Inhibition of Multimolecular RNA–Protein Interactions Using Multitarget

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Inhibition of Multimolecular RNA−Protein Interactions Using Multitarget-Directed Nanohybrid System Woo-jin Jeong, Mahnseok Kye, So-hee Han, Jun Shik Choi, and Yong-beom Lim* Department of Materials Science & Engineering, Yonsei University, Seoul 03722, Korea S Supporting Information *

ABSTRACT: Multitarget-directed ligands (MTDLs) are hybrid ligands obtained by covalently linking active pharmacophores that can act on different targets. We envision that the concept of MTDLs can also be applied to supramolecular bioinorganic nanohybrid systems. Here, we report the inhibition of multimolecular RNA−protein complexes using multitarget-directed peptide−carbon nanotube hybrids (SPCHs). One of the most well-characterized and important RNA−protein interactions, a Rev-response element (RRE) RNA:Rev protein:Crm1 protein interaction system in human immunodeficiency virus type-1, was used as a model of multimolecular RNA−protein interactions. Although all previous studies have targeted only one of the interaction interfaces, that is, either the RRE:Rev interface or the RRE−Rev complex:Crm1 interface, we here have developed multitarget-directed SPCHs that could target both interfaces because the supramolecular nanosystem could be best suited for inhibiting multimolecular RNA−protein complexes that are characterized by large and complex molecular interfaces. The results showed that the single target-directed SPCHs were inhibitory to the single interface comprised only of RNA and protein in vitro, whereas multitarget-directed SPCHs were inhibitory to the multimolecular RNA−protein interfaces both in vitro and in cellulo. The MTDL nanohybrids represent a novel nanotherapeutic system that could be used to treat complex disease targets. KEYWORDS: multitarget-directed ligand, peptide−CNT hybrid, multivalency, scaffold-mediated peptide coassembly, supramolecular inhibitor

1. INTRODUCTION Two decades ago, RNA−protein complexes were regarded as new and attractive therapeutic targets;1,2 however, most efforts since then directed toward developing drugs against RNA targets have not been fruitful. One of the primary reasons for this difficulty is the involvement of multiple RNA and protein molecules in the complexes, which renders single-molecule drugs inadequate because of their inability to inhibit multiple targets at the same time. Meanwhile, multitarget-directed ligands (MTDLs) have recently been developed to combat multifactorial complex disease systems that are incurable with conventional single medications, such as Alzheimer’s, diabetes, cancer, and viruses, through simultaneous effects on pathologically relevant targets.3−6 MTDLs are hybrid ligands obtained by covalently linking active pharmacophores acting on different targets. Notably, recent reports have illustrated that dualtargeting bispecific antibodies directed against the human immunodeficiency virus type-1 (HIV-1) cell entry process have become the most potent and broad HIV-neutralizing antibodies to date.7,8 We envisioned that the concept of MTDLs could be applied to supramolecular bioinorganic nanohybrid systems, that is, the hybridization of multiple self-assembling peptides directed against different targets and a carbon nanotube (CNT) scaffold, © 2017 American Chemical Society

which could be more effective against multimolecular RNA− protein complexes compared to conventional approaches that use only a single species of peptide. This approach could be interesting because (1) combining the activity of individual ligands would produce strong synergistic activity, (2) peptides are known to be well-suited for targeting protein-related biointeractions mediated by spacious and shallow interfaces that are undruggable by small-molecule inhibitors,9 (3) the noncovalent hybrid structure would be able to interact with each heterogeneous target biomolecule more effectively than the conventional covalent MTDLs by virtue of the advantages of multivalency and adaptability in supramolecular interactions,10−12 and (4) the length of CNTs could be much greater than the size of typical biomacromolecules, thus indicating that a single hybrid could interact with many targets simultaneously. Additionally, the CNTs could provide a stable girder for adaptably stabilizing specific conformations of peptides.12 Here, we report the inhibition of multimolecular RNA− protein complexes using multitarget-directed peptide−CNT hybrids (SPCHs). One of the most well-characterized and Received: January 31, 2017 Accepted: March 13, 2017 Published: March 13, 2017 11537

DOI: 10.1021/acsami.7b01517 ACS Appl. Mater. Interfaces 2017, 9, 11537−11545

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Figure 1. (a) Development of multitarget-directed SPCHs through the hybridization of single-walled nanotubes (SWNT) scaffolds and peptides acting on different targets as well as the inhibition by the SPCHs of multimolecular RNA−protein interaction systems (RRE RNA:Rev protein:Crm1 protein) to prevent the nuclear export of RRE RNA. (b) Bioactive ligand display on a 1D scaffold and a planar scaffold. 11538

DOI: 10.1021/acsami.7b01517 ACS Appl. Mater. Interfaces 2017, 9, 11537−11545

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Figure 2. Supramolecular hybridization of peptides and SWNTs. (a) SDS-PAGE analysis to determine the amount of cyc-ARM bound to CNTs. Numbers indicate the concentration of cyc-ARM. (b) AFM images of the pristine SWNT bundles (left) and the SWNTs dispersed with cyc-ARM (right). (c) TEM image of homo-ARM-SPCH. (d) UV−vis−NIR spectra of the SWNT functionalized with cyc-ARM (red) and sodium dodecylbenzenesulfonate (SDBS, green). (e) Raman spectra of the SWNT functionalized with cyc-ARM (red) and sodium dodecyl sulfate (SDS, blue). (f) CD spectra of cyc-ARM (red) and homo-ARM-SPCH (blue). and chloramphenicol acetyltransferase (CAT) assay were performed as described previously.19 2.2. Tissue-Culture and Intracellular Experiments. For microscopic observation of the intracellular delivery of the peptide/ CNT hybrid structure, HeLa cells (1 × 104) were seeded in an eightwell Lab-Tek II chambered cover-glass system (Nunc) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% pen/ strep and cultured overnight at 37 °C with 5% CO2. The cells were washed with Dulbecco’s phosphate-buffered saline and treated with the hybrid structures for 4 h. Then the sample solution was removed, and the cells were further incubated for 1 h. The cells were visualized under a confocal microscope (LSM 710, Carl Zeiss, Germany). For fluorescence recovery after photobleaching (FRAP) analysis, the pyrene fluorescence in a small region of the cell was photobleached and observed for approximately 300 s at an interval of approximately one image per 2 seconds.

important RNA−protein interactions, a Rev-response element (RRE) RNA:Rev protein:Crm1 protein interaction system was used as a model of multimolecular RNA−protein interactions.13 Since the discovery that a short α-helical peptide derived from the HIV-1 Rev protein specifically recognized the high-affinity site in RRE,14 this RNA−protein interaction system has been a popular target for drug discovery. Rev binds the RRE in a multimeric fashion, and the RRE−Rev complex is then recognized by Crm1. The interaction between the Rev protein and RRE RNA is mediated by the arginine-rich motif (ARM). The RRE RNA contains multiple sites for ARM binding. Among these multiple sites, an RRE IIB site, which is known to be the highest binding site, is where the ARM binds in an αhelical conformation to the IIB RNA. Although many attempts have been made for drug development, none of them have progressed to preclinical development. Part of the reason for this dearth of successful cases is that all the previous studies have targeted only one of the interaction interfaces, that is, the RRE:Rev interface or the RRE−Rev complex:Crm1 interface.15−19 In the present study, we developed multitargetdirected SPCHs that could target both interfaces because supramolecular nanosystems could be best suited for inhibiting multimolecular RNA−protein complexes that are characterized by large and complex molecular interfaces (Figure 1a). The results showed that the single target-directed SPCHs were inhibitory to the single interface comprised only of RNA and protein only in vitro, whereas multitarget-directed SPCHs were inhibitory to the multimolecular RNA−protein interfaces both in vitro and in cellulo.

3. RESULTS AND DISCUSSION We first fabricated SPCHs using a single peptide species to observe the characteristics of the hybrid structure itself with a simplified system (homo-SPCH). To prepare a peptide with bioactivity and CNT-binding properties, we synthesized a macrocyclic peptide comprised of the ARM (14 amino acids) of Rev,22 a self-assembling and CNT-binding segment labeled with the pyrene fluorophore for intracellular tracking, and their linkers (cyc-ARM, Figure S1a of the Supporting Information). Rev facilitates the nucleocytoplasmic translocation (export) of unspliced and partially spliced HIV-1 RNA with the aid of Crm1, which is an essential process in the viral replication (Figure S2).23,24 During this process, Rev interacts with the RNA and Crm1 through the ARM and the nuclear export signal (NES), respectively. The peptide sequences have different structural properties, with the ARM being an α-helical peptide and the NES having an unordered conformation;25 thus, the RRE:Rev:Crm1 complex could be a suitable target for

2. EXPERIMENTAL DETAILS 2.1. General. Peptide synthesis and cyclization were performed as described previously.20,21 Electrophoretic mobility shift assay (EMSA) 11539

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Figure 3. Characterization of homo-SPCH in vitro and in cellulo. (a) EMSA of Rev−RRE RNA complexation solutions. The numbers shown above the gel are the Rev concentrations. [RRE] = 100 nM. (b) Competition of the RRE−Rev complex (RRE, 100 nM, Rev, 6 μM) with increasing concentrations of homo-ARM-SPCH. The numbers shown above the gel are the concentrations of homo-ARM-SPCH. (c) Intracellular distribution of homo-ARM-SPCH. Red fluorescence (rhodamine B) from SWNTs (left), blue fluorescence (pyrene) from cyc-ARM (middle), and a merged image showing the colocalization of cyc-ARM and SWNTs (right). (d) Quantitative analysis of fluorescence recovery after the photobleaching of cyc-ARM (red) or homo-ARM-SPCH (blue).

fixed positions on the SWNT surface. In contrast, a noncovalent approach offers the possibility to manipulate the detailed molecular states of functionalized peptides via supramolecular controls. In addition, the noncovalent approaches are more convenient for preparing the hybrid structure and controlling the ratio of the different types of immobilized cargos; the ratio can simply follow the concentrations of the peptides in the solution state used for hybridization (vide infra). To prepare the homo-SPCHs, the arc-produced SWNTs were functionalized with cyc-ARM in an aqueous sodium chloride (NaCl, 150 mM) solution with the aid of bath sonication as described previously to provide the desired product (homo-ARM-SPCH).38 For quantification of the maximum possible amount of cyc-ARM that could bind to the SWNT sidewall, we isolated unbound peptides from SPCH solutions prepared at various cyc-ARM concentrations using centrifugation and subjected them to gel electrophoresis (Figure 2a). Through densitometric analysis of the electrophoretic bands, we found that 1.4 nmol of cyc-ARM can bind to 1 μg of SWNTs. Considering the specific surface area of SWNTs and the calculated surface area of the CNT-binding segment, the coverage of CNT surface area with the peptide is ∼100% (Figure 2a and Figure S5).12 Under the conditions that enabled dense coverage of the SWNT surface without leaving unbound peptides, the hydrophobic inorganic scaffolds were effectively solubilized and debundled by the hybridization, as shown by atomic force microscopy (AFM, Figure 2b) and transmission electron microscopy (TEM, Figure 2c) micrographs. Characterization of homo-ARM-SPCH using UV−vis− NIR and Raman spectroscopy revealed that the binding of the peptide did not alter the intrinsic characteristics of SWNTs, possibly because they associated via noncovalent interactions

demonstrating the efficacy of the multitarget-directed SPCH, with ARM and NES used as the multitarget-directed bioactive ligands (Figure S3). As a scaffold of artificial biomacromolecules developed by bioinorganic hybridization, CNTs have attracted much interest owing to their high capacity to incorporate guest materials,26 biocompatibility,27 in vivo stability,28 and cell-penetrating capability.29 Because CNTs can display the functional units of biomacromolecules, such as active peptide fractions isolated from proteins, on their surface in a multivalent manner, CNTbased bioinorganic hybrids have shown potential as powerful inhibitors of pathogenic biomolecular interactions.12,30,31 In addition, the intriguing physical,32 optical,33 and electrical34 characteristics of CNTs widen the range of applications for the hybrid structures to fields that would not be accessible with natural biomolecules. In this approach, the one-dimensional (1D) structure of CNTs could provide a significant benefit over the scaffolds that were much bigger than the size of peptides or planar because the biomolecular cargoes could be exposed on the CNT scaffold without steric hindrance due to the narrow diameter (∼1 nm) of single-walled nanotubes (SWNTs) (Figure 1b and Figure S4).12,35−37 For short bioactive peptides isolated from proteins, it is important to maintain the innate conformation, which is closely related to their functionality. In a previous study, we met this requirement and thus demonstrated that CNTs could provide an alternative to protein scaffolds in the context of relieving the intrinsic thermodynamic structural instability of the peptides.12 CNT functionalization can be achieved by covalent or noncovalent approaches. In a covalent approach, it is difficult to control the orientation and density of the functionalized peptides because reactive groups are randomly populated at 11540

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Figure 4. Coassembly of two different peptides on the SWNTs. (a) TEM image of hetero-(A&E)-SPCH. (b) EMSA assay. Dependence of coassembly ratio on the inhibition of RRE:Rev interactions by hetero-(A&E)-SPCH. M: RNA ladder; C: RRE−Rev complex. The numbers shown above the gel are the concentrations of the peptides. (c) Atomic force microscopy (AFM) image of lin-NES assembly (left), cyc-ARM assembly (middle), and the coassembled structure of lin-NES and cyc-ARM (right). (d) Normalized CD spectra of lin-NES (blue), the coassembly of linNES and cyc-ARM (green), and cyc-ARM (red). (e) Normalized CD spectra of homo-NES-SPCH (blue), hetero-(A&N)-SPCH (green), and homo-ARM-SPCH (red). (f) Fluorescence emission spectra of the coassembly of lin-NES and cyc-ARM (blue) and hetero-(A&N)-SPCH (red).

With the hybrid structure in hand, we investigated the possibility of using the SPCH as an inhibitor of RRE:Rev interactions in vitro. To prepare the stable RRE−Rev complex, we first determined the complex formation ratio between the Rev and full-length RRE (∼350 nt). RRE (100 nM) and various concentrations of Rev were mixed in HEPES-buffered saline (HBS) and subjected to an EMSA (Figure 3a). Because 10−12 Rev molecules can bind to an RRE RNA in a multivalent fashion,23 the RRE−Rev complex produced gradually broadening electrophoretic bands as the Rev concentration increased above a critical concentration (lanes 8−14). The broad band consisted of the RRE−Rev complexes with different numbers of bound Rev proteins.40 On the basis of this result, we used the ratio at which the stable RRE−Rev complex was stably

(Figure 2d,e). Next, the secondary structure of cyc-ARM bound to SWNTs was examined using circular dichroism (CD) spectroscopy. As shown in Figure 2f, the negative minimum at 222 nm, a signature of the α-helical conformation, was more intense in the CD spectrum of homo-ARM-SPCH than in the CD spectrum of only the peptide. The enhanced helicity was likely induced by the inorganic scaffold-mediated α-helix stabilization as demonstrated in our previous studies.12,39 This stabilization is highly important for the specific RRE recognition. As shown in Figure S6, with the stabilized ARM helices, homo-ARM-SPCH displayed selectivity toward RRE IIB RNA. In addition, the α-helical state of cyc-ARM remained stable when bound to the CNT surface, even at high temperature (85 °C), as revealed by a temperature-dependent CD study (Figure S5b). 11541

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suggested that the effective molecular mass of cyc-ARM had been increased by its binding to the SWNT. When these results were taken together, we concluded that the noncovalent hybrid structure remained intact inside of the cell. Using the information from studies on SPCHs with homogeneous ligands, we then progressed to a more complex system: an SWNT hybrid structure with different types of peptides (hetero-SPCH). We designed and synthesized a macrocyclic peptide, cyc-EAK, that could coassemble with cycARM on the SWNTs (Figure S1b).47 The Rev α-helix segment in cyc-ARM was substituted by a model α-helical peptide (HAEAAAKEAAAKA-OH) in cyc-EAK. Both cyc-ARM and cycEAK were first dissolved in pure water prior to the SWNT binding for homogeneous mixing, followed by the ionic forcemediated SWNT functionalization,38 which yielded discrete nano-objects, that is, hetero-A&E-SPCH (Figure 4a). We prepared hetero-A&E-SPCH solutions at different cyc-ARM/ cyc-EAK ratios while maintaining a constant total peptide concentration constant and tested their inhibition capability of RRE:Rev interactions in vitro. The competition experiments by EMSA revealed that the inhibition capability of the heteroA&E-SPCH decreased as the proportion of cyc-ARM was lowered (Figure 4b). This result provided evidence that the relative ratio of the coassembled peptides on the inorganic SWNT surface was a direct representative of the ratio of the peptides in solution. Similar to the results in Figure 3b, the bands at a lower part of the complex band disappeared first in this competition experiment. Another question regarding the development of heteroSPCHs was whether the hybridization method could be applied to peptides with different structural properties. To answer this question, we synthesized a linear peptide containing the Rev NES and SWNT-binding sequences and labeled it with a fluorescence resonance energy-transfer (FRET) pair of pyrene and fluorescein (lin-NES, Figure S1c). The morphologies of the cyc-ARM and lin-NES assemblies, in the absence of SWNTs, were spherical and fibrillary, respectively (Figure 4c). When cyc-ARM and lin-NES were coassembled, irregular aggregates were produced. In addition, the stabilization of αhelix was not observed in cyc-ARM, lin-NES, and the coassembly of both peptides (Figure 4d and Figure S12). By contrast, the CD spectrum of the hetero-SPCH fabricated by using the two peptides in a 1:1 ratio, called hetero-(A&N)SPCH, showed a high level of helix stabilization (Figure 4e). Thus, the hybridization with SWNTs increased the helicity of the peptides, which could not be attained in the assemblies formed in the absence of SWNTs. Characterization of hetero(A&N)-SPCH using AFM, UV−vis-NIR, and Raman spectroscopy revealed that the binding of both peptides (i.e., cyc-ARM and lin-NES) did not alter the intrinsic characteristics of the SWNTs, as had been observed for homo-ARM-SPCH (Figure S13). To verify that the ratio of the coassembled peptides on SWNT exactly matched the ratio of the peptide mixture in the solution used for hybridization, we performed the following control experiment. The hetero-(A&N)-SPCHs were prepared with different ratios of cyc-ARM and lin-NES. Then the peptides were detached from the SPCHs by the treatment with a mixed organic-aqueous solution (water and acetonitrile; 1:1, v/v) and subsequent sonication. Free SWNTs were removed by centrifugation and the supernatant containing the peptides was recovered. Comparison of the fluorescence spectra between the free peptide mixture and the peptides detached from SPCHs showed similar spectral profiles that depended on the ratio of

maintained as the condition for further competition experiments (RRE, 100 nM; Rev, 6 μM). We then asked the question of whether Rev in the already formed RRE−Rev complexes (vide ante) could be competitively exchanged with homo-ARM-SPCH. As shown in Figure 3b, there was a gradual disappearance of the bands corresponding to the complexes as the homo-ARM-SPCH concentration increased, confirming the competitive exchange reaction. Because homo-ARM-SPCH could not migrate through the gel, the exchange reaction resulted in a reduction in the band intensity of the complexes. Considering the fact that the complex consisted of multimeric Rev proteins and huge RNA molecules (∼350 nt), the competition with the hybrid based on the small cyc-ARM peptide was remarkable. The competition experiment data in Figure 3b also showed the bands at the lower part of the broad band, which corresponded to complexes with a smaller number of Rev proteins, disappeared first. This result was consistent with the fact that the complexes that contained a smaller number of Rev proteins would be more susceptible to the exchange reaction.41−43 The specificity of homo-ARM-SPCH was confirmed by performing the exchange experiment in the presence of nonspecific competitors (Figure S7). Interestingly, the concentrations of Rev and homo-ARM-SPCH at the complete exchange condition were 6 μM and 600 pM, respectively. Thus, the quantitative illustration of these results indicated that homoARM-SPCH was roughly 4 orders of magnitude stronger than the Rev protein in RRE binding. We interpreted that this outstanding in vitro inhibition capability of homo-ARM-SPCH was the result of strong α-helix stabilization by the formation of the self-assembled hybrid and the multivalent characteristics of the homo-ARM-SPCH. The statistical rebinding mechanism11 could account for the strong RRE RNA recognition by these multivalent and elongated 1D nanostructures. Because noncovalent interactions are reversible, the stability of the supramolecular peptide−CNT hybrids (SPCHs) in cellulo was not guaranteed. Despite the importance of this basic consideration, to the best of our knowledge, the intracellular stability of noncovalent peptide−CNT hybrids has not been characterized. To address this question, we examined the intracellular behavior of the supramolecular hybrid structure. To distinguish the inorganic scaffold from pyrene-labeled cycARM, the SWNTs were covalently labeled with rhodamine B, a red fluorescent probe (Figure S8). The fluorescent SWNTs were then noncovalently functionalized with the peptide, cycARM. Using confocal microscopy, after 4 h of incubation with the hybrid, we observed that the peptide (blue fluorescence) and SWNTs (red fluorescence) were widely distributed throughout the entire cell, including in the nucleus and nucleolus (Figure 3c, Figures S9 and S10).38,44−46 Colocalization of the blue and red fluorescences indicated that cyc-ARM did not dissociate from the SWNTs, even in the intracellular environment. To further verify the stability of the noncovalent immobilization of the peptides on the SWNTs in the intracellular environment, cells treated either with the peptide alone (i.e., cyc-ARM alone) or with homo-ARM-SPCH were analyzed by FRAP. The FRAP is a technique used to assess the size-related intracellular mobility of a material by measuring the time needed for fluorescence recovery in a photobleached region.45 As shown in Figure 3d and Figure S11, the FRAP analysis revealed the slower recovery of fluorescence from homo-ARM-SPCH than from cyc-ARM. Because the recovery rate is inversely proportional to the molecular mass, the results 11542

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Figure 5. Inhibition of multimolecular RNA−protein complexes using multitarget-directed peptide−SWNT hybrids. (a) Schematic illustration of the pDM128 plasmid. (b), (c) CAT assays measuring nucleocytoplasmic transport of RRE RNA. The numbers above the gels are the concentrations of LMB or SPCH. (d), (e) Densitometric analyses of the CAT assay data in (b) and (c), respectively.

interactions could be inhibited by the peptide−SWNT hybrids. As shown in Figure 5b−e, the CAT assay revealed that the bioinorganic hybrid inhibitor reduced the expression of the CAT gene dramatically in a dose-dependent manner. However, the SPCHs targeting only one of the interactions were not able to reduce the CAT activity when administered at the same concentration (Figure S15), indicating that the interactions had to be simultaneously regulated for effective inhibition of the multimolecular RNA−protein complexes. Compared to leptomycin B (LMB), a well-known small-molecule inhibitor of the HIV-1 Rev-mediated pre-mRNA export from the nucleus,49 hetero-(A&N)-SPCH showed an approximately 150-fold better performance as an inhibitor of the RNA−protein complexes; an LMB concentration of 100 nM was required for near complete prevention of the RRE export, while only 0.66 nM SPCH was needed to obtain similar activity. We ascribed the outstanding inhibition capability of the SPCHs to the simultaneous multivalent display of peptide ligands directed against two different targets and the adaptable nature of the noncovalent assembly. With this adaptable property, the assembly could be able to adjust the orientation and spacing of the ligand units according to the particular 3D environment of the target interfaces. When taken together, these results demonstrated that this type of hetero-SPCH has potential as an

peptides (Figure S14). Thus, the two peptides were not discriminated against during their noncovalent binding to the SWNTs. Moreover, hetero-(A&N)-SPCH exhibited higher energy-transfer efficiency in the FRET analysis than did the coassembled peptides, confirming that cyc-ARM and lin-NES were more effectively blended on the sidewalls of the CNTs than in the coassembled irregular aggregates (Figure 4f). Therefore, we concluded that bioactive peptides with different amino acid compositions, secondary structures, and conformations could be stably displayed on a hetero-SPCH through a noncovalent hybridization method. Finally, we examined the multitarget-binding and inhibition capabilities of the hetero-SPCHs. We used a nucleocytoplasmic export assay based on pDM128, a plasmid for RRE expression, to assess the inhibition of Rev-mediated RRE export (Figure 5a).19,48 Cotransfection of pDM128 with pSV2-Rev (a plasmid for Rev expression) in HeLa cells enabled the efficient nucleocytoplasmic export of RRE RNA, as revealed by the CAT assay (Figure 5b, lane 3). In contrast, negligible CAT activity was observed when the cell was cotransfected with pBluescript KS(+), a negative control plasmid (Figure 5b, lane 2). Next, we treated the cells with hetero-(A&N)-SPCH, which was fabricated with the peptides in a 1:1 ratio, to determine whether nucleocytoplasmic export mediated by RRE:Rev:Crm1 11543

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(6) Reker, D.; Seet, M.; Pillong, M.; Koch, C. P.; Schneider, P.; Witschel, M. C.; Rottmann, M.; Freymond, C. L.; Brun, R.; Schweizer, B.; Illarionov, B.; Bacher, A.; Fischer, M.; Diederich, F.; Schneider, G. Deorphaning Pyrrolopyrazines as Potent Multi-Target Antimalarial Agents. Angew. Chem., Int. Ed. 2014, 53, 7079−7084. (7) Bournazos, S.; Gazumyan, A.; Seaman, M. S.; Nussenzweig, M. C.; Ravetch, J. V. Bispecific Anti-HIV-1 Antibodies with Enhanced Breadth and Potency. Cell 2016, 165, 1609−1620. (8) Huang, Y.; Yu, J.; Lanzi, A.; Yao, X.; Andrews, C. D.; Tsai, L.; Gajjar, M. R.; Sun, M.; Seaman, M. S.; Padte, N. N.; Ho, D. D. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity. Cell 2016, 165, 1621−1631. (9) Verdine, G. L.; Hilinski, G. J. Stapled Peptides for Intracellular Drug Targets. Methods Enzymol. 2012, 503, 3−33. (10) Kitov, P. I.; Bundle, D. R. On the Nature of the Multivalency Effect: A Thermodynamic Model. J. Am. Chem. Soc. 2003, 125, 16271−16284. (11) Dam, T. K.; Brewer, C. F. Effects of Clustered Epitopes in Multivalent Ligand-Receptor Interactions. Biochemistry 2008, 47, 8470−8476. (12) Jeong, W. J.; Choi, S. J.; Choi, J. S.; Lim, Y. B. Chameleon-like Self-Assembling Peptides for Adaptable Biorecognition Nanohybrids. ACS Nano 2013, 7, 6850−6857. (13) Pollard, V. W.; Malim, M. H. The HIV-1 Rev Protein. Annu. Rev. Microbiol. 1998, 52, 491−532. (14) Battiste, J. L.; Mao, H.; Rao, N. S.; Tan, R.; Muhandiram, D. R.; Kay, L. E.; Frankel, A. D.; Williamson, J. R. α-Helix-RNA Major Groove Recognition in an HIV-1 Rev Peptide-RRE RNA Complex. Science 1996, 273, 1547−1551. (15) Hamasaki, K.; Ueno, A. Aminoglycoside Antibiotics, Neamine and its Derivatives as Potent Inhibitors for the RNA-Protein Interactions Derived from HIV-1 Activators. Bioorg. Med. Chem. Lett. 2001, 11, 591−594. (16) DeJong, E. S.; Chang, C. E.; Gilson, M. K.; Marino, J. P. Proflavine Acts as a Rev Inhibitor by Targeting the High-Affinity Rev Binding Site of the Rev Responsive Element of HIV-1. Biochemistry 2003, 42, 8035−8046. (17) Fornerod, M.; Ohno, M.; Yoshida, M.; Mattaj, I. W. CRM1 is an Export Receptor for Leucine-Rich Nuclear Export Signals. Cell 1997, 90, 1051−1060. (18) Daelemans, D.; Afonina, E.; Nilsson, J.; Werner, G.; Kjems, J.; De Clercq, E.; Pavlakis, G. N.; Vandamme, A. M. A Synthetic HIV-1 Rev Inhibitor Interfering with the CRM1-Mediated Nuclear Export. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14440−14445. (19) Choi, J. S.; Han, S. H.; Kim, H.; Lim, Y. B. Cyclic PeptideDecorated Self-Assembled Nanohybrids for Selective Recognition and Detection of Multivalent RNAs. Bioconjugate Chem. 2016, 27, 799− 808. (20) Jeong, W. J.; Lee, M. S.; Lim, Y. B. Helix Stabilized, Thermostable, and Protease-Resistant Self-Assembled Peptide Nanostructures as Potential Inhibitors of Protein-Protein Interactions. Biomacromolecules 2013, 14, 2684−2689. (21) Han, S. H.; Lee, M. K.; Lim, Y. B. Bioinspired Self-Assembled Peptide Nanofibers with Thermostable Multivalent α-Helices. Biomacromolecules 2013, 14, 1594−1599. (22) Fang, X. Y.; Wang, J. B.; O’Carroll, I. P.; Mitchell, M.; Zuo, X. B.; Wang, Y.; Yu, P.; Liu, Y.; Rausch, J. W.; Dyba, M. A.; Kjems, J.; Schwieters, C. D.; Seifert, S.; Winans, R. E.; Watts, N. R.; Stahl, S. J.; Wingfield, P. T.; Byrd, R. A.; Le Grice, S. F. J.; Rein, A.; Wang, Y. X. An Unusual Topological Structure of the HIV-1 Rev Response Element. Cell 2013, 155, 594−605. (23) Rausch, J. W.; Le Grice, S. F. J. HIV Rev Assembly on the Rev Response Element (RRE): A Structural Perspective. Viruses 2015, 7, 3053−3075. (24) Daugherty, M. D.; Liu, B.; Frankel, A. D. Structural Basis for Cooperative RNA Binding and Export Complex Assembly by HIV Rev. Nat. Struct. Mol. Biol. 2010, 17, 1337−1342. (25) DiMattia, M. A.; Watts, N. R.; Stahl, S. J.; Rader, C.; Wingfield, P. T.; Stuart, D. I.; Steven, A. C.; Grimes, J. M. Implications of the

effective therapeutic agent for diseases involving interactions among multiple pathogenic biomolecules.

4. CONCLUSION We have devised a novel strategy for regulating multiple biomolecular interactions simultaneously using multivalent MTDLs. Instead of utilizing small-molecule inhibitors, peptides, a promising class of molecules in the drug discovery field, were displayed on a CNT scaffold, which has a length that is much longer than that of most biomolecules. The bioinorganic hybrid structures were prepared via a simple method using noncovalent interactions and showed good stability in cellulo. With the multivalent display of the peptide ligands, SPCHs showed outstanding target inhibition capabilities, even in the multitarget-directed assay. Because the efficacy of drugs is often limited by the low rate of accumulation in diseased areas after administration, obtaining high therapeutic activity from a single medication is an important goal in drug discovery. From this perspective, we expect that the outstanding performance of the multivalent supramolecular structures will provide new insight into MTDL-based therapeutics.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01517. Chemical structures, mass spectra, additional fluorescence emission and CD spectra, and EMSA and CAT assay results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yong-beom Lim: 0000-0001-6590-7373 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Yonsei-Carl Zeiss Advanced Imaging Center, Yonsei University College of Medicine, for technical assistance. This work was supported by grants from the National Research Foundation (NRF) of Korea (2014R1A2A1A11050359, 2014M3A7B4051594) and the Yonsei University FutureLeading Research Initiative.

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