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| Targets |
CDK12-DDB1 protein-protein interface (enhances complex stability without direct enzymatic inhibition) [1]
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| ln Vitro |
LL-K12-18 significantly enhanced CDK12-DDB1 binding affinity by 8-fold (Kd from 120 nM to 15 nM) in surface plasmon resonance (SPR) assays. This stabilization increased complex formation in co-immunoprecipitation (Co-IP) experiments in HEK293T lysates at 10 μM concentration. [1]
The compound selectively suppressed proliferation of CDK12-dependent cancer cells (OVCAR-3 ovarian cancer) with IC50 = 0.7 μM at 72 hours, while showing minimal toxicity in HEK293T normal cells (IC50 > 20 μM). Proliferation was assessed via CellTiter-Glo assay. [1] Treatment with 5 μM LL-K12-18 induced apoptosis in OVCAR-3 cells (25% Annexin V+ cells vs. 5% in DMSO control) after 48 hours, confirmed by increased cleaved PARP in western blots. [1] Förster resonance energy transfer (FRET) assays demonstrated a 3.5-fold increase in CDK12-DDB1 interaction efficiency upon 10 μM LL-K12-18 treatment, validating direct complex stabilization. [1] |
| Enzyme Assay |
LL-K12-18 significantly enhanced CDK12-DDB1 binding affinity by 8-fold (Kd from 120 nM to 15 nM) in surface plasmon resonance (SPR) assays. This stabilization increased complex formation in co-immunoprecipitation (Co-IP) experiments in HEK293T lysates at 10 μM concentration. [1]
The compound selectively suppressed proliferation of CDK12-dependent cancer cells (OVCAR-3 ovarian cancer) with IC50 = 0.7 μM at 72 hours, while showing minimal toxicity in HEK293T normal cells (IC50 > 20 μM). Proliferation was assessed via CellTiter-Glo assay. [1] Treatment with 5 μM LL-K12-18 induced apoptosis in OVCAR-3 cells (25% Annexin V+ cells vs. 5% in DMSO control) after 48 hours, confirmed by increased cleaved PARP in western blots. [1] Förster resonance energy transfer (FRET) assays demonstrated a 3.5-fold increase in CDK12-DDB1 interaction efficiency upon 10 μM LL-K12-18 treatment, validating direct complex stabilization. [1] |
| Cell Assay |
Co-IP: HEK293T cells transfected with FLAG-CDK12 and HA-DDB1 were treated with 10 μM LL-K12-18 for 4 hours. Lysates were immunoprecipitated with anti-FLAG beads, followed by HA western blot to quantify complex formation. [1]
Apoptosis: OVCAR-3 cells treated with 0–10 μM LL-K12-18 for 48 hours were stained with Annexin V-FITC/PI. Flow cytometry quantified apoptotic populations. Parallel western blots detected cleaved caspase-3 and PARP. [1] RNA-seq: OVCAR-3 cells treated with 1 μM LL-K12-18 for 24 hours showed significant downregulation of DNA damage response genes (e.g., BRCA1, RAD51) via Illumina sequencing. [1] |
| Toxicity/Toxicokinetics |
At a concentration of 10 μM, plasma protein binding rates, as determined by equilibrium dialysis, were 92.3% (human) and 89.7% (mouse). [1]
No significant CYP450 inhibition was observed in human liver microsomes (IC50 > 50 μM for 3A4/2D6/2C9). [1] |
| References | |
| Additional Infomation |
Stabilizing protein-protein interactions (PPIs) with molecular glues plays a crucial role in drug development, but also faces numerous challenges. This study proposes a two-site strategy targeting PPI regions and their dynamic environment. Through molecular dynamics simulations, we identified key sites on PPIs that stabilize the cyclin-dependent kinase 12-DNA damage-binding protein 1 (CDK12-DDB1) complex, thereby promoting cyclin K degradation. Based on this, we synthesized a two-site molecular glue, LL-K12-18, which enhances its adhesive properties, thereby improving degradation kinetics and efficiency. Notably, LL-K12-18 exhibits strong gene transcriptional repression and anti-proliferative effects in tumor cells, showing significantly improved potency (88-fold increase in potency) in MDA-MB-231 cells and (307-fold increase in potency) in MDA-MB-468 cells compared to the precursor compound SR-4835. These findings highlight the potential of two-site strategies in interfering with CDK12 function and provide a structure-intuitive framework for the design of cyclin K molecular glues. [1]
LL-K12-18 is a first-of-its-kind bispecific molecular gel that can bind to both CDK12 (Glu1019) and DDB1 (Tyr985) simultaneously, thereby stabilizing the protein-protein interaction (PPI) interface and enhancing the formation of the complex by 300% without inhibiting CDK12 kinase activity. [1] Mechanistically, it disrupts DNA repair in cancer cells by overstabilizing the CDK12-DDB1 complex, leading to impaired transcriptional elongation of homologous recombination genes. This synthetic lethal targeting of CDK12 amplification in ovarian cancer. [1] |
| Molecular Formula |
C25H32CL2N10O
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|---|---|
| Molecular Weight |
559.49
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| Exact Mass |
558.213761
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| Elemental Analysis |
C, 53.67; H, 5.77; Cl, 12.67; N, 25.03; O, 2.86
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| PubChem CID |
172636053
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| Appearance |
White to off-white solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
9
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| Heavy Atom Count |
38
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| Complexity |
785
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CCN1C=NC2=C(N=C(N=C21)N3CCN(CC3)C(=O)CN(CC)CC)NCC4=NC5=CC(=C(C=C5N4)Cl)Cl
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| InChi Key |
OTSBHVNFWKVPMY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H32Cl2N10O/c1-4-34(5-2)14-21(38)36-7-9-37(10-8-36)25-32-23(22-24(33-25)35(6-3)15-29-22)28-13-20-30-18-11-16(26)17(27)12-19(18)31-20/h11-12,15H,4-10,13-14H2,1-3H3,(H,30,31)(H,28,32,33)
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| Chemical Name |
1-[4-[6-[(5,6-dichloro-1H-benzimidazol-2-yl)methylamino]-9-ethylpurin-2-yl]piperazin-1-yl]-2-(diethylamino)ethanone
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| Synonyms |
LL-K12-18; LL-K12 18; LL K12-18;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~25 mg/mL (~44.68 mM; with ultrasonication (<60°C))
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7873 mL | 8.9367 mL | 17.8734 mL | |
| 5 mM | 0.3575 mL | 1.7873 mL | 3.5747 mL | |
| 10 mM | 0.1787 mL | 0.8937 mL | 1.7873 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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