WO2021249517A1 - Colle moléculaire régulant l'interaction cdk12-ddb1 pour déclencher une dégradation de la cycline k - Google Patents

Colle moléculaire régulant l'interaction cdk12-ddb1 pour déclencher une dégradation de la cycline k Download PDF

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WO2021249517A1
WO2021249517A1 PCT/CN2021/099517 CN2021099517W WO2021249517A1 WO 2021249517 A1 WO2021249517 A1 WO 2021249517A1 CN 2021099517 W CN2021099517 W CN 2021099517W WO 2021249517 A1 WO2021249517 A1 WO 2021249517A1
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hydrogen
cdk12
ring
unsubstituted
substituted
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PCT/CN2021/099517
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Xiangbing QI
Ting HAN
Peihao Chen
Lu LV
Yamei LI
Longzhi CAO
Qingcui WU
Jiao LI
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National Institute Of Biological Sciences, Beijing
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D277/82—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • Molecular glues are a class of small molecules that induce the formation of protein-protein interactions to elicit biologic or therapeutic effects.
  • the name “molecular glue” was first coined to describe the mechanism of action of the plant hormone auxin, which bridges an interaction between the E3 ubiquitin ligase TIR1 and IAA transcription repressor proteins, leading to IAA destruction by the ubiquitin proteasome system and activation of auxin-response gene expression.
  • auxin which bridges an interaction between the E3 ubiquitin ligase TIR1 and IAA transcription repressor proteins, leading to IAA destruction by the ubiquitin proteasome system and activation of auxin-response gene expression.
  • auxin Unlike conventional small molecules that modulate their target proteins by binding to pockets linked to protein functions (known as druggable pockets) , molecular glues promote the interaction between a target protein and a regulatory protein, and thereby influencing the activity or the fate of the target protein.
  • molecular glues are rare and were discovered serendipitously. Only a handful of molecular glues have been documented over the past four decades. For example, macrocyclic nature products cyclosporin A, FK506, and rapamycin recruit FKBP proteins to the phosphatase calcineurin or the kinase mTORC1, interfering with their enzymatic activities to control intracellular signal transduction. Similarly, the thalidomide class of immunomodulatory drugs (IMiDs) binds to the E3 ubiquitin ligase cereblon to recognize and degrade several zinc finger transcription factors so as to mediate IMiDs’ therapeutic and side effects.
  • IiDs immunomodulatory drugs
  • molecular glues typically bind to one protein with modest or even undetectable affinity; only when two proteins are present, enhanced affinities are observed. Structural studies of molecular glue-bound protein complexes have revealed induced protein-protein interactions and pre-existing complementarity as the underpinning of molecular glue’s binding characteristics. Second, molecular glues can target transcription factors and splicing factors, doing so by either targeting relatively flat surfaces or disordered regions of the proteins. Third, molecular glues possess favorable pharmacological properties to serve as drug candidates.
  • the compactness of the thalidomide scaffold allowed the development of bivalent proteolysis target chimeras to degrade disease-relevant proteins via cereblon recruitment.
  • the limited availability of molecular glues has stymied rational design of molecular glues to expand their impact in therapeutic development.
  • the present invention relates to a new kind of molecular glues for triggering polyubiquitination and degradation of CCNK (cyclin K) .
  • the present invention relates to a new kind of molecular glues for modulating CDK12 protein so as to bind DDB1 of DDB1-CUL4-RBX1.
  • the molecular glues of the present invention convert CDK12 into a substrate-specific receptor to trigger the polyubiquitination and subsequent degradation of CDK12’s partner protein CCNK.
  • the present invention provides a kind of molecular glues having the following general formula:
  • X 1 ⁇ X 11 are independently selected from O, S, wherein R 1 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen;
  • Y 1 ⁇ Y 4 are independently selected from O, S, wherein R 2 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen;
  • Z 1 and Z 2 are independently selected from -O-and wherein R 4 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen;
  • Z 3 is wherein R 5 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen; or Z 3 , L and Z 1 form a ring;
  • n 0, 1, 2 or 3;
  • n 1, 2 or 3;
  • p 1, 2 or 3;
  • ring A, ring B and ring C are independently selected from a substituted or unsubstituted 4-6 membered aryl, heteroaryl, cycloalkyl or heterocycle ring, wherein hetero-forms comprise 1-5 heteroatoms selected from the group consisting of N, O, P, B, Si, Se and S;
  • R 1 ⁇ R 5 are independently selected from methyl, ethyl, -CF 3 , -NO 2 , -CN, -F, -Cl, -Br, amino, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl ethoxycarbonyl and hydrogen.
  • R 1 is methyl, ethyl, -CF 3 , -F, -Cl, -Br or hydrogen.
  • R 2 is methyl, ethyl, -CF 3 , -F, -Cl, -Br or hydrogen.
  • R 3 is methyl, ethyl, -CF 3 , -F, -Cl, -Br or hydrogen.
  • R 4 is methyl, ethyl, -CF 3 , -F, -Cl, -Br or hydrogen.
  • R 5 is methyl, ethyl, -CF 3 , -F, -Cl, -Br or hydrogen.
  • p is 1.
  • X 1 , X 3 and X 5 is selected from O, S, wherein R 1 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen, and X 2 , X 4 and X 6 are all carbon.
  • X 1 , X 3 and X 5 is selected from O, S, wherein R 1 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen, and X 2 , X 4 and X 6 are all carbon.
  • the ring composed by X 1 ⁇ X 6 is
  • X 8 and X 11 is selected fromO, S, wherein R 1 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen, and X 7 , X 9 and X 10 are all carbon.
  • X 8 and X 11 is selected from O, S, wherein R 1 is selected from substituted or unsubstituted C1-C5 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl, ethoxycarbonyl and hydrogen, and X 7 , X 9 and X 10 are all carbon.
  • the ring composed by X 7 ⁇ X 11 is
  • Y 2 and/or Y 4 is and other Ys are -CH 2 -; n is 1 or 2.
  • Y 2 and/or Y 3 is and other Ys are -CH 2 -; m is 1, 2 or 3.
  • the ring composed by Y 1 ⁇ Y 4 is
  • L is selected from
  • ring A is selected from a substituted or unsubstituted 5-6 membered heteroaryl or heterocycle ring, comprising 1-3 heteroatoms selected from the group consisting of N, O and S.
  • ring A is selected from a unsubstituted 5-6 membered heteroaryl, comprising 1-3 heteroatoms selected from the group consisting of N, O and S.
  • ring B is selected from a substituted or unsubstituted 5-6 membered heteroaryl or heterocycle ring, comprising 1-3 heteroatoms selected from the group consisting of N, O and S.
  • ring B is selected from a substituted 5-6 membered heteroaryl, comprising 1-3 heteroatoms selected from the group consisting of N, O and S.
  • ring B is selected from
  • the molecular glues having the following general formula:
  • W and V are independently selected from the group consisting of N, O and S.
  • ring A is selected from
  • the molecular glues having the following general formula:
  • R 6 is selected from substituted or unsubstituted C1-C2 alkly, -NO 2 , -CN, amino, halogen, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl and ethoxycarbonyl and R 7 is hydrogen; or R 6 and R 7 form a substituted or unsubstituted aryl, heteroaryl, cycloalkyl or heterocycle ring; or R 6 is hydrogen and R 7 is substituted or unsubstituted C1-C2 alkly,
  • R 6 is methyl, ethyl, -CF 3 , -NO 2 , -CN, -F, -Cl, -Br, amino, hydroxyl, methoxyl, ethoxyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethy, methoxycarbonyl or ethoxycarbonyl and R 7 is hydrogen.
  • R 6 is hydrogen and R 7 is methyl.
  • R 6 and R 7 form a aryl, methylphenyl, cyclobutyl, cyclopentyl or cyclohexyl.
  • R 6 is hydrogen and R 7 is
  • ring C is selected from a substituted or unsubstituted 5-6 membered heteroaryl or heterocycle ring, comprising 1-3 heteroatoms selected from the group consisting of N, O and S.
  • ring C is selected from wherein X and Y are independently selected from hydrogen, halogen, hydroxyl, amino, substituted or unsubstituted C1-C2 alkly, methoxyl, hydroxymethyl, aminomethyl, pyrazolyl, vinyl and styrene.
  • X is F, Br or Cl.
  • X is methyl, ethyl or trifluoromethyl.
  • Y is selected from methyl, vinyl and styrene.
  • Y is methyl or styrene.
  • ring C is and X is a hydrogen, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and X is a hydrogen, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is a halogen, particularly F, Br or Cl, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and X is a halogen, particularly F, Br or Cl, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is a hydroxyl
  • R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl
  • R 7 is hydrogen
  • ring C is and X is a hydroxyl, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is an amino
  • R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl
  • R 7 is hydrogen
  • ring C is and X is an amino, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is C1-C2 alkly, particularly methyl or ethyl, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and X is C1-C2 alkly, particularly methyl or ethyl, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is substituted C1-C2 alkly, particularly methyl, wherein the substituents are at least one halogen, particularly one, two or three F, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen. Particularly, wherein the X is trifluoromethyl (-CF 3 ) .
  • ring C is and X is substituted C1-C2 alkly, particularly methyl, wherein the substituents are at least one halogen, particularly one, two or three F, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is methoxy
  • R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl
  • R 7 is hydrogen
  • ring C is and X is methoxy, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is hydroxymethyl, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and X is hydroxymethyl, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is aminomethyl, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and X is aminomethyl, R 6 is hydrogen and R 7 is particularly
  • ring C is and X is a pyrazolyl
  • R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl
  • R 7 is hydrogen
  • ring C is and X is a pyrazolyl R 6 is hydrogen and R 7 is particularly
  • ring C is and Y is methyl or styrene, R 6 is substituted or unsubstituted C1-C2 alkly, particularly methyl, and R 7 is hydrogen.
  • ring C is and Y is methyl or styrene, R 6 is hydrogen and R 7 is particularly
  • the compound has a structure selected from any of following:
  • the present invention provides a modified CDK12 protein or its kinase domain binding the molecular glue.
  • the modified CDK12 protein or its kinase domain has the molecular glue binding to the ATP-binding pocket therein, and has a surface for binding DDB1.
  • the CDK12 protein has a kinase domain and the kinase domain binds the molecular glue. More preferably, the CDK12 protein has G731 located in the kinase domain.
  • the present invention provides a modified CDK13 protein or its kinase domain binding the molecular glue.
  • the CDK13 protein has a kinase domain and the kinase domain binds the molecular glue. More preferably, the CDK13 protein has G709 located in the kinase domain.
  • the present invention provides a modified CDK12 carrying G731E or G731R substitution.
  • the modified CDK12 binds the molecular glue.
  • the present invention provides a modified CDK13 carrying G709E or G709R substitution.
  • the modified CDK13 binds the molecular glue.
  • the present invention provides an inter-protein peptide pair, crosslinking lysine 745 of CDK12 and lysine 867 of DDB1.
  • CDK12KD/CCNK ⁇ C/DDB1 complex was crosslinked using lysine-specific crosslinkers. And the only inter-protein peptide pair, lysine 745 of CDK12 was crosslinked to lysine 867 of DDB1.
  • the present invention provides use of the molecular glues for triggering polyubiquitination and/or degradation of CCNK.
  • the molecular glues promote the recruitment of CDK12/CCNK to DDB1-CUL4-RBX1 for polyubiquitination.
  • the new molecular glues convert CDK12 into a substrate-specific receptor to trigger the polyubiquitination and subsequent degradation of CDK12's partner protein Cyclin K.
  • the present invention provides use of the molecular glues for inhibiting ARE-luc2p activity.
  • the present invention provides use of the molecular glues for inhibiting TK-luc2P activity.
  • the present invention provides use of the molecular glues for triggering downregulation and/or cytotoxicity of NRF.
  • the human cell lines A549, HCT-116, and HEK293T were obtained from Dr. Deepak Nijhawan’s lab at University of Texas Southwestern Medical Center. Free style 293-F was a gift from Dr. Linfeng Sun at China University of Science and Technology.
  • the insect cell lines Sf9 and High Five were gifts from Dr. Sanduo Zheng from National Institute of Biological Sciences, Beijing.
  • the identities for A549 and HCT-116 were confirmed by short tandem repeat (STR) analysis. All cell lines were confirmed to be mycoplasma free on a weekly basis using a PCR-based assay with primers: 5’-GGGAGCAAACAGGATTAGATACCCT-3’ (SEQ ID NO.
  • A549, HCT-116, and HEK293T cells were grown in tissue-culture incubators with 5%CO 2 at 37°C.
  • A549 were grown in PRMI-1640 medium with 10%fetal bovine serum (FBS) and 2 mM L-glutamine.
  • HCT-116 and HEK293T cells were grown in DMEM medium with 10%FBS and 2 mM L-glutamine.
  • Regular suspension cell culture methods were used to grow 293-F, Sf9 and high five cells.
  • Free style 293-F cells were grown in SMM 293-TII Expression Medium (Sino Biological, M293TII) in a shaker incubator at 150 rpm, 37°C, 5%CO 2 .
  • Sf9 and high five cells were cultured in ESF 921 medium (Expression Systems, 96-001-01) in a shaker at 140 rpm, 27°C.
  • A549 cells were transiently transfected with the plasmid pGL4.37 [luc2P/ARE/Hygro] (Promega) and selected with 500 ⁇ g/ml of hygromycin to obtain a stable cell line harboring the ARE-luc2P reporter.
  • the TK-luc2P reporter sequence was cloned into the pLVX-IRE-Puro backbone by replacing its CMV promoter with HSV TK promoter fused to luc2P.
  • the resulting pLVX-TK-luc2P-IRES-Puro plasmid was packaged into lentiviral to transduce A549 cells.
  • A549 cells stably expressing TK-luc2p were then obtained by selection of transduced cells with 2 ⁇ g/ml of puromycin.
  • Luminescence was recorded by EnVison multimode plate reader (Perkin Elmer) .
  • IC50 was determined with GraphPad Prism using baseline correction (by normalizing to DMSO control) , the asymmetric (five parameter) equation, and least squares fit.
  • a sequence encoding an sgRNA targeting NRF2 (5’-TATTTGACTTCAGTCAGCGA-3’) was cloned into the lentiCRISPR v2 vector (Addgene #52961) . Lentiviral packaging was performed by co-transfecting the resulting plasmid with psPAX2 (Addgene 12260) and pMD2. G (Addgene 12259) into 293T cells. Media collected from transfected 293T cells was used to infect A549 cells followed by selection with 5 ⁇ g/ml puromycin. Cells surviving puromycin selection were then plated sparsely on 10-cm plates and clones were isolated. NRF2 knockout clones were identified by NRF2 western blotting.
  • the human CRISPR knockout pooled library (Brunello) was a gift from Dr. Feiran Lu at University of Texas Southwestern Medical Center.
  • the sgRNA library was packaged into lentiviral vector for delivery into A549 cells as described.
  • the screening parameters were as follows. Thirty million A549 cells were infected at a multiplicity of infection (MOI) of ⁇ 0.3. Infected cells were passaged every two days in the presence of 10 ⁇ g/ml puromycin for one week with a population size of at least 200 million.
  • MOI multiplicity of infection
  • A549 cells transduced with the sgRNA library were passaged every two days in the presence of 4 ⁇ M glue or the vehicle DMSO (0.1%v/v) for three weeks with a population size of at least 10 million. Two biological replicates were performed for both glue and DMSO treatment.
  • genomic DNA was transferred by a pipet tip to a tube containing 75%(v/v) ethanol. This process was repeated twice to ensure complete removal of residual organic solvents. Afterwards, genomic DNA was dissolved in 1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA by incubation at 50°C for three hours.
  • DNA fragment containing sgRNA sequences were amplified from isolated genomic DNA by two rounds of PCR using NEBNext Ultra II Q5 master mix (NEB) .
  • NEB NEBNext Ultra II Q5 master mix
  • For the first round of PCR forty-eight 50- ⁇ L PCR reactions (each containing 2 ⁇ g of genomic DNA template) were performed with the forward primer NGS-Lib-KO-Fwd-1 (5’-CCTACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNGCTTTATATATCTTG TGGAAAGGACGAAACACC-3’) (SEQ ID NO. 04) and the reverse primer NGS-Lib-KO-Rev-0 (5’-CAGACGTGTGCTCTTCCGATCTCCGACTCGGTGCCACTTTTTCAA-3’) (SEQ ID NO.
  • Products of the first-round PCR were pooled and purified by a DNA clean and concentrator kit (Zymo) and diluted to 2 ng/ ⁇ L.
  • a DNA clean and concentrator kit Zymo
  • CRISPR screening sequencing data were analyzed by MAGeCK (Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout, v0.5.9.2) to discover candidate genes by comparing experimental condition (glues) with control condition (DMSO) .
  • Raw Fastq files were directly loaded into MAGeCK and the “count” command was used to collect read counts from Fastq files and to generate the sgRNA read count table.
  • the “test” command was used to perform statistical test from the count table, outputting log 2 fold change, p value, and false discovery rate (FDR) .
  • the result table generated by MAGeCK was loaded into ggplot2 in R to plot the volcano plot.
  • Two sgRNA were chosen from the Brunello library for DDB1 (5’-CATTGTCGATATGTGCGTGG-3’ (SEQ ID NO. 08) and 5’-CTACCAACCTGCGATCACCA-3’ (SEQ ID NO. 09) ) , RBX1 (5’-AGTACACTCTTCTGAAGTAG-3’ (SEQ ID NO. 10) and 5’-ATGGATGTGGATACCCCGAG-3’ (SEQ ID NO. 11) ) , and UBE2G1 (5’-ACTTACTAAAGTGTATCTGG-3’ (SEQ ID NO. 12) and 5’-ATGAAAAGCCAGAGGAACGC-3’ (SEQ ID NO. 13) ) .
  • Annealed sgRNA oligos were cloned into the lentiCRISPR v2 vector. Lentiviral packaging was performed by co-transfecting the resulting plasmids with psPAX2 (Addgene 12260) and pMD2. G (Addgene 12259) into 293T cells. Media collected from transfected 293T cells was used to infect A549 cells at MOI ⁇ 5 for four days. The resulting cells were then tested for their sensitivity to glues using methods describe in the previous session cell viability assay.
  • 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 15 ⁇ M glue continuously for two weeks, with media change every 3 to 4 days. Glue resistant clones emerged from 5 out of the 10 populations. These clones were isolated, expanded, and tested for their sensitivity to glue using methods describe in the previous session cell viability assay. Five glue-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 glue selection were also pooled for whole-exome sequencing.
  • Genomic DNAs isolated from glue-resistant clones were used as template for amplification of a DNA sequence flanking glycine 731 of CDK12 with primers 5’-GTAAAACGACGGCCAGTGTTGTCCTCGTTATGGAGAAAGAA-3’ (SEQ ID NO. 14) and 5’-CTGTGTCTTTGTCCTTGGCTTTAT-3’ (SEQ ID NO. 15) .
  • PCR amplicons were Sanger sequenced with the M13F sequencing primer: 5’-GTAAAACGACGGCCAGT-3’ (SEQ ID NO. 16) .
  • sgRNA targeting CDK12 (5’-GTGGACAAGTTTGACATTAT-3’ (SEQ ID NO. 17) ) was cloned into the pSpCas9 (BB) -2A-eGFP (PX458) vector (Addgene 48138) .
  • CDK12 G731E/R knock-in 1 million A549 cells were nucleofected (using 4D-Nucleofector, Lonza) with PX458-sgCDK12 and single-stranded oligodeoxynucleotides (G731E: 5’- ttatatacttggccataggttccttctccaataatTTCaataatgtcaaacttgtccacacagcgtttcccccagtc-3’ (SEQ ID NO.
  • G731R 5’-ttatatacttggccataggttccttctccaataatGCGaataatgtcaaacttgtccacacagcgtttcccccagtc-3’ (SEQ ID NO. 19) ) .
  • CDK12 isoform 2 (NM_015083.3) was PCR amplified from A549 cDNA and cloned into a pCDNA3.1-N-3xFLAG vector which was derived from the pCDNA3.1 vector. G731E and G731R mutations in CDK12 were introduced by overlap extension PCR. After sequencing verification, wild-type and mutant 3xFLAG-CDK12 sequences were subcloned into lentiCas9-Blast (Addgene 52962) by replacing Cas9 ORF with 3xFLAG-CDK12.
  • lenti-EF1 ⁇ -3xFLAG-CDK12-P2A-BSD WT/GE/GR constructs were packaged into lentiviral vectors to transduce A549 cells.
  • Stable cell lines were obtained by a selection with 20 ⁇ g/ml of blasticidin.
  • Standard SDS-PAGE and western blotting procedures were used with the following modifications.
  • cells were rinsed with DPBS to remove residual medium and then lysed in 20 mM HEPES pH 8.0, 10 mM NaCl, 2 mM MgCl 2 , 1%SDS freshly supplemented with 0.5 units/ ⁇ L of benzonase and 1x cOmplete, Mini, EDTA-free protease inhibitor (Roche) .
  • Protein concentrations of the resulting lysates were quantified by the BCA method. Between 30 to 60 ⁇ g of proteins were resolved on SDS-PAGE and transferred to nitrocellulose membranes with a pore size of 0.5 ⁇ m.
  • Membranes were blocked in 5%nonfat milk PBST (0.1%v/v Tween-20) for 30 minutes before blotting with antibodies.
  • the following primary antibodies were used by dilution in 5%nonfat milk PBST: anti-NRF2 (Abcam, ab62352, 1: 4,000) , anti- ⁇ -Actin-HRP (Huaxingbio, HX18271, 1: 10,000) , anti- ⁇ -Tubulin-HRFP (MBL Life Science, PM054-7, 1: 10,000) , anti-DDB1 (Abcam, ab109027, 1: 10,000) , anti-RBX1 (Proteintech, 14895-1-AP, 1: 5,000) , anti-UBE2G1 (Proteintech, 12012-1-AP, 1: 2,000) , anti-CDK12 (Cell Signaling Technology, 11973S, 1: 4,000) , anti-CCNK (Bethyl lab, A301-939A-T, 1: 4,000)
  • HRP linked secondary antibodies were used by dilution in PBST: anti-rabbit IgG (Cell Signaling Technology, 7074S, 1: 10,000) , anti-mouse IgG (Zsbio, ZB-2305, 1:10,000) , and anti-rat IgG, (Sino Biological, SSA005, 1: 10,000) .
  • M5 HiPer ECL Western HRP Substrate (Mei5bio, MF074-01) was used for detection of HRP enzymatic activity. Western blot images were taken with a VILBER FUSION FX7 imager.
  • pCMV-8 ⁇ His-Ub was a gift from Dr. William Kaelin at Dana Farber Cancer Institute of Harvard.
  • To set up cells for the in vitro ubiquitination assay 0.6 million HEK293T cells were seeded per well in 6-well plates.
  • these cells were transfected with 500 ng of pCMV 8 ⁇ His Ub, 25 ng of pCDNA3.1-CCNK-3xHA, and 50 ng of pCDNA3.1-3xFLAG-CDK12. Two days later, cells were pretreated with 1 ⁇ M of bortezomib for 2 hours, and then treated with DMSO or three doses of glue (1, 3, 10 ⁇ M) for 4 hours.
  • Tubes containing the beads-lysate mixtures were rotated for 4 hours at room temperature followed by two washes with buffer 1, one wash with a 1: 3 mixture of buffer 1: buffer 2 (25 mM Tris-HCl, pH 6.8, 20 mM imidazole) , and one wash with buffer 2. His-ubiquitin conjugated proteins were eluted from the beads by boiling in 50 ⁇ L of 1x SDS sample buffer supplemented with 300 mM imidazole.
  • ORFs encoding amino acid residues of 715-1052 of CDK12 and residues 693-1030 of CDK13 were cloned into a pCDNA3.1-N-3xFLAG vector.
  • 0.6 million 293T cells were seeded per well in 6-well plates and allowed to attach to plates overnight. These cells were then transfected with 50 ng of pCDNA3.1-3xFLAG-CDK12/13 kinase domain, 50 ng of pCDNA3.1-CCNK-3xHA, and 400 ng of the empty pCDNA3.1 vector. Twenty-four hours post transfection, pCDNA3.1-CCNK-3xHA transfected cells were treated with glue for 8 hours, and lysates were collected for western blotting with anti-FLAG and anti-HA antibodies.
  • sgRNA sequence (5’-GCCCAATTCAGAGAGACATG-3’ (SEQ ID NO. 20) ) targeting the genomic region immediately downstream the CDK12 start codon was cloned into the PX458 vector (Addgene 48138) .
  • the 3xFLAG knock-in repair template was constructed in a pTOPO-TA vector (Mei5bio) and contained a BSD-P2A-3xFLAG sequence flanked by two 500 bp homology arms matching upstream and downstream sequences of the CDK12 genomic locus.
  • Anti-FLAG M2 antibody (Sigma F3165) was coupled to magnetic epoxy beads (Beijing Yunci Technology Co. ) at the ratio of 10 ⁇ g of anti-FLAG antibody/mg of beads in the presence of 1 M ammonium acetate in 0.1 M sodium phosphate, pH 7.4 at 37°C overnight.
  • A549 3xFLAG-CDK12 knock-in cells were detached from plates by scraping, washed in DPBS, and then frozen in liquid nitrogen. Frozen cells were pulverized using a mixer mill MM 400 (Retsch) with two rounds of 1-minute milling at 30 Hz.
  • Bound proteins were eluted with 1 mg/ml 3 ⁇ FLAG peptide (Sigma F4799) at 4°C for 30 min followed by SDS-PAGE and western blotting with anti-FLAG-HRP, anti-CCNK, and anti-DDB1.
  • ORFs encoding UBA1 (residues 49-1058) , UBE2G1, UBE2D3, CUL4A were cloned into pPB-CAG-1xFLAG vector.
  • ORF encoding RBX1 was cloned into pPB-CAG vector without a tag. Plasmids were transfected into 293-F cells at the condition of 0.1 mg of plasmid for 100 million 293-F cells at a density at 1 million cells per ml. FLAG-UBA1, FLAG-UBE2G1, FLAG-UBE2D3 were separately expressed, whereas FLAG-CUL4A was co-expressed with RBX1.
  • FLAG-tagged proteins were eluted from beads by 0.2 mg/ml FLAG peptide (Sangon Biotech, T510060-0005) .
  • the protein-containing fractions were concentrated by ultracentrifugation and fractionated by gel filtration using an Enrich650 size-exclusion chromatography column (Bio-Rad) in binding buffer. Fractions containing the target proteins were pooled, concentrated by ultrafiltration, flash frozen in liquid nitrogen and stored at -80°C.
  • 3xFLAG-CDK12 in complex with CCNK was purified from 1 million A549 stably expressing 3xFLAG-CDK12 (A549 lenti-3xFLAG-CDK12) using FLAG antibody conjugated magnetic beads. Glues or DMSO were added to the reaction before the addition of enzymes.
  • in vitro ubiquitination reactions were performed by mixing 3xFLAG-CDK12/CCNK captured on magnetic beads with 0.2 ⁇ M UBA1, 0.5 ⁇ M UBE2G1, 0.5 ⁇ M UBE2D3, 0.8 ⁇ M CUL4A-RBX1, 1 ⁇ M DDB1, and 100 ⁇ M ubiquitin in a buffer containing 50 mM HEPES, pH 7.5, 5 mM MgCl 2 , 5 mM ATP, 75 mM sodium citrate, and 0.1%Tween-20. After incubation of 1 hour at 30°C with agitation, the reactions were quenched with SDS sample buffer, followed by SDS-PAGE and western blotting with anti-CCNK antibody.
  • Human CDK12KD (residues 715-1052) and human CCNK ⁇ C (residues 11-267) were cloned into the baculoviral transfer vector pFastBac with an N-terminal 6xhis tag and a PreScission Protease cleavage site. G731E and G731R mutations were introduced to CDK12KD by overlap extension PCR.
  • Sf9 or high five cells were coinfected with baculovirus for CDK12KD (WT or G731E/R mutants) , Cyclin K, and yeast CAK1.
  • Cells were lysed by sonication in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5%glycerol) supplemented with 20 mM imidazole and 1x cOmplete, Mini, EDTA-free protease inhibitor (Roche) .
  • the lysate was clarified by centrifugation at 15,000 g for 1 hour at 4°C.
  • Recombinant proteins were purified by Ni NTA Beads (Smart Lifesciences, SA004010) with 30 mM imidazole in binding buffer for washing and 300 mM imidazole in binding buffer for elution. Fractions containing CDK12KD/CCNK ⁇ C were treated with recombinant PreScission Protease at an enzyme to substrate ration of 1: 100 at 4°C overnight to remove 6xHis tag. The cleaved proteins were buffer exchanged using 30 KDa MWCO ultra centrifugal filters (Amicon) to 50 mM MES pH 6.5, 100 mM NaCl, 0.5 mM TCEP and manually loaded onto a 5 mL HiTrap Q column (GE Healthcare) .
  • the flow-through containing CDK12KD/CCNK ⁇ C was concentration by ultrafiltration, loaded onto Bio-Rad ENrich SEC650 size exclusion column, and then fractionated in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP. Fractions containing proteins were pooled and concentrated by ultrafiltration, flash frozen in liquid nitrogen, and stored at -80°C. For the AlphaScreen assay, His-tag removal was omitted from the purification procedures.
  • Human DDB1 ⁇ BPB (removing residues 400-704) was cloned into pFastBac with an N-terminal Strep-tag and an 8 ⁇ His tag.
  • Sf9 cells infected with DDB1 ⁇ BPB were lysed by sonication in binding buffer (50 mM Tris pH 7.5, 500 mM NaCl, 10%glycerol, 2 mM TCEP, 1 mM PMSF, 10 mM imidazole) supplemented with 1x cOmplete, Mini, EDTA-free protease inhibitor (Roche) .
  • binding buffer 50 mM Tris pH 7.5, 500 mM NaCl, 10%glycerol, 2 mM TCEP, 1 mM PMSF, 10 mM imidazole
  • DDB1 ⁇ BPB was purified from clarified lysates by Ni NTA Beads and eluted in 50 mM Tris pH 7.5, 500 mM NaCl, 10%glycerol, 2 mM TCEP, 1 mM PMSF, 300 mM imidazole.
  • the fractions containing DDB1 ⁇ BPB were pooled, concentrated, and then subjected to gel filtration on an ENrich SEC650 size exclusion column (Bio-Rad) in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP.
  • the protein-containing fractions were pooled and concentrated by 50 KDa MWCO Ultra centrifugal filters (Amicon) and flash frozen in liquid nitrogen and stored at -80°C.
  • Full-length human DDB1 was cloned into a pPB-CAG vector with an N-terminal FLAG-tag (for purification) and an Avi-tag (for biotinylation) .
  • the resulting pPB-CAG-FLAG-Avi-DDB1 construct was co-transfected with pPB-BirA (1: 1 mass ratio) into 293-F cells at the condition of 0.1 mg of plasmid for 100 million 293-F cells at a density at 1 million cells per ml.
  • D-Biotin (Targetmol, T1116) was added to the transfected cells at a final concentration of 50 ⁇ M.
  • Bind Cells were collected 48 hours later by 1000 g centrifugation and resuspended in binding buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10%glycerol, 2 mM TCEP, 1 mM PMSF) supplemented with cOmplete, Mini, EDTA-free protease inhibitor (Roche) . After sonication, the lysates were clarified by centrifugation at 15,000 g for 1 hour at 4°C. The supernatant containing recombinant proteins were incubated with anti-FLAG M2 affinity gel (Sigma, A2220) for 2 hours at 4°C on a rotator.
  • binding buffer 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10%glycerol, 2 mM TCEP, 1 mM PMSF
  • cOmplete, Mini, EDTA-free protease inhibitor Roche
  • FLAG-Avi-DDB1 was eluted from beads by 0.2 mg/ml FLAG peptide (Sangon Biotech, T510060-0005) .
  • the protein- containing fractions were concentrated by ultracentrifugation and flash frozen in liquid nitrogen and stored in -80°C.
  • FLAG-DDB1 CDK12KD/CCNK ⁇ C pull down assay
  • FLAG-Avi-DDB1 (18.2 ⁇ g) was mixed with CDK12KD/CCNK ⁇ C (10 ⁇ g) at 1: 1 molar ratio in 500 ⁇ L of pulldown assay buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP.
  • the protein mix was supplemented with DMSO or glue. After 30 minutes of incubation on ice, the protein-compound mix was incubated with anti-FLAG M2 affinity gel for another 30 minutes on a rotator at 4 °C. Protein-bound beads were washed three times with pulldown assay buffer and bound proteins were eluted with 0.2 mg/mL FLAG peptide dissolved in assay buffer. Eluted proteins were analyzed by standard SDS-PAGE followed by coomassie blue staining.
  • Biotinylated FLAG-Avi-DDB1 (3 ⁇ M) and 6xHis-CDK12KD/6xHis-CCNK ⁇ C (WT or G731E/R mutants) were mixed in an assay buffer (50 mM HEPES pH7.5, 300 mM NaCl, 0.5 mM TCEP, 0.1%NP-40, 0.1%BSA) and a serial dilution of glue.
  • the protein-compound mix was transferred to 384-well plates (Corning 3572) with 15 ⁇ L per well.
  • Ni-NTA nickel chelate
  • AlphaScreen acceptor beads PerkinElmer, 6760619C
  • 10 ⁇ L of streptavidin donor beads PerkinElmer, 6760619C
  • 10 ⁇ L of streptavidin donor beads PerkinElmer, 6760619C
  • AlphaScreen signal was measured on the EnVision multimode plate reader (PerkinElmer) .
  • a mixture of 23.4 ⁇ g of FLAG-Avi-DDB1 and 12.2 ⁇ g of CDK12KD/CCNK ⁇ C (1: 1 molar ratio) was incubated in the presence of 10 ⁇ M glue for 30 minutes on ice, followed by crosslinking with 1 mM DSS (Thermo Fisher, A39267) or 1 mM BS3 (Thermo Fisher, A39266) .
  • the crosslinking reactions were incubated for 1 hour on a horizontal rotator at room temperature, followed by quenching with 20 mM ammonium bicarbonate for 20 minutes.
  • Crosslinked samples were precipitated by 20%trichloroacetic acid (final concentration) for 30 minutes at -20 °C.
  • the resulting pellets were air-dried and then dissolved, assisted by sonication, in 8 M urea, 20 mM methylamine, 100 mM Tris, pH 8.5. After reduction (5 mM TCEP, room temperature, 20 minutes) and alkylation (10 mM iodoacetamide, room temperature, 15 minutes in the dark) , the samples were diluted to 2 M urea with 100 mM Tris, pH 8.5. Denatured proteins were digested by trypsin at 1/50 (w/w) enzyme/substrate ratio at 37°C overnight and the reactions were quenched with 5%formic acid (final concentration) .
  • the LC-MS/MS analysis was performed on an Easy-nLC 1000 HPLC (Thermo Fisher Scientific) coupled to a Q-Exactive HF mass spectrometer (Thermo Fisher Scientific) .
  • Peptides were loaded on a pre-column (75 ⁇ m ID, 4 cm long, packed with ODS-AQ 12 nm-10 ⁇ m beads) and separated on an analytical column (75 ⁇ m ID, 12 cm long, packed with Luna C18 1.9 ⁇ m resin) with a 60 min or 120 min linear gradient at a flow rate of 250 nl/min.
  • the top fifteen most intense precursor ions from each full scan (resolution 60,000) were isolated for HCD MS2 (resolution 15,000; NCE 27) with a dynamic exclusion time of 30 s. Precursors with 1+, 2+, more than 7+ or unassigned charge states were excluded.
  • the pLink2 search parameters were as follows: instrument, HCD; precursor mass tolerance, 20 ppm; fragment mass tolerance 20 ppm; cross-linker, BS3/DSS (cross-linking sites K and protein N terminus, cross-link mass-shift 138.068, mono-link mass-shift 156.079) ; peptide length, minimum 6 amino acids and maximum 60 amino acids per chain; peptide mass, minimum 600 and maximum 6,000 Da per chain; enzyme, Trypsin, with up to three missed cleavage sites per chain; Carbamidomethyl [C] , Oxidation [M] , Deamidated [N] and Deamidated [Q] as variable modification. The results were filtered by requiring FDR ⁇ 1%, E-value ⁇ 0.0001, spectra count > 2.
  • CDK12KD/CCNK ⁇ C (WT or G731E/R) and DDB1 ⁇ BPB were diluted to 5 ⁇ M with DSF assay buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP) .
  • 50 ⁇ M glue or 10 ⁇ M HBx was added to diluted proteins (final DMSO concentration 0.25%) .
  • Twenty ⁇ L of protein-compound mix was aliquoted per qPCR tube (Labtidebiotech, P01-0803E) followed by the addition of 5 ⁇ L of 1: 400 diluted SYPRO Orange (Sigma, S5692) .
  • the ORF for CCNK was fused with the firefly luciferase sequence and inserted into the pCDNA3.1 vector.
  • 0.6 million 293T cells were seeded per well in 6-well plates and allowed to attach to plates overnight. These cells were then transfected with 50 ng of pCDNA3.1-3xFLAG-CDK12 kinase domain, 50 ng of pCDNA3.1-CCNK-luc, and 400 ng of the empty pCDNA3.1 vector. Twenty-four hours later, transfected cells were dissociated by trypsin digestion and then plated in 96-well white plates.
  • the product was purified by column chromatography on silica gel to obtain 2- (2-aminothiazol-4-yl) -N- (4- (furan-2-yl) thiazol-2-yl) acetamide (glue01-013) (124 mg; 0.41 mmol, 65%) .
  • the top-ranking genes include the E2 ubiquitin activating enzyme G1 (UBE2G1) , three members of the DDB1-CUL4-RBX1 E3 ubiquitin ligase complex (DDB1, RBX1, DDA1, CUL4B) , and multiple regulators of DDB1-CUL4-RBX1 activity (GLMN, NAA30/35/38, NAE1, and UBE2M) .
  • G1 E2 ubiquitin activating enzyme G1
  • DDB1, RBX1, DDA1, CUL4B three members of the DDB1-CUL4-RBX1 E3 ubiquitin ligase complex
  • GLMN multiple regulators of DDB1-CUL4-RBX1 activity
  • HCT-116 We performed gain-of-function genetic screening in the colorectal cancer cell line HCT-116. HCT-116 cells are defective in mismatch repair, and therefore harbor a high rate of random point mutations. Previous studies suggest that the identification of recurrent mutations present in multiple independent drug resistant clones may reveal the direct drug target. To ensure the isolation of independent glue01-resistant clones, we derived 10 clonal isolates from HCT-116 that were initially sensitive to glue01 (glue01 S ) . Five of these clones evolved glue01 resistance (glue01 R ) during the expansion from a single cell into a large cell population.
  • the top-ranking variant was a G to A substitution resulting in a non-synonymous substitution of glycine to glutamate (G731E) in the gene Cyclin-dependent kinase 12 (CDK12) .
  • This variant occurred at an allele frequency of ⁇ 30%in glue01 R versus 0%in glue01 S .
  • Deconvolution of pooled samples by Sanger sequencing of CDK12 revealed a heterozygous CDK12 G731E mutation in three out of five clones, consistent with an overall allele frequency of 30%in a diploid cell line.
  • Sanger sequencing of CDK12 to examine additional glue01 R clones, we discovered another CDK12 mutation affecting the same codon which replaces glycine with arginine (G731R) .
  • CDK12 is a large protein with a central kinase domain flanked by an arginine serine rich (RS) domain and two proline rich motifs.
  • the G731 hotspot for glue01 resistance mutations is located in the kinase domain of CDK12. This structural position for the mutation led us to speculate that CDK12 might be a protein substrate destabilized by DDB1-CUL4-RBX1 following glue01 treatment and that G731E or G731R substitution in CDK12 might interfere with such destabilization.
  • CRISPRi CRISPR interference
  • CDK12 is known to interact with CCNK via its kinase domain, and this domain was sufficient to mediate both CCNK polyubiquitination and degradation. Similar to full-length CDK12, co-expression of CDK12’s kinase domain with G731E/R mutations blocked CCNK polyubiquitination and degradation.
  • CDK13 is a paralog of CDK12 with a similar domain architecture and its kinase domain shares 90.5%identity to CDK12’s kinase domain. Expression of the wild-type kinase domain of CDK13 also supported CCNK degradation. Mutating CDK13’s glycine 709 to glutamate or arginine (G709E or G709R) , which is located at an equivalent position of glycine 731 in CDK12, also blocked CCNK degradation.
  • DDB1-CUL4-RBX1 requires a type of substrate-specific receptor protein that known as a DDB1 CUL4 associated factor (DCAF) .
  • DCAF DDB1 CUL4 associated factor
  • CCNK in complex with CDK12 G731E or G731R was resistant to glue01-induced polyubiquitination.
  • glue01 promotes the recruitment of CDK12/CCNK to DDB1-CUL4-RBX1 for polyubiquitination.
  • Example 6 Glue01 functions as a molecular glue between CDK12 and DDB1
  • CDK12KD/CCNK ⁇ C/DDB1 complex was crosslinked using two lysine-specific crosslinkers (DSS and BS3) , followed by digestion with trypsin and LC-MS/MS analysis.
  • DDS and BS3 lysine-specific crosslinkers
  • pLink 2 software 41 pairs of crosslinked peptides were identified, out of which only one pair was derived from inter-protein crosslinking; the remainder were all derived from intra-protein crosslinking.
  • lysine 745 of CDK12 was crosslinked to lysine 867 of DDB1.
  • CDK12 Like other kinases, CDK12’s kinase domain possesses an ATP-binding pocket sandwiched between an N-terminal ⁇ sheet-rich lobe and a C-terminal ⁇ helix-rich lobe. Both lysine 745 and glycine 731 reside in the N-terminal lobe atop the ATP-binding pocket of CDK12, suggesting that glue01 might bind to this cleft, creating a modified CDK12 protein surface to bind to DDB1.
  • DSF differential scanning fluorimetry
  • DDB1 is composed of three ⁇ propeller domains (BPA, BPB, and BPC) .
  • BPA ⁇ propeller domains
  • BPB ⁇ propeller domains
  • BPC ⁇ propeller domains
  • the residue lysine 867 on DDB1 that is crosslinked to CDK12 in the CXMS experiment is located in the BPC domain of DDB1, which is known to interact with a promiscuous ⁇ -helical motif present in DCAF proteins and viral proteins such as the hepatitis B virus X protein (HBx) .
  • HBx hepatitis B virus X protein
  • THZ531 is a specific covalent inhibitor of CDK12/CDK13 by irreversibly targeting a cysteine uniquely present in CDK12/13’s kinase domains. Using the AlphaScreen assay, we found that THZ531 inhibited glue01-dependent formation of the CDK12KD/CCNK ⁇ C/DDB1 complex. Dinaciclib is a non-covalent CDK inhibitor targeting CDK1, 2, 5, 9, 12, 13. Neither THZ531 nor dinaciclib treatment caused CCNK degradation.
  • %CCNK-luc degradation ranges are described as follows: A>80%, 20% ⁇ B ⁇ 80%, C ⁇ 20%.
  • Example 9 Glue01-dependent degradation of CCNK causes decreased NRF transcription and cell death
  • CCNK forms two distinct complexes with CDK12 and with CDK13, and is essential for the kinase activity of both complexes. Inactivation of CCNK therefore blocks the function of both CDK12 and CDK13. Indeed, treating cells with the dual CDK12/CDK13 inhibitor THZ531 also caused a reduction of the NRF2 level.
  • RNA polymerase II The C terminal domain of RNA polymerase II’s largest subunit is composed of 52 repeats of with the consensus heptad sequence YSPTSPS. Ser 2 of heptad repeat is a known phosphosubstrate of CDK12/13. Both THZ531 and glue01 reduced Ser 2 phosphorylation of POLII CTD without affecting total POLII levels. Inactivation of CDK12 is known to preferentially affect the expression of genes involved in DNA damage response (DDR) .
  • DDR DNA damage response
  • Example 11 DC 50 and IC 50 of molecular glues
  • DC 50 ranges are described as follows: A ⁇ 0.1 ⁇ M, 0.1 ⁇ M ⁇ B ⁇ 1 ⁇ M, 1 ⁇ M ⁇ C ⁇ 10 ⁇ M, D ⁇ 10 ⁇ M.
  • IC 50 ranges are described as follows: A ⁇ 2 ⁇ M, 2 ⁇ M ⁇ B ⁇ 10 ⁇ M, C ⁇ 10 ⁇ M.

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Abstract

L'invention concerne un nouveau type de colles moléculaires pour déclencher la polyubiquitination et la dégradation de CCNK (cycline K). Plus précisément, l'invention concerne un nouveau type de colles moléculaires pour créer une protéine CDK12 modifiée de manière à se lier au DDB1 de DDB1-CUL4-RBX1. Les colles moléculaires convertissent la CDK12 en un récepteur spécifique au substrat pour déclencher la polyubiquitination et la dégradation ultérieure de la protéine partenaire de CDK12', la CCNK.
PCT/CN2021/099517 2020-06-10 2021-06-10 Colle moléculaire régulant l'interaction cdk12-ddb1 pour déclencher une dégradation de la cycline k WO2021249517A1 (fr)

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