Nongenetic Reprogramming of the Ligand Specificity of Growth Factor

May 1, 2017 - Cite this:J. Am. Chem. Soc. 139, 19, 6554- ... The reprogramming of receptor–ligand interactions affords an opportunity to direct cell...
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Nongenetic Reprogramming of the Ligand Specificity of Growth Factor Receptors by Bispecific DNA Aptamers Ryosuke Ueki,* Saki Atsuta, Ayaka Ueki, and Shinsuke Sando* Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

they represent promising targets in regenerative therapy. Conversely, uncontrolled activation of RTK signaling could be oncogenic, as aberrant activity of RTKs has been implicated in cancer development.3 Therefore, a method that can regulate RTK signaling depending on a given external cue would represent a platform for designing smart and safer therapeutics that would function only where needed. Herein, we report DNA aptamer-mediated Reprogramming of the Interaction Partner of Receptor tyrosine kinases (DRIPaR). We selected oligonucleotides as a building block for this system because of their structural and functional programmability.4 An oligonucleotide-based molecular machinery that has molecular recognition ability can be constructed rationally and has demonstrated potential in mimicking or controlling biological processes.5 As shown in Figure 1, DRIPaR was designed to reproduce the growth-factor-induced activation process of RTK. The binding of growth factors induces the dimerization of RTK and the subsequent phosphorylation of the intracellular domain, which is a key step for triggering intracellular signaling cascades (Figure 1a).2 DRIPaR is based on bispecific DNA aptamers that consist of an aptamer sequence that binds to the RTK of

ABSTRACT: The reprogramming of receptor−ligand interactions affords an opportunity to direct cells to respond to user-defined external cues. Although this has often been achieved via the genetic engineering of receptors, an alternative, nongenetic approach is highly demanded. In this article, we propose the design of oligonucleotide-based synthetic switches that feature the ability to reprogram the ligand specificity of the growth factor receptor. We demonstrated that our synthetic switches induced growth factor signaling via the formation of the dynamic complex with specific external cues that would otherwise not induce the signaling. This chemical approach may be applied to designing a new class of chemical tools that can control the activities of native cells and represent smart and safer regenerative drugs.

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ells are one of the most sophisticated molecular assemblies that can respond to the surrounding environment. Receptor−ligand interactions at the cell surface play pivotal roles in such characteristic processes as the binding of ligand molecules to the extracellular domain of membrane receptors to trigger appropriate intracellular signaling. Receptors and ligands have evolved to interact with their partners specifically, thereby allowing cells to trigger the responses depending on environmental cues from the extracellular space. Reprogramming the ligand specificity of a given receptor has provided the opportunity to customize cellular signaling and to direct cells to respond to external stimuli in a user-defined manner. To date, this has been achieved via the genetic engineering of existing receptors or the development of chimeric receptors containing antibody fragments.1 These methodologies have been applied in basic research aimed at elucidating the physiological roles of receptor signaling and in cell engineering for cell-based therapy and sensing. However, genetic modification may perturb other biological processes in cells. In addition, the need for genetic modification often creates obstacles in practical therapeutic applications. In this context, a robust and rational chemical approach that can control the activities of receptors of interest in a user-defined manner is highly demanded. In the present study, we propose a nongenetic approach that features the ability to reprogram the ligand specificity of receptor tyrosine kinases (RTKs),2 which are receptors for growth factors. RTKs regulate fundamental cellular activities, such as cell proliferation, migration, and differentiation; thus, © 2017 American Chemical Society

Figure 1. Schematic representation of (a) the activation mechanism of RTK induced by growth factor binding and (b) the working mechanism of DRIPaR. The image of the receptor was depicted using the data from the Protein Data Bank (PDB IDs 2UZY and 3A4P). Received: March 10, 2017 Published: May 1, 2017 6554

DOI: 10.1021/jacs.7b02411 J. Am. Chem. Soc. 2017, 139, 6554−6557

Communication

Journal of the American Chemical Society

Figure 2. (a) Sequence and (b) schematic representation of DRIPaR for inducing thrombin-dependent Met activation. The structure of thrombin was taken from the Protein Data Bank (PDB ID 1HUT). (c) Western blotting analysis of the phosphorylation level of Met in A549 cell lysates. A549 cells were cultured in the presence or absence of TS15, TS29 (10 nM each), and thrombin (1−10 nM) for 15 min.

Figure 3. (a) Sequence and (b) schematic representation of DRIPaR for inducing PDGF-dependent Met activation. The structure of PDGF was taken from the Protein Data Bank (PDB ID 3MJG). (c) Western blotting analysis of the phosphorylation level of Met in A549 cell lysates. A549 cells were cultured in the presence or absence of bispecific aptamers (20 nM each) and PDGF (10 nM) for 15 min. (d) The phosphorylation level of Met in A549 cell lysates measured by ELISA assay. A549 cells were cultured in the presence of the bispecific aptamer (20 nM) and PDGF (1−10 nM) for 15 min. (e) Western blotting analysis of the phosphorylation level of Gab1 (Y627), Akt (S473), and ERK1/2 (T202/Y204) in A549 cell lysates. A549 cells were cultured in the presence of appropriate ligands for 15 min. (f) PDGF-inducible DU145 cell scattering. The cells were cultured in the presence or absence of the ligands for 24 h. The phase contrast (PH) images were captured after 24 h of culture. Scale bars: 200 μm. (g) The migration trajectories of randomly selected cells (n = 30) during DU145 cell scattering assay shown in panel f. (h) The migration distance of each cell (n = 30), as determined from trajectories shown in panel g. The bars indicate the average migration distances.

interest (shown in red in Figure 1b) and another aptamer sequence that binds to a given cue from the extracellular space (shown in blue in Figure 1b). If the bispecific aptamers are appropriately designed to form a 2:1 ternary complex with the external cue, RTK dimerization and activation are induced depending on the external cue.

As a proof of concept of DRIPaR, we targeted the Met receptor, which is a cognate receptor for the hepatocyte growth factor (HGF). Because of its mitogenic and antiapoptotic effect, Met signaling has been one of the most extensively studied RTK signaling pathways in regenerative therapy.6 A 50-mer Met-binding DNA aptamer, CLN0003_SL1 (SL1),7 was 6555

DOI: 10.1021/jacs.7b02411 J. Am. Chem. Soc. 2017, 139, 6554−6557

Communication

Journal of the American Chemical Society

original Met aptamer,7b,8 the bispecific aptamer inhibited HGFinduced Met activation in the absence of PDGF (Figure S1). The results shown in Figure 3c suggest the presence of a linker-length dependency regarding Met-activating potential, as we observed stronger signals in cells that were treated with bispecific aptamers with a shorter linker length (lanes 4−7 in Figure 3c). ELISA was used to confirm further the Metactivating potential of each bispecific aptamer (Figure 3d). Consistent with the results of the Western blotting experiment, bispecific aptamers with a shorter linker exhibited higher Metactivating potential in the presence of 10 nM PDGF. The bispecific aptamer PS0 was the most effective among the aptamers tested here. The Met phosphorylation level induced by PS0 (20 nM) in the presence of PDGF (10 nM) was almost comparable with the putative maximal level induced by the natural ligand, HGF (1 nM).9 We also evaluated by Western blotting the activation of intracellular signaling molecules downstream of Met, such as Gab-1, Akt, and ERK (Figure 3e). In cells that were treated with HGF (lane 2 in Figure 3e) and PS0 (20 nM) in the presence of PDGF (lane 3 in Figure 3e), these signaling molecules showed increased phosphorylation levels. This result supports the contention that the ternary complex formed among PDGF and bispecific aptamers transduces intracellular signaling in a manner that is similar to that of the natural HGF. Having developed a potent bispecific aptamer, we checked whether it could reproduce the cellular activities that are induced by Met signaling. We focused on the migration of epithelial cells because it is a fundamental process during wound healing. We performed a DU145 cell scattering assay. The cells were incubated in the presence of ligands for 24 h (Figures 3f and S2) and the cell migration trajectory (Figures 3g,h, and S3) was traced using time-lapse imaging (Supporting Information movie). When the cells were treated with PS0 (20 nM) in the presence of PDGF (10 nM), a significant increase in cell motility was observed (522 ± 269 μm) compared with that of untreated cells (139 ± 61 μm) and that of PDGF-treated cells (191 ± 57 μm). This increased cell motility was comparable with that induced by HGF (1 nM) treatment (562 ± 251 μm). It was demonstrated that the aptamer evoked cellular activities in an external cue-dependent manner, as the increased cell migration was not observed when the cells were incubated with other protein inputs (Figure S4). In summary, we propose DRIPaR, which is a nongenetic method that can be used for reprogramming the ligand specificity of growth factor receptors. The bispecific aptamer strategy induced receptor activation via a user-defined molecule that otherwise did not work as an agonist of the receptor. As a model system, we designed bispecific aptamers that can induce cell migration at the injury site-specific biomarker, PDGF. It should be noted that expression of PDGF has been also implicated in other proliferative disease states14 and thus may not provide sufficient selectivity to the injury sites. As demonstrated in this work, however, the aptamer domain of the bispecific aptamer may be customized for specific external cues. This modularity would allow researchers to design desired oligonucleotide-based synthetic switches. Selection of appropriate biomarkers would be a key point in future therapeutic application of the concept of DRIPaR. Importantly, we have found that the activity of the receptor can be regulated via the diverse molecular interaction patterns, such as DNA−DNA hybridization and protein−small molecule interaction (Figure S5). Therefore, the concept of the present

selected as a component of the DRIPaR targeting the Met receptor. We and other research groups have reported that these aptamer and truncation variants work as a Met antagonist, as they compete with HGF for Met binding.7b,8 In addition, we have recently reported that the aptamer can induce Met activation when used in covalently linked dimer form.9 This result is consistent with the fact that other dimerized Met ligands work as Met agonists.10 However, it has not been examined so far whether the activity of the receptor can be regulated by programmable input molecules via dynamic formation of the signaling complex on the cells. We first tested the feasibility of DRIPaR by designing bispecific aptamers that consisted of SL1 and thrombin-binding aptamers (TBAs). TBA15 and TBA29 are known to recognize distinct epitopes of thrombin, exosite-1 and exosite-2, respectively.11 These TBAs were tethered to the 5′ end of SL1 and termed TS15 and TS29 (Figure 2a). We anticipated that these bispecific aptamers and thrombin would form a 2:1 ternary complex on the Met receptors and induce receptor dimerization and subsequent activation (Figure 2b). To test this hypothesis, Met-expressing A549 cells were incubated with thrombin (1−10 nM) in the presence or absence of bispecific aptamers (10 nM each). We conducted Western blotting analysis to check the Met phosphorylation level. In cells that were treated with bispecific aptamers (lane 2 in Figure 2c) or thrombin alone (lane 3 in Figure 2c), no significant changes in the Met phosphorylation level were observed. However, when the cells were incubated with both bispecific aptamers and thrombin, Met phosphorylation was observed in a thrombin-concentration-dependent manner (lanes 4−6 in Figure 2c). Importantly, Met phosphorylation occurred only when both types of bispecific aptamers existed in the media (lane 6 vs lanes 7 and 8 in Figure 2c). This supported our hypothesis that Met phosphorylation is induced by the formation of a ternary complex between two types of bispecific aptamers and thrombin. After confirming that DRIPaR worked using the bispecific aptamer approach, we next designed a bispecific aptamer that can control cell activities depending on a biologically relevant extracellular cue. As a model system, we focused on cell migration in the injury-healing process, which is induced by Met signaling. HGF stimulates the Met receptor on epithelial cells to induce cell migration to the injury site, to facilitate injury repair.6b However, the oncogenic nature of Met signaling causes inevitable risks for the administration of recombinant HGF or other synthetic Met agonists as regenerative medicines. Therefore, there is an increasing focus on molecular therapeutics that are capable of site-specific Met activation.12 In the subsequent experiments, we targeted platelet-derived growth factor (PDGF) as an external cue for injury lesion, because PDGF expression is increased after injury.13 To construct PDGF-responsive DRIPaR (Figure 3a,b), a previously reported 39-mer DNA aptamer14 to PDGF was tethered to the 5′ end of SL1 via an X-mer thymidine linker (PSX, Figure 3a). As PDGF is expressed as a homodimer, we anticipated that two bispecific aptamers would form a ternary complex with one PDGF homodimer to induce Met activation (Figure 3b). A Western blotting experiment confirmed that these bispecific aptamers (20 nM) induced Met activation in the presence of PDGF (lanes 3−7 in Figure 3c). When the cells were treated with PDGF alone, the level of Met phosphorylation was not affected, as there is no cross-reactivity between PDGF and the Met receptor (lane 2 in Figure 3c). As with the 6556

DOI: 10.1021/jacs.7b02411 J. Am. Chem. Soc. 2017, 139, 6554−6557

Communication

Journal of the American Chemical Society

A.; Comoglio, P. M. Nat. Rev. Mol. Cell Biol. 2010, 11, 834. (c) Matsumoto, K.; Funakoshi, H.; Takahashi, H.; Sakai, K. Biomedicines 2014, 2, 275. (7) (a) Boltz, A.; Piater, B.; Toleikis, L.; Guenther, R.; Kolmar, H.; Hock, B. J. Biol. Chem. 2011, 286, 21896. (b) Ueki, R.; Sando, S. Chem. Commun. 2014, 50, 13131. (8) Piater, B.; Doerner, A.; Guenther, R.; Kolmar, H.; Hock, B. PLoS One 2015, 10, e0142412. (9) Ueki, R.; Ueki, A.; Kanda, N.; Sando, S. Angew. Chem., Int. Ed. 2016, 55, 579. (10) (a) Niemann, H. H.; Jäger, V.; Butler, J. G.; van den Heuvel, J.; Schmidt, S.; Ferraris, D.; Gherardi, E.; Heinz, D. W. Cell 2007, 130, 235. (b) Tolbert, W. D.; Daugherty, J.; Gao, C.; Xie, Q.; Miranti, C.; Gherardi, E.; Vande Woude, G.; Xu, H. E. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 14592. (c) Ferraris, D. M.; Gherardi, E.; Di, Y.; Heinz, D. W.; Niemann, H. H. J. Mol. Biol. 2010, 395, 522. (d) Simonneau, C.; Leclercq, B.; Mougel, A.; Adriaenssens, E.; Paquet, C.; Raibaut, L.; Ollivier, N.; Drobecq, H.; Marcoux, J.; Cianférani, S.; Tulasne, D.; de Jonge, H.; Melnyk, O.; Vicogne, J. Chem. Sci. 2015, 6, 2110. (e) Ito, K.; Sakai, K.; Suzuki, Y.; Ozawa, N.; Hatta, T.; Natsume, T.; Matsumoto, K.; Suga, H. Nat. Commun. 2015, 6, 6373. (11) (a) Bock, L. C.; Griffin, L. C.; Latham, J. A.; Vermaas, E. H.; Toole, J. J. Nature 1992, 355, 564. (b) Tasset, D. M.; Kubik, M. F.; Steiner, W. J. Mol. Biol. 1997, 272, 688. (12) Landgraf, K. E.; Steffek, M.; Quan, C.; Tom, J.; Yu, C.; Santell, L.; Maun, H. R.; Eigenbrot, C.; Lazarus, R. A. Nat. Chem. Biol. 2014, 10, 567. (13) Werner, S.; Grose, R. Physiol. Rev. 2003, 83, 835. (14) Green, L. S.; Jellinek, D.; Jenison, R.; Ö stman, A.; Heldin, C. H.; Janjic, N. Biochemistry 1996, 35, 14413.

work may be integrated with the existing DNA-based logic circuits and protein based molecular devices. One potential application would be the development of smart and safer regenerative medicines that can induce growth factor signaling and cell activities such as growth, migration, and differentiation in a specific biological environment. Our future works will be conducted along these lines.

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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/jacs.7b02411. Experimental details and Figures S1−S5 (PDF) Time-lapse imaging of cell migration (AVI)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Ryosuke Ueki: 0000-0002-5433-5354 Shinsuke Sando: 0000-0003-0275-7237 Notes

The authors declare the following competing financial interest(s): The authors have filed a patent application (WO2016-039371).

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ACKNOWLEDGMENTS This work was supported by a Core Research for Evolutional Science and Technology (CREST) of Molecular Technology (No. JPMJCR13L4), Japan Science and Technology Agency (JST), grant to S.S and a Grant-in-Aid for Research Activity Start-up (No. 15H06136), Japan Society for the Promotion of Science (JSPS), to R.U.

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REFERENCES

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DOI: 10.1021/jacs.7b02411 J. Am. Chem. Soc. 2017, 139, 6554−6557