Construction of a Sequenceable Protein Mimetic Peptide Library with

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Construction of a Sequenceable Protein Mimetic Peptide Library with a True 3D Diversifiable Chemical Space Zhonghan Li,† Shiqun Shao,† Xiaodong Ren,† Jianan Sun,† Zhili Guo,† Siwen Wang,† Michelle M. Song,‡ Chia-en A. Chang,† and Min Xue*,† †

Department of Chemistry, University of California, Riverside, Riverside, California 92521, United States Martin Luther King High School, Riverside, California 92508, United States

‡

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S Supporting Information *

technical challenges. First, such library requires a bicyclic topology as the minimal complexity, where all three linkages must bear modular amino acid residues to satisfy the 3D spatial accessibility. As shown in Figure 1, conventional bicyclic

ABSTRACT: We present here a library of protein mimetic bicyclic peptides. These nanosized structures exhibit rigid backbones and spatially diversifiable side chains. They present modular amino acids on all three linkages, providing access to a true 3D diversifiable chemical space. These peptides are synthesized through a Cu-catalyzed click reaction and a Ru-catalyzed ringclosing metathesis reaction. Their bicyclic topology can be reduced to a linear one, using Edman degradation and Pd-catalyzed deallylation reactions. The linearization approaches allow de novo sequencing through mass spectrometry methods. We demonstrate the function of a particular peptide that was identified through a high throughput screening against the E363-R378 epitope on the intrinsically disordered c-Myc oncoprotein. Intracellular delivery of this peptide could interfere with the c-Mycmediated transcription and inhibit proliferation in a human glioblastoma cell line.

Figure 1. Comparison between the conventional bicyclic peptides and the newly designed structure. The translucent domes represent the diversifiable space accessible to the corresponding linkages, due to the modularity of amino acid residues. The conventional structure exhibits a constant region as the third linkage, therefore lacks the corresponding accessible dome.7,25

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peptide structures originate from monocyclic precursors, followed by ring closures between two side chains. Such constructs only present modular residues on two linkages, leaving the third linkage as a constant region.7,25 Therefore, they provide limited access to the 3D chemical space. Second, robust sequencing methods are required to identify the hits. In the conventional one-bead-one-compound (OBOC) premise, the bicyclic structure needs to be linearized to allow de novo sequencing procedures. Given that the cyclization or stapling methods are usually irreversible, it further encumbers the structural design. In this Communication, we present the construction of a unique sequenceable bicyclic peptide library that provides access to the true 3D diversifiable chemical space (Figure 1). Such a library mimics protein structures in two ways. First, the rigidified peptide backbones grant considerable stability to the structures. Second, the spatial arrangements of functional groups resemble the three-dimensional interaction interface that is commonly observed in proteins. The peptide structure contains modular amino acid positions on all three linkages, and its sequence can be determined through chemical linearization processes. We further demonstrate the function

roteins are the building blocks of life and the executors of biological functions.1,2 Their astronomical versatility derives from two parts: the plethora of functional groups provided by the 20 canonical amino acids, and the spatial arrangements of those groups diversified by the structural complexity. The quest of abiotically mimicking proteins to recreate such functional and structural panoply, especially using short peptides, is ever intriguing to chemists.3 The variety of functional groups and primary structures can be easily achieved by synthesizing a library of peptides using combinatorial methods.4,5 These libraries can include all possible combinations of amino acid sequences and are usually prepared in the solid-supported form. They can be standalone peptides, or as variable segments on synthetic proteins.6,7 High-throughput screening of these libraries has identified many sequences capable of recognizing specific targets.7−10 In terms of spatial diversity, cyclization and stapling strategies can generate peptides with complex structures.11−20 Additionally, nanoparticles and supramolecular interactions can facilitate 3D arrangements of peptides.21−24 Nevertheless, these structural manipulations are usually performed on known sequences, either to stabilize a specific structure or to generate intricate topologies.7,8,25,26 Currently, there lacks a peptide library that covers a proteinlike true 3D diversifiable chemical space, mainly due to two © XXXX American Chemical Society

Received: August 4, 2018

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DOI: 10.1021/jacs.8b08338 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Communication

Journal of the American Chemical Society

Figure 2. (a) Construction of the bicyclic peptide through sequential cyclization reactions. In the cartoon illustration, each ball represents an amino acid residue; the nonspheres are the functional groups that react during cyclization. HG-II: Hoveyda−Grubbs 2nd generation catalyst. (b) Chemical linearization of the bicyclic peptide structure achieved through deallylation and Edman degradation. (c) Photolabile linkers allow facile cleavage of the linearized peptide from the resin. (d, e) MS and MS/MS spectra of the linearized peptide with the sequence of LEPAVZIFE. * GluPro induced fragmentation. Z represents the PTH derivative of triazole linker after Edman degradation.

of a particular bicyclic peptide (NT-A1), which binds with an epitope of the c-Myc transcription factor. Similar to the process of protein folding, the bicyclic peptides were generated from their linear peptide precursors (Figures 2a and S1). Two cyclization strategies were employed to form the bicyclic topology: copper-catalyzed azide−alkyne cycloaddition (CuAAC) and Ru-catalyzed ring-closing meta-

thesis (RCM) reactions. These reactions have few side products and are orthogonal to the traditional solid phase peptide synthesis schemes. They have been widely utilized to construct stapled peptides, due to the excellent biocompatibility of the products.8,27−29 The cyclization handles (residues containing the azide, alkyne and alkene moieties) in the linear precursor peptides were separated by modular amino acid B

DOI: 10.1021/jacs.8b08338 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Communication

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Figure 3. (a) Library screening process utilizing the binding-promoted in situ click reaction. (b) Structure of the identified best hit. (c) Some conformations of the NT-A1 ligand from the molecular dynamics (MD) trajectory. The structures in orange are the conformations evenly extracted from the simulation (every 2 ns), and the one in blue is one of the most populated conformations by manual investigation. The thicker sticks represent the NT-A1 backbone, while the thinner lines are the amino acid side chains. (d) Binding affinity between surface-immobilized epitope and solubilized NT-A1 ligand. e. Binding affinity determination using recombinant c-Myc protein. The data were obtained through the enzymelinked immunosorbent assay methods. (f) Intracellularly delivered NT-A1 inhibited U87 cell proliferation. NT-A1 group (liposome 0.4 mg/mL; NT-A1 1.4 μM); Vehicle group (liposome 0.4 mg/mL). (g) LDH expression level decreased upon NT-A1 treatment, while the c-Myc level remained unchanged, indicating the interference to c-Myc transcription activities.

nine was not compatible with the metal catalysts used in our case, and an alternative approach was required. Herein, we employed a photolabile linker. As shown in Figures 2c, the 3amino-3-(2-nitrophenyl)propionic acid (ANP, Figure S2) linker could be cleaved by UV irradiation with high efficiency. 34 The resulting liberated peptide could be sequenced through MS/MS methods (Figure 2d,e). We prepared an OBOC bicyclic peptide library using the strategies above. We incorporated five modular amino acid positions using 18 natural amino acids to construct the library (Cys and Met were excluded due to the incompatibility with the catalysts). The library was synthesized on Tentagel S resins and sequentially cyclized using CuAAC and RCM reactions (Figure S1).8 In order to validate the quality of the library, we randomly picked resins out to perform chemical linearization followed by photocleavage and MS/MS sequencing (Figures S3−S17). As protein mimetics, the bicyclic peptides were expected to perform designated functions. We reasoned that the threedimensionally arranged residues and the bicyclic topology could lead to prominent binding affinities toward highly disordered proteins. Herein, we chose the c-Myc transcription factor as a target to demonstrate such an application. The cMyc protein is an oncoprotein that directly contributes to oncogenesis and tumor maintenance in many cancers.35 It is a notoriously “undruggable” protein due to the highly disordered monomeric structure and the lack of obvious binding pockets.36 We performed screening against the E363-R378 epitope on c-Myc, which is highly involved in the formation of the Myc-Max dimer and the associated DNA recognition (Figure S18). We envisioned that a specific ligand toward this epitope could interfere with the c-Myc transcriptional activities and have potential therapeutic applications.

residues. Because of these arrangements, the fully assembled bicyclic peptides presented variable functional groups on every linkage (Figure 2a). Such structures dramatically expanded the access to additional spatial diversities, compared to the conventional bicyclic peptide libraries. In order to implement a peptide library, robust sequencing methods must be available. To this end, we incorporated two design features in the bicyclic peptide structure to enable chemical linearization, which allowed de novo sequencing using mass spectrometry methods. As shown in Figure 2b, the alkyne moiety for the CuAAC cyclization was placed at the Nterminus. Consequently, the corresponding triazole-linked ring could be opened through Edman degradation.30 The resulting phenylthiohydantoin group would stay on the sequence (Figure 2b). Such a strategy has been previously demonstrated in preparing monocyclic peptide libraries.8 Regarding the RCM reaction, it is generally not considered reversible under normal conditions. Therefore, an additional chemical design was required to open the RCM ring. Here we used the Glu-allyl esters to perform the RCM reaction (Figure 2a).31 The resulted 5,5′-2-butene-1,4-diyl-diglutamate was stable under standard peptide synthesis and library screening conditions. However, it was very labile to the Pd-catalyzed deallylation reaction.32 This property granted pseudoreversibility to this RCM linkage and enabled complete linearization of the bicyclic peptides (Figure 2b). Conventional high throughput screening methods using OBOC libraries involve resin-immobilized sequences. Because of the harsh conditions during library synthesis and screening, the linkage between the peptide and the resin must be relatively inert. On the other hand, mass spectrometry-based sequencing requires that the peptides being liberated from the resin.10 A common strategy is to implement the cyanogen bromide-mediated methionine cleavage.33 However, methioC

DOI: 10.1021/jacs.8b08338 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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We followed well-established protocols to perform the screening (Figure 3a).8 A bicyclic peptide library bearing an additional alkyne group at the N-terminus was first precleared (undergone mock screening processes) to remove the nonspecific binders. This precleared library was then incubated with the biotinylated E363-R378 epitope, where the K371 residue was replaced by an azido lysine (Figures S18−S21). This epitope was structure-less without the binding partners (Max and DNA), and the biotinylation and azide-substitution retained the random coil feature (Figures S22−S24). The strong interactions between the epitope and potential hit sequences permitted proximity-driven in situ click reactions and appended biotin groups on the corresponding resins. These biotinylated hits were identified through enzymeamplified colorimetric reactions. The hit resins were separated from the library, undergone chemical linearization and photocleavage, and their sequences were determined through MS/MS methods (Figure 3a). We identified two constructs that could potentially recognize the E363-R378 epitope (NT-A1 NT-A2, Figures 3b and S25). In order to validate the hits, we resynthesized the identified bicyclic peptide at mg scale and evaluated their binding toward the E363-R378 epitope (Figures S26−S34). NT-A1 exhibited a superior binding affinity. Computational studies revealed that NT-A1 exhibited substantial structural rigidity on the peptide backbone (Figures 3c and S35). This feature may contribute to its ability to recognize a structure-less epitope with a prominent binding affinity (Figure 3d). Further validation experiments also confirmed that it was capable of binding with the c-Myc recombinant protein, albeit with a relatively weaker affinity (Figure 3e). Further tests revealed that the 3D conformation of NT-A1 was crucial to its c-Myc binding ability. (Figure S36). We then sought to test the biological activity of the identified c-Myc-targeting NT-A1 on U87 cells. U87 is a glioblastoma cell line where elevated c-Myc activities are essential for metabolic maintenance and cell survival.37 In principle, ligands that target the E363-R378 epitope would interfere with the Myc-Max dimer formation and the DNA binding, therefore inhibiting cell proliferation. Because NT-A1 was not cell-permeable, we employed a wellestablished liposome formulation for its delivery to U87 cells (Figure S37).38 As shown in Figure 3f, intracellularly delivered NT-A1 significantly inhibited cell proliferation (Figures S38 and S39). In addition, we found that NT-A1 caused a considerable decrease in the lactate dehydrogenase (LDH) expression level (Figure 3g). These results are consistent with the fact that LDH is a target of the c-Myc transcription, and that c-Myc regulates the aerobic glycolytic activities in U87 cells.37 The newly designed bicyclic peptide library reported here provides access to a true 3D diversifiable chemical space by introducing modular amino acid residues on all three linkages. The rigid and spatially diverse structures could be exploited to develop ligands targeting intrinsically disordered proteins, as exemplified in this Communication. This bicyclic peptide library could easily be expanded through manipulations of the stereochemistry as well as the introduction of unnatural amino acids. Also, we envision that implementing additional pseudoreversible cyclization methods could lead to more complex topologies while retaining the well-defined spatial arrangements of residues.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b08338. Procedures for bicyclic peptide synthesis and chemical linearization, examples of randomly picked sequences from the bicyclic peptide library, cell culture and other experimental details (PDF) Structure models (ZIP)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Chia-en A. Chang: 0000-0002-6504-8529 Min Xue: 0000-0002-8136-6551 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. David N. Bunck and Dr. JingXin Liang for valuable discussions. We thank the U.S. National Science Foundation MCB-1350401 for financial support.

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REFERENCES

(1) Hunter, T. Cell 2000, 100 (1), 113−27. (2) Ouzounis, C. A.; Coulson, R. M. R.; Enright, A. J.; Kunin, V.; Pereira-Leal, J. B. Nat. Rev. Genet. 2003, 4, 508. (3) Groß, A.; Hashimoto, C.; Sticht, H.; Eichler, J. Front. Bioeng. Biotechnol. 2015, 3, 211. (4) Gray, B. P.; Brown, K. C. Chem. Rev. 2014, 114 (2), 1020−1081. (5) Lam, K. S.; Lebl, M.; Krchňaḱ , V. Chem. Rev. 1997, 97 (2), 411− 448. (6) Gates, Z. P.; Vinogradov, A. A.; Quartararo, A. J.; Bandyopadhyay, A.; Choo, Z.-N.; Evans, E. D.; Halloran, K. H.; Mijalis, A. J.; Mong, S. K.; Simon, M. D.; Standley, E. A.; Styduhar, E. D.; Tasker, S. Z.; Touti, F.; Weber, J. M.; Wilson, J. L.; Jamison, T. F.; Pentelute, B. L. Proc. Natl. Acad. Sci. U. S. A. 2018, 115 (23), E5298. (7) Lian, W.; Upadhyaya, P.; Rhodes, C. A.; Liu, Y.; Pei, D. J. Am. Chem. Soc. 2013, 135 (32), 11990−11995. (8) Das, S.; Nag, A.; Liang, J.; Bunck, D. N.; Umeda, A.; Farrow, B.; Coppock, M. B.; Sarkes, D. A.; Finch, A. S.; Agnew, H. D.; Pitram, S.; Lai, B.; Yu, M. B.; Museth, A. K.; Deyle, K. M.; Lepe, B.; RodriguezRivera, F. P.; McCarthy, A.; Alvarez-Villalonga, B.; Chen, A.; Heath, J.; Stratis-Cullum, D. N.; Heath, J. R. Angew. Chem., Int. Ed. 2015, 54 (45), 13219−24. (9) Cho, C.-F.; Behnam Azad, B.; Luyt, L. G.; Lewis, J. D. ACS Comb. Sci. 2013, 15 (8), 393−400. (10) Trinh, T. B.; Pei, D. Screening One-Bead-One-Compound Peptide Libraries for Optimal Kinase Substrates. Kinase Screening and Profiling: Methods and Protocols; Springer: New York, NY, 2016; pp 169−181. (11) Walensky, L. D.; Bird, G. H. J. Med. Chem. 2014, 57 (15), 6275−6288. (12) Verdine, G. L.; Hilinski, G. J. Stapled Peptides for Intracellular Drug Targets. Methods Enzymol. 2012, 503, 3−33. (13) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509. (14) Bandyopadhyay, A.; Gao, J. J. Am. Chem. Soc. 2016, 138 (7), 2098−2101. (15) Zhang, L.; Navaratna, T.; Thurber, G. M. Bioconjugate Chem. 2016, 27 (7), 1663−1672. (16) Wang, C. K.; Craik, D. J. Nat. Chem. Biol. 2018, 14 (5), 417− 427. (17) Kale, S. S.; Villequey, C.; Kong, X.-D.; Zorzi, A.; Deyle, K.; Heinis, C. Nat. Chem. 2018, 10 (7), 715−723. D

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Journal of the American Chemical Society (18) Rojas, A. J.; Zhang, C.; Vinogradova, E. V.; Buchwald, N. H.; Reilly, J.; Pentelute, B. L.; Buchwald, S. L. Chem. Sci. 2017, 8 (6), 4257−4263. (19) Noisier, A. F. M.; García, J.; Ionuţ, I. A.; Albericio, F. Angew. Chem., Int. Ed. 2017, 56 (1), 314−318. (20) Hill, T. A.; Shepherd, N. E.; Diness, F.; Fairlie, D. P. Angew. Chem., Int. Ed. 2014, 53 (48), 13020−13041. (21) Ikemi, M.; Kikuchi, T.; Matsumura, S.; Shiba, K.; Sato, S.; Fujita, M. Chem. Sci. 2010, 1 (1), 68−71. (22) Zaramella, D.; Scrimin, P.; Prins, L. J. J. Am. Chem. Soc. 2012, 134 (20), 8396−8399. (23) Montet, X.; Funovics, M.; Montet-Abou, K.; Weissleder, R.; Josephson, L. J. Med. Chem. 2006, 49 (20), 6087−6093. (24) Qian, E. A.; Wixtrom, A. I.; Axtell, J. C.; Saebi, A.; Jung, D.; Rehak, P.; Han, Y.; Moully, E. H.; Mosallaei, D.; Chow, S.; Messina, M. S.; Wang, J. Y.; Royappa, A. T.; Rheingold, A. L.; Maynard, H. D.; Král, P.; Spokoyny, A. M. Nat. Chem. 2017, 9, 333. (25) Rhodes, C. A.; Pei, D. Chem. - Eur. J. 2017, 23 (52), 12690− 12703. (26) Bartoloni, M.; Jin, X.; Marcaida, M. J.; Banha, J.; Dibonaventura, I.; Bongoni, S.; Bartho, K.; Gräbner, O.; Sefkow, M.; Darbre, T.; Reymond, J.-L. Chem. Sci. 2015, 6 (10), 5473−5490. (27) Castro, V.; Rodríguez, H.; Albericio, F. ACS Comb. Sci. 2016, 18 (1), 1−14. (28) Angell, Y.; Burgess, K. J. Org. Chem. 2005, 70 (23), 9595−9598. (29) Reichwein, J. F.; Versluis, C.; Liskamp, R. M. J. J. Org. Chem. 2000, 65 (19), 6187−6195. (30) Matsueda, G. R.; Haber, E.; Margolies, M. N. Biochemistry 1981, 20 (9), 2571−2580. (31) Araki, Y.; Topolovčan, N.; Kotora, M. Eur. J. Org. Chem. 2017, 2017 (13), 1736−1739. (32) Jeffrey, P. D.; McCombie, S. W. J. Org. Chem. 1982, 47 (3), 587−590. (33) Gross, E. The cyanogen bromide reaction. Methods Enzymol. 1967, 11, 238−255. (34) Brown, B. B.; Wagner, D. S.; Geysen, H. M. Mol. Diversity 1995, 1 (1), 4−12. (35) Dang, C. V. Cell 2012, 149 (1), 22−35. (36) Dang, C. V.; Reddy, E. P.; Shokat, K. M.; Soucek, L. Nat. Rev. Cancer 2017, 17, 502. (37) Tateishi, K.; Iafrate, A. J.; Ho, Q.; Curry, W. T.; Batchelor, T. T.; Flaherty, K. T.; Onozato, M. L.; Lelic, N.; Sundaram, S.; Cahill, D. P.; Chi, A. S.; Wakimoto, H. Clin. Cancer Res. 2016, 22 (17), 4452− 4465. (38) Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S. W.; Zarghami, N.; Hanifehpour, Y.; Samiei, M.; Kouhi, M.; Nejati-Koshki, K. Nanoscale Res. Lett. 2013, 8 (1), 102−102.

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DOI: 10.1021/jacs.8b08338 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX