J26

Amino Acid Sequence

3D Structure (/structures/AF-J7MA22-F1-model_v6.cif)

Function

Essential for the formation and stability of microtubules . Required for the organization of the mitotic spindle and for the proper attachment of kinetochores to MTs. Promotes the recruitment of NUDC to the mitotic spindle for mitotic progression.

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

Furthermore, to investigate the degradation mechanism, we assessed the protein degradation pathways. Pretreatment with MG-132, a proteasome inhibitor, effectively blocked J26 induced ALK degradation, while chloroquine, an autophagy/ lysosome inhibitor, had no effect (Figure 6H). Additionally, coimmunoprecipitation (co-IP) pulldown experiments suggested that engagement of Hsp70 and ubiquitin are essential for the observed ALK degradation (Figure 6I).

Description

The degradation potency and antiproliferative activity of four optimal degraders, J2, J18, J21, and J26, capable of degrading over 80% of EML4-ALK at 0.5μM, were evaluated through Western blot and MTT assay in H3122 cells. Among the four lead degraders, J26 was the most potent, with a DC50 of 0.080 μM and an IC50 of 0.025μM, followed byJ18(DC50= 0.169 μM; IC50= 0.044μM),J21(DC50= 0.268μM; IC50= 0.193 μM), andJ2(DC50= 0.286μM; IC50= 0.260μM), illustrating that J26 not only drives the most efficient ALK degradation but also exerts the strongest antiproliferative effect (Figure 4A−C).

Description

The degradation potency and antiproliferative activity of four optimal degraders, J2, J18, J21, and J26, capable of degrading over 80% of EML4-ALK at 0.5μM, were evaluated through Western blot and MTT assay in H3122 cells. Among the four lead degraders, J26 was the most potent, with a DC50 of 0.080 μM and an IC50 of 0.025μM, followed byJ18(DC50= 0.169 μM; IC50= 0.044μM),J21(DC50= 0.268μM; IC50= 0.193 μM), andJ2(DC50= 0.286μM; IC50= 0.260μM), illustrating that J26 not only drives the most efficient ALK degradation but also exerts the strongest antiproliferative effect (Figure 4A−C).

Description

we evaluated J26’s antitumor efficacy in an H3122 xenograft mouse model. Mice bearing H3122 tumors ( ∼ 90 mm3 ) were randomly assigned to six groups: vehicle control, two positive control groups (A1, 20 mg/kg, P.O.; 5 mg/kg, I.V.), and three J26-treated groups (20 mg/kg, P.O.; 5 and 10 mg/kg, I.V.). Mice received treatment every other day for 21 days at the indicated doses and routes (Figure 7C). Both positive control and J26-treated groups showed significant tumor volume and weight reductions, with tumor growth inhibition rates of approximately 55.9%−70.0% compared to the vehicle control (Figures 7D and S8). Importantly, no weight loss or other signs of toxicity were observed in any treatment group (Figures 7E and S9).