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| Status |
Public on Jul 03, 2020 |
| Title |
Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation [WES] |
| Organism |
Homo sapiens |
| Experiment type |
Genome variation profiling by high throughput sequencing
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| Summary |
Molecular glues are small molecules that exert their biologic or therapeutic activities by inducing gain-of-function interactions between pairs of proteins. In particular, molecular-glue degraders, which mediate interactions between target proteins and components of the ubiquitin proteasome system to cause targeted protein degradation, hold great promise as a unique modality for therapeutic targeting of proteins that are currently intractable. Here, we report a new molecular glue HQ461 discovered by high-throughput screening of small molecules that inhibited NRF2 activity. Using unbiased loss-of-function and gain-of-function genetic screening followed by biochemical reconstitution, we show that HQ461 acts by promoting interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and proteasomal degradation of CDK12’s interacting protein Cyclin K (CCNK). Degradation of CCNK mediated by HQ461 compromised CDK12 function, leading to reduced phosphorylation of CDK12 substrate, downregulation of DNA damage response genes, and cell death. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. Our studies reveal a new molecular glue that engages its target protein directly with DDB1 to bypass the requirement of a substrate-specific receptor, presenting a new strategy for targeted protein degradation.
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| Overall design |
Parental HCT-116 were plated sparsely on 10-cm plates to allow the isolation of ten individual clones. Each of these ten clones were expanded to one confluent 15-cm plate to establish ten independent cell populations. One million cells from each population were then plated on one 10-cm plate and treated with 20 μM HQ461 continuously for two weeks, with media change every 3 to 4 days. HQ461 resistant clones emerged from 5 out of the 10 populations. These clones were isolated, expanded, and tested for their sensitivity to HQ461 using cell viability assay. Five HQ461-resistant clones selected from independent populations were subjected to genomic DNA isolation as described in the previous session. Their genomic DNAs were then pooled in equal amounts for whole-exome sequencing at 200x average coverage (Novogene). As a control, genomic DNAs isolated from the corresponding cell populations before HQ461 selection were also pooled for whole-exome sequencing.
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| Contributor(s) |
Lv L, Chen P, Cao L, Li Y, Zeng Z, Cui Y, Wu Q, Li J, Wang JH, Dong MQ, Qi X, Han T |
| Citation(s) |
32804079 |
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| Submission date |
Jul 02, 2020 |
| Last update date |
Oct 06, 2020 |
| Contact name |
Zhi Zeng |
| E-mail(s) |
zengzhi@nibs.ac.cn
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| Organization name |
National Institute of Biological Sciences, Beijing
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| Lab |
Han Ting Lab
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| Street address |
7 Science Park Road, ZGC life science park
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| City |
Beijing |
| ZIP/Postal code |
102206 |
| Country |
China |
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| Platforms (1) |
| GPL20301 |
Illumina HiSeq 4000 (Homo sapiens) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE153708 |
Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger Cyclin K degradation |
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| Relations |
| BioProject |
PRJNA643782 |
| SRA |
SRP269754 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE153707_HQ461_HCT116_Whole_Exome_Sequencing.xlsx |
7.9 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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