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monitored at 370 nm after interrupting irradiation at 740 nm showed that the cis-to-trans thermal isomerization was very slow, and the photo-switched cis conformer had a half-time exceeding 3 h at 25 °C and a first-order rate constant kcis to trans = 0.3 h–1 at 37 °C (Supplementary Figs. 1 and 2). Furthermore, the cis and trans conformers of the AB conjugates were found to be stable under reductive conditions13 (Eredox = −210 mV) and in whole blood cell lysate (Supplementary Figs. 4 and 5).

Although trans-to-cis photoisomerization of the AB-conjugated CsA derivative 3 resulted in altered inhibitory potencies toward Cyp18 and CaN (Supplementary Figs. 6 and 7), the relatively simple branching group would account for the moderate functional differ-ence between the two isomers. We hypothesized that the introduc-tion of an additional protein-binding moiety on AB and the resulting recruitment of a macromolecule to the photoswitchable side chain on CsA residue 8 would increase the topological effect induced by AB isomerization. Two ligands (two CsA in 1 or one CsA and one biotin in 2) were linked by AB (Fig. 1a). Notably, cis-1 showed a sevenfold greater inhibitory potency toward the PPIase activity of Cyp18 (half-maximal inhibitory concentration (IC50) = 30 ± 3 nM) compared to the non-irradiated trans-1 (IC50 = 206 ± 22 nM) (Fig. 1b). The cis-1 underwent thermal cis-to-trans isomerization, and the Cyp18 inhibi-tory profile was reversed after keeping the irradiated sample over-night in darkness. Although the Cyp18 inhibition by cis-1 (30 nM) is lower than that by CsA (3.7 nM)9, the photoswitched isomer is still a potent inhibitor and causes complete Cyp18 inhibition at 200 nM. Notably, the inhibition can be switched back to the low potency of the trans isomer. We have measured the CaN inhibitory efficiencies of cis-1 and trans-1 (ref. 14) in the presence of Cyp18. We observed a considerable increase of CaN inhibition upon light irradiation (Supplementary Fig. 7a).

In order to demonstrate the advantage of two-photon photoisomer-ization for potential biological and medical applications, we mea-sured the inhibition of endogenous cyclophilins in blood lysate by 1 (ref. 15) (Fig. 1c). Cyp18 is the most abundant cyclophilin in cells and the major binding protein for CsA2. Because the absorption of near-UV by blood lysate is much higher than that of near-IR, irra-diation at 370 nm caused little change of endogenous PPIase activ-ity, whereas light of 740 nm induced a marked decrease of PPIase activity, due to the production of photoswitched cis-1, which has higher inhibitory potency. The decrease in total PPIase activity is smaller than that obtained in the Cyp18 assays using purified enzyme, because of the presence of CsA derivative–resistant PPIases in the blood2.

Because the formation of either binary Cyp18-1 or ternary Cyp18-1- Cyp18 could not be strictly controlled, 2 was designed to overcome this difficulty. Since the affinity of biotin for streptavidin (SA) (Kd ~ 10 fM)16 is much higher than that of CsA for Cyp18 (Kd = 9 nM)17, and the CaN inhibition (IC50 = 100 nM) by CsA is mediated through a gain-of-function mechanism upon Cyp18 binding1, the sequential

Augmented photoswitching modulates immune signalingYixin Zhang1,2, Frank Erdmann1,2 & Gunter Fischer1

Reversibleandnon-invasivephotoswitchingoftheimmunosuppressiveeffectofadrugwouldbeaveryvaluabletoolforpreciselyregulatingtheimmunesystem.Usingacombinationofproteinborrowingandtwo-photonphotoisomerization,wedesignedandsynthesizedderivativesofcyclosporinA.Herewedemonstratephotoswitchingofthelocalconformationwithinsmallmolecules,whichweusedtomodulateinhibitorypotenciesforcyclophilin,influenceternaryandquaternarycomplexformationsandregulateT-celltranscriptionalactivationin situ.

Phosphatase calcineurin (CaN) has a pivotal role in T-cell signal-ing and is inhibited by treatment with the immunosuppressive drugs cyclosporin A (CsA) and tacrolimus (FK506)1. The gain-of-function mechanism of CsA in immunosuppression and its structure-activity relationship are well understood2–4: CsA binds to and inhibits the peptidyl prolyl cis-trans isomerase (PPIase) cyclophilin 18 (Cyp18; also called Cyp A), and the resulting Cyp18-CsA complex targets CaN1. Though the binary Cyp18-CsA complex is capable of CaN inhibition and can therefore lead to immunosuppression, neither CsA nor Cyp18 alone cause any detectable inhibition of CaN.

Azobenzene (AB) was chosen as the photoresponsive group to con-jugate with CsA. Photoisomerization of AB has been used success-fully to generate conformational changes within biomolecules5–8. The residue 8 chosen for modification on CsA is known to exhibit subtle effects on Cyp18-CsA-CaN complex formation9 (Fig. 1a), whereas most changes on other positions abolish its immunosuppressive activ-ity. In order to achieve a photoswitching of CsA derivatives under biocompatible conditions, two-photon photoreaction with near-IR light10–12 has been investigated.

The CsA derivatives (Fig. 1a) CsA-AB-CsA (1), CsA-AB-biotin (2) and CsA-AB (3) underwent trans-to-cis photoisomerization under irradiation with light of either 370 nm or 740 nm, and the resulting cis conformers had completely different UV-vis spectra from their corre-sponding trans counterparts (Supplementary Results, Supplementary Figs. 1 and 2 and Supplementary Methods). Whereas the photo-stationary equilibrium (cis/trans ≈ 95/5) was reached within 3 min under irradiation at 370 nm, 45 min were required for completing the same reaction at 740 nm (Supplementary Fig. 3). The UV time courses

1Max Planck Research Unit for Enzymology of Protein Folding, Halle, Germany. 2These authors contributed equally to this work. Correspondence should be addressed to G.F. (fischer@enzyme-halle.mpg.de).

Received 12 May; accepted 6 July; published online 6 September 2009; doi:10.1038/nchembio.214

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interactions and resulting regulations are unidirectional in the following order: SA-biotin binding → Cyp18-CsA interaction → CaN inhibition.

Applying two orthogonal conditions (light irradiation and addi-tion of SA) on 2 resulted in four different states. Uncomplexed 2 and binary SA-2 complex in either the trans or photoproduced cis state yielded distinct inhibitory profiles on Cyp18 PPIase activity (Fig. 2a). While each cis conformer was more potent than the trans counter-part, SA bindings decreased the inhibitory efficiencies of both cis-2 and trans-2. Notably, in the absence of SA, the difference between the inhibitory capacity of cis-2 (IC50 = 2.21 ± 0.02 nM) and trans-2 (IC50 = 2.96 ± 0.68 nM) was smaller than that between cis-2 (IC50 = 4.45 ± 0.55 nM) and trans-2 (IC50 = 9.37 ± 0.75 nM) bound to the borrowed protein SA.

In CaN inhibition assays, Cyp18-2 in cis and trans and Cyp18-2-SA in cis and trans exhibited considerably different inhibitory potencies (Fig. 2b). Whereas binary Cyp18-2 in cis was most efficient in CaN

inhibition (IC50 = 0.95 ± 0.11 µM), the trans form of the Cyp18-2-SA complex showed only 30% inhibition at 10 µM. The augmentation of difference in activity between the reversibly switchable 2 resulting from borrowing SA was noteworthy. In contrast to its CaN-inactive trans counterpart, the photoswitched Cyp18-2-SA exhibited a moder-ate inhibitory effect on CaN (IC50 = 5.17 ± 1.28 µM). In contrast, the simple photoswitchable side chains, such as those of 2 in the absence of SA, and of derivative 3 (Supplementary Fig. 7b), have shown that the conformational difference of AB between the cis and trans states caused only a minor change of CaN inhibition.

To demonstrate that different CaN inhibitory efficacies between cis and trans conformers of 2 could lead to distinct signaling pat-terns in activated T cells, we measured their suppressive effects in an NFAT (nuclear factor of activated T cell) coupled luciferase reporter gene assay. Here, we refer to the well-known CaN-mediated dephosphorylation and transcriptional activation of NFAT (Fig. 2c and Supplementary Fig. 8)18. Without borrowing a protein through

Figure 1 Structures of CsA derivatives and photoswitching of 1. (a) Structure-activity relationship of CsA and chemical structures of CsA derivatives conjugated to AB. (b) Inhibition of Cyp18 PPIase activity by non-irradiated 1 (–––– and ), irradiated 1 (– – – and ) and irradiated 1 kept in darkness overnight (– – and ). PPIase activities were measured with a protease coupled assay24, using Suc-Ala-Phe-Pro-Phe-NH-Np as substrate and chymotrypsin at 7 °C. Cyp18 concentration is 2.5 nM. DMSO was used as control. Irradiations were performed with light of 740 nm for 45 min. (c) Inhibition of PPIase activity in human blood lysate. Whole blood lysates treated with 1 or DMSO as negative control were kept in darkness or irradiated with light of 370 nm (for 3 min) or 740 nm (for 45 min). PPIase activities were measured with a protease coupled assay as described above24. All measurements were performed as triplicates. All data are means ± s.d.

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Figure 2 Orthogonal switching of compound 2. (a) Orthogonal regulation of Cyp18 inhibition by 2. The concentrations of SA (calculated as monomer) were in a fourfold molar excess to those of 2. PPIase activities were measured with a protease coupled assay24, using Suc-Ala-Phe-Pro-Phe-NH-Np as substrate and chymotrypsin at 7 °C. Cyp18 concentration is 2.5 nM. Irradiations were performed with light of 740 nm for 45 min. (b) Orthogonal regulation of CaN inhibition by 2. CaN phosphatase activities were measured with 33P-labeled phosphocasein at 30 °C, in the presence of 10 µM irradiated (– – –) or non-irradiated (––––) 2 and Cyp18 of desired concentrations, with () or without () SA25. The inhibitor concentration designates the concentration of the Cyp18-2 complex. CaN concentration was 3.5 nM. (c) Orthogonal regulation of CaN activity in cells. Jurkat cells were transfected with an NFAT-luciferase reporter plasmid. Cells without () or with () SA cotransfection were pretreated with non-irradiated 2, and the cells were irradiated with light of 740 nm (– – –) for 1 h or kept in dark (––––) before stimulation with PMA and ionomycin. Luciferase activity in lysed cells was determined. (d) For a mixed lymphocyte reaction, human PBMCs were transfected with () or without () SA, incubated with 2 and stimulated with γ-irradiated allogenic PBMCs from another individual. Photoswitching was performed by sequential irradiation at 740 nm every 4 h for 45 min (– – –), or cells were kept in dark (––––). At day 4 a [3H]thymidine pulse was applied for the last 5 h of culture, and incorporated radioactivity was measured. All data are means ± s.d.

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high-affinity interaction, both CsA derivatives 2 and 3 exhibited minor differences between the cis and trans conformers in suppress-ing NFAT transcriptional activation, with a trans/cis ratio of activity of 1.5 (Supplementary Fig. 8). Similar ratios of 1.4 and 1.5 have also been observed in the CaN inhibition assay by 2 (in the absence of SA) and 3, respectively (Fig. 2b and Supplementary Fig. 7). In cells trans-fected with SA (Supplementary Fig. 8), photoswitched cis-2 exhibited a substantially higher suppressive activity compared to that of trans-2. Protein borrowing through the binding of SA to the biotin moiety of 2 led to high isomer specificity approaching a trans/cis ratio of activ-ity of 12. These results correlate well with the photoresponsiveness of IL-2 production in peripheral blood mononuclear cells (PBMCs) stimulated by PMA and ionomycin (Supplementary Fig. 9).

The effects of irradiation (740 nm) on cells pretreated with 2 were evaluated with the NFAT reporter gene assay (Fig. 2c). In cells without SA transfection, the enhancement of inhibition induced by irradiation was minor (1.5-fold). In contrast, irradiation caused an 11.5-fold increase in inhibitory efficacy in the cells transfected with SA. Near-IR light was able to selectively isomerize an individual bond (AB in 2) in the cells and to modulate the transcriptional activation of stimulated T cells in situ.

In order to demonstrate the photoswitchable effect of 2 in an immunologically relevant system, we have performed mixed lym-phocyte reaction with human PBMCs19,20. PBMCs from an individual were transfected with or without SA, incubated with compound 2, and stimulated with γ-irradiated allogenic PBMCs from another indi-vidual. Only in PBMCs transfected with SA, near-IR light irradia-tion induced a substantial increase of inhibition of cell proliferation (Fig. 2d). Notably, given that only the recipient cells were pretreated with the photoswitchable compound and were transfected with SA, the photoswitching of drug activity can occur only in the recipient cells, because the photoswitchable effect is dependent on the presence of derivative 2 and cellular SA. This new method could be used to selectively regulate a subset of cells in a mixture, and to distinguish their function from a complex of biological reactions.

In summary, we have established a model system to illustrate how a microscopic local conformational change such as cis-trans isomerization can influence various structural and functional events of distinct biological levels. Such conformational changes can influence recognition of ligands by enzymes (for example, CsA derivatives and Cyp18) and multicomponent complex formations (such as Cyp18-1-Cyp18 and Cyp18-2-SA-CaN, a quaternary complex of 140 kDa), and they can also affect signal transduction pathways in T cells (for example, NFAT transcriptional activation and IL-2 production). In order to achieve a high functional difference between cis and trans conformers and to tune the inhibitory efficacy of CsA

derivatives through non-invasive optical methods, we have applied a “protein borrowing” strategy21–23 to augment the structural difference between the photoswitchable conformers. This approach offers an attractive avenue for achieving modulation of molecular and cellular functions.

Note: Supplementary information and chemical compound information is available on the Nature Chemical Biology website.

AcknowlEdGmEntsWe thank the SFB610 of the Deutsche Forschungsgemeinschaft for supporting the work and M. Heidler and I. Kunze for their excellent technical assistance.

AUtHoR contRIBUtIonsY.Z. and F.E. performed and analyzed experiments. Y.Z. synthesized all CsA derivatives and measured the enzymatic activities together with F.E. F.E. designed and performed all bioassays. G.F. prepared the manuscript and planned all experiments together with Y.Z. and F.E.

Published online at http://www.nature.com/naturechemicalbiology/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.

1. Liu, J. et al. Cell 66, 807–815 (1991).2. Fischer, G. Angew. Chem. Int. Edn. Engl. 33, 1415–1436 (1994).3. Jin, L. & Harrison, S.C. Proc. Natl. Acad. Sci. USA 99, 13522–13526 (2002).4. Huai, Q. et al. Proc. Natl. Acad. Sci. USA 99, 12037–12042 (2002).5. Behrendt, R. et al. Angew. Chem. Int. Edn. Engl. 38, 2771–2774 (1999).6. Kumita, J.R., Smart, O.S. & Woolley, G.A. Proc. Natl. Acad. Sci. USA 97,

3803–3808 (2000).7. Vollmer, M.S., Clark, T.D., Steinem, C. & Ghadiri, M.R. Angew. Chem. Int. Edn.

Engl. 38, 1598–1601 (1999).8. Westmark, P.R., Kelly, J.P. & Smith, B.D. J. Am. Chem. Soc. 115, 3416–3419

(1993).9. Zhang, Y., Erdmann, F., Baumgrass, R., Schutkowski, M. & Fischer, G. J. Biol.

Chem. 280, 4842–4850 (2005).10. Churikov, V.M. et al. Opt. Commun. 209, 451–460 (2002).11. Furuta, T. et al. Proc. Natl. Acad. Sci. USA 96, 1193–1200 (1999).12. Cahalan, M.D., Parker, I., Wei, S.H. & Miller, M.J. Nat. Rev. Immunol. 2, 872–880

(2002).13. Boulegue, C., Loweneck, M., Renner, C. & Moroder, L. Chem. Bio. Chem. 8,

591–594 (2007).14. Baumgrass, R. et al. J. Biol. Chem. 279, 2470–2479 (2004).15. Kullertz, G., Luthe, S. & Fischer, G. Clin. Chem. 44, 502–508 (1998).16. Green, N.M. Methods Enzymol. 184, 51–67 (1990).17. Fanghanel, J. & Fischer, G. Biophys. Chem. 100, 351–366 (2003).18. Kiani, A., Rao, A. & Aramburu, J. Immunity 12, 359–372 (2000).19. van Besouw, N.M. et al. Transplantation 70, 136–143 (2000).20. van Besouw, N.M. et al. Transpl. Int. 9 (suppl. 1): S345–S347 (1996).21. Briesewitz, R., Ray, G.T., Wandless, T.J. & Crabtree, G.R. Proc. Natl. Acad. Sci.

USA 96, 1953–1958 (1999); comment in 96, 1826–1827 (1999).22. Gestwicki, J.E., Crabtree, G.R. & Graef, I.A. Science 306, 865–869 (2004).23. Liu, J.O. Chem. Biol. 6, R213–R215 (1999).24. Fischer, G., Bang, H. & Mech, C. Biomed. Biochim. Acta 43, 1101–1111

(1984).25. Waelkens, E., Goris, J., Di Salvo, J. & Merlevede, W. Biochem. Biophys. Res.

Commun. 120, 397–404 (1984).