LL-K8-22

Amino Acid Sequence

3D Structure (/structures/AF-P49336-F1-model_v6.cif)

Function

Component of the Mediator complex, a coactivator involved in regulated gene transcription of nearly all RNA polymerase II-dependent genes. Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional pre-initiation complex with RNA polymerase II and the general transcription factors. Phosphorylates the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAp II), which may inhibit the formation of a transcription initiation complex. Phosphorylates CCNH leading to down-regulation of the TFIIH complex and transcriptional repression. Recruited through interaction with MAML1 to hyperphosphorylate the intracellular domain of NOTCH, leading to its degradation.

Amino Acid Sequence

3D Structure (/structures/AF-P24863-F1-model_v6.cif)

Function

Component of the Mediator complex, a coactivator involved in regulated gene transcription of nearly all RNA polymerase II-dependent genes. Mediator functions as a bridge to convey information from gene-specific regulatory proteins to the basal RNA polymerase II transcription machinery. Mediator is recruited to promoters by direct interactions with regulatory proteins and serves as a scaffold for the assembly of a functional preinitiation complex with RNA polymerase II and the general transcription factors. Binds to and activates cyclin-dependent kinase CDK8 that phosphorylates the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAp II), which may inhibit the formation of a transcription initiation complex.

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 further investigate the potential mechanism of the CDK8−cyclin C complex degraded by LL-K8-22, we co treated cells with the proteasome inhibitor MG132 and the HSP90 inhibitor 17-AAG together with LL-K8-22 and performed immunoblotting assays. As shown in Figure 4C,D. MG132 reversed the degradation of CDK8 and cyclin C by inhibiting the cellular UPS pathway, whereas 17-AAG potentiated LL-K8-22-mediated CDK8−cyclin C degradation (Figure 4E,F), demonstrating that the proteasome plays a central role in the HyT-mediated degradation mechanism. In addition, we examined the mRNA levels of CDK8 and CCNC in MDA-MB-468 cells after treatment with different concentrations of LL-K8-22 using RT-qPCR (Figure 4G) and found that LL-K8-22 did not have any influences on the mRNA levels of CDK8 and CCNC. RNA-seq data also demonstrated that CDK8 and CCNC were unaffected in the transcriptome level upon LL-K8-22 treatment (Figure S4C). These together suggested that LL-K8-22 induced CDK8−cyclin C degradation in a proteasome-dependent way

Description

With the optimized degrader, LL-K8-22 in hand, we further evaluated the dose−response relation of LL-K8-22 on CDK8−cyclin C degradation after treating MDA-MB-468 cells with varying concentrations of LL-K8-22. As demonstrated in Figure 3A,B, LL-K8-22 induced a significant reduction of CDK8 and cyclin C protein expression in a dose-dependent manner with DC50 of 2.52 and 2.64 μM, respectively

Description

With the optimized degrader, LL-K8-22 in hand, we further evaluated the dose−response relation of LL-K8-22 on CDK8−cyclin C degradation after treating MDA-MB-468 cells with varying concentrations of LL-K8-22. As demonstrated in Figure 3A,B, LL-K8-22 induced a significant reduction of CDK8 and cyclin C protein expression in a dose-dependent manner with DC50 of 2.52 and 2.64 μM, respectively

Description

To compare the anti-proliferative effects of the HyT degrader LL-K8-22 with the inhibitor BI-1347, a 4 day anti proliferation assay was conducted (Figure 5A−C). As expected, LL-K8-22 showed stronger proliferation inhibition against the three cell lines mentioned above, with IC50 values of 5.73±0.97, 5.87±0.64, and6.22±0.36 μM against the proliferation of MDA-MB-468, HCT-116, and MDA-MB-231 cells, respectively, which were 5.03-, 5.61-, and 4.61-fold lower than the IC 50 values of the parental compound BI-1347 respectively (Figure 5A−C).

Description

To compare the anti-proliferative effects of the HyT degrader LL-K8-22 with the inhibitor BI-1347, a 4 day anti proliferation assay was conducted (Figure 5A−C). As expected, LL-K8-22 showed stronger proliferation inhibition against the three cell lines mentioned above, with IC50 values of 5.73±0.97, 5.87±0.64, and6.22±0.36 μM against the proliferation of MDA-MB-468, HCT-116, and MDA-MB-231 cells, respectively, which were 5.03-, 5.61-, and 4.61-fold lower than the IC 50 values of the parental compound BI-1347 respectively (Figure 5A−C).

Description

To compare the anti-proliferative effects of the HyT degrader LL-K8-22 with the inhibitor BI-1347, a 4 day anti proliferation assay was conducted (Figure 5A−C). As expected, LL-K8-22 showed stronger proliferation inhibition against the three cell lines mentioned above, with IC50 values of 5.73±0.97, 5.87±0.64, and6.22±0.36 μM against the proliferation of MDA-MB-468, HCT-116, and MDA-MB-231 cells, respectively, which were 5.03-, 5.61-, and 4.61-fold lower than the IC 50 values of the parental compound BI-1347 respectively (Figure 5A−C).