Molecular Glue that Spatiotemporally Turns on ProteinâProtein

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Molecular Glue that Spatiotemporally Turns on Protein–Protein Interactions Rina Mogaki, Kou Okuro, Ryosuke Ueki, Shinsuke Sando, and Takuzo Aida J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b02427 • Publication Date (Web): 12 Apr 2019 Downloaded from http://pubs.acs.org on April 12, 2019

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Journal of the American Chemical Society

Molecular Glue that Spatiotemporally Turns on Protein–Protein Interactions Rina Mogaki,† Kou Okuro,*,† Ryosuke Ueki,† Shinsuke Sando,† and Takuzo Aida*,†,‡ †

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ‡ Riken Center for Emergent Matter Science, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

Supporting Information Placeholder

We developed a dendritic molecular glue Glue-NBD that can serve universally to “turn on” protein– protein interactions (PPIs) spatiotemporally. PCGlue-NBD carrying multiple guanidinium ion (Gu+) pendants can adhere strongly to target proteins and cover their surfaces including the PPI interface regions, thereby suppressing PPIs with their receptor proteins. Upon irradiation with UV light, PC Glue-NBD on a target protein is photocleaved at butyratesubstituted nitroveratryloxycarbonyl (BANVOC) linkages in the dendrimer framework, so that the multivalency for the adhesion is reduced. Consequently, the guest protein is liberated and becomes eligible for a PPI. We found that hepatocyte growth factor HGF, when mixed with PCGlueNBD, lost the affinity toward its receptor c-Met. However, upon exposure of the PCGlue-NBD/HGF hybrid to LED light (365 nm), the PCGlue-NBD molecules on HGF were photocleaved as described above, so that HGF was liberated and retrieved its intrinsic PPI affinity toward c-Met. The “turn-on” PPI, thus achieved for HGF and c-Met, leads to cell migration, which can be made spatiotemporally with a mm-scale resolution by pointwise irradiation with UV light. ABSTRACT: PC

Needless to say, protein–protein interactions (PPIs) play essential roles in many biological events.1 If spatiotemporal modulation of PPIs is possible, one may expect to develop low-side-effect therapeutics and also to elucidate how biological events involving such PPIs proceed at the molecular level.2 From this point of view, PPI inhibitors that photochemically alter their affinity to target proteins may be promising. As a successful example of such PPI inhibitors, an azobenzene-attached peptide inhibitor was reported, where photoisomerization of the azobenzene unit from its trans-to-cis and cis-to-trans forms allowed for reversible modulation of PPIs.3 Although the reversible modulation is interesting, an essential issue to consider is an insufficient stability of the azobenzene unit both chemically and photochemically. For

one-way turning off PPIs, a ‘caged’ antibody for brainderived neurotrophic factor (BDNF) was developed.2 BDNF is known to induce neurotransmission through the PPI with its receptor TrkB.4 Because the ‘caged’ antibody is designed to be poorly affinitive toward BDNF, it barely affects the PPI between BDNF and TrkB. However, when the cage part was cleaved off photochemically, the antibody part with a strong affinity toward BDNF was exposed, thereby “turning off” the PPI between BDNF and TrkB spatiotemporally.2 Here we report a dendritic molecular glue PCGlue-NBD

Figure 1. Molecular structure of photocleavable molecular glue PC Glue-NBD, carrying 9 guanidinium ion (Gu+) pendants and butyrate-substituted nitroveratryloxycarbonyl (BANVOC) linkages. The BANVOC linkage is known to be cleaved off by exposure to UV or near-IR light.8b

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Figure 2. Schematic illustration of the mechanistic role of PCGlue-NBD for turning off and on a protein–protein interaction (PPI) between hepatocyte growth factor HGF and its receptor c-Met. Large PCGlue-NBD molecules adhere onto HGF and cover its surface, including the interface regions, thereby suppressing the PPI with c-Met. Upon exposure to UV light, PCGlue-NBD on HGF is degraded at the photocleavable linkages to reduce the multivalency for the adhesion and consequently liberates HGF. Then, HGF binds to cMet to cause its dimerization and phosphorylation, thereby enabling cell migration.

(Figure 1) that serves as the first inhibitor to “turn on” PPIs. Previously, we developed water-soluble molecular glues5–9 bearing multiple guanidinium ion (Gu+) pendants,10 which tightly adhere to proteins,6 nucleic acids,7 phospholipid membranes,8 and clay nanosheets9 via the formation of multiple salt bridges between their Gu+ pendants and oxyanionic groups on the above targets. In 2010, we reported that actin and myosin, when pre-treated with molecular glues, could not form their heterotropic conjugate.6b This finding prompted us to utilize molecular glues as universal inhibitors for PPIs. PCGlue-NBD (Figure 1) was designed as a new class of dendritic molecular glue carrying in its dendrimer framework nine Gu+ pendants and photocleavable linkages (butyrate-substituted nitroveratryloxycarbonyl; BANVOC).11 This compound strongly adheres to target proteins by a multivalent salt-bridge interaction between its Gu+ pendants and the oxyanionic groups of the proteins, and utilizes its large dendritic wedge to cover the protein surfaces, including their interface regions, to suppress PPIs. However, upon UV exposure, PCGlue-NBD is cleaved off at the carbamate bonds to give smaller dendrons carrying only three Gu+ pendants. The reduced multivalency for the adhesion supposedly results in the liberation of the target protein,6a which can then take part in PPIs. As a proof-of-concept study, we chose a PPI between the hepatocyte growth factor (HGF) and its receptor c-Met as a target (Figure 2). The PPI between HGF and c-Met is known to initiate the signaling pathways for cell proliferation, survival, and migration,12 which promote wound healing.13 In this communication, we report an interesting finding that HGF, when bound to PCGlue-NBD, barely interacts with its receptor c-Met, but can retrieve its

high affinity toward c-Met by exposure to LED light (365 nm). We also highlight that the resulting HGF/c-Met interaction allows for the dimerization followed by phosphorylation of c-Met, thereby enabling spatiotemporal cell migration. PC Glue-NBD (Figure 1) was synthesized by an azide– alkyne “click” reaction14 between a dendron carrying both a focal core appended with an alkyne-conjugated BANVOC and three Boc-protected Gu+ pendants and a dendron carrying both a nitrobenzoxadiazole (NBD)-substituted focal core and three azide (N3) pendants, followed by the removal of the Boc groups. The photocleavage of PCGlue-NBD was investigated by electronic absorption spectroscopy: When a HEPES buffer (50 mM HEPES, pH 7.2) solution of PCGlueNBD (10 µM; Figure S9)15 at 37 °C was exposed to UV light at 365 nm (1.1 mW/mm2), the absorbance at 430 nm increased, suggesting the formation of nitroso ketone upon photolysis.16 The spectral change leveled off after the UV exposure for 2 min (Figure S9),15 where PCGlue-NBD was photocleaved in 82% conversion, as estimated by thin layer chromatography (TLC; Figure S10).15 Prior to the investigation of PPI switching, we evaluated the adhesion and photo-triggered detachment of PCGlueNBD using bovine serum albumin (BSA) as a model protein. The association constant (Kassoc) of PCGlue-NBD with BSA was estimated by fluorescence spectral titration: Addition of BSA (0–10 µM) to a HEPES buffer (50 mM, pH 7.2) solution of PCGlue-NBD (5 µM) at 37 °C resulted in an increase in the NBD fluorescence at 540 nm (λex = 480 nm; Figure 3a), which is most likely due to a microenvironmental change around the NBD unit upon adhesion to the protein


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Figure 3. (a) Fluorescence spectra (λex = 480 nm) of PCGlueNBD (5 µM) at 37 °C in HEPES buffer (50 mM, pH 7.2) upon titration with bovine serum albumin (BSA, 0–10 µM) and (b) its binding profile. The fractions of BSA-bound PCGlue-NBD were calculated from the fluorescence intensity at 540 nm.15 (c) Zeta potential profiles at 25 °C in HEPES buffer (2.5 mM, pH 7.2) of BSA (20 µM; top) and a mixture of PCGlue-NBD (50 µM) and BSA (20 µM) before (middle) and after UV exposure at 365 nm for 2 min (1.1 mW/mm2; bottom).

surface.17,18 According to the reported method,19 we estimated the association constant (Kassoc) of PCGlue-NBD with BSA to be 6.3 × 105 M–1 (EC50 = 1.6 µM) by fitting the fraction of bound PCGlue-NBD to the Hill equation (Figure 3b).19 This value is as large as those of the reported dendritic molecular glues with proteins (~105 M–1).6a Accordingly, the zeta potential of BSA (20 µM; ζ = –18 ± 5 mV; Figure 3c, top) increased upon addition of PCGlue-NBD (50 µM; ζ = –7 ± 4 mV; Figure 3c, middle). This is most likely due to the charge neutralization of the carboxylate anions on BSA by the saltbridge formation with the Gu+ pendants on PCGlue-NBD. In contrast, after 2 min exposure of the resultant mixture to UV light (1.1 mW/mm2) at 365 nm, the zeta potential decreased (ζ = –15 ± 4 mV; Figure 3c, bottom), suggesting the successful liberation of BSA by the photocleavage of PCGlue-NBD.

Figure 4. (a–c) Phase-contrast micrographs of DU145 cells in RPMI1640 (0.5% FBS) containing HGF (500 pM) at 37 °C. Micrographs recorded in the absence (a) and presence (b) of PC Glue-NBD (2 µM) before (left) and after (right) 2 min UV exposure at 365 nm (1.1 mW/mm2). Scale bars = 100 µm. (c) Average total migration distances of DU145 cells (n = 30) after 12 h at 37 °C in RPMI1640 (0.5% FBS) in the absence (gray) and presence of HGF (500 pM; orange) or both PCGlue-NBD (2 µM) and HGF (500 pM) before (blue) and after (purple) 2 min UV exposure at 365 nm. (d) Western blotting profiles of (i) phosphorylated c-Met (p-Met), (ii) total Met (c-Met and pMet), and (iii) β-actin obtained from DU145 cells. The cells were incubated for 15 min at 37 °C in RPMI1640 (0.5% BSA) in the absence and presence of HGF (100 pM) or both PCGlueNBD (5 µM) and HGF (100 pM) before and after 2 min UV exposure (1.1 mW/mm2).

We then examined the possibility of switching PPIs with Glue-NBD. Binding of HGF to c-Met induces its dimerization followed by phosphorylation, which is known to result in the downstream signal transduction (Figure 2).12 Although the full crystal structure of the HGF/c-Met complex has not been reported, the structural information of the NK1 and SP domains of HGF, which are considered to be in-

PC



volved in the interaction with c-Met, is available.20 By visualizing the surface charge distribution of the NK1 and SP domains, we confirmed that both of them bear negatively charged regions (Figure S16)15 where PCGlue-NBD may preferentially adhere. As an output of the c-Met signaling, we observed cell migration.12 Human prostate carcinoma DU145 cells were immersed in a Roswell Park Memorial Institute medium (RPMI1640; 0.5% fetal bovine serum, FBS) containing HGF (500 pM) and subjected to phasecontrast microscopy. As shown in Figure 4a, the cells scattered after 12 h at 37 °C under 5% CO2. Accordingly, the average cell migration distance (dave), calculated from the micrographs over a period of 12 h by using the ImageJ software, was 281 µm (Figure 4c, orange). In sharp contrast, when treated with a mixture of HGF (500 pM) and PCGlueNBD (2 µM) under otherwise identical conditions to the above, the cells moved only a short distance (dave = 35 µm; Figures 4b and 4c, blue), analogous to those in the absence of HGF (dave = 52 µm; Figures 4c, gray and S12).15 Interestingly, when the above PCGlue-NBD/HGF hybrid was added to the cells after 2 min exposure to UV light at 365 nm (1.1 mW/mm2), the cells migrated actively (dave = 300 µm; Figures 4b and 4c, purple). These results suggest that PCGlueNBD adheres to HGF and temporarily suppresses its interaction with c-Met but photochemically detaches from HGF, which subsequently binds to c-Met and induces cell migration. To further confirm the possibility of switching the HGF/cMet interaction with PCGlue-NBD, we examined the phosphorylation of c-Met by Western blotting analysis. DU145 cells were incubated in RPMI1640 (0.5% BSA) for 15 min at 37 °C in the absence and presence of HGF (100 pM), PC Glue-NBD/HGF ([PCGlue-NBD] = 5 µM, [HGF] = 100 pM), or UV-exposed PCGlue-NBD/HGF (λ = 365 nm, 2 min, 1.1 mW/mm2; [PCGlue-NBD] = 5 µM, [HGF] = 100 pM). After being rinsed with Dulbecco’s phosphate buffered saline (D-PBS), the cells were lysed, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene fluoride (PVDF) membrane.15 For the visualization of phosphorylated c-Met (p-Met), the resultant PVDF membrane was treated with an antibody for p-Met, a secondary antibody, and then ImmunoStar LD chemiluminescent reagent.15 As expected, pMet was detected from lysates of DU145 cells treated with HGF but not from those of untreated DU145 cells [Figure 4d(i)]. Notably, HGF, when conjugated with PCGlue-NBD, phosphorylated c-Met a little [Figure 4d(i)]. In contrast, UV-exposed PCGlue-NBD/HGF phosphorylated c-Met as efficiently as HGF did [Figure 4d(i)]. As shown in Figure 4d(ii–iii), the expression levels of total Met (c-Met and pMet) and β-actin, a reference protein stably expressed in DU145 cells, remained substantially unchanged by the treatment with HGF, PCGlue-NBD/HGF, and UV-exposed PC Glue-NBD/HGF, thereby excluding the possibility of cell damage. Accordingly, PCGlue-NBD did not cause significant

Figure 5. (a, b) Schematic illustration of the experimental setup for in situ turning on the HGF/c-Met interaction using PC Glue-NBD in the presence of DU145 cells. Left half (x = – 13.5–0 mm) and right half (x = 0–13.5 mm) are UV-exposed (365 nm, 2 min, 1.1 mW/mm2) and non-exposed areas, respectively. (c, d) Confocal laser scanning micrographs (λex = 552 nm, λobs = 560–620 nm) of CellBrite Orange-labeled DU145 cells at 37 °C in RPMI1640 (0.5% FBS) containing PCGlueNBD (2 µM) and HGF (500 pM) at (c) x = –3.5 mm and (d) x = 3.9 mm. Scale bars = 100 µm. (e) Average total migration distances of DU145 cells (n = 20) after 12 h at 37 °C in RPMI1640 (0.5% FBS) containing PCGlue-NBD (2 µM) and HGF (500 pM) at different positions of the dish (x = –10.2, – 8.2, –5.4, –3.5, 3.9, 5.2, 6.6, and 7.7 mm).

cytotoxicity at 5 µM before and after the UV exposure (Figure S15).15 Therefore, it is most likely that the decrease and increase in the amount of p-Met observed above with PC Glue-NBD/HGF before and after the UV exposure, respectively, are due to the switching of the HGF/c-Met interaction with PCGlue-NBD.


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Of particular interest, in situ “turn-on” of the HGF/c-Met interaction using PCGlue-NBD is possible in a spatially specific manner. DU145 cells fluorescently labeled with CellBrite Orange were incubated in RPMI1640 (0.5% FBS) containing a mixture of PCGlue-NBD (2 µM) and HGF (500 pM), exposed at 365 nm for 2 min (1.1 mW/mm2) through a plastic tape mask (Figures 5a and 5b), and then subjected to confocal laser scanning microscopy (λex = 552 nm). After 12 h at 37 °C under 5% CO2, the cells in the UV-exposed area scattered (Figure 5b, x = –13.5–0 mm) as shown in Figures 5c and 5e. In sharp contrast, the cells located in non-exposed area (Figure 5b, x = 0–13.5 mm) barely migrated (Figures 5d and 5e). Namely, the photoinduced "turn-on" of the HGF/c-Met interaction, which leads to cell migration, could be pointwisely achieved with a mm-scale resolution. In conclusion, we developed PCGlue-NBD as the first inhibitor to “turn on” protein–protein interactions (PPIs) in a spatiotemporal manner. It is likely that PCGlue-NBD is widely applicable, considering that its parent dendritic molecular glue is highly affinitive to a wide variety of proteins.6 By turning on the PPI between HGF and its receptor c-Met using PC Glue-NBD, we pointwisely induced cell migration with LED light as a consequence of the signal transduction with a mm-scale resolution. Considering that molecular glues are readily taken up into cells,7a,7c,8b photoswitching of intracellular PPIs with PCGlue-NBD is an interesting subject worthy of further investigation. ASSOCIATED CONTENTS Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Synthesis of PCGlue-NBD; 1H NMR, 13C NMR, and MALDITOF-MS spectral data; electronic absorption spectra; and related experimental procedures (PDF) Movie showing migration of CellBrite Orange-labeled DU145 cells in the presence of PCGlue-NBD/HGF (MPG) Web Enhanced A WEO is available in the HTML version of the paper.

AUTHOR INFORMATION Corresponding Authors

[email protected]; [email protected] Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Early-Career Scientists (18K14353) to K.O., WINGS/GPLLI Collaboration Project (the University of Tokyo) to R.M., and partially supported by Grant-in-Aid for Scientific Research (S) (18H05260) to T.A. We appreciate Prof. H. Cabral (the Uni-

versity of Tokyo) for zeta-potential measurements. R.M. thanks the Research Fellowships of Japan Society for the Promotion of Science (JSPS) for Young Scientists and the Program for Leading Graduate Schools (GPLLI).

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