Hansen MJ et al. (2018), Photoactivation of MDM2 Inhibitors: Controlling...

XB-ART-56273

J Am Chem Soc 2018 Oct 17;14041:13136-13141. doi: 10.1021/jacs.8b04870.

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Photoactivation of MDM2 Inhibitors: Controlling Protein-Protein Interaction with Light.

Hansen MJ , Feringa FM , Kobauri P , Szymanski W , Medema RH , Feringa BL .

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Selectivity remains a major challenge in anticancer therapy, which potentially can be overcome by local activation of a cytotoxic drug. Such triggered activation can be obtained through modification of a drug with a photoremovable protecting group (PPG), and subsequent irradiation in the chosen place and time. Herein, the design, synthesis and biological evaluation is described of a photoactivatable MDM2 inhibitor, PPG-idasanutlin, which exerts no functional effect on cellular outgrowth, but allows for the selective, noninvasive activation of antitumor properties upon irradiation visible light, demonstrating activation with micrometer, single cell precision. The generality of this method has been demonstrated by growth inhibition of multiple cancer cell lines showing p53 stabilization and subsequent growth inhibition effects upon irradiation. Light activation to regulate protein-protein interactions between MDM2 and p53 offers exciting opportunities to control a multitude of biological processes and has the potential to circumvent common selectivity issues in antitumor drug development.

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Species referenced: Xenopus laevis
Genes referenced: mdm2 tp53

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	Figure 1. A schematic representation of the principles behind phototriggered p53 stabilization. Caged inhibitor (PPG-idasanutlin) is not able to inhibit the MDM2–p53 protein–protein interaction, which results in p53 ubiquitylation and degradation. Irradiation with 400 nm light releases the active inhibitor idasanutlin which prevents MDM2–p53 binding and as a consequence increases the p53 level, leading to senescence or cell death.
	Figure 2. Strategy toward photocleavable nutlin derivatives. (a) Idasanutlin, a potent MDM2 inhibitor allowing the stabilization of p53 levels in tumor cells. (b) Molecular docking showcases the possible interaction with Lys90 as a potential site to alter the activity (PDB: 4JRG).29 (c) Irradiation of PPG-idasanutlin led to the formation of idasanutlin and PPG(6) as the sole products. (d) Absorption spectra of PPG-idasanutlin, idasanutlin and PPG(6) in buffer.30 (e) UV–vis spectra of PPG-idasanutlin upon exposure to 400 nm light showing a clean photochemical conversion (isosbestic point at 350 nm) to the desired products, see SI for detailed UPLC–MS studies.
	Figure 3. Functional p53 induction upon λ = 400 nm irradiation in PPG-idasanutlin treated cells. (a) RPE-1 cells were treated with indicated compounds (all 10 μM final) and fixed 4 h after 5 min (∓ 400 nm) irradiation.34 (b) Quantification of the mean p53 intensity per nucleus in cells treated as in (a).35 (c) Representative Western blot showing p53 protein levels in cells 4 h after addition of DMSO or PPG-idasanutlin and irradiation for indicated time periods. Hsp90 is used as a loading control. (d) Selective outgrowth disadvantage in RPE-1 cells 6 days after PPG-idasanutlin treatment +400 nm irradiation for 5 min. (e) Representative Western blot showing p53 protein levels in three cell lines (U2OS, RKO, BJhTert) 4 h after indicated treatments. (f) Selective outgrowth inhibition in indicated cell lines 6 days after PPG-idasanutlin treatment +400 nm irradiation for 5 min.36
	Figure 4. Spatiotemporal control of PPG-idasanutlin. (a) Schematic representation of microwell setup for laser irradiation of individual RPE-1 cells to activate PPG-idasanutlin. Laser target area (represented by red circle) for single pulse (0.1 s irradiation at 5 μm interspaced position) indicated with scale. Individual irradiated cells followed by measuring nuclear p53-venus levels (fluorescence) every 15 min for 3 h after laser irradiation. Approximately 200 cells in each microwell. (b) Percentage of cells that divide within 8 h after indicated treatments.38 (c,d) p53-venus fluorescent signal in individual RPE-1 cells tracked over time after indicated treatments as represented in (a).39

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