Turning a Luffa Protein into a Self-Assembled Biodegradable

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Turning a Luffa Protein into a Self-Assembled Biodegradable Nano-Platform for Multi-Targeted Cancer Therapy Wangxiao He, Jin Yan, Fang Sui, Simeng Wang, Xi Su, Yiping Qu, Qingchen Yang, Hui Guo, Meiju Ji, Wuyuan Lu, Yongping Shao, and Peng Hou ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07079 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 16, 2018

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Turning a Luffa Protein into a Self-Assembled Biodegradable Nano-Platform for Multi-Targeted Cancer Therapy

Wangxiao He†,‡, Jin Yan§, Fang Sui†, Simeng Wang†, Xi Su†, Yiping Qu†, Qingchen Yang‡, Hui Guo†, Meiju Ji∥, Wuyuan Lu⊥, Yongping Shao# and Peng Hou†

†

Key Laboratory for Tumor Precision Medicine of Shaanxi Province and Department of

Endocrinology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China ‡

Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life

Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China. §

Frontier Institute of Science and Technology, State Key Laboratory for Mechanical Behavior of

Materials, Xi’an Jiaotong University, Xi’an 710049, China ∥

Center for Translational Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an

710061, China ⊥

Institute of Human Virology and Department of Biochemistry and Molecular Biology, University

of Maryland School of Medicine, Baltimore, MD 21201, USA. #

Frontier Institute of Science and Technology, Center for Translational Medicine, School of Life

Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China.

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ABSTRACT Peptide-derived self-assembly platform has attracted increasing attention for its great potential to develop into multi-targeting nanomedicines as well as its inherent biocompatibility and biodegradability. However, their clinical application potentials are often compromised by low stability, weak membrane penetrating ability and limited functions. Herein, inspired by a natural protein from the seeds of Luffa cylindrica, we engineered via epitope grafting and structure design a hybrid peptide-based nanoplatform, termed Lupbin, which is capable of self-assembling into a stable superstructure and concurrently targeting multiple protein-protein interactions(PPIs) located in cytoplasm and nuclei. We showed that Lupbin can efficiently penetrate cell membrane, escape from early endosome-dependent degradation, and subsequently disassemble into free monomers with wide distribution in cytosol and nucleus. Importantly, Lupbin abrogated tumor growth and metastasis through concurrent blockade of the Wnt/βcatenin signaling and reactivation of the p53 signaling, with a highly favorable in vivo biosafety profile. Our strategy expands the application of self-assembled nanomedicines into targeting intercellular PPIs, provides a potential nano-platform with high stability for multi-targeted cancer therapy, and likely reinvigorate the development of peptide-based therapeutics for the treatment of different human diseases including cancer.

KEYWORDS: self-assembled protein-based nanoparticles, engineered protein, protein-protein interactions, multi-targeted cancer therapy, hazard-free therapy

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Targeted therapy is always challenged by adaptive or acquired resistance, mainly because efficacies of single-target drugs are often compromised by the compensatory mechanism.1 While this limitation can be overcome by attacking the disease on multiple fronts using combinational therapy,2 simultaneous administration of multiple drugs inevitably complicates the design of clinical trials, and increase the risk of drug-drug interactions.1 Thus, it is clinically desirable to develop multi-targeted therapeutic agents with single entity. Indeed, some multi-kinase inhibitors such as Sorafenib and Regorafenib have been successfully applied in the treatment of cancers and other complex diseases.3 However, to our knowledge, almost all the multi-targeting therapeutics in clinical applications are small-molecule kinase inhibitors.4, 5 In comparison to the 538 protein kinases encoded by human genome3, there are ~650,000 protein-protein interactions (PPIs) in the human proteome6. The PPIs are the fundamental basis of almost all cellular process.7 This complex network of PPIs will provide numerous potential therapeutic targets for various diseases including malignancies. Small molecule inhibitors are generally not suitable to block PPIs that usually bear flat and large binding interface.8 Peptides, however, with their large interacting interfaces of chemical and structural diversity serve as ideal candidates for inhibitors of PPIs.9 However, their clinical applications, especially targeting intracellular PPIs, are severely limited by intrinsic in vivo instability and weak membrane penetrating ability.9, 10 To overcome these pharmacological hurdles, nanotechnology provides a perspective to “bottomto-up” engineer versatile peptide into highly ordered nanostructures with stable structure and cellmembrane penetrability.10, 11 To date, many peptide-based nanosystems such as, but not limited to, liposome encapsulating peptide nanomicelles, peptide-coating nanoparticles and self-assembled peptide nanostructure, exhibit both sophisticated superstructures as well as fascinating biological effects 3


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including longer blood circulation time, higher targeting ability, greater stability and better therapeutic efficacy.10-12 Among them, benefited from inherent biocompatibility, biodegradability and high loading capacity, peptide-derived self-assembly nanoplatform has attracted more and more attention in nanotherapeutic construction and drug delivery.13 Therefore, designing self-assembled peptide-based nanomedicine may be of great interests for next generations of PPI-targeted therapies. Current self-assembly nanotechnologies are challenged by three common hurdles: (1) how to form well-controlled superstructures with high stability, (2) how to incorporate one or more of pharmic functions into nanoscale building blocks, and (3) how to escape from endosome or lysosome-dependent degradation.13-15 Fortunately, as a consequence of millenniums of evolution, life already possesses an unique set of principle to control the self-assembly and oligomerization of the proteins, which provide versatile self-assembled scaffolds with high stability and desirable biological properties, including intracellular response and distribution.15 More importantly, epitope grafting offers a viable strategy to endow the scaffold with binding activity for targeted PPIs by transferring critical binding residues of therapeutic peptides onto the structurally permissible region of the surrogate protein.16,17 In this context, a scaffold with multiple grafting sites, stable hierarchical structures and endosome escaping capability will enable the design of multi-targeting nanotherapeutic for intracellular PPIs. To address this, Luffin P1, a small protein from the seeds of Luffa cylindrica,18 called our attention. Luffin P1 consists of a cyclized helix-loop-helix motif stabilized by two disulfide bridges,18 therefore serving as an ideal template for multi-targeted epitope grafting. In addition, its arginine-rich sequences and oligomerization tendency provide a potential possibility for the design of self-assembled nanotherapeutics with efficient endosomal escape.19, 20 For proof of concept, we introduced two anti4


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cancer peptides into the topologically equivalent positions of Luffin P1: (1) PMI, a potent dodecameric peptide inhibitor for the interaction between p53 and MDM2/MDMX;21 (2) Bcl9p, a peptide that inhibits the Wnt/β-catenin signaling by blocking β-catenin-Bcl9 interaction.22 Preferably, the hydrophobic residues from PMI and Bcl9 peptides further increased the intermolecular hydrophobic interactions among the grafted protein molecules, thereby inducing the oligomerization of the grafted proteins into a stable high-ordered structure at nanoscales. More interestingly, intracellular glutathione will trigger the disassembly of the nanostructure into smaller monomers of amorphous form through rupturing the disulfide bridge, thereby resulting in widespread distribution not only in the cytoplasm but also in the nucleus. In this case, the Luffin P1-derived p53-MDM2/MDMX and beta-catenin-Bcl9 inhibitor, termed Lupbin, can concurrently block the interactions between p53 and MDM2/MDMX in the cytoplasm, and between β-catenin and Bcl9 in the nucleus, thereby potently inhibiting tumor growth and metastasis in vitro and in vivo. Notably, intracellular amorphous monomers of Lupbin that bind to target proteins has high stability, while the unbound Lupbin will be rapidly degraded, thereby guaranteeing its safety. Therefore, this viable strategy will provide a potential nano-platform to develop peptide-based nanomedicines for multi-targeted cancer therapy.

RESULTS AND DISCUSSION Rational design of the self-assembled nanotherapeutic, Luffin P1. To engineer the Luffin P1 for simultaneous antagoniztion of the p53-MDM2, p53-MDMX and β-catenin-Bcl9 interactions, we used a dodecameric peptide PMI,21 and a natural peptide (Bcl9p) intercepted from the β-catenin -binding domain of Bcl9 (residues 351 to 374) as the grafts. PMI binds to MDM2 and MDMX with low nM 5


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affinities mainly through three critical residues including Phe19, Trp23 and Leu26 (Fig. 1a, b).21 In the Bcl9 peptide, five key residues (H358, R359, L366, I369 and F374) mediate its high affinity binding to the hydrophobic groove of β-catenin (Fig. 1c).23 Sequence and structure alignment revealed some levels of similarity between the PMI and Luffin P1 (residues 6-17) or the Bcl9 and Luffin P1 (residues 23-42) pairs. Therefore, we simply replaced the Luffin P1 amino acids with those key residues (together with their neighboring residues) identified in PMI or Bcl9 peptides at corresponding positions (Fig. 1d). In addition, the GS sequence at the N-terminus of Luffin P1 was replaced with a double arginine sequence (RR) to compensate for the loss of electric charges due to residue swapping (Fig. S1). The resulting hybrid protein Lupbin containing 43 amino acid residues was synthesized chemically via a twofragment ligation strategy as illustrated in Fig. 1e.

Owing to the hydrophobic residues grafted from

PMI and Bcl9 peptides, the intermolecular hydrophobic interactions among Lupbin monomers will be enhanced, thereby facilitating their oligomerization into a stable structure at nanoscales (Fig. 1e). These stable nanostructures are expected to dissemble in the reductive intracellular environment into smaller monomers of free-form structure, leading to a widespread distribution in both cytoplasm and nucleus (Fig. 1f).

Preparation of the self-assembled Luffin P1. The final product was purified by high performance liquid chromatography (HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS) (Fig. 2a). As a control, Luffin P1 was chemically synthesized in the same way as described for Lupbin (Table S1). Next, we folded the two synthetic proteins by dissolving them in PBS. As shown in Fig. 2b, both Lupbin and Luffin P1 adopted similar alpha-helical conformations in 6


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solution, as evidenced by their similar circular dichroism (CD) spectra showing three characteristic absorption peaks of α-helix at 195, 208 and 222 nm, which was in line with the reported structure of Lupbin.19 In addition, we characterized the surface charge (Zeta potential) of the two proteins (Fig. 2c), and the data unambiguously demonstrated that the two proteins exhibited strong and similar levels of positive charge in PBS buffer (pH 7.4). Luffin P1 has been reported to have a tendency to oligomerization in a hydrophobic condition,18 while grafting multiple hydrophobic residues of PMI and Bcl9 into Luffin P1 will exacerbate this process. To test it, small angle X-ray scattering (SAXS), an excellent technique that are particularly sensitive to the shape of the objects in a size at nanoscale,24, 25 was used to interrogate the morphological details of the Lupbin particles. Because of the individual random orientation of Lupbin in aqueous media, the scattering results had the isotropism, thus it is acceptable for us to azimuthally average 2D data to 1D patterns.25, 26 At a concentration of 0.1 mg/mL, the scattering spectra of Lupbin consisted solely of undulations that are characteristic of irregular elliptic nanorods (Fig. 2d). It was noted that the observed average radius-of-gyration (Rg) values (2.3 nm) for Lupbin particles was larger than the calculated values of 1.1 nm for monomers, indicating that Lupbin molecules probably form oligomeric nanoellipsoids (Fig. 2d). Using an orientationally averaged rod model, we predicted three different sizes of nano-ellipsoids, and calculated the lengths of principal axes of these ellipsoids, which were 1.15 nm, 2.2 nm and 5.12 nm, respectively (Fig. 2e, f). Through measuring the hydrate diameters of protein particles by dynamic light scattering, we demonstrated that the average sizes of Lupbin and Luffin P1 were 4.9 nm and 2.7 nm, respectively (Fig. 2g). In addition, the average molecular weight of Lupbin was determined to be 25.4 kDa, which is around 4 times as large as that of single Luffin P1 molecule 7


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(5.8 kDa) (as per Mark-Houwink equation27), further supporting that Lupbin exists as tetramers in physiological buffer (Fig. 2h). Notably, the size of tetrameric form of Lupbin is appropriate to penetrate and distribute in the dense tumor matrix owing to the EPR effect.28, 29 Peptides are generally susceptible to proteolysis, hampering their application in clinics.9,

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Grafting structurally flexible peptides (PMI and Bcl9p) into the conformationally-constrained Luffin P1 scaffold may improve the stability of peptides through blocking the access of proteolytic enzymes via steric hindrance.10 To evaluate the stability of free peptides versus Lupbin, protein/peptide samples were incubated in PBS with oxidized glutathione (GSSG), 10% serum and chymotrypsin (10 μg/mL), a protease specific degrades hydrophobic residues. As expected, Lupbin was more resistant to proteolysis (half-life of ~ 44 h) than the PMI (half-life of ~ 0.1 h) or Bcl9p (half-life of ~ 0.3 h) peptides (Fig. 2i).

Lupbin specifically targets intracellular complexes of p53/MDM2, p53/MDMX and Bcl9/βcatenin. To explore the structural features of the free Lupbin monomer, we performed molecular dynamics (MD) simulations (Fig. 3a-e). The simulation results revealed an antiparallel monomeric αhelical structure for the Lupbin monomer with an overall helicity of 70%, which was consistent with the results determined by CD spectroscopy (Fig. 2b). The overall cyclized helix-loop-helix structure of Luffin P1 was maintained in Lupbin and two-original antiparallel α-helices overlapped nicely with the on-complexation structure of PMI (RMSD =1.0 Å) and Bcl9p (RMSD =0.5 Å) (Fig. 3a). To further explore the affinity of Lupbin to MDM2, MDMX and β-catenin, a molecular docking simulation was performed to calculate the binding free energy. As expected, the binding interfaces of PMI/MDM2, PMI/MDMX and Bcl9p/β-catenin were fairly well reserved in the Lupbin/MDM2, Lupbin/MDMX and 8


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Lupbin/β-catenin structure, respectively (Fig. 3b). Upon complexation, PMI-derived residues Phe8, Tyr11, Trp12 and Leu15 of Lupbin docked their side chains into a hydrophobic cavity of MDM2/MDMX (Fig. 3c, d). Similarly, Bcl9p-derived residues of Lupbin interacted with β-catenin in a fashion closely resembling that of the Bcl9p/β-catenin complex (Fig. 3e). The inhibitory effects of Lupbin on the interactions between p53 and MDM2/MDMX or between Bcl9 and β-catenin were measured by fluorescence polarization-based competition assays, in which different concentrations of Lupbin were preincubated with MDM2/18-26p53-FITC, MDMX/18-26p53-FITC or β-catenin /356-376Bcl9FITC complexes, respectively (Fig. 3f-h). Also shown in Fig. 3f-h, the half-maximal inhibitory concentrations (IC50 values) of Lupbin were less than those of PMI or Bcl9p, indicating the stronger affinity of the Lupbin to the three targeted proteins than the free peptide PMI or Bcl9p. According to the law of thermodynamics, the high affinity means the low free energy loss. Fortunately, the formed αhelix in Lupbin decreased the free energy loss during the PPIs, while free peptides must consume more free energy to form pre-defined structure for binding to targeted proteins. Besides, we almost did not find inhibitory effects of the scaffold protein Luffin P1 on the above interactions. Collectively, our data indicate that Luffin P1-based grafting strategy successfully targets multiple PPIs.

Excellent cell penetration, whole-cell distribution and endosomal escape of Lupbin. We evaluated cellular uptake of Lupbin and free peptides using FITC-labeling and Laser Scanning Confocal Microscopy (LSCM). As shown in Fig. 4a, both Lupbin and Luffin P1 showed efficient cellular uptake and intracellular diffusion in HCT116 and Hep3B cells within 6 h of incubation, whereas the free PMI or Bcl9p peptides barely penetrated into cancer cells. This was also supported by flow cytometry analysis 9


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(Fig. 4b). The superior membrane traversing ability of Lupbin likely results from efficient internalization of Lupbin via electrostatic interactions between the positive-charged Lupbin particles and the negative-charged cell membrane.30, 31 It was noted that the peptide-based nanoparticles spread over the cytoplasm as well as the nucleus in Lupbin-treated cells (Fig. 4a), indicating that the reduced Lupbin can effectively traverse the nuclear envelope and target intranuclear PPIs. Next, we investigated the intracellular distribution of Lupbin to explore its ability to escape from endosome/lysosome-dependent degradation. To address this, HCT116 cells were incubated with lowconcentration FITC-labeled Lupbin (10 μg/mL) for 6 h and then dyed with known markers for early endosomes (EEA1), late endosomes (RAB), Golgi apparatus (GP73) and lysosomes (Lysotracker). As shown in Fig. 4c, the image of red-dye-labeled subcellular organelles and FITC-labeled nanoparticles indicated that Lupbin did not colocalize with late endosomes, Golgi apparatus or lysosomes, but did partially overlap with early endosomes. These results demonstrate that the Lupbin can escape from early endosomes, thereby effectively avoiding the sequestration and degradation in lysosomes.

Lupbin inhibits tumor growth in vitro through blocking the activity of Wnt/β-catenin pathway and reactivating p53 signaling. Given that Lupbin effectively penetrates into cell membrane and blocks multiple PPIs, we next tested its cytotoxicity towards cancerous cells. As shown in Figs. 5a-c, Lupbin, but not the free PMI or Bcl9p peptides, dose-dependently inhibited the viability of A375 (carrying wild-type p53 and lowly expressing β-catenin) and HepG2 (carrying wild-type p53 and mutant β-catenin) cell lines, and its cytotoxicity was similar to Luffin P1 grafted with PMI alone (Lupin), further validating the intracellular functionality of the PMI graft. In addition, Lupbin also inhibited the 10


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proliferation of HCT116 p53-/- and Hep3B cells (both of them carrying deleted p53 and wild-type βcatenin) (Figs. 5d-f and Fig. S2), proving the functional blockade of Wnt signaling by the BCL9p graft. To determine multi-targeting ability of Lupbin, a HCT116 p53+/+ cells (containing wild-type β-catenin and p53) were incubated with Lupbin, Lupin, Lubin (Luffin P1 grafted with BCL9p alone) and Nutlin3 (a micromolecular inhibitor of p53-MDM2 interaction)32, respectively. As shown in Fig. 5g, Lupbin, Lupin, Lubin and Nutlin3 dose-dependently inhibited the viability of HCT116 p53+/+ cells with IC50 values of 2.5, 7.3, 90 and 5.0 μM, respectively. These findings demonstrate that Lupbin with multitarget function outplays its single-target counterparts. To explore the mechanism of intracellular Lupbin inhibiting cancer cell growth at the molecular levels, western blot analysis was performed to explore the level of p53, p21, β-catenin and Cyclin D in HCT116 p53+/+cells. After a 24-h treatment, Lupbin and Nutlin3 significantly up-regulated p53 and its downstream target p21 (function of inducing cycle arrest)33,

34,

indicating the reactivation of p53

pathway (Fig. S3a and b). Also shown in Fig. S3a and b, the expressions of β-catenin and a downstream target of Wnt signaling, Cyclin D1, was significantly decreased upon Lupbin treatment, further supporting the inhibition of the Wnt/β-catenin pathway by Lupbin. Correspondingly, similar to Nutlin3 treatment, cell cycle was blocked in the G1/S phase in Lupbin treated-HCT116 p53+/+ cells compared to Lupin, Luffin P1 and free PMI plus Bcl9p peptides (Fig. 5h). In addition, we found that Lupbin significantly induced the apoptosis of HCT116 p53+/+ cells compared to control, Lupin, Luffin P1 and free PMI plus Bcl9p peptides, and this effect was even better than Nutlin3 treatment (Fig. 5i, S3c). Expectedly, as shown in Fig. S4, Lupbin also efficiently inhibited the growth of MCF7 Homo breast carcinoma cell line and F16B10 Mus musculus skin melanoma cell line (both of them carrying wild11


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type p53 and β-catenin), demonstrating a wide spectrum anti-tumor activity of Lupbin. Notably, we did not find any significant anti-tumor activities of the scaffold protein Luffin P1 and the free PMI/Bcl9p peptides, even at a very high concentration in these cells. Taken together, our results demonstrate that Lupbin is capable of viability inhibition towards malignant cells via simultaneously targeting the p53 and Wnt/β-catenin signaling pathways.

Lupbin in vivo suppresses tumor progression through concurrently targeting the p53 and Wnt/β-catenin pathways. 30 mice xenografting tumors (about 50 mm3) growing from HCT116 p53+/+ were divided equally in six groups to receive a 13-day administration: Lupbin, Lupin, Luffin P1, free PMI/Bcl9p combo, doxorubicin (DOX, as a positive control) and PBS (as a negative control). Treatment was adopted every other day by intraperitoneal injection at a dosage of 2.5 mg/kg. On day 13, relative to the PBS-treated mice, we barely detected any therapeutic efficacy in the PMI/Bcl9p combo- or Luffin P1-treated mice, whereas Lupin treatment significantly inhibited tumor growth by 74.1%, which was close to inhibition efficiency (74.5%) of the first-line chemotherapy drug DOX (Fig. 6a). More importantly, we found that Lupbin exhibited potent tumor inhibitory activity (inhibition efficiency of 91.3% in comparison with the PBS control), much better than DOX (Fig. 6a). This was mainly attributed to multi-targeting function of Lupbin. At the 13th day, all tumors were stripped and weighted, and measurement results further supported the above conclusion that Lupbin possesses an excellent antitumor activity (Fig. 6b). To further determine anti-tumor efficacy of Lupbin at pathological levels, we examined the above xenograft tumor tissues by the H&E and TUNEL staining. As shown in Fig. 6c and S5, the number of 12


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apoptotic or necrotic cells in tumors upon Lupbin treatment were much more than the PBS control, Lupin or DOX treatment. Next, we analyzed the expression of p53, p21, β-catenin, Cyclin D1 and Ki67 (a marker of proliferative cells) in tumor tissues from mice with different treatments by immunohistochemistry staining (Fig. 6d). Consistent with in vitro findings, the expressions of p53 and p21 were obviously up-regulated in Lupin- or Lupbin- treated tumors compared to the PBS-, Luffin P1or PMI/Bcl9p combo-treated tumors, indicating reactivation of p53 pathway by Lupin or Lupbin (Fig. 6d-f). Conversely, levels of β-catenin, Cyclin D1 and Ki67 were lower in the Lupbin-treated tumors than other groups (Fig. 4d, g-i), mainly resulting from concurrent targeting of the p53 and Wnt/β-catenin pathways. In addition, it was noted that Luffin P1 performed similarly to the PBS control, which showed almost no inhibition of tumor growth, further demonstrating that potent anti-tumor activity of Lupbin is mostly endowed by the grafted peptides rather than the scaffold. Altogether, our data suggest that Lupbin protein-based nanoparticles potently inhibit tumor growth in vivo through concurrently targeting the p53 and Wnt/β-catenin pathways.

Lupbin inhibits tumor metastasis in vitro and in vivo. Tumor metastasis is closely bound up with ~90% cancerous death.35 The aberrant activation of Wnt/β-catenin cascade has been well demonstrated to promote tumor metastasis.36 Upon the activation of Wnt pathway, β-catenin translocated into the nucleus to turn on the transcription of a number of metastasis-related genes.37 In addition, tumor suppressor protein p53 also prevents tumor metastasis by negatively regulating some metastasis-related genes.38, 39 Thus, we consider that Lupbin can effectively inhibit tumor metastasis through concurrently targeting p53 and Wnt pathways. As expected, by the transwell assays, we found 13


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that Lupbin significantly inhibited the migration/invasion potential of HCT116 and F16B10 cells compared to the control or Lupin treatment (Fig. 7a-d). Next, to further evaluate the anti-metastasis activity of Lupbin in vivo, mice with lung metastasis were generated by intravenously inoculating B16F10 cells and were intraperitoneally administered with Lupbin (1.5 mg/Kg) on a daily basis for one week, starting from day 3 after cell injection. As shown in Fig. 7e and f, the number of metastatic nodules in the lung was significantly reduced in Lupbin-treated mice compared to control mice. The H&E-stained tissue sections also confirmed a reduction in size and number of the lung metastases upon Lupbin treatment (Fig. 7g). Cancer stem cells are one of the key driving forces for tumorigenesis and progression, particularly tumor metastasis.43, 44 To elucidate the anti-metastasis mechanism of Lupbin, the expressions of β-catenin, CD133 (a cancer stem cell marker)40, 41

and MMP9 (a target of β-catenin, which is involved in tumor metastasis)42 was analyzed by

immunohistochemistry staining. In line with the metastasis phenotype, Lupbin treatment reduced the expressions of β-catenin, CD133 and MMP9 in metastatic lesions compared to the control (Fig. 7h-i). Taken together, these observations suggest that Lupbin suppresses cancer metastasis probably through the distinct mechanisms (such as inhibition of Wnt signaling, MMP9 expression and cancer cell stemness) via its multi-target function.

In vivo safety evaluation of Lupbin. As mentioned above, the antagonism of MDM2, MDMX and β-catenin will be an effective strategy for cancer therapy by reactivating p53 signaling and blocking

Wnt pathway. In recent years, many selective small-molecular antagonists of MDM2/MDMX have been widely developed including a class of imidazoline compounds termed nutlins, some of which are being 14


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tested in clinical trials.45, 46 However, the off-target effects severely limited their clinical application.47 In a clinical trial of a ramification of nutlin, termed RG7112, thrombocytopaenia and neutropenia were seen in many patients.48 These adverse events are mainly caused by off-target toxicity of nutlin, which directly induces DNA damage.49 Similar to the MDM2/MDMX inhibitors, to date, none of β-catenintargeted drugs has been approved for clinical application due to their tremendous off-target side effects.50, 51 Therefore, when successfully targeting the aberrant p53 and Wnt signaling pathways in cancer cells, the potential off-targeted effects on normal cells should ideally be abrogated. To our satisfaction, Lupbin can provide a viable method to overcome this pharmacological hurdle. Lupbin consists of cyclized helix-loop-helix structure globally stabilized by two disulfide bridges (Cys10-cys32 and Cys15-Cys28). In the cytoplasm, the reducing environment will break the disulfide bonds and result in structural perturbation of Lupbin and loss of its proteolytic stability (Fig. S6). In cancer cells, Lupbin, in the unpacked form, will bind to MDM2, MDMX and β-catenin to form a stable complex, thereby inhibiting Wnt/β-catenin cascade and reactivating p53 signaling in cancer cells (carrying wild-type p53 and overexpressed MDM2/MDMX/β-catenin). In normal cells where the binding partners are mutated or expressed at low levels, reduced Lupbin molecules will be rapidly degraded for the lack of protection from these binding partners (Fig. 8a). These features of Lupbin will greatly reduce the side effects, thereby guaranteeing its biosafety. To test it, peripheral blood mononuclear cells collected from C57 mice were firstly incubated with Lupbin and DOX. After a 48 hincubation, relative to the control, Lupbin treatment almost did not affect cell viability, even at a high concentration (up to 40 μM), whereas DOX treatment dramatically inhibited cell viability at the indicated concentrations (Fig. S7). To further address in vivo biosafety, comprehensive evaluation of 15


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the drug toxicity was carried out during and after the Lupbin treatment. After a 13-day administration, there existed no significant difference in body weights between Lupbin-treated mice and control mice, whereas DOX-treated mice markedly lost their body weights (Fig. S8). In addition, relative to control or healthy mice, Lupbin-treated mice did not show the symptoms of leukopenia or thrombopenia (Fig. 8b-d), two common side effects of small-molecular therapeutics targeting the p53 and Wnt/β-catenin pathways.48 Liver and kidney have vital function on drug metabolism, thus we attempted to evaluate the chronic toxicity of Lupbin to them. After the 13-day administration, the average liver weight in the PBSor DOX-treated mice was significantly decreased compared to the healthy one, whereas Lupbin-treated mice maintain their weight permanently (Fig. 8e). In addition, although aspartate aminotransferase (AST) showed no significant difference in activity among different treatments (Fig. 8f), the levels of another important liver enzyme, alanine transaminase (ALT), was significantly increased in the blood of DOX-treated mice (Fig. 8g). These results were supported again by histopathology of liver sections (Fig. 8h), collectively indicate that Lupbin treatment has no apparent adverse effects on liver weight and function. Kidney examination, including blood urea nitrogen (BUN), serum creatinine (CRE) and histological H&E staining, were performed at the same time as liver examination. As shown in Fig. 8ik, DOX-treated mice were attacked by acute kidney inflammation and glomerular lesions, while Lupbin-treated mice kept kidney healthy. Notably, except for DOX, control and free PMI/Bcl9p-treated mice also showed an increase in CRE levels compared to healthy mice (Fig. 8j). In addition, unlike DOX, Lupbin didn't cause any toxicology, including but not limited to spleen failure (Fig. S9), myocardial damage (Fig. S10), acute sepsis (Fig. S11) or allergic lung resistance (Fig. S12) compared 16


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to healthy mice. Overall, our data demonstrate that Lupbin -based nanoparticles are sufficiently safe for potential clinical use.

CONCLUSIONS Multi-targeted therapies have emerged as an effective strategy to overcome the resistance of cancer cells to conventional single-targeted therapies.52 The major concern on the design of multi-target, proteinbased nanotherapeutics is the appropriate choice of the scaffold, which should fit different epitopes for targeted protein binding and have proper properties as pharmic carrier.53 With two independent helices, Luffin P1 is an ideal scaffold that provides two grafting areas to substitute solvent-exposed amino acids. Moreover, the simple hairpin-shaped helix-loop-helix structure of Luffin P1 can easily accommodate its binding to two different targets. In addition, Luffin P1 can be easily produced in large quantity through chemical or recombinant approaches.18 Based on these advantages, we have grafted the epitopes of PMI and Bcl9p into the Luffin P1 to concurrently target the p53 and Wnt/β-catenin signaling pathways. Tumor suppressor p53 is a major anti-cancer factor that induces apoptosis and cell cycle arrest of cancer cells, and high levels of wild-type p53 also promotes the degradation of deregulated β-catenin to block the Wnt/β-catenin signaling, thereby inhibiting tumor growth.54 Conversely, the deregulated β-catenin not only promotes tumorigenesis by itself, but also, in parallel, inactivates p53 through upregulating MDMX/MDM2.55 Thus, concomitant activation of p53 signaling and inhibition of Wnt/β-catenin pathway will have synergistic anti-tumor efficacy compared to single therapy alone, which was supported by our data that Lupbin exhibits much better anti-tumor efficacy than Lupin (only grafting PMI into Luffin P1). 17


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Besides, self-assembly of Lupbin will provide a strategy to overcome the drawbacks of small size of the grafted miniportein. Self-assembly of peptides and proteins are modulated by noncovalent interactions mainly including van der Waal forces, hydrophobic effect and coulomb force,56 all of which can be controlled by varying the amino acid sequences.57 Based on this principle, the self-assembly of proteins can be elaborately modulated to form diverse multilevel nanostructures.58 Owing to the high hydrophobicity of the key amino acids in the epitopes of PMI and Bcl9p, the increased intermolecular hydrophobic interaction will facilitate the self-assembly of Lupbin into an oligomeric nano-ellipsoids with appropriate sizes to specifically target tumor via the EPR effect and postpone their renal excretion. In summary, we successfully used Luffin P1, a cyclized helix-loop-helix scaffold protein, to generate a hybrid protein Lupbin, which is capable of self–assembling into protein-based nanoparticles to concomitantly target the p53 and Wnt/β-catenin pathways, thereby suppressing tumor progression potently. The data presented here provide compelling evidence that Lupbin is advantageous in overcoming important obstacles to clinical application of therapeutic peptides and pioneers a powerful strategy to target multiple intracellular PPIs in a single chemical entity. Considering that this strategy can accommodate multiple therapeutic α-helix peptides, and has excellent biosafety profile and favorable cost-effectiveness, we anticipate that Luffin P1 holds great potential as a grafting scaffold for future clinical translation of therapeutic peptides and the development of multi-target intracellular PPI inhibitors.

EXPERIMENTAL SECTION Synthesis of Lupbin, Lupin, Lubin and Luffin P1. The peptides were synthesized by Fmoc 18


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chemistry as our described previously.59 Crude products were collected by ether precipitation and purified by reverse-liquid-HPLC methods according to our previous protocol.11 ESI-MS was used to ascertain molecular masses of peptides, and analytical HPLC was used to identify the purity via a 30min gradient from 5% to 65% acetonitrile (0.1% trifluoroacetic acid). Lupbin, Lupin, Lubin and Luffin P1 were synthesized by a two-segment strategy ligated by a hydrazides-induced Native chemical ligation (NCL) reactions.60 NCL was catalyzed by 200 mM 4mercaptophenylacetic acid as described previously.60,61 After that, linear Lupbin (or other synthesized proteins) can be obtained after HPLC purification. Of note, for the correct matching of the cysteine during the disulfide bond oxidation, Acm-protected Fmoc-Cys-OH was used to extend the peptide chain at Cys10 and Cys31. The disulfide bond between Cys14 and Cys27 can be formed by dimethyl sulfoxide as our precious protocol.59 And the intermediate with Cys14 - Cys27 disulfide was then treated with 10 mM iodine aqueous solution including 20% methanol at pH 4.0 to remove Acm and oxidize the native disulfide between Cys10 and Cys31. Lupin, Lubin and Luffin P1 were synthesized as the same protocol as Lupbin.

CD spectroscopy. CD spectrum of Lupbin variants at a concentration of 20 μM were obtained on a Jasco J-810 spectropolorimeter at room temperature in 10 mM pH 7.4 PB (pH 7.4). The detailed protocol was described as previously.62, 63

Fluorescence polarization (FP)-based competitive binding assay. To perform fluorescence polarization (FP) assay, fluorescein isothiocyanate (FITC) was conjugated to 15-29p53 and Bcl9p via its 19


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N-terminal amino group. The resultant products

15-29p53-FITC

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and Bcl9p-FITC were HPLC-purified

and lyophilized. MDMX was synthesized and purified as previously described.64 The MDM2 and 137781β-catenin

were expressed in E. coli BL21(DE3) star cells using the pET28a expression vector. Stable

overexpression of target proteins was obtained by inducing with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) when the cultures were grown to OD 600 around 0.6. These proteins were puriﬁed (> 98%) by reverse-phase HPLC as previously described.65 Fluorescence polarization-based competitive binding assay was then performed as described previously,64 and the readings were taken using a fluorescence microplate reader (Tecan M2000, λex = 470 nm; λem = 530 nm. ). Lupbin and corresponding peptides were serially 2-fold diluted in 10 mM Tris-HCl buffere (pH 7.0) containing 150 mM NaCl and 1 mM EDTA, and subsequently incubated with 200 nM Bcl9p-FITC/β-catenin, 15-29p53FITC/MDM2 or 15-29p53-FITC/MDMX for 2 h in a total volume of 150 μL per well. IC50 values were calculated by nonlinear regression as described previously.64

Xenograft tumor model. Xenograft tumor model was used the same protocol as our previous reports,66 and approved by the Animal Ethics Committee of Xi’an Jiaotong University. The detailed protocols for measuring tumor volume and weight, and performing H&E/immunohistochemical staining were similarly described as previously.11 In addition, to evaluate the biosafety of different treatments, blood routine examinations as well as liver and kidney function, including the number of white blood cells, red blood cells and thrombocyte, and the levels of aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN) and serum creatinine (CRE), were performed according to the previously reported protocols.66 20


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Tail vein metastasis model. Suspend B16F10 cells in serum free medium after washed by PBS, and then injected 200 μL cell suspension (~5×105 cells) into the tail vein of C57BL/6 mice. After that, randomly divided them into the control and treatment groups (4 mice per group), and the weeklong injection of medication (1.5 mg/kg Lupbin or same volume of PBS per two days) was then started at the 3th day after implantation. After the treatment, the mice were euthanized, following the lungs collection and photographing. Next, all lung tissues were fixed, embedded, cut and prepared for H&E staining and IHC analysis of β-catenin, CD133 and MMP9 as described previously.11 Additional details of the methods are described in Supplementary Data.

ASSOCIATED CONTENT The authors declare no competing financial interest.

Supporting Information Supplementary methods (including general information for reagents, necessary details for nanoprotein characterization, necessary protocols for cell experiments and statistical approach), 12 supplementary figures and 2 supplementary tables were presented in supporting information. This material is available free via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding authors: Email (P. Hou): [email protected] 21


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Email (W. He): [email protected] Email (Y. Shao): [email protected]

ORCID Wuyuan Lu: 0000-0003-1318-9968 Peng Hou: 0000-0001-7010-7944

Author Contributions W. He, J. Yan and F. Sui contributed equally to this work.

ACKNOWLEDGEMENTS This work was supported by the Clinical Research Award of the First Affiliated Hospital of Xi’an Jiaotong University (No. XJTU1AF-CRF-2017-003 to P. H. and W. H.), and the National Natural Science Foundation of China (No. 81572627 to P. H.).

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Figure 1. Schematic diagram for the design and synthesis of Lupbin. (a, b) The anti-cancer peptide PMI (brown) is capable of binding MDM2/MDMX (gray) with low nM affinity to restore the function of p53. (c) The Bcl9 (green) specificly bind to a groove on the surface of β-catenin (gray). (d) Structure design of Lupbin. PMI and residues 6-17 of Luffin P1 as well as 356-376Bcl9 and residues 23-43 of Luffin P1 share some degrees of sequence identity and structural similarity. (e) Strategy for synthesis and folding of Lupbin. (f) Schematic diagram for the function of the Lupbin.

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Figure 2. Synthesis and properties of self-assembled Lupbin to resist proteolysis. (a) Characterization of Lupbin by HPLC and ESI-MASS. (b) Circular dichroism spectra of Lupbin (red) and Luffin P1 (black) at concentration of 20 µM in chloridion-free aqueous solution. (c) The surface charge (Zeta potential) of Lupbin and Luffin P1 in pH 7.4 PBS buffer at 37 ºC. (d) Small Angle X-ray scattering (SAXS) diffractograms of Theoretical monomer and Lupbin protein-based nanoparticle measured at the concentration 0.1 mg/ml in PBS buffer. The orange line (Lupbin protein-based nanoparticle) is the least squares fit to the data (green points) using a rod model. (e) Corresponding chord length distributions were calculated from (d). P(r) =chord length distribution function; R =radius. (f) The schematic illustration of the size of Lupbin protein-based nanoparticle, which obtained from (e). (g) Hydrodynamic distributions of Lupbin protein-based nanoparticle and Luffin P1. (h) The molecular weight of Lupbin and Luffin P1. (i) The proteolysis resistance of Lupbin, PMI and Bcl9 under PBS containing 10mM oxidized glutathione, 10% serum and chymotrypsin, showing that Lupbin has higher resistance against enzymolysis than PMI or Bcl9.

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Figure 3. The structural and functional characteristics of Lupbin. (a) Structural alignment of simulated structures of the Lupbin with PMI and Bcl9p peptide in unbound form. (b) The binding interfaces and binding free energy (ΔiG) were calculated from a molecular docking simulation followed by MD simulations. Molecular docking simulated structures of Lupbin-MDM2 (c), Lupbin-MDMX (d) and Lupbin-β-catenin (e), aligned to PMI-MDM2 (c), PMI-MDMX (d) and Bcl9p-β-catenin (e), respectively. Competitive FP-based binding assay of Luffin or PMI to MDM2/p53 complex (f), PMI or Luffin to MDMX/p53 complex (g) and Bcl9p or Luffin to Bcl9/β-catenin complex (h). IC50 values were averaged from three independent experiments and showed as mean ± s.e.

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Figure 4. Lupbin efficiently penetrates plasma membrane and escapes from the endosome. (a) CLSM images of HCT116 and Hep3B cells after a 6-h incubation with 2 μM FITCLupbin, FITCLuffin P1, FITCBcl9 and FITCPM, all photos were taken under the same settings. (scale bar: 60 μm) (b) Flow cytometry analysis of cellular uptakes of 2 μM FITCLupbin, FITCLuffin

P1,

FITCBcl9

and

FITCPMI

after a 6-h incubation, showing that Lupbin can overcome the inability of free

peptides to traverse the cell membrane. Statistical test was performed by t-test (**, p < 0.01; ***, p < 0.001). (c) Colocalization of FITC-labeled AuNp-DPA with lysosome, early and late endosomes. The subcellular organelles were marked in red, and the enlargement factor is ×500.

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Figure 5. Anti-cancer activities of Lupbin in vitro. Dose–response curves of different nanoparticles formulations against A375 (a) and HepG2 (b) cells after a 72-h incubation with Luffin P1, Lupbin, Lubin and PMI&Bcl9p, which were measured by the MTT assay (n =4, mean ± s.d.). (c) The MTT tetrazolium assay was performed to monitor the effects of Luffin P1, Lupbin, Lubin and PMI&Bcl9p on the growth of A375 and HepG2 cells. Each set of data is expressed as mean ± s.d. (n =4). Dose–response curves of different nanoprotein formulations against HCT116 p53-/-(d) and Hep3B (e) cells after a 72-h incubation with Luffin P1, Lupbin, Lubin and PMI&Bcl9p, which were measured by the MTT assay (n =4, mean ± s.d.). (f) The level of Cyclin D and total β-catenin in Hep3B cells treated with 5 μM Lupbin, Luffin P1, PMI&Bcl9p and Nutlin3 for 24 h, measured by Western blot analysis. (internal reference: β-actin). Relative protein levels of different nanoprotein formulations against HCT116 p53+/+ cells were analyzed by image J. (g) Dose-dependent viability suppression of HCT116 p53+/+ cells upon the indicated treatments as determined by the MTT assays (mean ± s.d., n =4). (h) HCT116 p53+/+ cell cycle analysis of after a 24-h treatment (n=3, mean ± s.d.). (i) HCT116 p53+/+ cell apoptosis determined by FACS after treatments. Statistical test was performed by t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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Figure 6. In vivo anti-tumor activity of Lupbin by concurrently targeting the p53 and Wnt/β-catenin pathways. (a) Growth curves of HCT116 p53+/+ xenografted tumor in nude mice. (mean ± s.e.; n =5). (b) Weight distribution of tumor in mice with different treatments. (c) TUNEL (apoptosis) assay in tumor tissues (×200). (d) Representative IHC staining for p53, p21, β-catenin, Cyclin D and Ki67 in tumor (scale bar: 50 μm). (e-i) IHC scores for p53, p21, β-catenin, Cyclin D and Ki67 in tumor tissues (scale bar: 50 μm). Statistical test was performed by t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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Figure 7. In vitro and in vivo anti-metastasis activity of Lupbin. (a, b) Representative pictures of migrated/invaded HCT116 or F16B10 cells. (c, d) Histograms, in line with (a) and (b), showed means ± s.d. of the number of migrated/invaded cells from three independent assays. (e) Representative photographs of the lungs from mice treated with PBS and Lupbin for 15 days after injection of B16F10 cells. (f) The number of superficial macroscopic metastases in the lungs (n =4). (g) Representative photographs of the H&E-stained tissue sections of pulmonary metastases. (h, i) Representative IHC staining of β-catenin, CD133 and MMP9 for three serial sections from pulmonary metastasis tumors (scale bar: 100 μm). Statistical test was performed by t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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Figure 8. In vivo safety evaluation of Lupbin. (a) Abridged general view of anti-tumor mechanism of Lupbin through simultaneous reactivating p53 pathway and blocking Wnt pathway. The count of white blood cells (WBCs, b), red blood cells (RBCs, c) and Thrombocyte (d) in mice after 13-day treatments. (e) Liver weight of mice after 13-day treatments. Levels of liver function index measured in mice blood after 13-day treatments: aspartate aminotransferase (AST, f) and alanine transaminase (ALT, g). (h) Representative histological H&E staining images of liver in mice after 13-day administration (scale bar: 50 µm). (i, j) Renal function indicators in mice blood after 13-day administration (BUN, blood urea nitrogen; CRE, serum creatinine). (k) Representative histological H&E staining images of kidney after 13-day administration (scale bar: 50 µm). Statistical test was performed by t-test in this figure (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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Table of Contents

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Turning a Luffa Protein into a Self-Assembled Biodegradable

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