14b

Amino Acid Sequence

3D Structure (/structures/AF-Q71U36-F1-model_v6.cif)

Function

Tubulin is the major constituent of microtubules, a cylinder consisting of laterally associated linear protofilaments composed of alpha- and beta-tubulin heterodimers. Microtubules grow by the addition of GTP-tubulin dimers to the microtubule end, where a stabilizing cap forms. Below the cap, tubulin dimers are in GDP-bound state, owing to GTPase activity of alpha-tubulin.

Amino Acid Sequence

3D Structure (/structures/AF-P07437-F1-model_v6.cif)

Function

Tubulin is the major constituent of microtubules, a cylinder consisting of laterally associated linear protofilaments composed of alpha- and beta-tubulin heterodimers. Microtubules grow by the addition of GTP-tubulin dimers to the microtubule end, where a stabilizing cap forms. Below the cap, tubulin dimers are in GDP-bound state, owing to GTPase activity of alpha-tubulin.

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 this degradation process relied on the ubiquitin-proteasome system (UPS), proteasome inhibitors bortezomib (5 μM) and MG132 (5 μM) were independently pre-treated 2 h prior to treatment with compound 14b (5 μM) for an additional period of 24 h. Both proteasome inhibitors effectively blocked the degradation of α-tubulin, confirming the proteasome-dependent activity of compound 14b, as depicted in Fig. 4B. Furthermore, we conducted a competitive binding study using colchicine to explore the binding site of compound 14b. MCF-7 cells were pre-treated with colchicine (5 μM) for 2 h before exposure to compound 14b (5 μM) for another 24 h. Notably, pretreatment with colchicine significantly reduced the rate of α-tubulin degradation compared to no pretreatment, providing strong evidence that compound 14b binds similarly to tirbanibulin at the colchicine-binding site [16]. Overall, our findings confirm that compound 14b induces dose-dependent reduction in α- and β-tubulin levels through proteasomal-mediated mechanisms by specifically targeting the colchicine-binding site.

Description

Compound 14b inhibited purified recombinant tubulin polymerization in a dose-dependent manner, similarly to colchicine. Western blot analysis identified that compound 14b degraded α-tubulin with a DC50 value of 4.9 μM in MCF-7 cells, and approximately 44 % degradation of β-tubulin was observed at 5.0 μM.

Description

Compound 14b inhibited purified recombinant tubulin polymerization in a dose-dependent manner, similarly to colchicine. Western blot analysis identified that compound 14b degraded α-tubulin with a DC50 value of 4.9 μM in MCF-7 cells, and approximately 44 % degradation of β-tubulin was observed at 5.0 μM.

Description

As shown in Table 1, most of the target compounds exhibited significant potency against the tested tumor cell lines with IC 50 values ranging from tens of nanomole to a few micromolar. In particular, compound 14b (IC 50 values ranging from 29 to 60 nM, equivalent to tirbanibulin and slightly weaker than that of colchicine) was identified as the most promising candidate for subsequent in vitro and in vivo investigations.

Description

As shown in Table 1, most of the target compounds exhibited significant potency against the tested tumor cell lines with IC 50 values ranging from tens of nanomole to a few micromolar. In particular, compound 14b (IC 50 values ranging from 29 to 60 nM, equivalent to tirbanibulin and slightly weaker than that of colchicine) was identified as the most promising candidate for subsequent in vitro and in vivo investigations.

Description

As shown in Table 1, most of the target compounds exhibited significant potency against the tested tumor cell lines with IC 50 values ranging from tens of nanomole to a few micromolar. In particular, compound 14b (IC 50 values ranging from 29 to 60 nM, equivalent to tirbanibulin and slightly weaker than that of colchicine) was identified as the most promising candidate for subsequent in vitro and in vivo investigations.

Description

The antitumor efficacy of compound 14b was further evaluated in vivo using BALB/c female mice bearing derived from the mouse mammary 4T1 tumor cells. Once the tumor volume reached approximately 130 mm3, three different doses (5 mg/kg, 10 mg/kg and 20 mg/kg) of compound 14b were administered via tail intravenous injected once every three days for a continuous period of 18 days. PTX (5 mg/kg) was used as a comparative control group and administered in the same manner. Treatment with compound 14b distinctly inhibited tumor growth in an dose-dependent manner (Fig. 8C).Treatment with a high dose (20 mg/kg) of compound 14b resulted in a significant tumor growth inhibition (TGI) values of 74.27 % compared to the control group, as evidenced by contrasting tumor weights (Fig. 8B). Lower doses of compound 14b (5 mg/kg and 10 mg/kg) also exhibited commendably effects on tumor suppression (TGI = 56.21 % and 66.89 %, respectively), although weaker than PTX (5 mg/kg) group. In Fig. 8D, mice body weight showed a slight decrease only at high dose of compound 14b, while other groups didn’t exhibit noticeable changes in body weight.