# Light-Activating PROTACs in Cancer: Chemical Design, Challenges, and Applications
## 4 Photoswitches in PROTACs
### Photoswitchable PROTACs in Bromodomain Proteins  
A collaboration of the [PERSON] research group (Department of Chemistry, New York University, NY, USA) and the [PERSON] research group (Department of Biochemistry and molecular pharmacology, New York School of Medicine) incorporated an azobenzene photoswitch to form photoswitchable PROTACs, called PHOTACs. In these photoswitchable PROTACs, **(+)-JQI** (a high-affinity inhibitor of BET proteins BRD2-4) was selected as a POI inhibitor, alongside two different types of E3- ligase ligands (with lenalidomide: **PHOTAC-I-1** to **8**, and ponalidomide **PHOTAC-I-9** to **13**) [50], to target CRBN E3 ligase, as shown in Figure 12. Among azobenzene-photoswitch PROTACs (**PHOTAC-I-1-12**), diazocine-based photoswitch PROTACs (**PHOTAC-I-13**) were also designed to degrade the bromodomain proteins, where **PHOTAC-I-3** emerged as a lead compound. The **PHOTAC-I-3** switched to the _cis_-configuration at a certain wavelength (\(\lambda\) = 390 nm, PSS > 90%) and achievedsimilar photostationary states (PSSs) at a range of 380-400 nm. An efficient _trans_-to-_cis_ configuration of **PHOTAC-I-3** photoisomerization was observed at 370 and 450 nm, while in the absence of light _cis_-configuration of **PHOTAC-I-3**, **it** gradually relaxed back to _trans_-configuration of **PHOTAC-I-3** (T\({}_{1/2}\) = 8.8 h at 37 \({}^{\circ}\)C in DMSO). However, when irradiated (\(\lambda\geq\) 500 nm), the chemical equilibrium shifted to _trans_-configuration of **PHOTAC-I-3** (\(\lambda\geq\) 450 nm, PSS of \(>\)70%). To evaluate the optical qualitative nature of these photoswitchable PROTACs (**PHOTAC-I-1 to 13**), human acute leukemia cell lines (RS4;11) in a 96-well plate were treated with increasing concentrations of these PROTACs. Later, they were irradiated with either 390 nm light pulses (100 ms/10 s) for 72 h or as a control incubated in dark conditions. Interestingly, **PHOTAC-I-3** showed an optical response where a sevenfold difference was measured in median effective concentration (EC\({}_{50}\)): EC\({}_{50}\) = 88.5 nM when irradiated with 390 nm light, whereas EC\({}_{50}\) = 631 nM in the dark conditions. Moreover, similar optical responses were noted for **PHOTAC-I-1,2,4** to **8,10**. Interestingly, **PHOTAC-I-9, 11** to **13**, and (+)-JQ1** (**+)-JQ1** was used as a control in these experiments) did not show differences in their EC\({}_{50}\) from irradiated samples against those samples incubated in the dark, suggesting a role  
Figure 12: PHOTACs: PROTACs based on azobenzene photoswitches (**PHOTAC-I-1-12**) and PROTACs based on diazocrine photoswitch (**PHOTAC-I-13**) for bromodomain proteins (BRD2, BRD3, and BRD4). of E3-ligand chemical specificity as **PHOTAC-I-1** to **8** (having lenalidomide chemicals), as opposed to **PHOTAC-I-9** to **13** (having pomalidomide as E3 ligand chemical structural features). To understand light-dependent targeted BET protein BRD2-4 degradation for these PROTACs, Western blotting was used to determine the cellular protein levels. The authors treated RS4;11 cells with increasing concentrations of **PHOTAC-I-3** for 4 h and pulse irradiation (\(\lambda\) = 390 nm, pulse = 100 ms/10 s). A significant decrease in BRD4 levels when irradiated (\(\lambda\) = 390 nm), but not in the dark, was observed for **PHOTAC-I-3** in the range of 100 nm to 3 uM. At 10 uM, a \"_hook effect_\" was observed, a common issue with PROTAC strategies [17; 55; 56]. Similar cellular protein level reduction was also noted for BRD3 at 100 nm to 3 uM under violet light irradiation but not in the dark. Conversely, a moderate BRD2 degradation was observed in a narrower concentration range. To characterize the degradation mechanism of these photoswitchable PROTACs, several biochemical experiments were conducted using competitive assays. Upon irradiation, **PHOTAC-I-3** (1 uM) was used along with CRL inhibitor (**MLN4924** at 2.5 uM). **MLN4924** inhibits medylation and, subsequently, cellular CRL (including CRL4 CRBN) activities.