8E

Amino Acid Sequence

3D Structure (/structures/AF-P08151-F1-model_v6.cif)

Function

Acts as a transcriptional activator. Binds to the DNA consensus sequence 5'-GACCACCCA-3'. Regulates the transcription of specific genes during normal development. Plays a role in craniofacial development and digital development, as well as development of the central nervous system and gastrointestinal tract. Mediates SHH signaling. Plays a role in cell proliferation and differentiation via its role in SHH signaling.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

Having identified 8e as an effective Gli1 degrader, we continued to investigate its protein degradation mechanism. Theoretically, the hydrophobic tag-mediated degradation does not require the participation of E3 ligase [38], therefore, two proteasome inhibitors, MG132, a 26S proteasome inhibitor, and PS341 (Bortezomib), a 20S proteasome inhibitor, were employed to explore the Gli1 degradation mechanism of 8e. As shown in Fig. 4C–E, the two proteasome inhibitors almost completely reversed the Gli1 degradation effect, suggesting the proteasome-dependent degradation by 8e

Description

Subsequently, the HyT Gli1 degrader 8e, which exerted potent inhibitory effect on the proliferation of HT29 cells, was applied for western blotting analysis in HT29 cells to examine the Gli1 protein degradation effects. As shown in Fig. 4A, the hydrophobic tagging molecule 8e can concentration-dependently downregulate the protein levels of Gli1 at 24 h incubation, resulting in a DC50 value of 5.4 μM (Fig. S-3). In addition, 8e also rapidly degraded the Gli1 protein in a time-dependent manner Fig. 4B).

Description

As shown in Table 1, all the bifunctional molecules exhibited improved cell proliferation inhibitory activities compared to Gli1 inhibitor 4 or 5. Among them, the best HyT degrader 8e (IC50 =0.462 μM).