CHEMBIOCHEM COMMUNICATIONS DOI: 10.1002/cbic.201402237

Proteasome Inhibitors with Photocontrolled Activity Mickel J. Hansen,[a] Willem A. Velema,[a] Gerjan de Bruin,[b] Herman S. Overkleeft,[b] Wiktor Szymanski,*[a, c] and Ben L. Feringa*[a] Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity.

Cancer is often treated with a combination of surgery, radiation therapy, and chemotherapy.[1] Although in many cases chemotherapy has proven to cure cancer and to alleviate symptoms, it is notorious for its often severe adverse effects. Such effects can be caused by poor selectivity of the chemotherapeutic agent, but also because the primary biological function targeted by the drug is essential to healthy tissue as well.[2] One option to overcome adverse effects is by developing chemotherapeutic agents with biological activity that can be dynamically and externally controlled in space and time.[3] Photopharmacology,[4, 5] a strategy that relies on the incorporation of a photoswitchable moiety into the molecular structure of a bioactive molecule,[6, 7] holds great promise for gaining spatiotemporal control over cancer drug activity. Through irradiation with light, photoresponsive molecules can be switched between two isomeric states, which show differences in their biological activities. This method has been used for the [a] M. J. Hansen, W. A. Velema, Dr. W. Szymanski, Prof. B. L. Feringa Centre for Systems Chemistry Stratingh Institute for Chemistry, University of Groningen Nijenborgh 4, 9747 AG, Groningen (The Netherlands) E-mail: [email protected] [email protected] [b] G. de Bruin, Prof. H. S. Overkleeft Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre Einsteinweg 55, 2333 CC Leiden (The Netherlands) [c] Dr. W. Szymanski Department of Radiology, University of Groningen University Medical Center Groningen Hanzeplein 1, 9713 GZ, Groningen (The Netherlands) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201402237.

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photocontrol of, inter alia, antibiotic activity[8] and pain perception.[9, 10] For exploring the photopharmacological approach, it is possible to envisage a chemotherapeutic agent that is switched between a state of high and low activity through the action of light. The advantages of such an approach are twofold: 1) low activity can be assured in healthy tissue, thus avoiding adverse effects, and 2) a higher concentration of a bioactive chemotherapeutic agent can be established at the site of the tumor, thus making the treatment more effective. Proteasome inhibitors are promising chemotherapeutic agents, and bortezomib (Velcade, Scheme 1 A) is the first drug of this group to be clinically approved—for the treatment of multiple myeloma and mantle cell lymphoma.[11, 12] Proteasome inhibitors block one or more of the catalytic sites of the proteasome, which is a complex of proteolytic enzymes responsible in cells for the degradation of redundant, ubiquitin-tagged proteins.[13–15] The catalytic sites, which can be classified as displaying trypsin-like (b2), chymotrypsin-like (b5), or PGPH-like (b1, PGPH refers to peptidyl-glutamyl peptide-hydrolyzing) activity, are located inside the tunnel-shaped structure of the 20S proteasome core particle. These catalytic sites and their immunoproteasome equivalents (upregulated by stimuli such as oxidative stress and proinflammatory cytokines[16]), are instrumental in the degradation of the majority of cytosolic and nuclear proteins, as well as of misfolded proteins dislocated from the endoplasmic reticulum (ER) to the cytoplasm, in most cell types throughout the kingdoms of life. Inhibition of one or more of the proteasome active sites leads to accumulation of redundant proteins and peptides inside the cell, and this is particularly damaging to tumorous cells.[17, 18] In tumor tissue, protein biosynthesis rates are often greatly elevated, particularly also in the ER. As a result, a large proportion of the newly synthesized ER proteins do not pass the quality control and so become proteasome substrates. Blocking of proteasomal functioning leads to accumulation of these proteins, which—when the burden becomes too high—trigger apoptosis. Proteasomes are essential to healthy tissue, so it is not surprising that proteasome inhibitors exhibit an adverse effect on healthy cells as well, resulting in frequent side effects such as peripheral neuropathy and gastrointestinal complications.[11, 19–21] Here we describe the design, synthesis, and inhibitory activities of photoswitchable proteasome inhibitors. Six different compounds (Scheme 1) were prepared, and their biological activities were assayed by means of competition experiments on cell lysates and cell toxicity tests. Substantial differences in proteasome inhibitory activity were found between the two photoisomeric forms of all compounds. The concept presented here with photoswitchable chemotherapeutic agents might be ChemBioChem 2014, 15, 2053 – 2057

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Scheme 1. Molecular structures of bortezomib and photoswitchable proteasome inhibitors. A) Structure of bortezomib. B) Structures of photoswitchable proteasome inhibitors 1–6. UV irradiation switches the trans isomers to the cis isomers. Subsequent irradiation with visible light, or thermal relaxation, switches the cis isomers back to the trans isomers.

useful for more effective cancer treatment and reduction of pounds with light of different wavelengths, the ratios between side effects. Furthermore, the responsive inhibitors might also trans and cis isomers can be changed repetitively. Ideally, be used as tools in chemical biology studies to provide dea large difference in ratio—that is, switching between samples tailed insight into proteasome function. consisting of almost purely trans and almost purely cis Bortezomib (Scheme 1 A) is a widely used proteasome inhibiisomer—can be achieved. Using UV–visible and 1H NMR spec[22] tor. Its structure consists of a boronic acid “warhead”—this troscopy, we determined the trans/cis ratios (Table 1) and studied the photochemical isomerization of the photoswitchable binds reversibly to the catalytic hydroxy group in the active proteasome inhibitors. site of the proteasome—and a leucine-phenylalanine dipeptide that facilitates recognition and uptake by the proteasome (Scheme 1 A).[23] The leucine binds to the S1 pocket inside the Table 1. trans/cis ratios of compounds 1–6 before and after irradiation 20S proteasome, whereas the phenylalanine binds to the S2 (lirr = 365 nm, in [D6]DMSO/D2O), together with half-lives [in H2O/DMSO and the pyrazine moiety to the S3 pocket. Altogether, this pep(1 %vol) at 37 8C] of the cis isomers. tide backbone forms an antiparallel b-sheet through hydrogen Compound 1 2 3 4 5 6 bonding with the active sites. The major effect of bortezomib non-irradiated (trans/cis) 97:3 89:11 93:7 91:9 97:3 65:35 is observed on the b1/b1i and b5/b5i active sites, whereas only irradiated (trans/cis) 28:72 13:87 10:90 11:89 3:97 47:53 [13] little inhibition of the b2/b2i active site is observed. Co-cryshalf-life [h] 6.8 7.7 5.4 6.6 3.8 5.8 tal structures of proteasome-bound bortezomib reveal that the 329 332 339 337 360 324 lmax [nm] peptidic part of the bound bortezomib molecule fills a tight gap in the proteasome protein, as exemplified for the binding to the b5 active site shown in Figure S8 in the Supporting InThe UV–visible absorption spectra (Figure 1 and Figures S1– formation.[24, 25] S6) of compounds 1–6 each show an absorption maximum at  340 nm, characteristic of trans-azobenzene.[30] After irradiaOn the basis of these data and also of SAR studies by Zhu [26] et al., we envisioned that the attachment of an azobenzene tion with l = 365 nm light, the absorption band decreases, and a new absorption maximum, distinctive of cis-azobenzene, apphotoswitch to the peptidic part of the bortezomib molecule pears at  430 nm (Figures 1 A and S1–S6).[30] All six commight provide a bioactive compound with photocontrollable pounds could be switched reversibly between the trans and cis activity. More specifically, we expected that the flat trans isomer of the compound should be able to fit in the tight gap in the active site. Upon photoisomerization to the cis form of the azobenzene, a large conformational change from a planar to a bent structure is observed.[27–29] We envisioned that the large change in geometry and steric effects caused by the nonplanar cis conformation of the molecule would prevent its binding to the proteasome active sites. On the basis of these considerations, a series of six different, azobenzene-containing proteasome inhibitors were synthesized (1–6, Scheme 1 B, for details on synthesis see the Supporting Information). Molecules that contain azobenzene moieties usuFigure 1. A) UV/Vis absorption spectra of compound 1 in DMSO/H2O (0.03 mm, < 5 % v/v ally consist of mixtures of trans and cis isoDMSO). c: non-irradiated, a: 365 nm for 5 min, ····: white light, 2 min. B) Reversible mers,[3, 30–33] with the trans isomers being the thermophotochromism of compound 1 (20 mm) at pH 7.4 in PBS (10 mm), GSH (10 mm), and dynamically stable forms. By irradiating the comDMSO (1 % v/v) on irradiation with 365 nm or white light for 1 min.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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forms at least four times without showing significant fatigue (Figures 1 B and S1–S6). A major concern with incorporation of azobenzene switches into biomolecules is their potential metabolic instability.[4] This instability is caused by reduction of the azobenzene to the hydrazobenzene, mediated either by enzymes[34] or by glutathione (GSH).[35] GSH concentrations of up to 10 mm can be found in the cytosol;[36] this makes GSH-mediated reduction a serious concern for the use of azoFigure 2. Results of fluorescence-based competition experiments for non-irradiated and benzenes in bioapplications.[4] To determine the stairradiated compound 1. IC50 values were obtained for all compounds for b1, b1i, and b5 bilities of the photoswitchable proteasome inhibitors active sites before and after irradiation (l = 365 nm). For non-irradiated compound 1, disto glutathione reduction,[37] reversible photochromism appearance of the fluorescent band is observed at 30 nm for the b1i and b5 active sites. After irradiation with l = 365 nm light, disappearance of these bands was observed at of compound 1 was determined in phosphate-buf100 nm. fered saline (PBS, 10 mm, pH 7.4) containing 10 mm GSH. As was expected, in view of earlier work by To obtain quantitative data for the differences in activity beWoolley et al.,[38, 39] only little photobleaching was observed in tween the non-irradiated and irradiated forms of compounds the presence of GSH (Figure 1 B), thus showing the applicability 1–6, IC50 values were determined (Table 2). By plotting the fluoof incorporated azobenzene switches for cell studies. Table 1 shows that the trans/cis ratios for compounds 1–5 rescent intensities against inhibitor concentration, dose–recan be changed efficiently by irradiating the compounds at sponse curves could be obtained and IC50 values were calculatl = 365 nm. Compound 5 shows almost complete bidirectional ed (Table 2). Experiments were performed in triplicate, and simswitching (97:3$3:97) between the pure trans and pure cis ilar non-irradiated/irradiated activity ratios were found (see isomer; this has also been observed previously for 4-alkoxyTable S1). substituted azobenzenes.[40] Compound 6 shows a smaller Table 2. IC50 values for compounds 1–6 and bortezomib at b1, b1i, and change in trans/cis ratio upon l = 365 nm light irradiation. b5 active sites before and after irradiation (365 nm light), with all comThe half-lives of the cis forms were determined by measurpounds (except bortezomib) showing differences in activity at the b1 ing the changes in absorbance at the absorption maximum at active site before and after irradiation (l = 365 nm). 37 8C in water (< 1 %vol DMSO) and were in the multi-hour Active Compound IC50 [nm] range for all six compounds. This ensured only minor changes site 1 2 3 4 5 6 Bort. in the trans/cis ratios during the competition assays (vide b1 non-irradiated (trans) 29 41 25 26 46 70 80 infra), which include a 90 min incubation step. irradiated (cis) 59 91 42 37 93 96 90 To study the biological activities of the photoswitchable prob1i non-irradiated (trans) 14 16 16 16 16 16 34 teasome inhibitors, competition experiments were performed irradiated (cis) 26 42 16 15 44 46 38 on cell lysates. For this purpose we performed an activityb5 non-irradiated (trans) 13 16 6 8 22 14 10 irradiated (cis) 23 22 9 10 32 11 12 based protein profiling assay, based on competition between the proteasome inhibitors and fluorescent, mechanism-based inhibitors, which specifically, covalently, and irreversibly bind to Table 2 shows the active-site-specific differences in inhibitory one or more of the active sites of the proteasome (see Figactivity between the non-irradiated and irradiated forms of ure S9).[41–43] Cell lysates were incubated with increasing concompounds 1–6 and bortezomib. Depending on the substitution pattern of the aryldiazo group, specific differences for the centrations of compounds 1–6 for 1 h, after which the fluoresinhibition of the b1, b1i, or b5 active sites could be observed. cent probes were added, and incubation was continued for anHowever, the general trend is that compounds that show difother 30 min. Subsequently, the cell lysates were loaded onto ferences in activity at the b1 active site before and after irradiaan SDS gel. The fluorescence intensities of the bands on the tion show similar differences in activity at the b1i active site, gel can be related to the activities of the proteasome inhibiwhereas smaller differences are observed at the b5 active site. tors: that is, weak inhibitors will compete with the fluorescent Compound 1 (without substituents) shows an over twofold difprobes only at high concentrations, whereas strong inhibitors ference in activity between the non-irradiated and the irradiatwill compete at lower concentrations (Figure 2). A clear differed forms at all the active sites; compound 2 (m-methyl) shows ence in activity between the non-irradiated and the light-irradian over twofold difference in activity between the non-irradiatated (l = 365 nm) compound 1 could be observed (Figure 2). ed and irradiated forms at the b1 active site whereas it shows The non-irradiated form, which contains 97 % trans-1 (Table 1), an almost threefold difference in activity at the b1i active site. is a stronger inhibitor of the b1i and b5 active sites than the However, compound 2 shows only a minor difference in activilight-irradiated (l = 365 nm) form, which contains 72 % cisty between the isomers at the b5 active site. These results 1 (Table 1). This is consistent with our hypothesis that the trans show that a certain specificity can be obtained for the effect of form would be able to fit in the tight gap of the active site, cis/trans isomerization at one of the active sites. The same whereas the conformation of the cis form would hinder the trend is observed for compound 5 (p-methoxy), which shows binding.  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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CHEMBIOCHEM COMMUNICATIONS the largest difference in activity before and after irradiation at both b1 (over twofold) and b1i (almost threefold) active sites, whereas almost no difference is observed for the b5 active site. Compounds 3 and 4, with a methyl group at the para- and the ortho-position, respectively, show the smallest changes in activity: still, substantial differences in activity between non-irradiated and irradiated forms at the b1 active site were observed, whereas no or only minor differences were observed for the activity of both compounds towards the b1i and b5 active sites. These results demonstrate not only how, by modifying the substitution pattern on the aromatic ring, the changes in activity between non-irradiated and irradiated forms can be increased, but also how the specificity for the effect on one of the active sites can be gained or lost. Moreover, the control experiments with bortezomib (Table 2) have shown that the incorporation of azobenzene moieties does not compromise the activities of the proteasome inhibitors. These results constitute a first step towards photoswitchable inhibitors with specificity for one of the active sites, and by designing the compounds in a suitable way it might even be possible to change the specificity from one active site to another through photoisomerization. To determine whether or not similar differences in inhibitory activity between non-irradiated and light-irradiated (l = 365 nm) forms could be observed in live cancerous cells, cytotoxicity assays were performed. By means of a standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,[44] the cytotoxicities of compounds 1–6 were determined with HeLa cells. In these preliminary studies distinct differences in activity prior to and after irradiation were observed for all compounds (Figure S10). Remarkably, photoisomerization resulted in substantial differences in curve shape, whereas only minor changes in inflection point (IC50) were observed. This is subject to further studies because different factors such as solubility, cellular uptake, etc. might influence the observed effects. In conclusion, we have successfully developed a set of proteasome inhibitors, the activities of which could be altered by irradiation with light. The photoswitchable behavior of the compounds was characterized by UV–visible and 1H NMR spectroscopy, giving information on trans/cis ratios and half-lives of the cis isomers under the assay conditions. Changes in inhibitory activity on the proteasome were determined through competition assays on cell lysates. Two- to threefold changes in activity at all three active sites before and after exposure to light (l = 365 nm) were observed, depending on the substitution pattern. The changes in activity observed after cis/trans isomerization were further studied by means of cytotoxicity (MTT) assays. These results provide the basis for the development of proteasome inhibitors with activities that can be controlled with light, enabling control of cytotoxic activity. By this approach it could be possible to develop a cytotoxic agent that would be turned on and off inside the body. This could help to avoid severe side effects, by turning the chemotherapeutic agent off at sites of healthy tissue. Secondly, it might help the therapy to be more effective, because higher doses could be obtained at the sites of the cancerous cells, without  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chembiochem.org causing the severe side effects that usually stem from high dosing. In addition, by a similar approach, photoswitchable proteasome inhibitors might be used as chemical biology tools to address the different active sites of the proteasome specifically and might help in obtaining more pharmacodynamic data relating to the mode of action of proteasome inhibitors. Future research after this proof of concept should focus on the optimization of substitution patterns on the azobenzene switch to obtain larger differences in activity. This might be achieved by the use of larger substituents or by incorporating the azobenzene into the bortezomib backbone: that is, by using it to replace the phenyl group of the phenylalanine moiety instead of the pyrazine moiety. In light of the control experiments with bortezomib, it can be concluded that incorporation of an azobenzene moiety does not compromise the activity of the inhibitor. However, a major improvement would be to address photoisomerization with more biocompatible red light, which is nontoxic and allows deeper tissue penetration.[38, 45–47] Even though further optimization might be required for future applications, these results constitute a proof of principle for developing proteasome inhibitors with specificities towards one or more of the active sites that might be changed through the action of light.

Experimental Section Synthesis of photoswitchable inhibitors: The synthesis of the photoswitchable inhibitors is described in the Supporting Information. Photoswitching experiments: Irradiation experiments in H2O/ DMSO were performed with a Spectroline ENB-280C/FE UV lamp (365 nm) and a Thor Labs OSL1-EC Fiber Illuminator (white light). Competition experiments: Lysates of RAJI cells (10 mg/9 mL) were prepared in Tris (pH 7.5, 50 mm), MgCl2 (5 mm), ATP (2 mm), DTT (2 mm), and glycerol (10 %). The cell lysates (9 mg total protein) were incubated with the different concentrations of the synthesized inhibitors 1–6 for 1 h at 37 8C in the dark. Next, the cell lysates were incubated with BODIPY-NC001 (1 mm, Figure S9) and BODIPY-NC005 (0.1 mm, Figure S9) for an additional 30 min at 37 8C in the dark. This was followed by 5 min boiling with a reducing gel-loading buffer (4 mL) (20 % SDS (1 mL), Tris·HCl (0.6 m), pH 6.8, 1 mL), 87 % glycerol (2.1 mL), b-mercaptoethanol (0.4 mL), 10 % BPB (0.1 mL), H2O (MiliQ), and fractionation on 12.5 % SDS-PAGE gel. In-gel detection of residual proteasome activity was performed on the wet gel slabs with a Typhoon Variable Mode Imager (Amersham Biosciences) and use of the Cy2 settings (to detect BODIPYFL-Ala-Pro-Nle-Leu-EK, BODIPY-NC001) and Cy3 settings (to detect BODIPY-TMR-MeTyr-Phe-Leu-VS, BODIPY-NC005).[43] Intensities of b1/b1i and b5 bands were measured by fluorescent densitometry and normalized to mock-treated samples, and the values of three independent experiments were plotted. Average IC50 (inhibitor concentrations giving 50 % inhibition) values and standard deviations were obtained from fitted dose-response curves. Cell culture: HeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with fetal calf serum (10 %), penicillin (100 units mL1), streptomycin (100 mg mL1) and l-glutamine (2 mm). Cells were cultured at 37 8C, 5 % CO2 and 95 % relative humidity. ChemBioChem 2014, 15, 2053 – 2057

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Cytotoxicity assay: The cytotoxicities of the studied compounds were determined by a standard MTT assay.[44] In short, HeLa cells were dispensed in a sterile 96-well plate at a cell density of 10 000 cells per well and were incubated for 8 h at 37 8C, 5 % CO2 and 95 % relative humidity. Next, the compounds were added to the cells at various concentrations, and the systems were incubated for 16 h. MTT was then added to each well to a final concentration of 0.5 mg mL1, and the systems were incubated for 4 h. After this period, all medium was removed and DMSO (100 mL) was added. The absorbance was measured at 570 nm with the aid of a microplate reader (SynergyMX, BioTek). Cell survival was expressed as relative viability of cells relative to control cultures that were incubated with medium only.

Acknowledgements This work was financially supported by the Netherlands Organization for Scientific Research (NWO-CW), The Royal Netherlands Academy of Arts and Sciences Science (KNAW) and the European Research Council (ERC) advanced grant 227897 (to B.L.F.) and the Ministry of Education, Culture and Science (Gravity programme no. 024.001.035). We thank Dr. Martin D. Witte for helpful discussions. Keywords: azobenzenes · cytotoxicity · inhibitors photocontrol · photopharmacology · proteasome

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Received: May 15, 2014 Published online on August 14, 2014

ChemBioChem 2014, 15, 2053 – 2057

2057

Proteasome inhibitors with photocontrolled activity.

Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, ...
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