Pluripotin (also known as SC1; SC-1) is a dual inhibitor of extracellular signal-regulated kinase 1 (ERK1, MAPK3) and RasGAP as well as a sustainer of mES self-renewal. With an EC50 of 2.5±1.8 μM, pluripotin inhibits the in vitro kinase activity of RSK2. It keeps embryonic stem cells' (ESC) ability to renew itself. SC1 has an impact on mES cells only when both protein activities are simultaneously inhibited, which is both necessary and sufficient. Ras is activated by SC1 by blocking RasGAP activity. The C57BL/6-derived ES cells' derivation effectiveness and pluripotency were enhanced by SC1. When cultured with SC1, three different pluripotent stem cell types (fES, ntES, and iPS cells) of the C57BL/6 background were highly effective at producing full-term pups.

Physicochemical Properties

Molecular Formula	C27H25F3N8O2
Molecular Weight	550.53
Exact Mass	550.205
Elemental Analysis	C, 58.90; H, 4.58; F, 10.35; N, 20.35; O, 5.81
CAS #	839707-37-8
Related CAS #	839707-37-8
PubChem CID	12003241
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Index of Refraction	1.662
LogP	2.37
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	5
Heavy Atom Count	40
Complexity	925
Defined Atom Stereocenter Count	0
SMILES	0
InChi Key	NBZFRTJWEIHFPF-UHFFFAOYSA-N
InChi Code	InChI=1S/C27H25F3N8O2/c1-15-8-9-20(32-24(39)17-6-5-7-19(11-17)27(28,29)30)12-21(15)38-14-18-13-31-25(34-23(18)36(3)26(38)40)33-22-10-16(2)35-37(22)4/h5-13H,14H2,1-4H3,(H,32,39)(H,31,33,34)
Chemical Name	N-[3-[7-[(2,5-dimethylpyrazol-3-yl)amino]-1-methyl-2-oxo-4H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-(trifluoromethyl)benzamide
Synonyms	Pluripotin; SC-1; SC 1; SC1
HS Tariff Code	2934.99.9001
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)

Biological Activity

Targets	ERK1 (Kd = 98 nM); RasGAP (Kd = 212 nM); RSK1 (Kd = 0.5 μM); RSK2 (Kd = 2.5 μM); RSK3 (Kd = 3.3 μM); RSK4 (Kd = 10 μM)
ln Vitro	SC1 is a dual-purpose small molecule inhibitor of ERK1 and RasGAP, and it only affects mES cells when both protein activities are simultaneously inhibited. Ras is activated by SC1 by blocking RasGAP function[1]. The C57BL/6-derived ES cells' derivation efficiency and pluripotency were enhanced by SC1. When cultured with SC1, three different types of pluripotent stem cells (fES, ntES, and iPS cells) with a C57BL/6 background were highly efficient at producing full-term pups[1].
ln Vivo	Female NOD.SCID mice aged six weeks were randomly assigned to 20 treatment arms (n = 5 per group). Prior to being diluted in RPMI 1640 (at 10, 100, 1,000, and 10,000 cells/inoculum) for subcutaneous injection into the axillary region using a volume of 1 ml/mouse, treated and control colon tumor lines were enzymatically harvested and counted. All mice in the positive control groups developed tumors within a week or so, demonstrating the tumorigenicity of all tumor lines investigated. The positive control groups received cell inocula ranging from 1×106 to 2.5×106. Trypan blue exclusion was used to determine the cell viability of the cell stocks before they were diluted for injection, and it was regularly found to be >95%. Tumor formation was monitored weekly using caliper measurements and the tumor mass was calculated as weight in mg  = 1/2×(tumor length)×(tumor width)2. At 120 days or when any mouse's tumor weight exceeded 2000 mg, all experiments came to an end. Prior to injection, tumor cells in some studies were resuspended in Matrigel (50% concentration, BD Biosciences).
Enzyme Assay	Pluripotin (SC-1) inhibits in vitro kinase activity of RSK2 with EC50 of 2.5±1.8 μM. The RSK2 in vitro kinase inhibition assay used was a modification of the guidelines set forth by Nguyen et al. At Reaction Biology Corporation (Malvern, PA), additional protocols that verified the outcomes shown in Figure 4 and assessed kinase inhibitory activity in a randomly chosen protein kinase were carried out. It was a miniature 33P-based screening assay.
Cell Assay	mES cells are plated in six-well gelatin-coated plates at a density of 1.6×104 cells/cm2 and maintained with 3 μM SC1 in ESC-SR media, 1 μM SC1 in ESC-N2B27 media, or 300 nM SC1 in ESC-N2 media without LIF or feeder cells. As a positive control, mES cells maintained in ESC-N2B27 media with 103 units/ml LIF and 10 ng/ml BMP4 are used. As negative controls, mES cells are exposed to DMSO for two passages in ESC-SR, ESC-N2B27, or ESC-N2 media. Every three days, cells are divided and seeded at the same density (1.6×104 cells/cm2). FACS, immunocytochemistry, histocytochemistry, and RT-PCR are used to examine mES cells at passage 11.
Animal Protocol	NA Female NOD.SCID mice
References	[1]. Proc Natl Acad Sci U S A . 2006 Nov 14;103(46):17266-71. [2]. PLoS One . 2014 Sep 11;9(9):e106916. [3]. PLoS One . 2013;8(2):e57099.

Solubility Data

Solubility (In Vitro)

DMSO: ~100 mg/mL (~181.6 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.54 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 (4.54 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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.8164 mL	9.0822 mL	18.1643 mL
	5 mM	0.3633 mL	1.8164 mL	3.6329 mL
	10 mM	0.1816 mL	0.9082 mL	1.8164 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.