AZ-9

Amino Acid Sequence

3D Structure (/structures/AF-P50750-F1-model_v6.cif)

Function

Protein kinase involved in the regulation of transcription. Member of the cyclin-dependent kinase pair (CDK9/cyclin-T) complex, also called positive transcription elongation factor b (P-TEFb), which facilitates the transition from abortive to productive elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNAP II) POLR2A, SUPT5H and RDBP. This complex is inactive when in the 7SK snRNP complex form. Phosphorylates EP300, MYOD1, RPB1/POLR2A and AR and the negative elongation factors DSIF and NELFE. Regulates cytokine inducible transcription networks by facilitating promoter recognition of target transcription factors. P-TEFb is also involved in cotranscriptional histone modification, mRNA processing and mRNA export. Modulates a complex network of chromatin modifications including histone H2B monoubiquitination (H2Bub1), H3 lysine 4 trimethylation (H3K4me3) and H3K36me3; integrates phosphorylation during transcription with chromatin modifications to control co-transcriptional histone mRNA processing. The CDK9/cyclin-K complex has also a kinase activity towards CTD of RNAP II and can substitute for CDK9/cyclin-T P-TEFb in vitro. Replication stress response protein; the CDK9/cyclin-K complex is required for genome integrity maintenance, by promoting cell cycle recovery from replication arrest and limiting single-stranded DNA amount in response to replication stress, thus reducing the breakdown of stalled replication forks and avoiding DNA damage. In addition, probable function in DNA repair of isoform 2 via interaction with KU70/XRCC6. RPB1/POLR2A phosphorylation on 'Ser-2' in CTD activates transcription. AR phosphorylation modulates AR transcription factor promoter selectivity and cell growth. DSIF and NELF phosphorylation promotes transcription by inhibiting their negative effect. The phosphorylation of MYOD1 enhances its transcriptional activity and thus promotes muscle differentiation. Catalyzes phosphorylation of KAT5, promoting KAT5 recruitment to chromatin and histone acetyltransferase activity.

Amino Acid Sequence

3D Structure (/structures/AF-O60563-F1-model_v6.cif)

Function

Regulatory subunit of the cyclin-dependent kinase pair (CDK9/cyclin-T1) complex, also called positive transcription elongation factor B (P-TEFb), which facilitates the transition from abortive to productive elongation by phosphorylating the CTD (C-terminal domain) of the large subunit of RNA polymerase II (RNA Pol II). Required to activate the protein kinase activity of CDK9: acts by mediating formation of liquid-liquid phase separation (LLPS) that enhances binding of P-TEFb to the CTD of RNA Pol II. (Microbial infection) In case of HIV or SIV infections, binds to the transactivation domain of the viral nuclear transcriptional activator, Tat, thereby increasing Tat's affinity for the transactivating response RNA element (TAR RNA). Serves as an essential cofactor for Tat, by promoting RNA Pol II activation, allowing transcription of viral genes.

Mechanism

The autophagy-lysosomal pathway (ALP) is capable of degrading a broader range of substrates, including longer-lived and larger misfolded proteins and damaged organelles. ALP is classified into three subtypes: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. In microautophagy, substrates are directly sequestered and degraded by lysosomes and late endosomes via membrane protrusion and invagination. In CMA, HSPA8/HSC70 recognizes specific substrates containing KFERQ-like sequences; these substrates subsequently bind to LAMP2A on the lysosomal surface and are delivered into lysosomes for degradation. In macroautophagy, substrates are encapsulated through the formation of double-membrane autophagosomes, which subsequently fuse with lysosomes to degrade the substrates.

Description

Encouraged by the above results. We were strongly interested in the degradation mechanism of CDK9 by AZ-9 in HCT116 cells. First, the proteasome inhibitor MG132 and the lysosome inhibitor chloroquine (CQ) were used to confirm the subcellular localization of the degradation of CDK9 and Cyclin T1, and the results were shown in Fig. 5A and B, pretreatment with CQ significantly blocked CDK9 and Cyclin T1 degradation induced by AZ-9 in HCT116 cells but not MG132, suggesting that engagement of the autophagy–lysosome pathway is essential for the observed CDK9 and Cyclin T1 degradation. What’ more, in the cell viability assay, only lysosome inhibitor CQ effectively reduced the decrease in cell viability caused by the degradation of CDK9 and Cyclin T1 induced by AZ-9 (Fig. 5C). In addition, autophagosome-lysosome fusion inhibitor Bafilomycin A1 could also reverse CDK9 and Cyclin T1 degradation induced by AZ-9 (Supporting Information Fig. S13). To explore the molecular mechanism of AZ-9 induced autophagy–lysosome pathway degradation of CDK9 and Cyclin T1, Co-Immunoprecipitation Mass Spectrum (Co-IP/MS) was used to identify proteins that interact with AZ-9 and CDK9/Cyclin T1 complexes. And ATG101 was identified as a potential candidate (Fig. 5D), given that ATG101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes32, 33. To verify this discovery, Co-immunoprecipitation was performed to evaluate the possible involvement of ATG101. After treatment with 1 μmol/L AZ-9 for 48 h, we detected increased interactions between CDK9 and ATG101 (Fig. 5E), and an immunofluorescence colocalization assay confirmed these results (green fluorescence for CDK9 and red fluorescence for ATG101, Supporting Information Fig. S14). Moreover, we further explored the time and concentration dependence of the interaction between CDK9 and ATG101 under AZ-9 intervention. As shown in Supporting Information Fig. S15, after treatment for 4 and 8 h, AZ-9 could significantly enhance the interaction between CDK9 and ATG101 without showing a weakening trend (Supporting Information Figs. S15A and S16). When the administration concentration of AZ-9 is 3 or 6 μmol/L, the protein interactions between CDK9 and ATG101 induced by chemistry remain stable (Fig. S15B and S15C). Meanwhile, SNS-HYT and 205-HYT were used to verify the universality. As shown in Fig. S15D–S15I, CQ could still reverse the degradation effects of SNS-HYT and 205-HYT on CDK9 and Cyclin T1 (Fig. S15D, S15E, S15G, S15H), and in co-IP experiments, the enhanced effects of SNS-HYT and 205-HYT on the interaction between CDK9 and ATG101 could still be observed (Fig. S15F–S15I). Furthermore, in the ATG101-knockdown HCT116 cell model, the degradation effect of AZ-9 on CDK9 and Cyclin T1 had been greatly weakened, which suggested that ATG101 is essential for maintaining the CDK9 degradation effect of AZ-9 (Fig. 5F and G). However, in the cellular thermal shift assay (CETSA), AZ-9 did not directly bind to ATG101 as demonstrated by an insignificant increase in the thermal stability of the target protein compared with that of the DMSO group (Fig. 5H). We further synthesized an S-indacene-biotin conjugate named Ind-BIO to explore whether the S-indacene moiety directly binds to ATG101 or ATG101-recruiting (Fig. 5I and J). The results indicated that Ind-BIO did not directly bind to ATG101 as well, suggesting that AZ-9 enhances the interaction between CDK9 and ATG101 instead of binding to form a ternary complex, and the addition of AZ-9 is relatively conservative in the recruitment process of CDK9 and ATG101, which might be one of the reasons why AZ-9 can selectively degrade CDK9 and the partner protein Cyclin T1. In brief, we preliminary demonstrated the process that AZ-9 recruits ATG101 to initiate the autophagy–lysosome pathway and forms autophagosomes through the recruitment and overexpression of LC3, which then fuses with lysosomes to degrade CDK9 and its partner protein Cyclin T1.

Description

The HCT116 cell line (CDK9 high expression) was chosen to evaluate degradation effects and cell-inhibitory activity. The S-indacene moiety exposed on the surface of the CDK9/Cyclin T1 complex may cause increased degradation of CDK9 and Cyclin T1 together with increased antiproliferative activity (GI50 = 0.72 μmol/L).

Description

To investigate the degradation phenotype of AZ-9, we first confirmed the DC50 of AZ-9 in HCT116 cells. The results reveal that AZ-9 could significantly degrade CDK9 and Cyclin T1 in a dose-dependent manner, with DC50 values of 0.4073 and 1.215 μmol/L, respectively (Fig. 2A–C).

Description

To investigate the degradation phenotype of AZ-9, we first confirmed the DC50 of AZ-9 in HCT116 cells. The results reveal that AZ-9 could significantly degrade CDK9 and Cyclin T1 in a dose-dependent manner, with DC50 values of 0.4073 and 1.215 μmol/L, respectively (Fig. 2A–C).