# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 3 Intracellular Delivery and Controlling Activation/Deactivation ProtACs
### Photocaging PROTACs of Bromodomain Proteins
(**B**) Chemical transformation of thalidomide with NPOM photolabile group. (**C**) Photochemical transformation of **BRD4 PROTAC-4** to activate **BRD4 PROTAC 3**. tions of 51% and 39% in cell viability were found for BRD4 **PROTAC-3** and NPOM-caged BRD4 **PROTAC-4**, respectively (\(\lambda\) = 365 nm, 180 s). In addition, to explore the apoptosis mechanism of BRD4 **PROTAC-4**, caspase-3/7 activation was monitored. BRD4 **PROTAC 3**-treated 22 Rv1 cells showed a nearly threefold increase in caspase-3/7 activity, while NPOM-caged BRD4-**PROTAC-4**-treated 22 Rv1 cells showed no reasonable effect in the absence of light. However, when irradiated with UV light, caspase-3/7 activity was restored to similar levels to BRD4 **PROTAC 3**. Collaboratory work by [PERSON] et al. from Peking University and the Southern University of Science and Technology (Shenzhen, China) integrated the photo-control groups into PROTACs to develop photocaged PROTACs (pc-PROTACs) [41]. The Pc-PROTACs remain inactive until irradiated, but upon irradiation with a particular wavelength of light, photoc chemicals activate the PROTACs with the release of the photolabile group (or photodontrol groups). The authors targeted the bromodomain and extra-terminal (BET) protein bromodomain-containing protein 4 (BRD4) on the basis of the chemical design of **dBET1** proposed by the [PERSON] research group (Dana-Farber Cancer Institute, Harvard Medical School (Boston, USA)), which is molecularly composed of thalidomide (as E3 ligase cereblon ligand) and **JQ1** (a BRD4 ligand) [25]. **dBET1** is a selective BET protein degrader. Numerous in vivo studies showed an efficient photocleavage of 4,5-dimethoxy-2-nitrobenzyl (DMNB) upon irradiation at 365 nm [26]. On the basis of the X-ray co-crystal structure of CRBN-thalidomide (PDB id: 4C1I) [42] and BRD4-JQ1 (PDB id: 3 MXF) [43], DMNB was tethered to an amide functionality (-CONH-) of the **JQ1** of **dBET1** to obtain **pc-PROTAC1**, while the amide nitrogen of the thalidomide moiety (CO-NH-CO) of **dBET1** was used to form **pc-PROTAC2**, as shown in Figure 6A,B. The photolysis studies by high-performance liquid chromatography (HPLC) showed the generation of **dBET1** upon irradiation (365 nm, 3 mW/cm2) from pc-PROTACs, where quantitative analysis of **pc-PROTAC1** and **pc-PROTAC2** showed a quick photohydrolysis of DMNB with half-times (T\({}_{1/2}\) = 60 and 105 s, respectively). The study also showed a 50% of **dBET1** from **pc-PROTAC1** (as shown in Figure 6C) and no reasonable amount of **dBET1** produced from **pc-PROTAC2**. Furthermore, the stability of **pc-PROTAC1** was studied in phosphate-buffered saline (PBS) buffer solution for 24 h in dark conditions, where \(\approx\)88% of **pc-PROTAC1** and **no dBET1** release were observed. These studies led the authors to examine the **pc-PROTAC1** for the photocaging PROTAC mechanism [41]. As anticipated, an increase in the molecular size of **pc-PROTAC1** reduced approximately 100 and 300 times its BRD4-binding affinity (IC\({}_{50}\) = 7.6 \(\upmu\)M) when compared to the **JQ1** (IC\({}_{50}\) = 71 nM) and **dBET1** (IC\({}_{50}\) = 22 nM), respectively. Ramos cells were used to study the dose-dependent BRD4 protein degradation of **pc-PROTAC1**. The **pc-PROTAC1** at 3 \(\upmu\)M did not show a reasonable degradation, but upon UV irradiation (\(\lambda\) = 365 nm for 3 min), a dose-dependent BRD4 protein degradation was observed. A comparative degradation profile of 0.3 \(\upmu\)M **pc-PROTAC1** was found to be equivalent to 0.1 \(\upmu\)M **dBET1**. A maximum Brd4 degradation efficacy (Dmax) of 93% was achieved with 1 \(\upmu\)M **pc-PROTAC1**. Later, irradiation time intervals were evaluated, where exposure of 0.3 min (at 365 nm) reduced the BRD4 protein cellular levels, while prolonged exposure (time = 3 min) completely degraded the BRD4 protein cellular level [41].