# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 4 Photoswitches in PROTACs
### Photoswitchable PROTACs in Bromodomain Proteins
The most plausible explanation for such an observation would be the higher binding affinity of cis-configuration for either BRD2 or VHL compared to trans-configuration of **photoPROTAC-1**, which prevents binding of the active trans-isomer and therefore abolishes the degradation. To study the time-dependent degradation profiles of trans-configuration and cis-configuration of **photoPROTAC-1**, various incubation time points (1-24 h) were selected with Ramos cells. No reasonable BRD2 degradation of trans-configuration of **photoPROTAC-1** was observed within 1 h, nor after 3 h. Conversely, degradation increased in the next 4 h, reached a maximum after 7 h, and remained unchanged for 17 h. A concomitant targeting of trans-configuration of **photoPROTAC-1** with **MLN-4924** (selective NEDDS inhibitor) showed a loss of BRD2 degradation potency, suggesting the proteasomalomal-mediated protein degradation activity. Interestingly, _cis_- or _trans_-configuration of photoPROTAC-1 did not show a substantial BRD4 degradation, while **ARV-771** was found to be an efficient degrader of BRD4 as well as BRD2. This observation might be explained on the basis of the structural differences of **ARV-771** and **photoPROTAC-1**, which affected their ternary complex stability for BRD4 protein degradation: (a) the presence of a reversed amide bond between **JQ-1** and o-F4-azobenzene moiety in **photoPROTAC-1** compared to polyether linkage of **ARV-771**, and (b) a higher intrinsic rigid character of azobenzene during their incorporation into **photoPROTAC-1**[69]. To evaluate the reversible photoswitching between _cis_-configuration and _trans_-configuration of **photoPROTAC-1**, two test tubes were irradiated by 415 and 530 nm LEDs for 30 mins to obtain _trans_-configuration and _cis_-configuration of **photoPROTAC-1**, respectively. _Trans_-configuration of **photoPROTAC-1** and _cis_-configuration of **photoPROTAC-1** were then subdivided (1:1) into two tubes each, whereas parent test tubes were stored separately. The new two test tubes were irradiated: the test tube that contained trans-configuration of photoPROTAC-1 was irradiated with \(\uplambda\) = 530 nm, while the test tube contained _cis_-configuration of **photoPROTAC-1** was irradiated with \(\uplambda\) = 415 nm. Varying concentrations of singly irradiated (415 nm, trans-photoPROTAC-1; or 530 nm, cis-photoPROTAC-1) or doubly irradiated (530/415 nm, trans-photoPROTAC-1; or 415/530 nm, cis-photoPROTAC-1) **photoPROTAC-1** solutions were incubated with Ramos cells to study the reversible photosomerization. The treated cells were lysed, and the cellular levels of BRD4 and BRD2 were estimated after 18 h. Interestingly, a similar pattern of BRD2/4 degradation was noticed for singly irradiated _cis_-configuration and _trans_-configuration of **photoPROTAC-1**, and reversed protein degradation activity was observed in doubly irradiated treatments, ensuring the reverse photoswitchablity of **photoPROTAC**. The spatiotemporal control of **photoPROTAC-1** was evaluated. Two sets of _trans_-configuration of photoPROTAC-1-treated Ramos cells were studied: one set of cells were located in the dark, while the second set of cells were irradiated at 530 nm. As predicted, _cis_-configuration recovered from the second set. Additionally, substantial degradation of BRD2 was recorded with _cis_-configuration of photoPROTAC-1-treated cells under 415 nm, indicating that incorporating _o_-F4-azobenzenes as photoswitches in the PROTAC linker region was successful in providing the reversible spatiotemporal control over protein degradation.