# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 4 Photoswitches in PROTACs
### Photoswitchable PROTACs in Bromodomain Proteins
The authors replaced the polyether linker from **ARV-771** skeleton with diamide azobenzene photoswitches, _o_-F\({}_{4}\)-azobenzene diacid (1, representing the _pull_-_pull_ azoswitch mechanism, Figure 13B), and _o_-F\({}_{4}\)-azobenzene amino acid (2, representing the _push_-_pull_ azoswitch mechanism, Figure 13B). The authors studied the photochemical properties. The photochemical properties of _pull_-_pull_ azoswitch-based **photoPROTAC-1** was studied in DMSO. Irradiation (\(\lambda\) = 415 nm) was found to be efficient in producing the photostationary state of 95% for _trans_-configuration of photoPROTAC-1 (separation was performed with the help of HPLC). However, irradiation (\(\lambda\) = 530 nm) showed the photostationary state of 68% for _cis_-configuration of **photoPROTAC-1**, which is comparatively lower than examples of _o_-F\({}_{4}\)-azobenzenes with higher _cis_-isomer photostationary states [67; 68]. From the spectra, pure _cis_-configuration, and _trans_-configuration of **photoPROTAC-1**, n-\(\pi^{\pm}\) absorption maxima of both isomers were found with a difference of 47 nm. The quantum yields for both isomerization reactions (\(\phi\)EZ (530 nm) = 0.28, \(\phi\)ZE (415 nm) = 0.65) were found in a similar order to previous literature for underativized _o_-F\({}_{4}\)-azobenzenes [67], suggesting an efficient isomerization upon photon absorption. Moreover, _pull_-_pull_ ortho-F\({}_{4}\) azobenzene reported bistability was retained in the photoPROTAC-1, and no reverse thermal isomerization of _cis_-configuration was noticed (DMSO, acetonitrile, aqueous buffer at 37 \({}^{\circ}\)C, even for several days), ensuring the suitability of a _pull_-_pull_ azoswitch as photoswitches for PROTAC strategies. Furthermore, a stability retention was noticed with a photoPROTAC-1 (50 uM) in reduced glutathione (10 mM) over 72 h, demonstrating its suitability for biological studies. To ensure stability in cellular systems, cells were incubated without or with photoPROTAC-1  
Figure 13: (**A**) Structure of **ARV-771**, a BET PROTAC. (**B**) Molecular azoswitch frames: _o_-F\({}_{4}\) azobenzene modular switches as “pull–pull” and “push–pull”. (**C**) Derivatized photoswitchable PROTAC with “pull–pull” diacid azoswitch. (25 \(\upmu\)M). Later, cells were harvested and lysed in phosphate-buffered saline by multiple freeze-thaw cycles in different time periods up to 24 h, and lysates were studied by LC-MS for reduced forms as well as non-reduced forms of **photoPROTAC-1**. During data processing, fragment masses matched with reduced form remained undetected, while isotopic mass distribution after 24 h matched with a nonreduced form of the **photoPROTAC-1**, assuring physiological stability of the **photoPROTAC-1**. For biological testing, Ramos cells were used. For initial 20 min irradiation with (\(\uplambda\) = 415) and (\(\uplambda\) = 530 nm), **PhotoPROTAC-1** solutions were used to obtain trans-configuration and cis-configuration, respectively. Then, 1 min of holding took place, followed by brief shaking (vortexing) and irradiating again for the next 10 min. _Trans_-configuration and _cis_-configuration of **photoPROTAC-1** solutions were diluted to the obtained the required molar concentrations, which were then added to Ramos cells. Incubating Ramos cells with a nanomolar concentration of trans-configuration of **photoPROTAC-1** for 6.5 h induced a significant BRD2 degradation, while cis-photoPROTAC-1 failed to induce BRD2 degradation in the tested range of molar concentrations. However, a prolonged incubation of 18 h with _cis_-configuration or _trans_-configuration of **photoPROTAC-1** failed to alter the BRD2 degradation efficiencies. Importantly, **photoPROTAC-1** did not degrade the BRD2 cellular level and required an initial 415 nm irradiation, suggesting a mixture of trans-configuration and cis-configuration of photoPROTAC-1 present in the solution.