B2

Amino Acid Sequence

3D Structure (/structures/AF-P07949-F1-model_v6.cif)

Function

Receptor tyrosine-protein kinase involved in numerous cellular mechanisms including cell proliferation, neuronal navigation, cell migration, and cell differentiation in response to glia cell line-derived growth family factors (GDNF, NRTN, ARTN, PSPN and GDF15). In contrast to most receptor tyrosine kinases, RET requires not only its cognate ligands but also coreceptors, for activation. GDNF ligands (GDNF, NRTN, ARTN, PSPN and GDF15) first bind their corresponding GDNFR coreceptors (GFRA1, GFRA2, GFRA3, GFRA4 and GFRAL, respectively), triggering RET autophosphorylation and activation, leading to activation of downstream signaling pathways, including the MAPK- and AKT-signaling pathways. Acts as a dependence receptor via the GDNF-GFRA1 signaling: in the presence of the ligand GDNF in somatotrophs within pituitary, promotes survival and down regulates growth hormone (GH) production, but triggers apoptosis in absence of GDNF. Required for the molecular mechanisms orchestration during intestine organogenesis via the ARTN-GFRA3 signaling: involved in the development of enteric nervous system and renal organogenesis during embryonic life, and promotes the formation of Peyer's patch-like structures, a major component of the gut-associated lymphoid tissue (By similarity). Mediates, through interaction with GDF15-receptor GFRAL, GDF15-induced cell-signaling in the brainstem which triggers an aversive response, characterized by nausea, vomiting, and/or loss of appetite in response to various stresses. Modulates cell adhesion via its cleavage by caspase in sympathetic neurons and mediates cell migration in an integrin (e.g. ITGB1 and ITGB3)-dependent manner. Also active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. Triggers the differentiation of rapidly adapting (RA) mechanoreceptors. Involved in the development of the neural crest (By similarity). Regulates nociceptor survival and size (By similarity). Phosphorylates PTK2/FAK1.

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

To investigate whether compound B2 degrades the RET fusion protein through specific cellular pathways, we employed a reverse validation strategy using MG132, a reversible proteasome inhibitor that blocks the 26S proteasome system, and hydroxychloroquine (HCQ), a reversible lysosomal acidification inhibitor that blocks the autophagy-lysosome pathway. Pretreatment of TPC-1 cells with 5 μM MG132 significantly antagonized B2induced RET fusion protein degradation, whereas 50 μM HCQ pretreatment showed no significant effect (Fig. S2). These results indicate that B2-induced degradation of the RET fusion protein depends on the ubiquitin-proteasome pathway, rather than the canonical autophagy-lysosome pathway, suggesting that compound B2 likely promotes RET fusion protein ubiquitination and subsequent proteasomal degradation

Description

As shown in Table 2, compound B2 demonstrated the highest degradation activity among this series. Specifically, when the carbon chain length was three and the HyTs type was norbornene, the RET protein degradation rate reached its peak at 91.4% at a concentration of 10 μM. Degradation rate of CCDC6-RET fusion protein in TPC-1 cells was evaluated using Western blotting assay after 48 h of treatment for each compound