CDK9- Cyclin T1 (IC50 = 44 nM); cdk2-cyclin A (IC50 = 2441 nM); cdk1-cyclin B1 (IC50 = 5513 nM); cdk4-cyclin D1 (IC50 = 9242 nM); GSK3A (IC50 = 1460 nM); HGK/MAP4K4 (IC50 = 820 nM); ABL2/ARG (IC50 = 3640 nM)
Cyclin-dependent kinase 9 (CDK9) [1]

LDC000067 is more selective for CDK9 than other CDKs, even more so than flavopiridol and DRB, which are recognized inhibitors of CDK9. In comparison to CDK2/1/4/6/7, LDC000067 showed 55/125/210/ >227/ >227-fold selectivity for CDK9. LDC000067 inhibits transcription in vitro in a dose-dependent and ATP-competitive manner. Gene expression profiling of LDC000067-treated cells shows a selective reduction of short-lived mRNAs, such as significant regulators of apoptosis and proliferation[1].

---

LDC000067 exhibits higher selectivity for CDK9 over other CDKs compared to known inhibitors flavopiridol and DRB [1]
- LDC000067 inhibits in vitro transcription in an ATP-competitive and dose-dependent manner [1]
- Gene expression profiling shows that LDC000067 selectively reduces short-lived mRNAs, including key regulators of proliferation and apoptosis [1]
- LDC000067 enhances pausing of RNA polymerase II on genes, a characteristic effect of CDK9 inhibition [1]
- LDC000067 induces apoptosis in various cancer cell lines and patient-derived acute myelogenous leukaemia (AML) blasts after 24 hours of treatment, as detected by flow cytometry of annexin V-propidium iodide double-stained cells [1]
- Treatment with LDC000067 leads to decreased global Ser2-P levels of RNA polymerase II (RNAPII) in mouse embryonic stem cells (mESCs) and HeLa cells, as analyzed by Western blot and normalized to RNAPII or loading controls [1]
- LDC000067 inhibits de novo RNA synthesis in a concentration-dependent manner, as demonstrated by RT-qPCR analysis using exon-intron primers in A549 cells, mESCs, and THP1 cells [1]
- In stably transfected HeLa cells, LDC000067 reduces luciferase reporter gene transcripts induced by doxycycline, as confirmed by RT-qPCR and luciferase assay [1]
- ChIP analysis reveals that LDC000067 increases RNAPII pausing at the MYC gene in HeLa cells, with altered distribution of RNAPII and its phosphorylated forms (Ser2-P, Ser5-P, Ser7-P) [1]

The LANCE Ultra KinaSelect Ser/Thr kit, which uses fluorescence resonance energy transfer (FRET) to calculate IC50 values, is used to measure the effectiveness of different CDK inhibitors. In summary, a CDK-cyclin pair phosphorylates a particular ULight MBP peptide substrate (final concentration of 50 nM) in a buffer containing ATP at the concentration of the K_m values of each individual kinase (50 mM HEPES-KOH pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 2 mM dithiothreitol) for one hour at room temperature. Phosphorylation is then identified by adding particular anti-phospho-antibodies (2 nM) labeled with Eu; these antibodies bind to the phosphopeptide and produce a FRET signal. After that, FRET signals are recorded[1].

---

A radiometric in vitro kinase assay is performed to evaluate the inhibitory effect of LDC000067 on kinase catalytic activity. The assay uses 10 μM LDC000067, with DMSO as the control, and measures residual kinase activity expressed as the percentage of remaining substrate phosphorylation. Each kinase is assayed in duplicate, and data are presented as means and range of the two measurements [1]
- A kinase assay using GST-CTD as the substrate is conducted in HeLa nuclear extract treated with 10 μM LDC000067 for 30 minutes. GST-CTD Ser2-P levels are quantified and normalized to Tubulin signals [1]

Specifically, LDC000067 exhibits selectivity for CDK9 over other CDKs ranging from 55-fold to over 230-fold, with an IC50 value of 44(10 nM). Its ATP-competitive kinase binding assay further demonstrates this selectivity. Apoptosis and the tumour suppressor protein p53 are two additional effects of LDC000067 on whole cells. Furthermore, gene expression profiling of cells treated with LDC000067 reveals a selective reduction of short-lived mRNAs, such as MCL1 and MYC, which encode regulators of proliferation and apoptosis.

---

Western blot analysis: mESCs are treated with 10 μM LDC000067 for 90 or 180 minutes (with DMSO and 1 μM flavopiridol as controls), and global Ser2-P levels are quantified using ImageJ, normalized to RNAPII signals. HeLa cells are treated for 60 minutes, with THAP1 as the loading control. MCF7 cells are treated with various concentrations of LDC000067 for 4 hours, with TBP as the loading control [1]
- RT-qPCR analysis for de novo transcription: A549 cells are treated with 1/2/4/6/10 μM LDC000067 for 90 minutes, and mESCs are treated with 2.5/5/10/20/40 μM for 90 minutes; target genes are analyzed using exon-intron (e-i) primers, and mESCs are additionally analyzed with exon-exon (e-e) primers. Stably transfected HeLa cells are treated with 10 μM LDC000067 or DMSO for 30 minutes, followed by 1 μg·mL⁻¹ doxycycline for 60 minutes, and luciferase reporter gene transcripts are analyzed [1]
- Luciferase assay: Dox-induced HeLa reporter cells are treated with increasing concentrations of LDC000067 for specified times, and luciferase activity is measured [1]
- Apoptosis assay: Various cancer cell lines and patient-derived AML blasts are treated with LDC000067 for 24 hours, and apoptosis is detected by flow cytometry using annexin V-propidium iodide double staining. Three biological replicates are used for cell lines, and three technical replicates for AML blasts [1]
- ChIP assay: HeLa cells are treated with 10 μM LDC000067 or DMSO for 1 hour, and the distribution of RNAPII, Ser2-P, Ser5-P, and Ser7-P at the MYC gene is determined using qPCR amplicons specific to different regions of the gene [1]
- Microarray analysis: THP1 cells are treated with LDC000067, and regulated genes are statistically analyzed; GO analysis is performed for down-regulated mRNAs, and RT-qPCR is used to verify unspliced de novo transcripts of selected genes [1]

[1]. Characterization of molecular and cellular functions of the cyclin-dependent kinase CDK9 using a novel specific inhibitor. Br J Pharmacol. 2014 Jan;171(1):55-68.

LDC000067 is a novel, highly specific CDK9 inhibitor designed to address the limitations of existing CDK9 inhibitors (low specificity and/or narrow therapeutic window) [1]. This study provides a framework for understanding the cellular mechanisms of CDK9 inhibition mediated by LDC000067 [1]. LDC000067 is a promising lead compound for the development of highly specific CDK9 inhibitors with clinical applications [1].

C18H18N4O3S

370.43

370.109

C, 58.36; H, 4.90; N, 15.13; O, 12.96; S, 8.65

1073485-20-7

25104564

White to off-white solid powder

1.4±0.1 g/cm3

604.1±65.0 °C at 760 mmHg

319.2±34.3 °C

0.0±1.7 mmHg at 25°C

1.646

2.11

2

7

6

26

539

0

S(C([H])([H])C1C([H])=C([H])C([H])=C(C=1[H])N([H])C1C([H])=C(C2=C([H])C([H])=C([H])C([H])=C2OC([H])([H])[H])N=C([H])N=1)(N([H])[H])(=O)=O

GGQCIOOSELPMBB-UHFFFAOYSA-N

InChI=1S/C18H18N4O3S/c1-25-17-8-3-2-7-15(17)16-10-18(21-12-20-16)22-14-6-4-5-13(9-14)11-26(19,23)24/h2-10,12H,11H2,1H3,(H2,19,23,24)(H,20,21,22)

[3-[[6-(2-methoxyphenyl)pyrimidin-4-yl]amino]phenyl]methanesulfonamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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