# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 3 Intracellular Delivery and Controlling Activation/Deactivation ProtACs
### Photocaging PROTACs of Estrogen Receptor  
Gaining precise control over the biological activity of smaller-sized probes has always interested chemical biologists and medicinal chemists. However, a light-controlled higher spatiotemporal resolution has been exploited as a chemical biology tool [26] and in phototherapies [27], where a specific wavelength of light activates the bioactive molecule. [PERSON]'s research group from the Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA, developed a coumarin-based photocaged VHL ligand. Initially, the authors investigated an X-ray co-crystal structure (PDB id: 4W9C) to find a tethering point for a photocleavable group onto the VHL ligand [28]. The authors noticed a hydroxyproline moiety buried into the binding cleft that had H-bond interactions with Ser111 and His115, which are critical amino acid residue interactions used to recognize VHL by HIF1-\(\alpha\) protein, as shown in Figure 4A [29; 30; 31]. Moreover, inverting the hydroxyl group stereochemistry of hydroxyproline moiety abolishes all protein degradability of PROTACs [17; 32]. On the basis of these facts, the authors rationalized the suitability of the tethering point for the photolabile group in a way that the tethering of the photolabile group would hinder VHL ligand binding to its VHL E3 ligase and can only be activated until irradiated (as shown in Figure 4B). The approach showcases an example of precise spatiotemporal control over photobiology. The authors used carbonate tethering to substitute the hydroxyl group of VHL with a diethylamino coumarin (DEACM) to form **ERR\(\alpha\) PROTAC-2** (as shown in Figure 4C). Moreover, the authors prepared a version of **ERR\(\alpha\) PROTAC-2** that does not have a DEACM photolabile group, namely, **ERR\(\alpha\) PROTAC-1**, as a control to assist in their biological study and ensure their photocaging PROTAC approach. Figure 3: Compilation of reported photocaged PROTACs by researchers (**A**). ERR\(\alpha\) PROTAC-2 targeting estrogen receptor. (**B**). PROTAC-4 as a bromodomain inhibitor (**C**). pc-PROTAC-1 and pc-PROTAC2 as bromodomain inhibitors. (**D**). pc-PROTAC-3 as a kinase inhibitor. Both PROTACs were designed to target an orphan nuclear hormone receptor (estrogen-related receptor \(\alpha\) (ERR\(\alpha\))) [17], typically overexpressed in malignant cancers [33]. Using HPLC and mass spectrometry, the DEACM caging group cleaved from the **ERR\(\alpha\) PROTAC 2** after 3 min of irradiation (\(\lambda\leq 405\) nm) and released the acidic functional groups with a pKa \(<5\)[34]. To understand the photocage PROTAC role of **ERR\(\alpha\) PROTAC-2**, MCF-7 cells (breast cancer cell line) were treated with **ERR\(\alpha\) PROTAC-1**, **ERR\(\alpha\) PROTAC-2**, and DMSO in the absence/presence of UV radiation (\(\lambda=365\) nm, 180 s). After 8 h of incubation, the authors used Western blotting to measure the extent of ERR\(\alpha\) cellular levels. As anticipated, **ERR\(\alpha\) PROTAC-1** showed a significant ERR\(\alpha\) protein reduction at the cellular level compared to the DMSO-based sample (used as control), which agrees with the previously reported literature [17]. Importantly, even a double concentration of **ERR\(\alpha\) PROTAC-2** compared to **ERR\(\alpha\) PROTAC-1** in the absence of UV light showed no change in ERR\(\alpha\) protein cellular levels, confirming that photocaging of the photolabile group prevented the binding conformation towards E3 ligase, exemplifying an idealistic example of photocaged-PROTACs. To understand the mechanism, competitive assays in the presence of either the proteasome inhibitor (**MG132**) or the nedydylation inhibitor (**MLN492**) prevented the degradation of **ERR\(\alpha\) PROTAC-2**, suggesting the proteasome- and E3- ligase-mediated degradation ability of **ERR\(\alpha\) PROTAC-2**.