# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 4 Photoswitches in PROTACs
### Photoswitchable PROTACs Targeting Kinase Protein  
[PERSON] et al., from the School of Pharmacy at China Pharmaceutical University, Nanjing (China) [48], studied the binding conformation of lenalidomide from an X-ray co-crystal structure (PDB id: 4 TZ4 [52]; as can be seen in Figure 10A) to design Azo-PROTACs. On the basis of the ligand-protein interaction of lenalidomide with E3 ligase, the binding pocket was relatively smaller and possessed a steric effect at the solvent boundary interface. On the basis of these observations, the authors rationalized incorporating an azobenzene photoswitch at the C3-position of the phenyl in lenalidomide. To develop a photoswitchable PROTAC against BCR-ABL fusion and ABL protein, **dasatinib** was selected as a POI warhead. **Dasatinib** is clinically used in acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and in CML patients with acquired **imatinib** resistance. It is a second-generation ATP-competitive tyrosine kinase inhibitor of BCR/ABL proteins. On the basis of the conformational binding of **dasatinib** with ABL protein in an X-ray co-crystal structure (PDB id: 2 GQG [53]; as shown in Figure 10B) and molecular docking of azobenzene-incorporated PROTACs with BCR-ABL fusion protein, these biophysical studies illustrated that only _trans_-form was combinatory due to steric (Figure 10C [48]), suggesting that the _cis_- and _trans_-forms could bring significant differences in the degradation activity of targeted proteins. To find a suitable linker length, the authors performed a chemical screening, wherein the resulting PROTACs were tested for their degradation (ABL  
Figure 10: (**A**) X-ray structure of _Homo sapiens_ cerebellum by DNA-damage-binding protein 1 and lenalidomide (PDB id: 4 TZ4) [52]. (**B**) X-ray structure of ABL protein with **dasatinib** (PDB id: 2 GQG) [53]. (**C**) Docking of **Azo-PROTAC-4C-trans** and CRBN. Subfigures (**A–C**) were adapted with permission from [PERSON] et al. [48]; Copyright © 2020 American Chemical Society. and BCR-ABL) activity on the K562 myelogenous leukemia cell line (ABL, BCR-ABL, and E3 ligase CRBN expressed cell line). Among all the synthesized photoswitchable PROTACs (**Azo-PROTACs-2C** to **6C**), **Azo-PROTAC-4C** had an appropriate linker length with higher BCR-ABL fusion protein degrading potency, as shown in Figure 11A. By UV-VIS spectroscopy photoisomerization, kinetic studies were performed to measure the photochemical control and stability over reverse switching of **Azo-PROTAC-4C**. The _trans_-configuration of **Azo-PROTAC-4C** showed \(\lambda_{\text{max}}\) at 361 nm. When **Azo-PROTAC-4C** was exposed to UV-C light, varying degrees of reduction of peak (\(\lambda_{\text{max}}\) = 361 nm) were recorded, indicating **Azo-PROTAC-4C** conversion into _cis_-configuration. Later, 1 h exposure was found to be sufficient to convert _trans_-configuration into _cis_-configuration. Further exposure with white light to **Azo-PROTAC-4C** showed _cis_-configuration within 4 h. **Azo-PROTAC-4C** is a T-type photoswitch [54], and a half-life of spontaneous thermal relaxation (T\({}_{1/2}\) = 620 min at 25 \({}^{\circ}\)C) was measured in dark conditions. In cellular assays, **Azo-PROTAC-4C** showed cell-selectivity against BCR-ABL-driven K562 cells (EC\({}_{50}\) = 28 nM in  
Figure 11: (**A**) Chemical design for Azo-PROTACs. (**B**) Linker length screening for Azo-PROTACs. cell viability assay, IC\({}_{50}\) = 68 nM in cellular proliferation assay), while no activity was found for non-BCR-ABL-derived cell lines (HCT116 colorectal carcinoma cells, A549 pulmonary carcinoma cells, and MCF-7 cells). Furthermore, negative control of **Azo-PROTAC-4C** was developed to determine the mechanism of targeted protein degradation. Methylation ofimide _(-NH-)_ of glutarimide (in the lenalidomide) of **Azo-PROTAC-4C** led to the formation of **PROTAC-4C** (-) (as a negative control) (as illustrated in Figure 11B).