PBS was added to the cells, and the cells were frozen and thawed repeatedly from liquid nitrogen in a constant temperature water bath 3\u0026ndash;5 times. The supernatant was placed into new EP tubes after centrifugation for 20 min at low temperature. The supernatant was added to 10 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eM \u003cem\u003eWS-384\u003c/em\u003e and incubated for 30 min at room temperature. The samples were placed into a PCR instrument, and a loading buffer was added; samples were denatured and used for western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Western blot analysis\u003c/h2\u003e \u003cp\u003eA549 and H975 cells were treated with different concentrations of \u003cem\u003eWS-384\u003c/em\u003e and incubated in an incubator for 48 h. Then, the cells were digested, centrifuged and washed with PBS. The total protein was extracted with RIPA. All samples were kept on ice for 30 min followed by centrifugation at 12000 rpm at 4℃ for 10 min. The supernatant was absorbed and quantified by a BCA protein assay kit (Beyoyime, Hainan, China). Next, the proteins of A549 and H975 cells were separated by sodium dodecyl sulfate 10% SDS-PAGE and transferred to NC membranes by the wet transfer method. After that, the protein in the membranes was incubated with primary antibodies and secondary antibodies, and finally, the protein expression was detected by ECL photoluminescence solution.\u003c/p\u003e \u003cp\u003e2.11. Comet assay\u003c/p\u003e \u003cp\u003eThe prepared suspension of cells in 0.6% low melting point agarose (LMA) dissolved in PBS was cast onto microscope slides with 0.6% average melting point agarose and laid LMA. The cells were lysed overnight at 4℃ in lysis buffer. DNA was allowed to unwind for 40 min in an electrophoretic solution. Electrophoresis was conducted for 30 min at 25 V and 300 mA. The slides were neutralized with Tris, pH 7.5. The slides were stained with ethidium bromide for 20 min and immediately examined under an Eclipse fluorescence microscope.\u003c/p\u003e \u003cp\u003e2.12. RNA extraction and quantitative RT-PCR\u003c/p\u003e \u003cp\u003eA549 and H1975 cells were spread in 12-well plates and treated with \u003cem\u003eWS-384\u003c/em\u003e for 48 h. Next, 500 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL TRIzol was added to each well. Then, 100 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL of chloroform was added to each EP tube and centrifuged by cryogenic centrifugation at 12000 rpm for 10 min to absorb the water phase. Isopropanol was added to the EP tube, placed at room temperature for 10 min, and centrifuged at 12000 rpm for 10 min at 4℃; the supernatant was discarded. The RNA precipitate was washed with 75% ethanol and centrifuged at 12000 rpm for 3 min at 4℃, and the supernatant was discarded to obtain RNA precipitate. RNA was dissolved with DEPC water. Then the concentration of nucleic acids was quantified. According to the steps of HiScript Ⅱ Q RT SuperMix for qPCR (+\u0026thinsp;gDNA wiper) (Vazyme, China), cDNA was obtained for PCR experiments. The cDNA was diluted according to the expression of the target gene, and PCR was carried out by quantitative fluorescence PCR.\u003c/p\u003e \u003cp\u003e2.13. Animal study\u003c/p\u003e \u003cp\u003e The SPF animal lab approved this study of Zhengzhou University. Female BALB/c nude mice (6\u0026ndash;8 weeks, 16\u0026ndash;18 g) were obtained from Hunan Shrek Jingda Experimental Animal Co. Ltd. Nude mice were inoculated with A549 cells at a density of approximately 1.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 10\u003csup\u003e7\u003c/sup\u003e cells/200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mu }\\)\u003c/span\u003e\u003c/span\u003eL. Then the nude mice were regrouped after 6 days. \u003cem\u003eWS-384\u003c/em\u003e was administered to nude mice by oral administration at 0, 25 mg/kg, and 50 mg/kg every day. When the average tumor volume reached approximately 900 mm\u003csup\u003e3\u003c/sup\u003e, the mice were euthanized with CO\u003csub\u003e2\u003c/sub\u003e, measured the tumor volume and removed the corresponding organs.\u003c/p\u003e \u003cp\u003eDuring the experiments, nude mice were cared for and handled strictly according to the guidelines of the Animal Ethics Committee of the School of Pharmacy, Zhengzhou University. All experimental protocols were approved by the Animal Ethics Committee of School of Pharmacy, Zhengzhou University.\u003c/p\u003e \u003cp\u003e2.14. Haematoxylin and eosin (H\u0026amp;E) staining\u003c/p\u003e \u003cp\u003eThe transplanted tumors and central organs of nude mice were fixed in paraformaldehyde for one week and then embedded in paraffin (Feldman, et al.2014). After paraffin slicing, dewaxing, dyeing, dehydration, sealing and other steps were carried out, a microscope was used to take pictures.\u003c/p\u003e \u003cp\u003e2.15. Molecular docking\u003c/p\u003e \u003cp\u003eChem3D software was used to build groups and optimize compound configurations through MM2 molecular mechanics. Then, Mgltools1.5.6 and AutodockTools1.5.6 were used to open the small ligand molecule in PMV, and hydrogen and charge were added to detect the root of the ligand. The 3D structure of the protein was downloaded from the RCSB Protein Data Bank and opened in Mgltools1.5.6 PMV. The original ligands and solvents were removed, all hydrogen atoms were added, the Gasteiger charge was calculated, and nonpolar hydrogen was merged. According to the coordinate file of the original ligand, we determined the grid box coordinates and box size of the docking site, and the Mark genetic algorithm generated the docking structure. The related energy in the grid was calculated by AutoGrid, and semiflexible docking was performed in Autodock. The composition with the lowest point was selected for the analysis of the docking structure pattern and the diagram was made using PyMOL.\u003c/p\u003e \u003cp\u003e2.16. Statistical analysis\u003c/p\u003e \u003cp\u003eAll experiments were conducted more than three times. The results were analyzed using GraphPad Prism version 8.0.1 to perform one-way ANOVAs and t-tests (GraphPad Software, San Diego, CA, USA). The results are given as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A p-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.1 \u003cem\u003eWS-384\u003c/em\u003e inactivated cullin1 and cullin3 neddylation and accumulated CRL substrates in A549 and H1975 cells.\u003c/h2\u003e\n \u003cp\u003eAccording to our previous reports, \u003cem\u003eWS-384\u003c/em\u003e is an inhibitor of the UBC12-DCN1 protein-protein interaction (Wang, et al.2019). The chemical formula of \u003cem\u003eWS-384\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA (Wang, et al.2019). To gain further insights into the interaction of \u003cem\u003eWS-384\u003c/em\u003e with target proteins, we conducted molecular modeling studies by docking the compound \u003cem\u003eWS-384\u003c/em\u003e into DCN1. The internal reorganization of binding is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, and \u003cem\u003eWS-384\u003c/em\u003e could bind well to the binding site of DCN1. \u003cem\u003eWS-384\u003c/em\u003e formed a hydrogen bond interaction with the amino acid residue PRO97 of DCN1 and fit relatively well into the hydrophobic cleft formed by DCN1. \u003cem\u003eWS-384\u003c/em\u003e inhibited the UBC12-DCN1 interaction with an IC\u003csub\u003e50\u003c/sub\u003e value of 14.81 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e0.95 nM.\u003c/p\u003e\n \u003cp\u003eAs reported, UBC12-DCN1 mainly mediates cullin1 and cullin3 neddylation (Zhou, et al.2017). The results found that \u003cem\u003eWS-384\u003c/em\u003e blocked the neddylation of cullin1 and cullin3 but had no effect on the neddylation of other cullin members in NSCLC cells (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cstrong\u003eand D\u003c/strong\u003e). Additionally, \u003cem\u003eWS-384\u003c/em\u003e increased the levels of p21 and Bim (substrates of cullin 1) and NRF-2 (substrate of cullin 3) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eE \u003cstrong\u003eand F\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eIn summary, these results suggested that \u003cem\u003eWS-384\u003c/em\u003e selectively inhibited the neddylation of cullin1 and cullin3 by binding to DCN1 and blocking the interaction between UBC12-DCN1 proteins but had no effect on the neddylation of other cullin family members in NSCLC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.2 Effects of \u003cem\u003eWS-384\u003c/em\u003e on LSD1 activity in A549 and H1975 cells\u003c/h2\u003e\n \u003cp\u003eNext, we explored whether \u003cem\u003eWS-384\u003c/em\u003e also had an inhibitory effect on LSD1. Therefore, we first conducted molecular modeling studies by docking the compound \u003cem\u003eWS-384\u003c/em\u003e into LSD1. The internal reorganization of the binding assay is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA. \u003cem\u003eWS-384\u003c/em\u003e could dock into the active binding pocket of LSD1 and form a hydrogen bond interaction with the amino acid residue HIS564 of LSD1. \u003cem\u003eWS-384\u003c/em\u003e fit relatively well into the hydrophobic cleft formed by LSD1. \u003cem\u003eWS-384\u003c/em\u003e was docked into the active binding pocket of LSD1 and \u003cem\u003eWS-384\u003c/em\u003e inhibited the activity of LSD1 with an IC\u003csub\u003e50\u003c/sub\u003e value of 338.79\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003e 2.53 nM.\u003c/p\u003e\n \u003cp\u003eSecond, we employed a cellular thermal shift assay (CETSA) (Martinez Molina, et al.2013) to assess the target engagement of \u003cem\u003eWS-384\u003c/em\u003e in A549 and H1975 cells. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, the thermal stability of LSD1 protein was enhanced by \u003cem\u003eWS-384\u003c/em\u003e at 56\u0026deg;C in H1975 cells and 52\u0026deg;C in A549 cells. Furthermore, \u003cem\u003eWS-384\u003c/em\u003e enhanced the stability of LSD1 protein in a dose-dependent manner, which indicated the cellular target engagement of \u003cem\u003eWS-384\u003c/em\u003e in NSCLC cells. LSD1 can catalytically remove methyl groups from LSD1 substrates H3K4me1/2 (Fu, et al.2017). Herein, we investigated the effects of \u003cem\u003eWS-384\u003c/em\u003e on the methylation of histone H3K4 in A549 and H1975 cells. As demonstrated in Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cstrong\u003eand D\u003c/strong\u003e, \u003cem\u003eWS-384\u003c/em\u003e induced the accumulation of H3K4me1/2 in A549 and H1975 cells in a concentration-dependent manner, which was consistent with GSK-LSD1 (an LSD1 inhibitor). These results indicated that \u003cem\u003eWS-384\u003c/em\u003e could also effectively inhibit the activity of LSD1.\u003c/p\u003e\n \u003cp\u003e3.3 \u003cem\u003eWS-384\u003c/em\u003e inhibited cell proliferation and induced cell apoptosis in a concentration-dependent manner in A549 and H1975 cells.\u003c/p\u003e\n \u003cp\u003eTo test whether \u003cem\u003eWS-384\u003c/em\u003e had cytotoxic effects on A549 and H1975 cells, we first conducted an MTT assay. The results showed that \u003cem\u003eWS-384\u003c/em\u003e significantly inhibited A549 and H1975 cell growth dose- and time-dependent (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cstrong\u003eand B\u003c/strong\u003e). The IC\u003csub\u003e50\u003c/sub\u003e values of \u003cem\u003eWS-384\u003c/em\u003e in A549 and H1975 cells are shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. In the clone formation assays, we observed a significant decrease in the colorogenic ability of A549 and H1975 cells with increased \u003cem\u003eWS-384\u003c/em\u003e concentrations and even complete disappearance of colony formation (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC \u003cstrong\u003eand D\u003c/strong\u003e). To further confirm the effect of \u003cem\u003eWS-384\u003c/em\u003e on cell proliferation, and EdU incorporation assay was performed. The data showed that \u003cem\u003eWS-384\u003c/em\u003e significantly reduced the number of EdU cells compared to the control group\u0026apos;s dose-dependent manner (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE \u003cstrong\u003eand F\u003c/strong\u003e). These data revealed that \u003cem\u003eWS-384\u003c/em\u003e inhibited the proliferation of NSCLC cells.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCytotoxicity of \u003cem\u003eWS-384\u003c/em\u003e in A549 and H1975 cells. Data are shown as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (\u0026micro;M)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eA549\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eH1975\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.87\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eApoptosis is the leading cause of cell death, so we next examined the effect of \u003cem\u003eWS-384\u003c/em\u003e on cell apoptosis by flow cytometry analysis. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, when A549 and H1975 cells were treated with \u003cem\u003eWS-384\u003c/em\u003e, cell apoptosis occurred obviously. At the same time, \u003cem\u003eWS-384\u003c/em\u003e treatment increased the expression of caspase family proteins such as cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, and cleaved PARP (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB \u003cstrong\u003eand C\u003c/strong\u003e). The results suggested that \u003cem\u003eWS-384\u003c/em\u003e could significantly promote NSCLC cell apoptosis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec16\"\u003e\n \u003ch2\u003e3.4 P21 acted as an important intermediate regulator responsible for the anticancer effect of \u003cem\u003eWS-384\u003c/em\u003e.\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eWS-384\u003c/em\u003e selectively inhibited neddylation of cullin1/3 over other cullin members and induced the accumulation of downstream proteins p21, Bim and NRF-2 in both A549 and H1975 cells. NRF-2, as a transcription factor, plays a vital role in the regulation of the antioxidant response and lowers ROS levels (Basak, et al.2017,Zhou, et al.2017). The ROS content is vital for cellular function and survival signaling. Therefore, we investigated the cellular effect of \u003cem\u003eWS-384\u003c/em\u003e on ROS generation in NSCLC cells. However, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cem\u003eWS-384\u003c/em\u003e had no significant effect on ROS levels in A549 and H1975 cells, which suggested that the proapoptotic effect of \u003cem\u003eWS-384\u003c/em\u003e was not caused by ROS and NRF-2 might not be an important regulator for the anticancer effect of \u003cem\u003eWS-384\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eIt has been reported that LSD1 inhibition increases H3K4 methylation on the CDKN1A promoter, which leads to the transcriptional activation of p21 and p21, the typical substrate protein of cullin1 (Liu, et al.2017,Zhu, et al.2019). Therefore, we further investigated the expression of p21, and the results showed that the gene and protein expression of p21 were both significantly increased after \u003cem\u003eWS-384\u003c/em\u003e treatment in A549 and H1975 cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, D, \u003cstrong\u003eand E\u003c/strong\u003e). Taken together, these results indicate that p21, not NRF-2, acts as an important intermediate regulator to inhibit A549 and H1975 cell proliferation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec17\"\u003e\n \u003ch2\u003e3.5 \u003cem\u003eWS-384\u003c/em\u003e induced cell cycle arrest and DNA damage in A549 and H1975 cells.\u003c/h2\u003e\n \u003cp\u003ep21 is an important protein that regulates the cell cycle, and dysregulation is a vital reason for cell proliferation inhibition. Therefore, we stained NSCLC cells with PI and analyzed the cell cycle using flow cytometry. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cem\u003eWS-384\u003c/em\u003e significantly induced G2/M phase arrest in both A549 and H1975 cells. It is generally acknowledged that p21 can bind and inhibit the activity of cyclin CDK4/6 complexes and then play a checkpoint regulator role in cell cycle arrest (Dongoran, et al.2020,Han, et al.2019,Liu, et al.2017,Vilgelm, et al.2019,Xie, et al.2018,Zhu, et al.2019). Therefore, we detected the levels of cell cycle regulatory proteins including cyclin B1, p-CDC2, CDK4 and CDK6. Figures\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB \u003cstrong\u003eand C\u003c/strong\u003e demonstrated that these proteins were downregulated in the \u003cem\u003eWS-384\u003c/em\u003e treated group. To further explore how \u003cem\u003eWS-384\u003c/em\u003e promoted cell cycle arrest and apoptosis, we next investigated the effect of \u003cem\u003eWS-384\u003c/em\u003e on DNA damage. The accumulation of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }\\)\u003c/span\u003e\u003c/span\u003e-H2AX (a marker protein of DNA damage) in A549 and H1975 cells was increased after treatment with \u003cem\u003eWS-384\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA \u003cstrong\u003eand B\u003c/strong\u003e), and this was further confirmed by the comet assay, which revealed an increased percentage of DNA damage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC \u003cstrong\u003eand D\u003c/strong\u003e). In summary, all these data suggested that \u003cem\u003eWS-384\u003c/em\u003e could induce cell cycle arrest and DNA damage in NSCLC cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e3.6 Dual inhibition of LSD1 and DCN1-UBC12 interaction led to proliferation inhibition and proapoptotic effects.\u003c/h2\u003e\n \u003cp\u003eTo further verify the proliferation inhibition and proapoptosis effects induced by the dual inhibition of LSD1 and DCN1, we performed a small interfering RNA (siRNA) transfection experiment (Zhou, et al.2019,Zhu, et al.2019). Quantitative analysis showed that the silencing efficiencies of siLSD1 #2 and siDCN1 #3 were 72.65% and 57.07%, respectively (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA \u003cstrong\u003eand B\u003c/strong\u003e). Moreover, silencing DCN1 by three siRNAs in both A549 cells decreased the DCN1 and neddylation of cullin1 and cullin3. Silencing LSD1 with two siRNAs in A549 cells decreased the LSD1 level and caused the accumulation of H3K4me2 (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eA \u003cstrong\u003eand B\u003c/strong\u003e). Therefore, siLSD1 #2 and siDCN1 #3 were used for subsequent experiments.\u003c/p\u003e\n \u003cp\u003eCell proliferation was also more pronounced after inhibition by siLSD1 and siDCN1, as measured by MTT and colony-forming assays (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eC \u003cstrong\u003eand D\u003c/strong\u003e). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eE, the protein level of p21 was significantly increased by LSD1 or DCN1 siRNA, and the relative level of p21 was higher in the combination LSD1 siRNA and DCN1 siRNA treatment group than in the LSD1 siRNA or DCN1 siRNA alone group, which was consistent with the increased expression induced by \u003cem\u003eWS-384\u003c/em\u003e. As indicated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eF, the levels of celaved caspase family proteins (cleaved caspase 3, cleaved caspase 9, and cleaved PARP) were significantly increased by pronounced LSD1 and DCN1 silencing, and proapoptotic, proliferation inhibition and DNA damage inducing effects were simultaneously verified by flow cytometry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eG). Moreover, the expression level of\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\gamma }\\)\u003c/span\u003e\u003c/span\u003e-H2AX and the percentage of DNA damage were significantly increased in cells treated with siLSD1 and siDCN1 compared with cells treated with siLSD1 or siDCN1 alone (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eH \u003cstrong\u003eand I\u003c/strong\u003e). Taken together, these data indicated that the pro-apoptosis, proliferation inhibition and DNA damage inducing effect of \u003cem\u003eWS-384\u003c/em\u003e on NSCLC cells was caused by dual inhibition of the LSD1 and DCN1-UBC12 interaction.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e3.7 Anti-tumor effect of \u003cem\u003eWS-384\u003c/em\u003e in A549 xenograft mice.\u003c/h2\u003e\n \u003cp\u003eTo further evaluate the anti-tumor activity of \u003cem\u003eWS-384 in vivo\u003c/em\u003e, xenograft models with subcutaneously implanted A549 cells were established. Tumor-bearing mice were treated with vehicle or \u003cem\u003eWS-384\u003c/em\u003e 25 mg/kg and 50 mg/kg by oral administration once a day for 39 consecutive days (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eA). After drug administration for 39 days, mice were killed, and tumors were harvested and weighed (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eB \u003cstrong\u003eand C\u003c/strong\u003e). From the results, tumor growth was significantly suppressed after 15 days in the 50 mg/kg group compared to the control. In addition, tumor weight in the 50 mg/kg group was significantly lower than that of the control group, and there were no significant changes in body weight during administration (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eD \u003cstrong\u003eand E\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eWe next homogenized the tumor tissue and extracted the tissue protein to further explore the dual-target inhibition effect of \u003cem\u003eWS-384 in vivo\u003c/em\u003e. We found that \u003cem\u003eWS-384\u003c/em\u003e suppressed the neddylation of cullin1 and cullin3 and increased the methylation of histone H3K4 by inhibiting DCN1 and LSD1, respectively (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eF). The expression level of p21 and \u0026gamma;-H2AX, as well as cleaved caspase family proteins, were all increased (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eG). Finally, we investigated the potential side effects of \u003cem\u003eWS-384\u003c/em\u003e on the main organs. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eH, H\u0026amp;E staining results demonstrated no apparent toxicity in the heart, liver, spleen, lung, or kidney after treatment with \u003cem\u003eWS-384\u003c/em\u003e. Additionally, there was no significant difference in ALT, AST, uric acid, or creatinine, consistent with the histopathological results (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003eI). Taken together, these results suggested that \u003cem\u003eWS-384\u003c/em\u003e could effectively inhibit the growth of NSCLC tumors with low toxicity.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eNSCLC is one of the most common human cancers with high morbidity and mortality. Novel therapeutic strategies and compounds with high efficacy and low toxicity are urgently needed. As reported, DCN1 is abnormally highly expressed in many squamous cell carcinomas and plays an essential role in the modulation of the neddylation pathway and tumorigenesis (Broderick, et al.2010,Sarkaria, et al.2006,Yoo, et al.2012). DCN1 could interact with both cullin1/3 and UBC12 and further facilitate cullin neddylation. Several studies have reported some inhibitors targeting the DCN1-UBC12 protein-protein interaction, and unlike MLN4924, these inhibitors showed no significant cytotoxicity (Wang, et al.2019,Zhou, et al.2017). Recently, our group also developed a potent and selective DCN1-UBC12 interaction inhibitor \u003cem\u003eWS-383\u003c/em\u003e (Wang, et al.2019). In this study, we further evaluated the inhibitory effect of \u003cem\u003eWS-384\u003c/em\u003e, an analog of \u003cem\u003eWS-383\u003c/em\u003e, on the DCN1-UBC12 protein-protein interaction and found that \u003cem\u003eWS-384\u003c/em\u003e could effectively inhibit cullin 1 and cullin 3 neddylation in NSCLC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have revealed that LSD1 is an important oncogene inducing tumorigenesis. However, the cytotoxicity of a single LSD1 inhibitor is usually limited in solid tumors, so it is necessary to enhance the anti-tumor effect of the small molecule inhibitor. Based on the structure of LSD1 and DCN1-UBC12, we speculate that it might be possible to design an inhibitor targeting LSD1 and DCN1. We found that \u003cem\u003eWS-384\u003c/em\u003e occupied the FAD active pocket in LSD1 and the DCN1 active pocket, so we inferred that \u003cem\u003eWS-384\u003c/em\u003e had a similar binding pattern at the active sites of LSD1 and DCN1 from the docking model. Moreover, molecular docking analysis and cellular activity experiment results showed that \u003cem\u003eWS-384\u003c/em\u003e could adapt to the ligand-binding pocket of LSD1 and inhibit the activity of LSD1. \u003cem\u003eWS-384\u003c/em\u003e could block the activity of the LSD1 and DCN1-UBC12 interaction at the same time. Interestingly, this finding suggested that \u003cem\u003eWS-384\u003c/em\u003e was a dual inhibitor targeting the LSD1 and DCN1-UBC12 interaction.\u003c/p\u003e \u003cp\u003eNext, we tested whether \u003cem\u003eWS-384\u003c/em\u003e had a potential anti-proliferative effect on lung carcinoma cells. First, we used the MTT assay to detect whether \u003cem\u003eWS-384\u003c/em\u003e had cytotoxic effect, and the results showed that \u003cem\u003eWS-384\u003c/em\u003e could significantly decrease cell viability. Second, the cell colony formation and EdU incorporation experiments showed that \u003cem\u003eWS-384\u003c/em\u003e significantly reduced the number of colon and EdU-positive cells compared to the control group. These data indicated that \u003cem\u003eWS-384\u003c/em\u003e obviously inhibited NSCLC cell proliferation. For most compounds that have cytotoxic effects, apoptosis is the main cause of cell death; thus, in our study, we detected cell apoptosis by flow cytometry analysis. When A549 and H1975 cells were treated with \u003cem\u003eWS-384\u003c/em\u003e, cell apoptosis was obvious. Taken together, \u003cem\u003eWS-384\u003c/em\u003e could significantly inhibit cell proliferation and induce NSCLC cell apoptosis. In addition, \u003cem\u003eWS-384\u003c/em\u003e also inhibited tumor growth and induced cell apoptosis \u003cem\u003ein vivo\u003c/em\u003e. Our findings also showed that the anti-tumor activity of simultaneous knockdown of LSD1 and DCN1 was the same as that of \u003cem\u003eWS-384\u003c/em\u003e treatment, which further confirmed that the anti-tumor effect of \u003cem\u003eWS-384\u003c/em\u003e was due to dual inhibition of the LSD1 and DCN1-UBC12 interaction.\u003c/p\u003e \u003cp\u003eThe cyclin-dependent kinase inhibitor p21 plays a vital role in cell cycle regulation, apoptosis and DNA damage. P21, as a downstream protein of cullin1 was significantly increased by \u003cem\u003eWS-384\u003c/em\u003e. We also found that \u003cem\u003eWS-384\u003c/em\u003e could elevate the gene expression of p21 in NSCLC cells, which is consistent with previous studies (Wang, et al.2019). Liangsong Zhu et al reported that LSD1 could directly bind to the p21 promoter, induce H3K4me2 demethylation, and further regulate p21 transcription (Zhu, et al.2019). Therefore, it was suggested that elevated p21 is an important regulatory factor in the synergistic anti-tumor effect of \u003cem\u003eWS-384\u003c/em\u003e. As p21 is an important protein that regulates the cell cycle, we next detected the cell cycle by flow cytometry. The data showed that \u003cem\u003eWS-384\u003c/em\u003e induced G2/M phase arrest and affected cell cycles regulatory proteins such as cyclin B1, p-CDC2, CDK4, and CDK6. From the results, we concluded that p21 was a critical intermediate regulator mediating the anticancer effect of \u003cem\u003eWS-384\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, we proposed for the first time the practical anti-tumor effect of \u003cem\u003eWS-384\u003c/em\u003e targeting LSD1 and the DCN1-UBC12 interaction. Assays of cullin protein neddylation, histone H3K4 methylation and knockdown of LSD1 or DCN1 demonstrated the highly active and selective inhibition of the LSD1 and DCN1-UBC12 interaction by \u003cem\u003eWS-384\u003c/em\u003e. Additionally, \u003cem\u003eWS-384\u003c/em\u003e could induce cell cycle arrest and apoptosis \u003cem\u003ein vitro\u003c/em\u003e by modulating the p21 signaling pathway. \u003cem\u003eIn vivo\u003c/em\u003e experiments further verified the anti-tumor effect of \u003cem\u003eWS-384\u003c/em\u003e in A549 bearing xenograft mouse models. Our results lay a theoretical foundation for the clinical application of LSD1 and DCN1-UBC12 protein-protein interaction inhibitors in the treatment of NSCLC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding \u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (No. 82020108030, U21A20416, 82104279, 81903770, 81973177 and 22277110), Program for Science \u0026amp; Technology Innovation Talents in Universities of Henan Province (No. 21HASTIT045), Key scientific and technological projects of Henan Province (No. 202102310162, 222102310125), China Postdoctoral Science Foundation (Nos. 2019M662550, 2019M662556) and the Natural Science Foundation of Henan Province (no. 222300420069).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions \u0026nbsp;\u003c/strong\u003eConception and design: Siqi Feng, Hongmin Liu, Ting Ma, Bin Yu and Anqi Li designed the research. Anqi Li, Ting Ma, Yueyang Guo and Qianqian Song performed the experiments and Anqi Li carried out data analysis. Anqi Li and Ting Ma wrote the manuscript and Siqi Feng revised the manuscript. All of the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability \u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u0026nbsp; Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical statement \u0026nbsp;\u003c/strong\u003eAll animal experiments and experimental procedures were approved by the Research Ethics Committee of the Drug Research Institute of Zhengzhou University and were in strict compliance with the Guide for the Care and Use of Laboratory Animals of the Drug Research Institute of Zhengzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate \u0026nbsp;\u003c/strong\u003eAll participants signed a document of informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest \u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen Access \u0026nbsp;\u003c/strong\u003eThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article\u0026rsquo;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article\u0026rsquo;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/licen ses/by/4. 0/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026apos;s note \u0026nbsp;\u003c/strong\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmbrosio S, Sacca CD, Majello B. Epigenetic regulation of epithelial to mesenchymal transition by the Lysine-specific demethylase LSD1/KDM1A. 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Acta Pharm Sin B. 2019;9(2):324-334.http://dx.doi.org/10.1016/j.apsb.2018.10.006\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"LSD1, DCN1, Non-small cell lung cancer, cancer therapy","lastPublishedDoi":"10.21203/rs.3.rs-2593334/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2593334/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAbnormally high expression of lysine-specific demethylase 1A (LSD1) and DCN1 plays a vital role in the occurrence, development, and poor prognosis of non-small cell lung cancer (NSCLC). Accumulating evidence has shown that the development of small-molecule inhibitors dually targeting LSD1 and the UBC12-DCN1 interaction probably have therapeutic promise for cancer therapy. This work reported that \u003cem\u003eWS-384\u003c/em\u003e dually targeted LSD1 and UBC12-DCN1 interactions and evaluated its antitumor effects \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Specifically, \u003cem\u003eWS-384\u003c/em\u003e inhibited A549 and H1975 cells viability and decreased colony formation and EdU incorporation. \u003cem\u003eWS-384\u003c/em\u003e could also trigger cell cycle arrest, DNA damage, and apoptosis. Moreover, \u003cem\u003eWS-384\u003c/em\u003e significantly decreased tumor weight and volume in A549 xenograft mice. Mechanistically, \u003cem\u003eWS-384\u003c/em\u003e increased the gene and protein level of p21 by suppressing the neddylation of cullin 1 and decreasing H3K4 demethylation at the CDKN1A promoter. The synergetic upregulation of p21 contributed to cell cycle arrest and the proapoptotic effect of \u003cem\u003eWS-384\u003c/em\u003e in NSCLC cells. Taken together, our proof of concept studies demonstrated the therapeutic potential of dual inhibition of LSD1 and the UBC12-DCN1 interaction for the treatment of NSCLC. \u003cem\u003eWS-384\u003c/em\u003e could be used as a lead compound to develop new dual LSD1/DCN1 inhibitors for the treatment of human diseases in which LSD1 and DCN1 are dysregulated.\u003c/p\u003e","manuscriptTitle":"Discovery of WS-384, a first-in-class dual LSD1 and DCN1-UBC12 protein-protein interaction inhibitor for the treatment of non-small cell lung cancer (NSCLC)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-20 15:37:12","doi":"10.21203/rs.3.rs-2593334/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"adab3891-b808-4ac7-b27d-0db1b8051589","owner":[],"postedDate":"February 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-17T15:13:47+00:00","versionOfRecord":{"articleIdentity":"rs-2593334","link":"https://doi.org/10.1016/j.biopha.2024.116240","journal":{"identity":"biomedicine-and-pharmacotherapy","isVorOnly":true,"title":"Biomedicine \u0026 Pharmacotherapy"},"publishedOn":"2024-04-01 15:13:47","publishedOnDateReadable":"April 1st, 2024"},"versionCreatedAt":"2023-02-20 15:37:12","video":"","vorDoi":"10.1016/j.biopha.2024.116240","vorDoiUrl":"https://doi.org/10.1016/j.biopha.2024.116240","workflowStages":[]},"version":"v1","identity":"rs-2593334","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2593334","identity":"rs-2593334","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}