A549 activity is inhibited by HQ461 with an IC50 of 1.3 µM[1] hour. In A549 cells, HQ461 (10 μM, 8) lowers the levels of CDK12 protein [1]. In A549 cells, HQ461 (0-10 μM) mediates CDK12/CCNK transfer.

A549 activity is inhibited by HQ461 with an IC50 of 1.3 µM[1] hour. In A549 cells, HQ461 (10 μM, 8) lowers the levels of CDK12 protein [1]. In A549 cells, HQ461 (0-10 μM) mediates CDK12/CCNK transfer.

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HQ461 induces the formation of a ternary complex between CDK12's kinase domain (in complex with the Cyclin box domain of CCNK) and DDB1, as demonstrated by pull-down assays and an AlphaScreen assay with an apparent EC50 of 1.9 µM. G731E and G731R mutants of CDK12 do not form this complex even at 20 µM HQ461. [1]
Chemical cross-linking mass spectrometry (CXMS) identified an inter-protein cross-link between lysine 745 of CDK12 and lysine 867 of DDB1 in the presence of HQ461, mapping the interaction interface. [1]
Differential scanning fluorimetry (DSF) showed that HQ461 stabilizes the CDK12 kinase domain/CCNK complex (increasing Tm by 1.4°C), indicating direct binding. This binding was also confirmed for G731E/R mutants via nanoDSF. HQ461 did not alter the thermal stability of DDB1. [1]
In an in vitro ubiquitination assay using purified recombinant ubiquitin, E1 (UBA1), E2 (UBE2G1/UBE2D3), and E3 (CUL4-RBX1-DDB1) components, HQ461 triggered polyubiquitination of CCNK in complex with wild-type CDK12, but not with CDK12 G731E or G731R mutants. CDK12 itself was not ubiquitinated. [1]
Structure-activity relationship (SAR) studies using a CCNK-luciferase degradation reporter identified a crucial 5-methylthiazol-2-amine pharmacophore. Analogue HQ005 (with a hydroxymethyl group replacing the methyl on the pyridine ring) showed improved potency with a DC50 of 41 nM, compared to 132 nM for the parent HQ461. [1]

AlphaScreen Assay for Protein-Protein Interaction: Biotinylated FLAG-Avi-DDB1 and 6xHis-tagged CDK12 kinase domain/CCNK complex (wild-type or G731E/R mutants) were mixed in an assay buffer containing HEPES, NaCl, TCEP, NP-40, and BSA. A serial dilution of HQ461 was added. After incubation, nickel chelate acceptor beads were added, followed by streptavidin donor beads after a further incubation. AlphaScreen signal was measured to quantify HQ461-dependent complex formation. For competition, proteins were pre-incubated with the CDK12 inhibitor THZ531. [1]
Differential Scanning Fluorimetry (DSF): The CDK12 kinase domain/CCNK complex or DDB1∆BPB protein was diluted in a buffer containing HEPES, NaCl, and TCEP. HQ461, DMSO (vehicle), or an HBx peptide control was added along with SYPRO Orange dye. Fluorescence was measured while increasing the temperature from 10°C to 95°C. The melting temperature (Tm) was determined from the resulting curve. [1]
Label-free nanoDSF: The CDK12 kinase domain/CCNK complex (wild-type or mutants) was diluted in buffer and mixed with increasing concentrations of HQ461. Tryptophan fluorescence at 330 nm and 350 nm was monitored during a temperature ramp from 35°C to 95°C using a nanoDSF instrument. The inflection temperature (Ti) was calculated from the 350nm/330nm ratio. [1]
In Vitro Ubiquitination Assay: FLAG-HA-tagged CDK12 complexed with V5-tagged CCNK (wild-type or mutant) was immunopurified from transfected cells. The complex was incubated with recombinant ubiquitin, E1 (UBA1), E2 (UBE2G1 and UBE2D3), E3 (CUL4-RBX1 and DDB1), ATP, and MgCl2 in a reaction buffer with or without HQ461. The reaction proceeded at 30°C and was stopped with SDS sample buffer. Polyubiquitination was assessed by western blotting using anti-HA and anti-V5 antibodies. [1]

Western Blot analysis [1]
Cell Types: A549 Cell
Tested Concentrations: 10 μM
Incubation Duration: 0-8h
Experimental Results: 8 hrs (hours) induced a 50% reduction in CDK12 protein levels, CCNK Reduces protein levels >8-fold at 4 hrs (hours).

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Cell Viability Assay (IC50 determination): A549 or HCT-116 cells were plated in 384-well plates. After overnight attachment, cells were treated with a serial dilution of HQ461 using a digital dispenser. Cell viability was measured 72 hours later using a luminescent ATP-based assay kit. Luminescence was recorded, and IC50 values were determined using non-linear regression analysis. [1]
Pooled Genome-wide CRISPR-Cas9 Knockout Screening: A549 cells were transduced with a genome-wide sgRNA library at a low MOI. After puromycin selection, cells were passaged for 3 weeks in the presence of a sub-lethal dose of HQ461 or DMSO control. Genomic DNA from surviving cells was isolated, sgRNAs were amplified by PCR and sequenced. Enriched sgRNAs (conferring resistance) were identified using the MAGeCK algorithm. [1]
Gain-of-function Mutational Screening: Ten independent clonal isolates of HCT-116 cells (which have a high mutation rate) were expanded and then subjected to selection with a high dose of HQ461. Resistant clones emerged from five populations. Genomic DNA from five resistant clones was pooled for whole-exome sequencing and compared to a pool of their non-selected counterparts to identify recurrent mutations. [1]
Co-immunoprecipitation (Co-IP): A549 cells with endogenously 3xFLAG-tagged CDK12 were pulverized under liquid nitrogen. Cell powder was solubilized in IP buffer, and lysates were treated with HQ461 or DMSO. 3xFLAG-CDK12 and associated proteins were immunoprecipitated using anti-FLAG antibody-conjugated magnetic beads. After washing, bound proteins were eluted with FLAG peptide and analyzed by western blotting for CDK12, CCNK, and DDB1. [1]
In Vivo Polyubiquitination Assay: HEK293T cells were co-transfected with plasmids encoding 8xHis-ubiquitin, HA-tagged CCNK, and FLAG-tagged CDK12 (wild-type or mutant). Cells were pre-treated with the proteasome inhibitor bortezomib, then treated with HQ461. Cells were lysed in a urea-based buffer, and His-tagged (ubiquitinated) proteins were purified using Ni-NTA beads. Ubiquitination of CCNK and CDK12 was detected by western blotting with anti-HA and anti-FLAG antibodies. [1]
Western Blotting for Protein Degradation: Cells (e.g., A549) were treated with HQ461 for various times. Cells were lysed in a buffer containing SDS and benzonase. Protein concentrations were quantified, and equal amounts were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific antibodies (e.g., against CCNK, CDK12, phospho-Ser2 RNA Pol II CTD, total RNA Pol II CTD). [1]
RT-qPCR for Gene Expression: Total RNA was extracted from HQ461-treated A549 cells using a phenol-based reagent. RNA was reverse transcribed into cDNA. Quantitative PCR was performed using a fluorescent dye-based master mix on a real-time PCR system. mRNA levels of DNA damage response genes (BRCA1, BRCA2, ATR, ERCC4) were normalized to GAPDH. [1]
Colony Formation Assay: Parental A549 cells or A549 cells expressing an sgRNA-resistant CCNK cDNA were infected with lentivirus expressing a non-targeting control sgRNA or an sgRNA targeting CCNK. One day post-infection, a low number of cells were plated per well and grown for 10 days with medium changes. Colonies were fixed, stained with crystal violet, and imaged. [1]

[1]. Discovery of a molecular glue promoting CDK12-DDB1 interaction to trigger cyclin K degradation. Elife. 2020 Aug 17;9:e59994.

HQ461 was discovered through high-throughput screening of phenotype-based NRF2 inhibitors. It represents a new class of molecular gel degraders that can directly recruit target proteins (CDK12/CCNK) to the adaptor protein DDB1 of the CRL4 E3 ligase, thereby bypassing the dependence of the classical substrate receptor (DCAF). [1]
Mechanically, HQ461 binds to the ATP-binding pocket of the CDK12 kinase domain, inducing conformational changes or the formation of a new protein surface that interacts with the BPC domain of DDB1. This leads to polyubiquitination and degradation of the CDK12 binding chaperone CCNK, but does not lead to the degradation of CDK12 itself. [1]
Degradation of CCNK impairs the kinase activity of CDK12 and CDK13, leading to decreased phosphorylation at serine 2 site of the C-terminal domain of RNA polymerase II and downregulation of key DNA damage response (DDR) genes (such as BRCA1 and BRCA2), thereby inducing a "BRCA-like" phenotype. [1]
This study included a structure-activity relationship (SAR) analysis of HQ461, which identified the 5-methylthiazol-2-amine moiety as the key pharmacophore and yielded an analogue with enhanced degradation ability (HQ005). [1]

C15H15N5OS2

345.442499399185

345.07

C, 52.16; H, 4.38; N, 20.27; O, 4.63; S, 18.56

1226443-41-9

2755241-76-8 (HBr);1226443-41-9;

49677967

Gray to light brown solid powder

3.1

2

7

5

23

413

0

QMRBCNQCRMTXBE-UHFFFAOYSA-N

InChI=1S/C15H15N5OS2/c1-9-4-3-5-12(17-9)19-15-18-11(8-22-15)6-13(21)20-14-16-7-10(2)23-14/h3-5,7-8H,6H2,1-2H3,(H,16,20,21)(H,17,18,19)

2-[2-[(6-methylpyridin-2-yl)amino]-1,3-thiazol-4-yl]-N-(5-methyl-1,3-thiazol-2-yl)acetamide

HQ-461; HQ461; HQ 461

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~62.5 mg/mL (~180.9 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)