In Silico Discovery of Stapled Peptide Inhibitor Targeting the Nur77‐PPARγ Interaction and Its Anti‐Breast‐Cancer Efficacy

Abstract The binding of peroxisome proliferator‐activated receptor γ (PPARγ) to the orphan nuclear receptor Nur77 facilitates the ubiquitination and degradation of Nur77, and leads to aberrant fatty acid uptake for breast cancer progression. Because of its crucial role in clinical prognosis, the interaction between Nur77 and PPARγ is an attractive target for anti‐breast‐cancer therapy. However, developing an inhibitor of the Nur77‐PPARγ interaction poses a technical challenge due to the absence of the crystal structure of PPARγ and its corresponding interactive model with Nur77. Here, ST‐CY14, a stapled peptide, is identified as a potent modulator of Nur77 with a K D value of 3.247 × 10−8 M by in silico analysis, rational design, and structural modification. ST‐CY14 effectively increases Nur77 protein levels by blocking the Nur77‐PPARγ interaction, thereby inhibiting lipid metabolism in breast tumor cells. Notably, ST‐CY14 significantly suppresses breast cancer growth and bone metastasis in mice. The findings demonstrate the feasibility of exploiting directly Nur77‐PPARγ interaction in breast cancer, and generate what to the best knowledge is the first direct inhibitor of the Nur77‐PPARγ interaction available for impeding fatty acid uptake and therapeutic development.


Introduction
Aberrant lipid metabolism plays key regulatory roles in the proliferation, aggressiveness, and metastasis of certain malignancies, especially breast cancer. [1,2]As the most diagnosed DOI: 10.1002/advs.2023084355] Lipocytes provide a predominant source for energy metabolism and biomembrane synthesis, and are conducive to breast cancer growth and metastasis. [5][8] However, peroxisome proliferator-activated receptor  (PPAR)-induced Nur77 degradation by recruiting the ubiquitin ligase (E3) Trim13 prevents the positive regulation of Nur77, which promotes aberrant lipid uptake in breast cancer. [6]Therefore, the interaction between Nur77 and PPAR could provide a therapeutic target to inhibit lipid metabolism in breast cancer.
Nur77 contains an N-terminal transcription-activating domain, a DNA-binding domain (DBD), and a ligand-binding domain (LBD). [9]Among them, the Nur77 LBD is responsible for PPAR DBD binding.A previous study showed that the small molecule cytosporone B (Csn-B) could indirectly impede Scheme 1.In silico approaches and chemical modifications are utilized to discover the first peptide inhibitor targeting the Nur77-PPAR interaction in the absence of cocrystal structures for breast cancer therapy.
the Nur77-PPAR interaction by promoting Nur77 homodimer formation, which in turn led to steric hindrance to prevent PPAR binding. [6]Given the pivotal role of mono Nur77 in breast cancer, directly inhibiting the Nur77-PPAR interaction would be extremely valuable for promoting mono Nur77 accumulation and binding to transcriptional corepressors to downregulate CD36 and FABP4 expression.Unfortunately, the unreported structural information currently available for the Nur77-PPAR complex has made the development of inhibitors directly targeting the Nur77-PPAR interaction extremely challenging.
13] Here, to discover the first direct inhibitor of the Nur77-PPAR interaction, the aforementioned in silico approaches were systematically adopted to predict the highly plausible and stable binding conformation of the Nur77-PPAR complex, followed by peptide rational design, binding affinity evaluation, and structural stapling modification.Encouragingly, a potent stapled peptide modulator, ST-CY14, bound to Nur77 with a K D value of 3.247 × 10 −8 M. By using ST-CY14 as a chemical probe, we found that directly blocking the Nur77-PPAR interaction promoted the accumulation of Nur77 and inhibited lipid absorption in breast tumor cells.Furthermore, animal experiments also validated the therapeutic potential of ST-CY14 to inhibit breast cancer tumor growth and bone metastasis.Our work provided crucial insights into the Nur77-PPAR PPI and a new strategy for the develop-ment of anti-breast-cancer peptides targeting lipid metabolism (Scheme 1).

Nur77 Inhibits Breast Cancer Proliferation, Lipid absorption, and Progression
To define the antitumor role of Nur77 in breast cancer, we knocked down Nur77 in human breast cancer MCF7 cells using a Nur77 siRNA (siNur77).Nur77 knockdown significantly promoted the clonal proliferation of MCF7 cells (Figure 1a).In addition, there were more long-chain FAs in Nur77-knockdown MCF7 cells than in wild-type (WT) MCF7 cells (Figure 1b), implicating the suppressive role of Nur77 in breast tumor cell proliferation and lipid uptake.Overall survival analysis also indicated that Nur77 had a positive impact on the overall survival time of postoperative patients with breast cancer, while patients with higher levels of PPAR expression had poorer prognoses (Figures S1, Supporting Information). [6]According to the opposing roles of Nur77 and PPAR in the clinical prognosis of breast cancer patients, we further found that the negative regulation of Nur77 protein expression by PPAR was observed in PPAR-knockdown MCF7 cells (Figure 1c).These findings strongly support the notion that Nur77 and PPAR are consistently linked to the clinical outcome of breast cancer, and that increasing Nur77 levels by blocking the Nur77-PPAR interaction holds promise for antibreast-cancer strategies.

In Silico Systematic Approach to Predict the Structure of the Nur77-PPAR𝜸 Heterodimer
To identify potent peptide inhibitors of the Nur77-PPAR interaction, we first determined the hot spots for Nur77-PPAR binding based on the Nur77 LBD spanning amino acids 351-598 (PDB: 6KZ5) and the PPAR DBD (residues 108-204) (Figure 1d), which have been previously reported as interaction domains. [6]We utilized AlphaFold2 [14] to predict the structure of PPAR.The structure of the PPAR DBD predicted by Al-phaFold2 had a very high per-residue confidence score (pLDDT >90) (Figure 1e; Figure S2a, Supporting Information).Both Al-phaFold2 and Molecular Operating Environment (MOE) [15] software were further employed to predict the binding conformation of the Nur77 LBD-PPAR DBD heterodimer.The predicted binding conformations of AlphaFold2 and the MOE (top 3 conformations, the ranking scores were shown in Figure S2b, Supporting Information) were evaluated using MD simulations by GROMACS. [16]Among them, the conformation predicted by AlphaFold2 was found to be unstable (Figure S2c, Supporting Information).Figure 1f-i demonstrated that the structures ranked 2 and 3 predicted by MOE showed conformational stability according to the C root mean square deviations (RMSDs), while the structure ranked 1 exhibited instability, as evidenced by fluctuating RMSD values (Figure S2d, Supporting Information).Thus, the rank 2 and rank 3 binding conformations of the Nur77-PPAR complex were chosen to design peptide inhibitors.

Identification of Inhibitors of the Nur77-PPAR𝜸 Interaction
Hotspot residues located on the protein-protein binding surfaces are commonly exploited for the design of peptide inhibitors. [17]ccording to the rank 2 conformation of the Nur77-PPAR complex (Figure S3a,b, Supporting Information), both a flexible loop of PPAR (residues Ile167-Leu178) named IL12 and an -helix of PPAR (residues Ser186-Val201) named SV16 bind one side of the Nur77 LBD, which indicates that IL12 and SV16 could be identified as potential peptide candidates.Similarly, another -helix of PPAR (residues Cys156-Leu169) named CL14 was obtained from the rank 3 binding conformation of the Nur77-PPAR complex (Figure S3c,d, Supporting Information).MD simulations revealed the stable binding of SV16 (Figure S3e, Supporting Information) and CL14 (Figure S3f, Supporting Information) to Nur77, whereas the binding between IL12 and Nur77 was found to be unstable (Figure S3g, Supporting Information).Therefore, SV16 and CL14 served as the starting points for our further design of peptide inhibitors.
Key interaction residues between the Nur77 LBD and CL14 or SV16 were determined using molecular mechanics with generalized born and surface area solvation binding free energy decomposition.The analysis demonstrated that SV16 Arg194 and The interaction between SV16 and Nur77 was depicted in both cartoon and stick forms, with SV16 colored orange and the Nur77 LBD colored blue.Interaction residues were labeled.b) The detailed interaction between SV16 and Nur77 was revealed, with the red dashed line representing the intermolecular bond between them.c) The binding free energy decomposition of SV16 and Nur77 was shown.d) The interaction between CL14 and Nur77 was depicted in both cartoon and stick forms, with CL14 colored pink and the Nur77 LBD colored blue.Interaction residues were labeled.e) The detailed interaction between CL14 and Nur77 was presented, with the red dashed line representing the intermolecular bond between them.f) The binding free energy decomposition of CL14 and Nur77 was illustrated.g) The free energy changes after peptide point mutations.h) The binding affinity of CY14 with Nur77 was depicted using MST.
Lys197 contributed more to the binding free energy (Figure 2c).SV16 Arg194 interacted with Nur77 Asp580 through a hydrogen bond, while SV16 Lys197 formed a hydrogen bond with Nur77 Glu579 and an H- interaction with Tyr575 (Figure 2a,b).For CL14, Arg164 contributed the highest proportion of the binding free energy (Figure 2f) and could interact with Nur77 Glu398 by H-bonds (Figure 2d,e).Additionally, the backbone of Cys156 and the side chain of Glu157 from CL14 engaged in hydrogen bonding interactions with Nur77 Phe598 and Gln571, respectively (Figure 2d,e).
To enhance the binding affinity of SV16 and CL14 with Nur77, amino acids near the binding surface that contributed less to the binding energy, as determined by energy decomposition, were mutated using Rosetta. [18]Specifically, Gln196 and Ala200 of SV16 were subjected to mutation, while Cys156 and Leu169 of CL14 were mutated.Moreover, given the low contribution of SV16 Val201 to the binding energy of Nur77 and the proximity of its binding surface, Val201 of SV16 was also mutated (Figure S4a, Supporting Information).The mutation scores were presented in Figure 2g, with a noticeable decrease in binding energy when CL14 Leu169 was mutated to Tyr (CY14), Phe (CF14), His (CH14), or Trp (CW14).Among the aforementioned mutated peptides, the binding free energy of CY14, in which Leu169 was mutated to Tyr, displayed the most significant change, with a decrease of 1.74 kcal mol −1 .Consequently, variants of the peptides, including SV16, CL14, CY14, CF14, CH14, and CW14, were generated and synthesized using standard Fmoc solid-phase peptide synthesis (SPPS) procedures.The binding affinity of these peptides to Nur77 was evaluated using microscale thermophoresis (MST).As depicted in Figure 2h, Figure S4b,c (Supporting Information), CY14, which resulted from the mutation of CL14, exhibited the highest affinity for Nur77 (K d CY14 = 8.70 × 10 −6 M , . We speculate that certain mutations (CF14, CH14, and CW14) may result in the instability of the peptide conformation, thereby reducing its binding affinity (Figure S4f−h).Furthermore, MD analysis confirmed the stable binding of CY14 with Nur77 (Figure S4d, Supporting Information).Notably, as illustrated in Figure S4e (Supporting Information), the presence of a benzene ring in Tyr facilitated - interactions with Nur77 Phe395, and the hydrophilic hydroxyl group of the tyrosine benzene ring was exposed to solvents.As a result, the mutated Tyr residue exhibited improved energy and geometric compatibility with Nur77.

Structural Optimization of CY14
To determine whether CY14 inhibits the Nur77-PPAR interaction in cells, we assessed its cell permeability by conjugating 5fluorescein isothiocyanate (5-FITC) to CY14 (Table S2, Supporting Information).Unfortunately, CY14 cannot cross cell membranes to interact with intracellular target (Figure 3a,b).Hence, we conjugated the transcription transactivating (TAT) sequence to the C-terminus of CY14 (Figure 3a) using SPPS.FITC-peptide conjugated with TAT (T-CY14) exhibited significantly enhanced membrane permeability in MCF7 cells (Figure 3b).Additionally, although AlphaFold2 predicted that CY14 adopted an helical conformation (Figure S4e, Supporting Information), neither CY14 nor T-CY14 exhibited representative -helical characteristics in circular dichroism (CD) spectroscopy (Figure 3c).This difference may be attributed to the inherent tendency of linear peptides to lose their native topology in aqueous solution, which could explain the relatively low affinity of CY14 toward Nur77.As one of the most widely applied peptide-stapling ap-proaches for PPIs, all-hydrocarbon stapling chemistry is an effective strategy for structurally stabilizing -helices and favoring protein binding. [19]We synthesized stapled CY14 and T-CY14 by incorporating S-pentenyl-alanine (S5) at the i and i + 4 positions (Phe163 & Ile167) along one face of the -helix exposed to the solvent surface (Figure S4j, Supporting Information), followed by ring-closing olefin metathesis (Figures 3a; S4k,l, Supporting Information).Both stapled -helical peptides, S-CY14 and ST-CY14, displayed relatively high -helicities, with -helical degrees of 15.6% and 12.01%, respectively (Figure 3a,c).To verify that the staple did not impair the interaction between the peptide and Nur77, the affinities of S-CY14 (K d = 3.96 × 10 −6 M) and ST-CY14 (K d = 6.68 × 10 −7 M) with Nur77 were determined using MST (Figure 3d,e), demonstrating higher affinities than T-CY14 (K d = 4.18 × 10 −6 M) (Figure S4i, Supporting Information).
To confirm the blocking function of ST-CY14 on the Nur77-PPAR complex in cells, plasmid DNA constructs containing luciferase SmallBit fusions of Nur77 and luciferase LargeBit fusions of PPAR were generated.These constructs were transiently expressed in HEK293T cells.The formation of the Nur77-PPAR complex contributes to luminescence due to the interaction between SmBit and LgBit upon the addition of the nanoluciferase substrate.The results showed that ST-CY14 effectively inhibited the formation of the Nur77-PPAR complex, with an EC 50 value of 3.15 × 10 −6 M, in comparison to T-CY14 (Figure S5, Supporting Information).Conversely, TAT and S-CY14 had minimal effects on the luminescence.(Figure 3f).All these results demonstrated the power of the stapled peptide and the necessity of the cell-penetrating sequence.Mutation of Nur77 at positions Gln571, Glu398, and Phe598 disrupted the interaction between Nur77 (Mut) and ST-Y14 (Figure S4i, Supporting Information), providing additional evidence that ST-CY14 primarily interacts with Nur77 through these three key residues.Surface plasmon resonance (SPR) analysis further confirmed that ST-CY14 bound to Nur77 with a K D value of 3.247 × 10 −8 M (Figure 3g).Subsequently, we examined the ability of ST-CY14 to interfere with the endogenous interaction between Nur77 and PPAR in HEK293T cells.Decreased PPAR expression was detected in the ST-CY14treated group, indicating that ST-CY14 inhibited the binding between Nur77 and PPAR.Trim13 was reported to mediate the ubiquitination of Nur77. [20]ST-CY14 obviously reduced the expression of Trim13 and attenuated the PPAR-induced ubiquitination of endogenous Nur77 (Figure 3h).These data suggested that ST-CY14 could bind to Nur77 and specifically inhibit the Nur77-PPAR interaction in cells.Owing to the preorganized stable -helix topology, ST-CY14 exhibited significantly greater proteolytic stability than T-CY14 (half-life 4.94 min vs 87.78 min), representing a seventeen-fold improvement (Figure 3i).

ST-CY14 Inhibits the Nur77-PPAR𝜸-Mediated Signaling Pathway
We  S7, Supporting Information).In addition to its antiproliferative activity, ST-CY14 exhibited a high solubility (>20 mg mL −1 ) in PBS, whereas the solubility of Csn-B was significantly lower at ≈1 mg mL −1 (Figure S8, Supporting Information).These results highlight the advantage and potential of targeting the Nur77-PPAR interaction interface with a peptide inhibitor rather than a Nur77 agonist.
Blockade of the Nur77-PPAR interaction could increase Nur77 protein levels via decreased PPAR-induced ubiquitination. [6]To this end, immunoblotting was employed and revealed that an increase in Nur77 expression in MCF7 and MDA-MB-231 cells following treatment with ST-CY14 and T-CY14 (Figure 4c,d).Moreover, ST-CY14 inhibited the protein expression of CD36 and FABP4, which are regulated by Nur77, in MCF7 (Figure 4c) and MDA-MB-231 cells (Figure 4d).T-CY14 also had some effect, but its efficacy was much lower (Figure 4c,d).Consistently, qPCR revealed that ST-CY14 dosedependently inhibited CD36 and FABP4 expression at the transcriptional level in MCF7 (Figure 4e,f) and MDA-MB-231 cells (Figure 4g,h).

ST-CY14 Inhibits Nur77-PPAR𝜸-Mediated Cell Metabolism
Nur77 acts as a suppressor of CD36 and FABP4, which regulate lipid metabolism during lipid uptake and lipid decomposition.To determine whether direct blockade of the Nur77-PPAR interaction is involved in lipid metabolism, we used ST-CY14 as a chemical probe to treat breast tumor cells.Examination of FA uptake by flow cytometry revealed that ST-CY14 induced a dose-dependent decrease in FA levels in MCF7 cells (Figure 5a).Immunofluorescence-based studies also showed that ST-CY14 inhibited FA uptake in MCF7 cells (Figure 5b).FAs are the main substrates for mitochondrial energy metabolism; thus, we further evaluated the oxygen consumption rate (OCR), an indicator of mitochondrial respiration, in MCF7 and MDA-MB-231 cells following ST-CY14 treatment.These results showed that the OCR was significantly reduced after ST-CY14 treatment, indicating that ST-CY14 reduced the rate of mitochondrial respiration in MCF7 (Figure 5c) and MDA-MB-231 cells (Figure 5d).
As the main component of the cell membrane and energy metabolism substrate, FAs are vital in cell migration and invasion in breast cancer metastasis.We wondered whether ST-CY14 might have inhibitory effects on the migration of breast tumor cells.Our transwell experiments showed that ST-CY14 and T-CY14 inhibited the transmigration of MCF7 (Figure 5e) and MDA-MB-231 (Figure 5f) cells.ST-CY14 and T-CY14 also suppressed the scratch healing of both breast tumor cell lines (Figure 5g,h).Taken together, these results suggest that direct blockade of the Nur77-PPAR interaction inhibits the metabolism, migration, and invasion of breast tumor cells.

ST-CY14 Suppresses Tumor Growth and Bone Metastasis of Breast Cancer in Mice
Following the above encouraging results observed for ST-CY14 in cells, we evaluated the antitumor efficacy of ST-CY14 in an MDA-MB-231-GFP xenograft model (Figure 6a).ST-CY14 suppressed tumor growth in a dose-dependent manner by 60.18% and 69.73% tumor growth inhibition values at the end of the thirtyday treatment after receiving 5 and 10 mg kg −1 doses, respectively, once every two days via the tail vein (Figure 6b), without significant body weight loss (Figure 6c) or other signs of organ toxicity (Figure S9, Supporting Information) in all treated mice.Moreover, bioluminescence imaging and tumor weight also indicated that ST-CY14 inhibited tumor progression in mice (Figure 6df).Consistent with the in vitro results, ST-CY14 effectively increased Nur77 protein levels, and decreased CD36 and FABP4 protein levels (Figure 6g) in MDA-MB-231-GFP tumors.Pathological and immunohistochemical analyses of tumor sections showed that, compared with the control group, ST-CY14 led to reduced tumor cell proliferation, increased necrosis area and apoptosis (Figures 6h; S10, Supporting Information).Considering the regulatory effect of Nur77 on lipid metabolism, lipid metabolome analysis was applied to detect the lipid content in tumor tissue.The relative abundance of various lipid Immunofluorescence staining images of the p) TRAP ratio in the mouse femoral head and q) relative statistics.For (b,e,f,h,m-o, and q), the quantified data from different experiments were presented as the mean ± SD.The P values were calculated by one-way ANOVA.
The bone is the most common site of breast cancer metastasis, accounting for ≈65 to 75% of all metastatic breast cancer patients.In particular, when metastasis occurs, the 5-year survival rate of breast cancer patients is as low as 10%. [21]In addition, enhanced lipid levels in the bone marrow benefit FA metabolism in breast cancer. [22]Thus, a bone metastatic breast cancer model was used to evaluate the anti-metastatic efficacy of ST-CY14.A bone metastasis model was constructed by injecting 1 × 10 5 MDA-BoM-1833 cells into the left cardiac ventricle of mice.Bone metastatic tumor progression was monitored by in vivo bioluminescence imaging (days 7, 14, 21, and 28) (Figure 6k).As shown in Figure 6l,m, significant bone metastasis was observed in the control group after the 28th day, while the fluorescence intensities of the ST-CY14 treated groups were lower, suggesting that ST-CY14 inhibited the metastasis of tumor cells.Tumor cells can secrete factors into the bone microenvironment, including IL-6 and PTH-related peptide, which activate osteoclasts when they are deposited in the bone marrow. [23]Beta C-terminal cross-linked telopeptides of type I collage (-CTx) and Bonesialo protein (BSP) are considered to be markers of osteoclasts.Lower levels of BSP (Figure 6n) and -CTx (Figure 6o) were detected in the ST-CY14-treated group than in the control group, indicating that ST-CY14 inhibited bone metastasis and the formation of osteoclasts in the bone marrow.None of the groups demonstrated notable changes in H&E staining of major organs, indicating satisfactory safety profiles (Figure S11, Supporting Information).Immunohistochemical analysis also showed that ST-CY14 treatment led to a decrease in the level of tartrate-resistant acid phosphatase (TRAP), the signature enzyme of osteoclasts (Figure 6p,q).Notably, the efficacy of ST-CY14 in inhibiting breast cancer bone metastasis was superior to that of Csn-B (Figure S12, Supporting Information).Taken together, these results demonstrated that ST-CY14 is a promising lead compound to inhibit breast cancer bone metastasis.

Conclusion
The highly metastatic nature of breast cancer results in poor prognosis and heightened mortality rates.Adjacent lipocytes establish a lipid-rich microenvironment, providing breast cancer cells with important substrates for proliferation, migration, and invasion.Thus, targeting lipid metabolism in breast cancer represents a promising direction for more effective therapeutic strategies.Extensive evidence has shown that Nur77 is a critical suppressor of lipid metabolism in breast cancer, and Nur77 defi-ciency often indicates a worse prognosis. [6]However, the activated Nur77-PPAR signaling pathway plays an antagonistic role in the Nur77-mediated process, and identifying direct inhibitors of the Nur77-PPAR interaction without a cocrystal structure is challenging.
Here, based on the optimal scaffolds of epitope peptides and advanced in silico approaches, we developed ST-CY14 as a highpotency PPI peptide inhibitor that effectively blocks the Nur77-PPAR interaction both in vitro and in vivo, which not only validates the feasibility of exploiting the Nur77-PPAR interaction as a target of breast cancer for drug discovery, but also provides an effective paradigm for computer-aided drug design in PPIpeptide-inhibitor research.In the present study, our data demonstrated that the binding of ST-CY14 to the Nur77 LBD could protect Nur77 from ubiquitination induced by PPAR, leading to the accumulation of Nur77 and low levels of CD36 and FABP4.More importantly, due to its ability to reduce lipid metabolism, ST-CY14 exhibited pronounced inhibitory efficacy on tumor growth and bone metastasis of breast cancer in mice.
In summary, we developed a potent and selective peptide inhibitor of Nur77-PPAR PPI, ST-CY14, with in vivo antitumor activities and no toxicity.The structural scaffold and binding mode of ST-CY14 make it a promising lead compound for further development in breast cancer treatment, and will facilitate the further design and modification of inhibitors in the Nur77-PPAR interface.

Experimental Section
Materials, Cell Culture, and Animal Models: Rink Amide MBHA resin (0.30 mmol g −1 loading) was purchased from Tianjin Nankai Hecheng Science & Technology Co., Ltd.;All the other chemical reagents and solvents used were purchased from Adamas-beta, GL Biotech, CSBio (Shanghai), Energy Chemical, and Sinopharm Chemical Reagent Co., Ltd, and were used without further purification.
The HEK293T, MCF7, and MDA-MB-231 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).The MDA-MB-231 cell line was labeled with EGFP and luciferase by Xinzhou.Additionally, the MDA-BoM-1833 cell line was provided by Professor Yu-Dong Zhou from the University of Mississippi and labeled with a luciferase tag in the lab.All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) medium supplemented with 10% (v/v) fetal bovine serum and antibiotics (100 mg mL −1 streptomycin and 100 units mL −1 penicillin), and maintained at 37 °C with 5% CO 2 .
Female BALB/c mice (4-5 weeks old, 16-18 g) and BALB/c mice (7-8 weeks old, 18-20 g) were provided by Shanghai Slake Experimental Animal Co., Ltd., and housed under specific pathogen-free conditions for use in orthotopic xenograft tumor model experiments and bone metastasis tumor model experiments.The animal experiment was approved by the ethical committee of Shanghai University of Traditional Chinese Medicine (approval number: PZSHUTCM220627036).
Molecular Dynamics Simulation: The MD simulations were performed using GROMACS [16] The Amber99SB-ILDN force field and TIP3P water model were set to generate the topology of the protein complex.And then a dodecahedron box was created to place the protein at the center with a minimum distance of 1.2 nm between the protein and the box edges.The system was subjected to energy minimization in vacuo using the steepest descent algorithm to remove any steric clashes or bad contacts.To mimic the physiological environment, the protein was solvated using a TIP3P water model, and the ions were added to neutralize the system.Energy minimization was performed again, and the system was equilibrated at NVT and NPT conditions to maintain constant temperature (310 K) and pressure (1 bar).Finally, the production simulation was carried out for a total of 100-150 ns with a time step of 2 fs.
Transient Transfection of siRNA: Cells were seeded 24 h prior to transfection.Transfection was performed with Transfect-Mate (GenePharma, G04009) according to the manufacturer's protocols.To transfect siRNA into cells seeded on 6 well cell culture plates, 150 pmol of siRNA was mixed with 8 μL Transfect-Mate, incubated at room temperature for 15 min, and then added to the cells.The medium was changed to DMEM after 6 h of culture, and the cells were incubated at 37 °C in a 5% CO 2 incubator.48 h later, the siRNA was successfully expressed and could be used for further experiments.The siRNAs used were listed in Table S1 (Supporting Information).
Colony Formation Assay: The colony formation assay was used to evaluate the proliferative ability of the MCF-7 cells.Transfected cells in the logarithmic growth phase were seeded into 6-well plates.After siRNA transfection the next day and 1-week incubation subsequently, visible colonies formed.Then, the cells were fixed and stained using methanol and Giemsa, respectively.Colonies were snaped under a microscope and counted at a wavelength of 590 nm.
Synthesis of Peptides: Peptides were synthesized using SPPS on Rink Amide MBHA resin.The resin (1 mmol) was swelled with dichloromethane (DCM) and then deprotected twice using a deprotect solvent (N,N-dimethylformamide, DMF, containing 20% piperidine and 0.1 mol L −1 Oxyma pure).The peptides were washed with DMF and DCM.A solution of Fmoc-AA-OH (1 mmol), N, N'-diisopropylcarbodiimide (DIC) (1 mmol), and Oxyma pure (1 mmol) in N-methylpyrrolidone (NMP) was added and reacted with the resin at 60 °C for 15 min.After the reaction, the resin was washed with DMF and DCM and deprotected for the next amino acid coupling.The 2 h coupling of Fmoc-S5-OH was carried out at 60 °C.After completion of all amino acid couplings, peptide cyclization was conducted using a 1 × 10 −2 M solution of the Grubbs firstgeneration ruthenium catalyst, benzylidene-bis(tricyclohexylphosphine) dichlororuthenium, in dichloroethane.The peptide was cleaved from the resin using a cocktail of trifluoroacetic acid (TFA), triisopropylsilane, and H 2 O (95:2.5:2.5) for 2 h at room temperature.
Fluorescent Labeling of Peptides: After elongation of the peptide from the resin according to a previous protocol, the Fmoc group was removed and the resin was treated with Fmoc-Ahx-OH (4 eq.), DIC (4 eq.), and Oxyma pure (4 eq.) in NMP for 15 min at 60 °C.Subsequently, the Nterminal amino group was deprotected and reacted with 5-FITC (2 eq.) and N,N-diisopropylethylamine (2 eq.) in DMF (6 mL) at room temperature overnight, resulting in the incorporation of a fluorescein label.
Analytical Methods: The detection wavelengths of the peptides were 214 and 254 nm.Solvent A: H 2 O (containing 0.1% TFA) and solvent B: acetonitrile (containing 0.1% TFA) were used as solvents in linear gradient mixtures.Peptide purification was performed by semi-preparative HPLC using an XBridge Prep C 18 column (19 × 250 mm, 10 μm particle size, flow rate of 20 mL min −1 ).Solvents A and B were the same as those in the peptide analysis procedure.The peptides were dissolved in H 2 O to a final concentration of 5 × 10 −5 M. The purified peptide was detected using a mass spectrometer (6410 Triple Quad LC/MS, Agilent, USA) (Table S2, Supporting Information).The solvent was 80% methanol and 20% H 2 O (containing 0.1% formic acid).The CD spectra were obtained with a 1 mm quartz cuvette on a BRIGHT TIME Chirasca (Applied Photophysics, Britain) spectropolarimeter with a scan wavelength between 185-260 nm at 25 °C.The helicity of each peptide was calculated by the literature equation. [24]bservation of FITC-peptide Translocation Using Confocal Microscopy: MCF7 cells (50 000 cells/well) were seeded in a 20 mm dish and treated with 1 × 10 −5 M FITC-labeled peptides for 1 h.After treatment, the cell nuclei were stained with Hoechst 33 342 (5 × 10 −5 M) for 8 min.The membrane detection of peptides was performed using a Leica TCS SP8 confocal microscope.The FITC-labeled peptide was excited at a wavelength of 488 nm, with an emission wavelength range of 505-530 nm, while the cell nuclei were excited at a wavelength of 350 nm, with an emission wavelength of 461 nm.
Surface Plasmon Resonance (SPR): ST-CY14 with Nur77-LBD was detected by SPR using the Biacore T200 system at 25 °C.Recombinant human Nur77-LBD protein was immobilized on an activated carboxymethylated 5 sensor chip (GE Healthcare) using the amine coupling method.Gradient concentrations of EB were injected at a flow rate of 30 μL min −1 in a running buffer (1% DMSO in PBS).The results were analyzed with Biacore evaluation software (T200 version 2.0).The data were fitted to the 1:1 Langmuir binding model, and kinetic parameters were derived.
Co-Immunoprecipitation (Co-IP): HEK293T cells were seeded overnight in 10 cm culture dishes and transfected with the Nur77 and PPAR plasmids for 48 h.After transfection, T-CY14 and ST-CY14 were incubated with the cells for 24 h.The cells were then collected and lysed in 500 μL precooled NP40 lysis buffer (Beyotime, P0013F), which contained 20x holoenzyme inhibitor and 50x phosphatase inhibitor.All the samples were prepared at a concentration of 1 mg mL −1 , and 0.5 mg of the sample was subsequently incubated overnight at 4 °C with 2 μg of Nur77 antibody (Proteintech, 12235-1-AP).The samples were then incubated with Protein A/G PLUS-Agarose (Santa Cruz, sc-2003) for 4 h at 4 °C.The beads were collected through centrifugation and washed five times with NP40 lysis buffer.Finally, 5x loading buffer was added to the buffer containing the beads for Western blot analysis of PPAR antibody (Proteintech, 16643-1-AP), Trim13 antibody (Abcam, ab234847), and -actin antibody (epizyme, LF201).
Protease Stability: The peptides were dissolved in PBS (pH 7.

Figure 1 .
Figure 1.The function of Nur77 in breast tumor cells and an in silico systematic approach to predict the structure of the Nur77-PPAR heterodimer.a) Cell proliferation assays of Nur77-knockdown MCF7 cells.The quantified data from different experiments were presented as the mean ± SD, and the P values were calculated using a two-tailed t-test.n = 3 biological replicates.b) Enhanced lipid uptake capabilities were observed in Nur77-knockdown MCF7 cells.c) Western blotting was used to determine the protein levels of Nur77 and PPAR after knocking down PPAR in MCF7 cells (PPAR siRNAs are represented by #1 and #2).The quantified data from different experiments were presented as the mean ± SD.The P values were calculated by one-way ANOVA.n = 3 biological replicates.d) Schematic representations of Nur77 and PPAR.e) The structure of the PPAR DBD predicted by AlphaFold2.f) Predicted binding conformation of the Nur77 LBD-PPAR DBD heterodimer (rank 2).g) The C RMSD for the rank 2 conformation.h) Predicted binding conformation of the Nur77 LBD-PPAR DBD heterodimer (rank 3).i) The C RMSD for the rank 3 conformation.

Figure 2 .
Figure 2. Identification of inhibitors targeting the Nur77-PPAR interaction.a)The interaction between SV16 and Nur77 was depicted in both cartoon and stick forms, with SV16 colored orange and the Nur77 LBD colored blue.Interaction residues were labeled.b) The detailed interaction between SV16 and Nur77 was revealed, with the red dashed line representing the intermolecular bond between them.c) The binding free energy decomposition of SV16 and Nur77 was shown.d) The interaction between CL14 and Nur77 was depicted in both cartoon and stick forms, with CL14 colored pink and the Nur77 LBD colored blue.Interaction residues were labeled.e) The detailed interaction between CL14 and Nur77 was presented, with the red dashed line representing the intermolecular bond between them.f) The binding free energy decomposition of CL14 and Nur77 was illustrated.g) The free energy changes after peptide point mutations.h) The binding affinity of CY14 with Nur77 was depicted using MST.

Figure 3 .
Figure 3. Structural optimization of CY14.a) Sequences and helicities of CY14, T-CY14, S-CY14, and ST-CY14.X, S5 (S-pentenyl alanine).H and NH 2 in the peptide sequence represent the N-terminal amino group and C-terminal primary amide, respectively.b) The cell transmembrane abilities of CY14 and T-CY14 were observed via confocal microscopy.c) CD spectra of T-CY14 and ST-CY14.d) The affinity of S-CY14 for Nur77 was depicted using MST.e) The affinity of ST-CY14 for Nur77 was depicted using MST.f) The blocking effect of ST-CY14 on Nur77 and PPAR was detected by NanoBit.The quantified data from different experiments were presented as the mean ± SD.The P values were calculated by one-way ANOVA.n = 3 biological replicates.g) The affinity of ST-CY14 for Nur77-LBD was determined using SPR.h) Immunoprecipitation of PPAR was performed after treatment with ST-CY14 (1 × 10 −5 M ) in 293T cells.IP: Nur77, IB: PPAR, Trim13.i) The proteolytic stability of T-CY14 and ST-CY14 in an -chymotrypsin solution.

Figure 4 .
Figure 4. ST-CY14 inhibits the Nur77-PPAR-mediated signaling pathway.The cell viability of a) MCF7 and b) MDA-MB-231 cells after treatment with T-CY14 or ST-CY14 for 24 h.The effects of ST-CY14 on increasing Nur77 protein levels and decreasing CD36 and FABP4 protein levels were more pronounced in c) MCF7 cells and d) MDA-MB-231 cells compared to those of T-CY14.ST-CY14 treatment inhibited the transcription of CD36 and FABP4 in e,f) MCF7 cells and g,h) MDA-MB-231 cells.The quantified data from different experiments were presented as the mean ± SD.The P values were calculated by two-way ANOVA.n = 3 biological replicates.* represents different concentrations of T-CY14 or ST-CY14 versus control group.# represents ST-CY14 versus T-CY14 at the same concentration.
next investigated the cell viability of MCF7, MDA-MB-231, and MDA-BoM-1833 cells treated with ST-CY14, T-CY14, and Csn-B, a reference control.ST-CY14 at 5 × 10 −6 M significantly attenuated breast tumor cell proliferation, and ST-CY14 was more potent for breast tumor cells than T-CY14 and Csn-B (Figure 4a,b: Figure S6, Supporting Information), which was in agreement with their respective binding affinities to Nur77.Moreover, normal breast cells (MCF-10A) and Nur77-knockdown breast tumor cells were less sensitive to ST-CY14 (Figure

Figure 5 .
Figure 5. ST-CY14 inhibits Nur77-PPAR-mediated cell metabolism.a) Flow cytometry was used to compare the lipid uptake abilities of MCF7 cells after treatment with T-CY14 or ST-CY14.b) Confocal microscopy images were taken to observe lipid uptake in MCF7 cells after 1 × 10 −5 M T-CY14 or ST-CY14 treatment.ST-CY14 significantly inhibited mitochondrial aerobic respiration in c) MCF7 and d) MDA-MB-231 cells.The quantified data from different experiments were presented as the mean ± SD.The P values were calculated by one-way ANOVA.n = 3 biological replicates.Representative images and statistics of Transwell migration assays of e) MCF7 and f) MDA-MB-231 cells following treatment with T-CY14 and ST-CY14.Representative images and statistics of wound-healing assays on g) MCF7 and h) MDA-MB-231 cells after treatment with T-CY14 and ST-CY14.For (e-h), the quantified data from different experiments were presented as the mean ± SD.The P values were calculated by two-way ANOVA.n = 3 biological replicates.* represents different concentrations of T-CY14 or ST-CY14 versus control group.# represents ST-CY14 versus T-CY14 at the same concentration.

Figure 6 .
Figure 6.ST-CY14 suppresses tumor growth and bone metastasis of breast cancer in mice.a) Flow diagram of the treatment regimen in the MDA-MB-231-GFP xenograft model.b) Tumor volume during treatment (n = 6).c) Time-course of body weight (n = 6).d) In vivo tumor imaging before sacrifice and e) accumulated optical density measurement.f) Tumor weight.g) Western blot analysis showing that ST-CY14 treatment inhibits Nur77 and its downstream signaling pathway-related target genes CD36 and FABP4 in vivo.h) Image-based quantitative results of Ki-67, H&E, and TUNEL staining of tumor samples (n = 3).i) Heatmap images showing a significant reduction in the levels of diverse lipid constituents within the group treated with ST-CY14.j) Heatmap images of the nearly twenty downregulated genes.k) Flow diagram of the treatment regimen in bone metastatic breast cancer model.l) In vivo imaging of mice every week.m) Bone metastasis tumor accumulated optical density measurement on day 28.n) BSP and o) -CTx levels in mouse blood detected through ELISA analysis.Immunofluorescence staining images of the p) TRAP ratio in the mouse femoral head and q) relative statistics.For (b,e,f,h,m-o, and q), the quantified data from different experiments were presented as the mean ± SD.The P values were calculated by one-way ANOVA.