# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 3 Intracellular Delivery and Controlling Activation/Deactivation ProtACs
### Photocaging PROTACs of Bromodomain Proteins  
To expand the photocaging PROTAC concept, the same authors ([PERSON]'s research group from the Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA) considered CRBN-based PROTACs. Similar to their chemical design of **ERR\(\alpha\) PROTAC-2**, the authors utilized the X-ray structure to find a suitable tethering position for incorporating the photolabile group on the CRBN ligand (as shown in Figure 5A). Through X-ray co-crystallize structure, the authors found imide functionality on the glutarimide ring buried into a hydrophobic pocket and that it had a H-bond interaction with the amide  
Figure 4: (**A**) Cocrystal VHL ligand binding to VHL E3 ligase, showing critical residue involvement in H-bonding interactions (reprinted (adapted) with permission from [35]; Copyright 2020 American Chemical Society). (**B**) Chemical transformation of VHL ligand into coumarin-photocaged VHL ligand. (**C**) Photochemical transformation of **ERR\(\alpha\) PROTAC-2** to **ERR\(\alpha\) PROTAC-1**. backbone of His380 (as shown in Figure 5A). A previous CRBN-based PROTAC study [36] reported that substituting even a smaller group such as methyl at imide functionality produces enough steric hindrance that abolishes the binding of overall thalidomide structure with CRBN E3-ligase. These studies led the authors to exploit imide (-NH-) functionality for installing the photolabile group (nitrophenyloxymethyl (NPOM)) (as shown in Figure 5B). The authors synthesized BRD4 **PROTAC-3**, which has a chemical frame of a reported CRBN-based PROTAC ([PERSON]'s research group from the Dana-Farber Cancer Institute, Harvard Medical School, Boston, USA, reported originally as _compound 3_, with BRD4 IC\({}_{50}\) = 37 nM by using luminescent-proximity-based AlphaScreen assay [37]). The authors installed the photolabile group (NPOM) onto the imide of glutarimide of BRD4 **PROTAC-3** to obtain the BRD4 **PROTAC-4**. Choosing of the NPOM group was based on its stability in aqueous conditions, as higher acidity of imide nitrogen (pKa = 14) compared to aliphatic amines (pKa = 30-40) was found to be problematic [38; 39]. Upon irradiation (\(\lambda\) = 365 nm), the NPOM undergoes photolysis and has already shown its use in previously reported cellular studies [40]. The authors monitored the light activation of BRD4 **PROTAC-4** through HPLC and mass spectrometry, where a non-caged version of PROTAC (**BRD4 PROTAC 3**) and a leaving cage group (nitrooketone) were observed upon irradiation (at 2 min) (as shown in Figure 5C). The results obtained from cellular experiments with NPOM-caged BRD4 **PROTAC-4** verified its optical control over CRBN-mediated degradation of BRD4, such as (a) a significant degradation of BRD4 in HEK293T cells within 5 h being found for BRD4 **PROTAC 3** by Western blotting; (b) no BRD4 degradation found for caged BRD4 **PROTAC 4** in the absence of light, suggesting installation of NPOM photolabile hindered its binding conformation with E3 ligase; (c) upon irradiation (\(\lambda\) = 365 nm, 6 mins), BRD4 degradation was observed; (d) no change in cellular BRD4 levels observed in the competitive assay (BRD4 **PROTAC-4** compared to with either **MG132** or **MLN4924**), suggesting proteasome- and E3-ligase-mediated degradation of BRD4 protein by BRD4 **PROTAC-4**; and (e) no effect on BRD4 levels observed for nitrooskeleton (caging group released fragment)-treated HEK293T cells, suggesting the entire BRD4 protein degradation by light-activated chemical transformed \"PROTAC\". To understand the optical control over BRD4 degradation at the cellular level, the authors treated 22 Rv1 cells (a castration-resistant prostate cancer cell line) with **BRD4 PROTAC-3** and NPOM-caged **BRD4 PROTAC-4** in the presence/absence of light. Reduc  
Figure 5: (**A**) Chemical design information revealed by X-ray cocrystallise thalidomide structure with CRBN E3-ligase (reprinted (adapted) with permission from [35]; Copyright 2020 American Chemical Society).