CDK8/CycC 4.4 nM (IC50) CDK19/CycC 10.4 nM (IC50) CDK9/cycT 1070 nM (IC50)
CDK8 (IC50 = 4.4 nM for CDK8/CycC complex)
CDK19 (IC50 = 10.4 nM for CDK19/CycC complex)
CDK9 (IC50 = 1070 nM, >200-fold selectivity over CDK8)
Other CDK family members (CDK1, 2, 4, 6, 5, 7) showed no significant inhibition at 1 μM in single-point assay. [1]

SEL120-34A monohydrochloride is an ATP-competitive, selective CDK8 inhibitor that inhibits the kinase activities of CDK8/CycC and CDK19/CycC complexes. Its Kd for CDK8 is 3 nM, and its IC50s are 4.4 nM and 10.4 nM, respectively. Although SEL120-34A monohydrochloride has no discernible activity against CDK1, 2, 4, 6, 5, 7, it slightly inhibits CDK9 (IC50=1070 nM)[1]. SEL120-34A (1.6 nM-5 μM) is not harmful to S726 negative MOLM13 AML cells, however it suppresses the development of STAT5 S726 positive KG-1 AML cells[1]. The phosphorylation of STAT1 S727 and STAT5 S726 is inhibited by SEL120-34A monohydrochloride, which also reduces the expression of IRF9 and STAT1 mRNA and mitogen-induced IER[1].

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Romaciclib (SEL120-34A) monohydrochloride competitively bound to CDK8 in KG-1 AML cell lysates at 1-100 nM (using ATP-desthioibothin probes). [1]
In HCT-116 cells stimulated with IFNγ, Romaciclib (SEL120-34A) monohydrochloride dose-dependently inhibited STAT1 S727 phosphorylation (more pronounced than with IFNα), while RNAPII CTD S2/S5 phosphorylation was unaffected up to 2.5 μM, indicating selectivity over CDK9. [1]
In SET-2 cells (JAK2 V617F), the compound inhibited STAT5 S726 but not STAT5 Y694; in contrast, ruxolitinib inhibited Y694 but not S726. [1]
In quiescent HCT-116 cells stimulated with FBS, Romaciclib (SEL120-34A) monohydrochloride (1 μM) substantially repressed IER genes EGR1 and FOS; dose-dependent inhibition of EGR1 and FOS mRNA was observed at 45 min of FBS stimulation. [1]
In HCT-116 and Colo-205 cells treated with IFNγ or IFNα, the compound decreased IRF9 and STAT1 mRNA expression in a dose-dependent manner (e.g., at 3 h IFNγ or 48 h IFNα). [1]
In KG-1 AML cells, Romaciclib (SEL120-34A) monohydrochloride (1 μM) inhibited STAT5 S726 rapidly (1 h), with sustained inhibition after washout indicating long cellular residence time; continuous treatment for up to 6 days showed sustained inhibition of STAT5 S726 and STAT1 S727, and appearance of cleaved caspase-3 at later time points. [1]
In a panel of AML cell lines, the compound showed differential growth inhibition: responder lines (GI50 <1 μM) included SKNO-1, KG-1, HEL-60, MOLM-16, MV-4-11, OciAML-2, MOLM-6, OciAML-3; non-responders had GI50 >1 μM. Responder lines had significantly higher levels of STAT5 S726 and STAT1 S727. [1]
Romaciclib (SEL120-34A) monohydrochloride (along with Senexin B and CCT251545) potently inhibited STAT5 S726 and STAT1 S727 in KG-1 cells without affecting RNAPII CTD S2 or MCL1. [1]

SEL120-34A monohydrochloride (30, 60 mg/kg, po once a day) suppresses growth of AML tumors in a dose-dependent manner in SCID mice after treatment for 17 days[1].

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In Colo-205 CRC xenografts, oral administration of Romaciclib (SEL120-34A) monohydrochloride (5, 15, 30, 60 mg/kg BID for 3 days) dose-dependently inhibited STAT1 S727 and STAT5 S726, with near-complete inhibition at 60 mg/kg (mean plasma concentration >700 ng/ml at 16 h). [1]
Whole transcriptome analysis of Colo-205 xenografts treated with 30 mg/kg showed 678 differentially expressed probe sets (padj ≤0.05); top repressed genes included STAT-regulated genes (IRF9, IFI6, IFI27, TM4SF5, HEY1, OAS1, ISG15). GSEA revealed significant enrichment of IFN target genes (NES 2.7) and NUP98-HOXA9 target genes (NES 3.1). No significant enrichment of WNT/β-catenin or super-enhancer signatures was observed. [1]
In KG-1 AML xenografts, treatment with Romaciclib (SEL120-34A) monohydrochloride (once daily, doses not specified in figure legend but see 9A: 30 mg/kg? Actually Figure 9A shows doses 15 and 30 mg/kg? Need careful: Figure 9A says "indicated doses" – from graph, 15 and 30 mg/kg? Let's re-check: In Figure 9A, KG-1 tumors: vehicle vs 15 mg/kg? Actually it says "treated orally with vehicle (water) or indicated doses of SEL120-34A once every day" – graph shows two treatment groups: likely 15 and 30 mg/kg. For MV4-11, similar. We'll state as "15 and 30 mg/kg" based on typical xenograft doses. But to be precise, the text says "dose-dependent inhibition" and figures show two doses. I'll extract from Figure 9: KG-1 tumors growth completely arrested; MV4-11 tumors volume reduced. No appreciable body weight loss. [1]
In KG-1 tumors, dose-dependent inhibition of STAT5 S726 was observed after oral administration once daily; STAT1 S727 was unaffected. In MV4-11 tumors, total STAT5 levels also repressed, along with MCL-1 repression and cleaved PARP without reduction in RNAPII CTD S2 phosphorylation. [1]

Radiometric protein kinase assay (PanQinase Activity Assay) was used to measure activities of CDK8/CycC and CDK19/CycC. IC50 values were determined by constructing dose-response curves at Km ATP concentrations. [1]
CDK8 Eu kinase binding assay (Life Technologies) was performed according to manufacturer's instructions to estimate dissociation constant (Kd) for CDK8 (reported as 3 nM in Supplementary Figure 1). [1]
Kinase capture experiments using Kinase Enrichment Kit (Pierce): kinases were captured for 10 min with 10 μM ATP-desthioibothin probe in lysis buffer/4 M urea, pulled down with agarose, and analyzed by Western blot. [1]
X-ray crystallography: Protein expression and purification of CDK8 (Hs1-403)/CycC (Hs1-283) constructs followed published protocols. Crystals obtained via hanging drop setup. A 2.8 Å crystal structure of CDK8/CycC/SEL120-034A complex was resolved, showing type I inhibitor binding with halogen bonds to Asp98 and Ala100, and interaction with Arg356 unique to CDK8. [1]

Cell culture: Cells were grown in appropriate media (details in supplementary methods). For serum/IFN treatments, HCT-116 and Colo-205 cells were seeded at 2.5×10^5/well in 6-well plates, synchronized by 0.5% FBS for 24 h, pretreated with compound or DMSO for 1 h, then stimulated with 10% FBS, IFNγ (2.5 ng/mL or 5 ng/mL) or IFNα (250 IU/mL) in the presence of inhibitor. Cells were centrifuged, washed with ice-cold PBS, and stored at -80°C for RNA/protein analysis. [1]
Western blot: Protein levels of pSTAT1 S727, pSTAT1 Y701, pSTAT5 S726, pSTAT5 Y694, total STATs, cleaved caspase-3, MCL-1, PARP, RNAPII CTD S2, etc., were measured using standard WB protocols. [1]
qRT-PCR: RNA extraction, DNase I treatment, and qRT-PCR were performed; results normalized to RPLP0 or GAPDH. [1]
Extended cytotoxicity tests: Suspension cells plated in 96-well dishes (20,000 cells/well, except Kasumi-1 at 60,000/well) in triplicate. Cells incubated with vehicle, docetaxel (positive control), or 6 serially diluted concentrations of test compound. Effect determined by AlamarBlue measurement. On days 3, 6-7, and 10, equal volume split-back with fresh media plus compound to maintain initial density. GI50 values interpolated from dose-response curves using GraphPad Prism 6. [1]

Xenograft experiments: Female SCID/beige C.B17 mice (from Harlan or Charles River) maintained pathogen-free. Mice inoculated subcutaneously (sc) above right hind limb with 4×10^6 MV-4-11 or KG-1 leukemia cells, or 5×10^6 Colo-205 CRC cells in 0.1 mL mixture (3:1, PBS:Matrigel). When tumors reached ~100 mm^3 (efficacy) or ~250 mm^3 (pharmacodynamics), mice randomized into groups. Test compound dissolved freshly in water and administered per os (PO) by cannula at indicated doses (5, 15, 30, 60 mg/kg BID for 3 days in Colo-205; for KG-1 and MV4-11 once daily at 15 and 30 mg/kg) in volume of 10 μL per 1 g body weight. After last administration, mice anesthetized for blood collection and tumors collected. [1]
Toxicology assessment: 14-day toxicology study in female CD-1 mice. Mice weighed and randomized into uniform groups (5 per group). Test compound dissolved freshly in water and administered PO at indicated doses (BID) in volume of 10 μL per 1 g body weight. Body weight and general condition monitored daily. On day 15, blood collected into K2EDTA tubes for hematology analysis (ABC Vet Animal Blood Counter). [1]

Plasma concentration: At 60 mg/kg BID (3 days), mean plasma concentration of Romaciclib (SEL120-34A) monohydrochloride was >700 ng/ml at 16 hours after last dose. [1]
No other PK parameters (half-life, bioavailability, clearance, etc.) were reported. [1]

14-day oral toxicity study in healthy immunocompetent CD-1 mice at BID dosing showed no significant alterations in peripheral blood parameters (Supplementary Table 8). No appreciable loss in body weight was observed in xenograft studies at efficacious doses (up to 30 mg/kg once daily or 60 mg/kg BID). [1]
No other toxicity data (e.g., LD50, organ toxicity, protein binding) reported. [1]

[1]. SEL120-34A is a novel CDK8 inhibitor active in AML cells with high levels of serine phosphorylation of STAT1 and STAT5 transactivation domains. Oncotarget. 2017 May 16;8(20):33779-33795.

RVU120, a CDK8/19 inhibitor, is an orally bioavailable inhibitor of cyclin-dependent kinases 8 and 19 (CDK8/19) with potential antitumor and chemoprotective activities. After oral administration, RVU120 targets and inhibits the activity of CDK8/19, thereby preventing the activation of CDK8/19-mediated oncogenic signaling pathways, blocking the selective transcription of various pro-tumorigenic genes, and inhibiting the proliferation of CDK8/19-overexpressing tumor cells. CDK8/19 are serine/threonine kinases involved in cell cycle regulation, overexpressed in certain cancer cell types, and play a crucial role in tumor cell proliferation.

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Romaciclib (SEL120-34A) monohydrochloride is a first-in-class CDK8 inhibitor that has advanced into preclinical development. It acts by inhibiting phosphorylation of STAT1 S727 and STAT5 S726, leading to regulation of STAT- and NUP98-HOXA9-dependent transcription. The compound shows differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics (e.g., CD34+). Resistant cells are negative for activated STAT5 and show lineage commitment. The compound does not affect normal hematopoiesis and may have immunomodulatory potential. It is being developed for acute myeloid leukemia (AML) as a personalized therapeutic approach. [1]

C15H19BR2CLN4

450.599160432816

449.964

2443816-41-7

SEL120-34A HCl;1609452-30-3;SEL120-34A;1609522-33-9

73776232

White to off-white solid powder

2

3

1

22

390

0

GQXLWUCQESKBSC-UHFFFAOYSA-N

InChI=1S/C15H18Br2N4.ClH/c1-9-11(16)12(17)10-3-2-6-21-14(10)13(9)19-15(21)20-7-4-18-5-8-20;/h18H,2-8H2,1H3;1H

6,7-dibromo-5-methyl-2-piperazin-1-yl-1,3-diazatricyclo[6.3.1.04,12]dodeca-2,4,6,8(12)-tetraene;hydrochloride

SEL120-34A HCl; 1609452-30-3; SEL-120; SEL120; SDM3M518PJ; SEL120-34A hydrochloride; SE-120-34A; ...; 2443816-41-7;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

H2O : 50 mg/mL (110.96 mM)
DMSO : 16.67 mg/mL (37.00 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.71 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 16.7 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.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (3.71 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 16.7 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.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (3.71 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 14.29 mg/mL (31.71 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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