Fig.1 Structure-based design of a light-activated PPARγ agonist. (a) Binding mode of the template agonist 1 in the PPARγ ligand binding domain (pdb ID: 6t6b) [32] with the Z-like sulfonamide engaging polar contacts with Tyr327 and His449 in the receptor’s activation function. (b) Design of azolog 2 as a light-activated PPARγ agonist. (c) Dose-response curves of dark-adapted E-2 and light-activated mostly-Z-2 (EC50 0.50 ± 0.09 µM) in a Gal4-PPARγ hybrid reporter gene assay (data as mean ± S.E.M., n = 4). (d) Selectivity profiles of E-2 and mostly-Z-2 (10 µM) on nuclear receptors related to PPARγ (data as mean ± S.E.M., n = 3). (e) PPARγ binding of 1, Z-2 and Z-5 evaluated by biolayer interferometry (BLI). Concentration ranges (light to dark color): 1: 0.75–1.5 µM; Z-2: 0.8–2.4 µM; Z-5: 1.2–8.0 µM. Data are the mean ± SD of two independent replicates. Data for E-2 and E-5 are shown in Figure S8. (f) Effects of 2 and 5 (1 or 3 µM) in a 21-day adipocyte differentiation assay (data as mean ± S.E.M. rel. Oil Red-O deposition versus pioglitazone (PIO, 1 µM); 24 h illumination on day 1 of each differentiation cycle; n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 (vs. the corresponding DMSO ctrl, ANOVA with Bonferroni multiple comparisons test)). (g) Light-dose dependent effects of 5 (3 µM) on adipogenesis (see Figure S10 for a schematic illustration of the model). Data are the mean ± S.E.M. Oil Red-O deposition; n = 3. A CellDisco [33] was used in (c,d,f,g) at the indicated wavelength for pulsed illumination (100 ms/10 s) to maintain the respective Z-isomers.#

为验证光控分子在生理过程中的时空调控能力，研究人员在脂肪来源干细胞（ASCs）成脂分化模型中评估了化合物5的作用。实验显示，5的促成脂分化效果完全依赖于光照，且其成脂效率对光照时长高度敏感；仅在周期第1天给予光照刺激即可获得与每日持续照射相近的表型效果，有力揭示了PPARγ在脂肪发育早期具有关键的时间窗口依赖性（如图1f、图1g）。

Scheme 1 Synthesis of 2–6. Reagents & Conditions: (a) Prepared according to ref [32].; (b) LiOH, H2O/THF, rt, overnight, 73%; (c) 2,4-dichloroaniline (10) or 4-chloro-2-methoxyaniline (11), oxone, CH2Cl2/H2O, rt, 2 h; then 9, HOAc, rt, 60 h, 32%–47%; (d) 2-((tert-butyldimethylsilyl)oxy)-4-chloroaniline (13), m-CPBA, CH2Cl2, 0°C, 60 min, then 9, HOAc, rt, 60 h; (e) TFA/H2O, rt, 2 h, 44% over two steps; (f) 9, HCl, NaNO2, H2O/THF, 0°C, 45 min, then NaOAc, 3-(N,N-dimethylamino)phenol (14) or 3-chloro-N,N-dimethylaniline (15), H2O/THF, rt, overnight, 27%–32%.#

针对更高时间精度的调控需求，团队进一步表征了热弛豫半衰期仅为47秒的快弛豫激动剂6。该分子可在435 nm可见光下迅速活化并在光照停止后极速失活，从而允许通过调节光脉冲占空比（0.5%至2%）实现PPARγ转录活性的线性功率剂量调控，并成功在HT29细胞中定量调控了内源性靶基因_FABP1_和_KLF4_的mRNA表达水平（如图2c、图2d、图2f）。

Fig.2 The photoswitchable agonist 6 enables high-precision optical control of PPARγ. (a) Chemical structure of 6. (b) UV-vis spectra of 6 in DMSO in the dark-adapted state and after illumination at the indicated wavelengths. (c) Thermal relaxation of 6 in PBS (1<1>) with a half-life of 47 s. (d) Dose-response curves for PPARγ activation by 6 at different light-doses (435 nm) with a CellDisco [33] demonstrating that the fast thermal relaxation of 6 allows for light-dose-dependent activation. % light-dose refers to the DISCO on-time; rel. PPARγ act. is relative to 1 µM pioglitazone; data are the mean ± S.E.M.; n ≥ 3. (e) Light-dose-dependent EC50 and % max. rel. PPARγ act. of 6; data are the mean ± S.E.M.; n ≥ 3. (f) Light-dose-dependent effects of 6 on the expression of the PPARγ-regulated genes fatty acid binding protein 1 (FABP1) and krüppel-like factor 4 (KLF4). Cells were treated with 6 (1 µM) and kept in the dark, or illuminated over 24 h with the indicated light-dose, and gene expression was determined by qRT-PCR after 24 h. Data are the mean ± S.E.M. relative mRNA levels normalized to the DMSO control in the respective dark or illuminated condition; n = 3; * p < 0.05, *** p < 0.001 (vs. the corresponding DMSO-treated control; ANOVA with Bonferroni’s multiple comparisons test).#