Chemical Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online EDGE ARTICLE View Journal | View Issue Photoswitchable dynasore analogs to control Cite this: Chem. Sci., 2020, 11, 8981 endocytosis with light† Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. All publication charges for this article Núria Camarero, ‡a Ana Trapero,‡ab Ariadna Pérez-Jiménez,a Eric Macia,c have been paid for by the Royal Society Alexandre Gomila-Juaneda,a Andrés Martı́n-Quirós,a Laura Nevola,§d Artur Llobet,e of Chemistry Amadeu Llebaria,b Jordi Hernando, f Ernest Giralt dg and Pau Gorostiza *ahi The spatiotemporal control of cellular dynamic processes has great fundamental interest but lacks versatile molecular tools. Dynamin is a key protein in endocytosis and an appealing target to manipulate cell trafficking using patterns of light. We have developed the first photoswitchable small-molecule inhibitors of endocytosis (dynazos), by a stepwise design of the photochromic and pharmacological properties of Received 13th July 2020 Accepted 13th August 2020 dynasore, a dynamin inhibitor. We have characterized their photochromism with UV-visible and transient absorption spectroscopy and their biological activity using fluorescence microscopies and flow DOI: 10.1039/d0sc03820b cytometry. Dynazos are water-soluble, cell permeable, and photostable, and enable fast, single- rsc.li/chemical-science wavelength photoswitchable inhibition of clathrin-mediated endocytosis at micromolar concentration. consequences, the longer they last and the more difficult they Introduction are to revert. Compared to genetic approaches to interfere with Most of the proteins in the cell require the interaction with the action of a protein, such as RNAi or the overexpression of other molecules or proteins to play their biological function. dominant negative proteins, the use of small molecules offers These interactions are crucial for the proper control of a wide several advantages in terms of controlling the dynamics and variety of cellular processes, such as cell division, membrane reversibility of the response. The effects of overexpressing trafficking, signaling, etc. A way to understand how these a protein can take hours or days to appear and could be irre- cellular processes work is by perturbing the activity of one key versible, whereas small molecules are oen fast acting and can component in the system and observing how the process is be readily removed. Acute stimulation with these compounds affected by this perturbation. Small molecules can be used to can also prevent the development of homeostatic or compen- interfere with the biological function of a protein, by inhibiting satory mechanisms by the cell, which plague the outcomes and or activating their function. These compounds are a powerful interpretation of genetic manipulations.2 tool to probe the molecular mechanisms involved in the Local administration of compounds can further improve transduction of specic biological signals and lead to the their efficacy and the reversibility of the elicited responses, e.g. development of novel therapeutic strategies.1 Potency and when studying the pharmacological prole of an ion channel selectivity to a target protein subtype are desirable qualities to with electrophysiological recordings and fast perfusion of avoid off-target effects, since the stronger the binding and its selective inhibitors.3 However, the use of diffusible molecules poses limitations on the temporal and spatial precision that can a Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science be achieved. Once the molecule is administered, it can be and Technology (BIST), Spain. E-mail: pau@icrea.cat diluted or distributed in tissue, and the control on spatial b Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Spain location and precise timing of its activity is lost. c Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), Université Nice Sophia This is important when investigating transient or irreversible Antipolis, France events (e.g. the cellular processes involved in membrane traf- d Institute for Research in Biomedicine (IRB Barcelona), Spain e cking) or when disturbing the activity of proteins essential for Bellvitge Biomedical Research Institute (IDIBELL), Spain f cell survival (whose consequences could be extended to other Departament de Quı́mica, Universitat Autònoma de Barcelona (UAB), Spain g cells or tissues).4,5 These limitations can also affect the efficacy Universitat de Barcelona (UB), Spain h CIBER-BBN, Spain and specicity of drugs used in pharmacotherapy. As an i ICREA, Spain example, the cytotoxic anti-neoplastic agents used in cancer † Electronic supplementary information (ESI) available. See DOI: treatment result in severe side effects due to the lack of speci- 10.1039/d0sc03820b city towards healthy tissue.6 ‡ NC and AT contributed equally. These problems can be addressed with compounds whose § Present address: IDP Discovery Pharma, Parc Cientı́c de Barcelona (PCB), biological activity can be turned on and off remotely and Spain. This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8981–8988 | 8981 View Article Online Chemical Science Edge Article reversibly, like light-regulated drugs used in photo- (Fig. 1a) and the photochromic properties of an azobenzene pharmacology. These molecules can be obtained by the intro- group, to yield a series of photoswitchable analogues of dyna- duction of a light-sensitive group (photoswitch) in the structure min inhibitors that we called dynazos (compounds dynazo-2, of a biologically active ligand.5,7 Careful molecular design and dynazo-3 and dynazo-4 in Fig. 1a). Here we present their design This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. extensive testing allow in some cases to use light as the external strategy, synthesis, and their photochromic and biological trigger to control the drug's pharmacologic activity with high characterization. spatiotemporal resolution.8 The azobenzene is one of the best- studied photoswitchable groups for this purpose and has Results and discussion been extensively used in photopharmacology to regulate the activity of ion channels,3,9–11 G protein-coupled receptors,12,13 We decided to partially integrate azobenzene into the structure and enzymes.14,15 It undergoes a trans/cis isomerization process Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. of dynasore as a constitutive part of the inhibitor pharmaco- upon irradiation with ultraviolet (UV) light, which can be phore (shaded in light blue in Fig. 1a). We expected that this reverted by illumination with visible light or by thermal relax- might allow preservation of the inhibitory activity to an extent ation of the less stable cis isomer.16 This results in light-induced that was sensitive to the photoisomer conguration, thereby changes of the molecular length, geometry and dipolar enabling control of inhibition with light. The number and the moment, which can be exploited to alter the pharmacological relative position of the hydroxyl groups in the catechol moiety of properties of an appended bioactive moiety. dynasore are important for its biological activity,25,30 and for this Among all fundamental biological processes, endocytosis reason the azobenzene unit was introduced at the position of greatly exemplies both the interest and challenges to manip- the naphthyl fragment. ulate the cellular spatiotemporal dynamics. It is used by all We had to take into account an additional constraint as an eukaryotic cells to internalize extracellular material along with integral part of the design, namely that dynasore absorbs in the portions of the plasma membrane through the formation of same spectral region as regular trans-azobenzenes16 (330 nm, vesicles. It regulates many physiological processes, including Fig. 1b, black plot) and, as a result, it photodegrades upon the uptake of nutrients, growth factors and transmembrane continuous direct excitation with the UV light required for receptors and it also constitutes the entry pathway of a variety of photoisomerization (e.g. 355 nm, ESI Fig. S1†). In order to pathogens.17 There are many different routes of endocytosis decouple the absorption spectra of dynasore and the azo- operating at the same time at the cell surface,18 which are crit- benzene moiety of the dyad, we selected azobenzene groups ical in controlling the membrane turnover and plasticity in with red-shied photoisomerization wavelengths, which have essential cellular functions such as mitosis, cell differentiation, the additional advantages of deeper penetration than UV light adhesion, migration, and transmembrane receptor signaling. and reduced cellular damage upon prolonged exposure.31,32 It is Endocytic processes involve the concerted action of many reported that azobenzenes with strong electron-donating proteins, complexes, cell membranes and compartments, and substituents at 2- or 4-position (e.g. –NH2 or –OH) absorb at constitute potential targets to prevent infections and to kill longer wavelengths and display fast thermal back isomerization cells. to the trans conguration in the dark.16,33 Although the main molecular players of endocytosis are well We thus took advantage of the hydroxyl group present in characterized, and several experimental methods have been the naphthyl moiety of the pharmacophore, which was developed to observe their dynamics, the molecular mecha- preserved in the azobenzene core of the dynazo compounds nisms are far from being understood due in part to the lack of designed in order to achieve the desired spectral red shi acute manipulation tools.19,20 One of the key molecular players (Fig. 1a). involved in most endocytic pathways is dynamin, a GTPase With these design principles in mind, we rst synthesized required for membrane scission during the endocytic vesicle dynazo-2, the simplest of the compounds prepared. Compared formation.21,22 to dynasore, the UV-visible absorption spectrum of dynazo-2 Dynamin is essential not only for clathrin-mediated endo- shows an additional absorption peak at 372 nm in phosphate cytosis (CME), one of the best-characterized endocytic pathways buffer saline (PBS) that conrms successful red shi of the which is dependent on the recruitment of clathrin cages to the absorption spectrum of its azobenzene photochrome (violet membrane, but also for other clathrin-independent routes for solid plot in Fig. 1b). This bathochromic displacement is even cellular endocytosis.23,24 The GTPase activity of dynamin repre- more pronounced in DMSO, the lowest energy azobenzene sents a highly efficacious target to control endocytosis and absorption band appearing at 447 nm (dark blue solid plot in a growing number of small molecules that specically inhibit ESI Fig. S2†). Based on previous reports on 4-hydrox- dynamin activity have been described.25–29 Nowadays these yazobenzenes, the larger spectral effect found in DMSO might inhibitors constitute not only useful research tools for the study be ascribed to intramolecular hydrogen bonding between the of endocytosis, but also a promising strategy for the treatment nearby hydroxyl and carbonyl substituents of the azo chromo- of viral and bacterial infections. Thus, dynamin is an appealing phore, which will not be favored in aqueous media.34 Next, we target to manipulate endocytosis in spatiotemporally desig- tested if the biological activity of dynasore was retained in nated patterns with light. dynazo-2. A well-established method to study dynamin inhibi- To photocontrol dynamin activity, we combined the phar- tors on endocytosis is to measure receptor-mediated transferrin macological properties of the dynamin inhibitor dynasore28 (Tf) uptake.35 Fluorescently labeled Alexa 563-Tf was allowed to 8982 | Chem. Sci., 2020, 11, 8981–8988 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. Fig. 1 Molecular design and biological activity of dynazos, photochromic dynasore analogs. (a) Chemical structures of dynasore, a well-known dynamin inhibitor,28 and dynazos. Highlighted in blue, the structure of dynasore that has been conserved in the design of dynazos-2, 3 and 4. There are also shown the electron-withdrawing substituents introduced on the azobenzene core of dynazos-3 and 4 (gray ovals) to improve their photochromic and biological properties with respect to dynazo-2. (b) UV-visible absorption spectra in PBS with 1% DMSO showing the red- shifted azobenzene absorption of dynazos-2, 3 and 4 compared to dynasore upon incorporation of strong electron-donating and electron- withdrawing substituents. (c) Confocal microscopy images of transferrin (Tf) uptake assays in COS-7 cells. The uptake of Alexa-568-Tf (red) is inhibited in cells treated with dynazo-2 (100 mM) to a similar extent as in cells treated with dynasore (80 mM) compared to control vehicle-treated cells. DRAQ5 staining shows nuclei (blue) (scale bar: 10 mm). bind to its endogenous receptor on the surface of COS-7 intact Two derivatives of dynazo-2 were thus designed and cells by incubation at low temperature to slow down endocy- synthesized. Dynazo-3 has an acetyl group at the 40 -position of tosis. Aer removal of the Tf excess, cells were transferred to the azobenzene core (Fig. 1a). We introduced this electron- 37  C for 15 minutes to allow internalization of the Tf-receptor withdrawing group to generate a push–pull azobenzene deriv- complex and cell surface-bound Tf was removed by an acidic ative with an even larger absorption bathochromic shi with wash. Aer nuclei staining and cell xation, internalized Tf- respect to dynazo-2. In addition, the incorporation of this group receptor complexes accumulated at the perinuclear-recycling was also intended to reduce the affinity of the cis form towards late endosomes and could be observed by uorescence dynamin by increasing the steric hindrance around the bio- confocal microscopy (Fig. 1c). Incubation with dynazo-2 (100 logically active fragment of the compound upon photo- mM) reduced Tf internalization to a similar extent than dynasore isomerization. This design concept was further exploited in (80 mM) compared to control vehicle-treated cells (Fig. 1c). dynazo-4, where a much bulkier electron-withdrawing amide These results conrmed that our approach for the integration of group was introduced. To facilitate chemical access to a variety the azobenzene moiety into the dynasore structure does not of potential candidates, the ketone group was replaced by an compromise its biological activity and prompted us to under- amide to which several commercially available N-substituents take further chemical modications of the azobenzene core could be attached. Importantly, this should not detrimentally away from the pharmacophore to improve its photochromic affect the optical properties of the resulting compound (i.e. response and activity modulation. absorption red-shi), as proven by our spectroscopic This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8981–8988 | 8983 View Article Online Chemical Science Edge Article measurements. The N-(2-morpholino-2-(pyridin-2-yl)ethyl) photoisomerization in dynazo-2, 3, 4 and allow estimating the amide substituent chosen for this purpose trades off steric most efficient wavelengths to induce this process:36 ca. 390 nm hindrance and aqueous solubility, compared to other options. (dynazo-2) and 405 nm (dynazo-3, 4) in PBS, which are in good For instance, for an N-(2-diphenylethyl)amide group, we agreement with the red-shied absorption bands shown by This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. observed a strong reduction in aqueous solubility that essen- these compounds with respect to dynasore in Fig. 1b. As such, tially prevented biological experiments (data not shown in the photoexcitation of the azobenzene and dynasore units in manuscript). The photophysical properties of the new dynazos dynazo-2, 3, 4 could be decoupled, which should enable trans/ were again analyzed by UV-visible spectroscopy (Fig. 1b). cis photoisomerization without degrading their pharmaco- Compared to parent compound dynazo-2, the substituents phore. This was demonstrated by illumination with violet light introduced in dynazo-3 and dynazo-4 caused an additional red at 405 nm, which should lead to efficient photoisomerization. Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. shi of the absorption spectra, especially for dynazo-3 (45 nm Remarkably, no photodegradation of dynasore or of any of the and 40 nm in PBS and DMSO respectively, Fig. 1d and ESI dynazos was observed under these excitation conditions (ESI Fig. S2†). Fig. S3†), thus conrming the success of our design approach. The synthesis of dynazo-4 was achieved as shown in Scheme In agreement with the behavior expected owing to their 1. Diazotization of 4-aminobenzoic acid and coupling of the azobenzene substitution pattern, fast thermal back isomeriza- resulting diazonium salt with methyl 2-hydroxybenzoate yielded tion of the cis isomers of dynazo-2, 3, 4 was clearly observed by azobenzene 5. The reaction of the obtained intermediated 5 monitoring the time evolution of transient absorption and the with hydrazine hydrate led to the formation of acid hydrazide 6, corresponding cis state lifetimes (s) were determined by tting which underwent condensation with 3,4-dihydroxybenzaldehye monoexponential functions to the recorded data. As it is shown affording hydrazine 7. Finally, amide coupling of acid 7 and 2- in the insets of Fig. 2b–d, dynazos' relaxation times are between morpholino-2-(pyridin-2-yl)ethanamine afforded dynazo-4. 0.6 and 0.7 ms, conrming that these compounds back isom- As reported for azobenzene derivatives with strong meso- erize to their trans conguration as soon as illumination is meric electro-donating and/or electron-withdrawing groups33 turned off. Continuous irradiation is thus required to maintain dynazo-2, 3, 4 displayed very fast cis to trans thermal isomeri- a signicant fraction of cis isomers, which is advantageous to zation rates, which prevented observation of the photo- spatiotemporally constrain dynamin photoresponses regardless conversion of these switches using steady state UV-visible of compound diffusion, in contrast to the behavior of slow- absorption spectroscopy. Instead, time-resolved absorption relaxing diffusible compounds. In addition, fast-relaxing spectroscopy allowed recording with nanosecond accuracy the compounds also allow single-wavelength operation to control time evolution of the cis states formed aer pulsed irradiation the target protein activity, thus simplifying the experimental of these compounds. resources needed. Fig. 2 shows the transient absorption spectra measured for To study the effect of dynazos on CME, we quantied the dynazo-2, 3, 4 in PBS right aer photoexcitation (DAbs at t ¼ 0), uptake of Alexa 488-labeled Tf by large populations of cells which display negative values in all cases that must be attrib- using ow cytometry. We used the same protocol than in the uted to the light-induced conversion of their initial trans experiments of Fig. 1c, with cell resuspension in complete isomers into less absorbing species. This, together with the fast medium aer the acidic wash, and propidium iodide staining to time decay of the negative DAbs signal to recover the initial exclude dead cells from the analysis. The uorescence of cells situation, prove reversible trans/cis azobenzene incubated in increasing amounts of each compound (ranging Scheme 1 Synthesis of dynazo-4. Reagents and conditions: (a) (i) NaNO2 (1.1 eq.), HCl/H2O (v/v ¼ 8 : 92), 5  C; (ii) NaOH (2 eq.), Na2CO3 (2 eq.), H2O, 5  C, 61% yield; (b) NH2NH2$H2O (4 eq.), EtOH, reflux, 62% yield; (c) EtOH, reflux, 83% yield; (d) 2-morpholino-2-(pyridin-2-yl)ethanamine (1.2 eq.), EDC (1.5 eq.), HOBt (1.5 eq.), TEA (2.6 eq.), DMF, RT, 57% yield. 8984 | Chem. Sci., 2020, 11, 8981–8988 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. Fig. 2 Characterization of dynazos' photochromism. (a) Photoisomerization of dynazos occurs upon illumination of the thermally stable trans isomer (gray ovals) with a wavelength between ca. 350–450 nm. The trans isomer is recovered by thermal back isomerization of the cis isomer (violet ovals). Transient absorption spectra of (b) dynazo-2, (c) dynazo-3 and (d) dynazo-4 after nanosecond pulsed laser excitation (l ¼ 355 nm, t ¼ 0) confirms efficient photoisomerization and bathochromic shift of the most efficient irradiation wavelength from 390 nm in dynazo-2 to 405 nm in dynazo-3, 4. The transient absorption decays and the thermal lifetimes of the cis isomers (s) retrieved from monoexponential fits are shown in the insets of (b) dynazo-2, (c) dynazo-3 and (d) dynazo-4. Measurements were conducted in PBS with 1% DMSO. from 0.5 to 10 mM) was measured and plotted as a percentage of must be noted that ow cytometry measurements are per- the mean uorescence of vehicle-treated cells (Fig. 3a–c, black formed in thousands of cells and that the signicance of the solid plots corresponding to dark conditions). These results observed differences is therefore very high (dynazo-2, P < 0.005, conrm that photoisomerizable dynazos inhibit Tf uptake in dynazo-3, P < 0.0001 and dynazo-4, P < 0.0005; 1 mM concen- a dose-dependent manner. To assess if the inhibitory activity of tration, two-tailed t-test). dynazos on CME could be remotely controlled with light, we Having determined the useful operating conditions, we repeated Tf uptake assays incubating dynazo-2, 3, 4 in separate further characterized the effect of illumination in Tf uptake cell plates in the dark and under optimal illumination condi- using confocal microscopy. We performed Alexa-563-Tf uptake tions in each case, i.e. 380 nm for dynazo-2, and 405 nm for assays as in Fig. 1c but separately incubating cells in the dark dynazo-3, 4 (Fig. 3a–c). Control experiments showed no effect of and under illumination (405 nm) in the presence of 1 mM light on Tf uptake assays performed in untreated cells (ESI dynazo-4. Representative confocal microscopy images of this Fig. S4 and Methods/ESI for further details†). A Tf uptake time- experiment are shown in Fig. 3d. In Fig. 3e, the average uo- course in untreated cells is also shown in ESI Fig. S5† to show rescence intensities of 112 to 153 cells for each condition are the progression of Tf internalization without addition of dyna- plotted relative to the control dark condition (100% internali- zos. The inhibitory activities of all dynazos displayed signicant zation). Dynazo-4 signicantly reduced Tf uptake by 47% (P < differences between dark and illuminated conditions, at 0.0163) in the dark compared to the 405 nm illumination concentrations of 1 mM for dynazo-2 and between 0.5 mM and 5 conditions or to vehicle-incubated controls, in good agreement mM for dynazo-3 and dynazo-4. The greatest relative differences with the cytometry experiments of Fig. 3c. These results in the inhibitory activities between the two conditions are demonstrate that CME can be remotely controlled in intact, 20% for 1 mM dynazo-2, 3 and 35% for 1 mM dynazo-4. It untransfected cells by means of small molecules (photochromic This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 8981–8988 | 8985 View Article Online Chemical Science Edge Article This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. Fig. 3 Characterization of dynazos' biological activity. (a) Dose–response curve of Tf uptake by flow cytometry in the dark and under illumination at 380 nm for dynazo-2 shows dose-dependent inhibition of Alexa-488-Tf uptake and significant differences in activity with light at 1 mM (*P < 0.05). (b) Dynazo-3 and (c) dynazo-4 dose–response curves in the dark and under illumination at 405 nm show dose-dependent inhibition and significant differences in activity with light between 0.5–5 mM (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). In all these cases, dose– response curves were normalized to those measured for vehicle-treated samples (100%). Data are representative of three independent experiments performed in duplicate and values are expressed in mean  SEM. (d) Confocal microscopy images of Tf uptake assays in the dark and at 405 nm irradiation shows a significant reduction in Alexa-568-Tf uptake only in cells treated with dynazo-4 (1 mM) in the dark condition. Internalized Tf is shown in red and DRAQ5 blue staining shows nuclei (scale bar: 10 mm). Quantification of Tf uptake assay from (d) [normalized to vehicle-treated in the dark (100%)] is shown in (e) (*P < 0.05, n ¼ 112 cells for vehicle/dark, n ¼ 122 cells for vehicle/405 nm, n ¼ 151 for dynazo-4 1 mM/dark and n ¼ 153 cells for dynazo-4 1 mM/405 nm). All data are expressed as the mean  SEM. inhibitors of dynamin) that are directly applied to the bath. expressing the eGFP-tagged adaptor protein AP-2 before and Dynazos are active at 1 mM concentration in the dark and can be aer the incubation with 25 mM dynazo-4 for 40 minutes. deactivated to different extents with violet illumination, which Following, we recorded the CCP dynamics under continuous may produce signicantly lower toxicity and less unspecic 405 nm illumination in the presence of dynazo-4 (ESI Fig. S6– effects than dynasore at the usual 80 mM concentration. In S8†). Dynazo-4 treatment signicantly increases the time particular, at 1 mM concentration dynazo-4 can inhibit CME up duration of the CCP at the plasma membrane, indicating to 47% and be photoswitched to a fully inactive state using an a slowdown of the endocytic process in full agreement with illumination wavelength available in most commercial laser transferrin uptake assays by ow cytometry and uorescence scanning microscopes. microscopy (Fig. 3). This effect is reversible under 405 nm We also assessed the capacity of dynazo-4 to photocontrol illumination in the case of the CCP with a time duration of the dynamics of clathrin-coated pit (CCP) formation during $120 seconds, but not at shorter time durations, probably due endocytosis. To this end, we used live-cell total internal reec- to the fast relaxation lifetime of dynazo-4, which limits the tion uorescence microscopy (TIRFM) to monitor the CCP effective percentage of (inactive) cis isomer that can be achieved lifetimes by tracking the uorescently labeled AP-2 protein in under illumination (ESI Fig. S7c†). These differences could also BSC-1 cells, using customized particle-tracking soware.37 be due to the absence of dynamin recruitment in some CCP Together with clathrin, AP-2 is the most abundant coat protein pools, which accelerates their disappearance from the plasma in the CCP and it is also present during the entire lifetime of membrane.40 Control experiments in untreated cells showed no a CPP.38,39 We recorded time lapse movies of BSC-1 cells effect of 405 nm illumination on CCP lifetime (ESI Fig. S7d†). 8986 | Chem. Sci., 2020, 11, 8981–8988 This journal is © The Royal Society of Chemistry 2020 View Article Online Edge Article Chemical Science Compared to traffic light peptides,37 dynazo-4 targets tools can be useful to dissect the role of endocytosis in cellular a protein bearing wider impact in endocytosis, it achieves functions where it exerts a crucial role, such as cell growth, comparable photocontrol of CME at 20-fold lower concentra- differentiation, cell motility and invasiveness, modulation of tion, it has 4.6-fold smaller molecular weight (609.63 Da transmembrane receptor signalling and synaptic transmission. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. compared to 2830.8 Da of TL-2) and simpler synthetic accessibility. Conflicts of interest Due to the rapid cis to trans relaxation of dynazos, we could not estimate the content of the cis isomer in the illuminated There are no conicts to declare. sample. According to the results in Fig. 2, it is likely that the cis/ trans mixture in the PSS of illuminated dynazos contains Acknowledgements Open Access Article. Published on 17 August 2020. Downloaded on 9/29/2022 4:54:56 PM. a residual fraction of the more active isomer (trans) that is sufficiently high to limit the range of concentrations displaying We acknowledge nancial support from the Ris3CAT-CECH light-dependent inhibition of Tf uptake. Reducing the trans project (001-P-001682, co-nanced by the European Union isomer content in the PSS of illuminated dynazos (i.e. photo- Regional Development Fund within the framework of the ERDF converting more efficiently to the cis state) would likely enhance Operational Program of Catalonia 2014-2020), from the CERCA the differences in activity between the cis/trans forms, thereby Programme and the 2017-SGR-1442 grant (Government of Cat- expanding the useful concentration range of dynazos. alonia), from the European Union’s Horizon 2020 Framework In addition, an enhancement of the photoswitchable steric Programme for Research and Innovation under the specic hindrance around the pharmacophore with even bulkier grant agreement no. 945539 (Human Brain Project SGA3), and substituents could further reduce the activity of cis isomers and from grant PID2019-111493RB-I00 (MINECO). A. G.-J., was increase cis/trans differential biological activity. It is worth supported by fellowship BES-2017-083025. We are grateful to noting that the inhibition of endocytosis by 1 mM dynazo-4 can Anna Lladó and Sebastian Tosi from the Advanced Digital be completely shut off under illumination, despite partial Microscopy Core Facility of Institute for Research in Biomedi- photoconversion (Fig. 3c). However, it would also be useful for cine (IRB) Barcelona for technical support in TIRF imaging and certain experiments that the inactive isomer was most abun- analysis, to Benjamı́n Torrejón from the Advanced Optical dant in the dark, and that endocytosis could be inhibited with Microscopy Unit for technical assistance and the Cytometry unit light (e.g. at focalized spots). This could in principle be achieved of the CCiT-UB. We also thank T. Kirchhausen for the AP-2 BSC- by means of a “reverse switch” with a bridged azobenzene which 1 cell line. adopts a more stable cis conguration in the dark,41 although in turn, the resulting compound may display partial (basal) inhi- Notes and references bition in the relaxed form instead of the complete inactivity of cis-dynazo-4. On the other hand, the large separation of the cis 1 S. L. Schreiber, J. D. Kotz, M. Li, J. Aubé, C. P. Austin, and trans absorbance bands in bridged azobenzenes should J. C. Reed, H. Rosen, E. L. White, L. A. Sklar, enable complete bidirectional switching with violet/green light. C. W. Lindsley, B. R. Alexander, J. A. Bittker, P. A. Clemons, A. De Souza, M. A. Foley, M. Palmer, A. F. Shamji, Conclusions M. J. Wawer, O. McManus, M. Wu, B. Zou, H. Yu, J. E. Golden, F. J. Schoenen, A. Simeonov, A. Jadhav, In summary, we have used rational design to obtain the rst M. R. Jackson, A. B. Pinkerton, T. D. Y. Chung, light-regulated small-molecule inhibitor of endocytosis. Inte- P. R. Griffin, B. F. Cravatt, P. S. Hodder, W. R. Roush, gration of substituted azobenzenes in the structure of dynasore E. Roberts, D. H. Chung, C. B. Jonsson, J. W. Noah, while preserving the integrity of its pharmacophore is W. E. Severson, S. Ananthan, B. Edwards, T. I. Oprea, a successful strategy to endow photosensitivity to a dynamin P. J. Conn, C. R. Hopkins, M. R. Wood, S. R. Stauffer and inhibitor. In a series of stepwise modications of the azo- K. A. Emmitte, Cell, 2015, 161, 1252–1265. benzene core, rst we have red-shied the photoisomerization 2 T. G. Iversen, G. Skretting, B. Van Deurs and K. Sandvig, Proc. wavelength of dynazos in order to uncouple it from dynasore Natl. Acad. Sci. U. S. A., 2003, 100, 5175–5180. absorption, prevent photodegradation, and allow the use of 3 M. Izquierdo-Serra, A. Bautista-Barrufet, A. Trapero, visible light to switch off their activity. And second, we have A. 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