# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 4 Photoswitches in PROTACs
### Photoswitchable PROTACs in Bromodomain Proteins
Therefore, BRD2-4 cellular level recovery was observed in this assay, suggesting a CRBN-dependent degradation. Similar results were also obtained from small interfering RNA (siRNA) knockdown of CRBN and methylation of the glutarimide [57]. To expand the optical responsive into different cancer cell types, light-dependent targeting BRD4 degradation of **PHOTAC-I-3** was evaluated in two breast cancer cell lines (MB-MDA-231 and MB-MDA-468). To access the reversible photoswitchablity of **PHOTAC-I-3**, a rescue experiment was performed. Continuous irradiation for an initial 1 min to **PHOTAC-I-3** with \(\lambda\) = 390 nm (activating wavelength), followed by pulse irradiation with \(\lambda\) = 525 nm (deactivating wavelength), resulted in an initial decrease in cellular BRD2 levels, but they recovered rapidly, even compared to when it was left in the dark conditions. One key characteristic of photopharmacology is _color dosing_[58; 59]. Color dosing is a photocontrol ability where the color intensity of the incident light can be compared with the respective concentration of the active isomer [60]. The color strength of the incident light can be used to estimate the extent of targeted protein degradation, where it is informative of the ratio of active _and_ inactive isomers in the photostionary states [61]. In cell viability assays, left-shifted curves were observed, where the color steadily approached 390 nm. Conversely, Western blotting showed maximum degradation of BRD4 at \(\lambda\) = 390 nm, while BRD4 levels recovered upon increasing the incident light wavelength, suggesting a reversible photoswitchablity over BRD4 protein degradation of **PHOTAC-I-3**. [PERSON] (from Yale University, USA) and [PERSON] (Laboratory of Organic Chemistry, ETH Zurich, Switzerland) and their coworkers rationalized the linker region of PROTACs [49]. Previous biophysical studies highlighted the small changes in length and chemical nature of the linker region affect the ternary complex formation [62; 63]. A sufficient length of linker is required for PROTACs so that both of the warheads are able to rotate and fit into the recruited protein (POI and E3 ligase) to form a ternary complex [64]. In the reported examples, the critical difference linker's length between inactive and active was calculated 3 A, which interestingly matched with typical distances (3-4 A) reported with _cis_- and _trans_-azobenzene photoswitches [45; 65]. On the basis of these key structural features, the authors were prompted with an idea \"of design of a _trans_-configuration of photoPROTAC that will ideally have an optimized linker length for efficient ternary complex while _cis_-configuration of photoPROTAC would have a shorter distance to reach the binding pocket of the second protein partner (which could be E3 ligase or POI)\". Therefore, the authors chemically transformed the polyether linker region of **ARV-771 (ARV-771** is a PROTAC of bromodomain proteins, as shown in the structure in Figure 13A) into azobenzene photoswitch in order to design a photoswitchable PROTAC (photo-PROATCs) [49]. Secondly, the authors envisioned an ideal azobenzene photoswitch having prolonged photostationary states (\(\approx\)days) that are populated during initial irradiation stimuli and must persist throughout the experiment so that continued pulse irradiation exposure can be avoided. This required an azobenzene whose _cis_-configuration shows configuration isomerization stability in biological settings, which led the authors to introduce bistable ortho-tetrafluoroacobenzene (o-F\({}_{4}\)-azobenzene) as azobenzene photoswitches [66], which had reported thermal \(\tau_{1/2}\)'s \(\approx\) 2 years at 25 \({}^{\circ}\)C as opposed to a few hours, as shown by its parent _cis_-azobenzene [67].