OSI-027 936890-98-1

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Molekulargewicht: 406,44 OSI-027 ist ein selektiver und potenter dualer Inhibitor von mTORC1 und mTORC2 mit IC50 von 22 nM und 65 nM und einer mehr als 100-fachen Selektivität für mTOR als PI3Kα, PI3Kβ, PI3Kγ oder DNA-PK. Phase 1.
OSI-027 zeigt die selektive und ATP-kompetitive Hemmungsaktivität gegen mTORC1 und mTORC2 mit IC50 von 22 nM bzw. 65 nM. Darüber hinaus hemmt OSI-027 die mTOR-Signalgebung von Phospho-4E-BP1 mit einem IC50 von 1 μM in zellbasierten Assays. OSI-027 zeigt dosisabhängig antiproliferative Aktivitäten gegen mehrere akute Leukämie-Zelllinien myeloischen/megakaryocytischen Ursprungs, darunter U937-, KG-1-, KBM-3B-, ML-1-, HL-60- und MEG-01-Zellen . Eine aktuelle Studie zeigt, dass die Hemmung von mTORC1/2 durch OSI-027 die Phosphorylierung von Akt (S473) und die Zellproliferation in Brustkrebszellen effektiv unterdrückt. In GEO kolorektalen Xenotransplantaten hemmt OSI-027 (65 mg/kg) sowohl die mTORC1- als auch die mTORC2-Effektoren, einschließlich der 4E-BP1-, Akt- und S6-Phosphorylierung. Darüber hinaus hemmt die mTORC1- und mTORC2-Hemmung zusammen durch OSI-027 das Tumorwachstum stärker als die mTORC1-Hemmung durch Rapamycin.

Biochemical assays

mTORC1 and mTORC2 inhibition is assayed using native enzyme complex immunoprecipitated from HeLa lysates at 1 mM ATP. To prepare whole cell lysates from HeLa cells, 25 g cell pellet is lysed in 60 mL of ice-cold buffer A [40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 0.5 mM orthovanadate, and EDTA-free protease inhibitors containing 0.3% CHAPS] for 30 minutes on a magnetic stirrer in a cold room. After clearing of the lysates by centrifugation at 13,000 g for 10 minutes, Protein G-coated 384-well plates are incubated with 0.25 μg of mTOR antibody in 15 μL of buffer A for 1 hour at 4 °C. To each well, 40 μg of HeLa cell lysate in 15 μL of buffer A is added and incubated overnight at 4 °C to immunoprecipitate mTOR complexes. Plates are washed 3 times with buffer A and twice with immunoprecipitation wash buffer [Buffer B: 50 mM HEPES (pH 7.5) and 150 mM NaCl]. OSI-027 is added at 10 μM concentration to each well and DMSO is added to the control wells. The reaction is started by adding 150 ng of His-tagged 4E-BP1 as a substrate in the presence or absence of 100 μM ATP to each well in 25 μL of freshly prepared kinase buffer [Buffer C: 20 mM HEPES (pH 7.5), 10 mM MgCl2, 4 mM MnCl2, 10 mM β-mercaptoethanol, and 200 μM vanadate] and incubated at room temperature (RT) for 30 minutes. The reaction is stopped by transferring 25 μL of reaction mixture from each well to corresponding wells of fresh Ni-chelate-coated plates and incubated overnight at 4 °C followed by 2 hours at 37 °C. To detect phosphorylation of 4E-BP1, the plates are washed once with TBST (Tris-buffered saline containing 0.1% Tween-20) containing 5% skim milk powder. To each well, 25 μL of 1:1,000 diluted phospho-4E-BP1 antibodies in TBST containing 5% skim milk are added and incubated for 1 hour at RT. The plates are washed once with TBST and then 25 μL of anti-rabbit HRP (diluted 1:10,000) in TBST containing 5% skim milk is added. The plates are incubated for 1 hour at RT and washed 5 times with TBST. For detection of phospho-4E-BP1, 25 μL of chemiluminescent reagents A+B is added and chemiluminescence is measured using an Analyst plate reader.

Cell lines	U937, KG-1, KBM-3B, ML-1, HL-60, and MEG-01
Concentrations	0-10 μM
Incubation Time	72 hours
Method	Inhibition of proliferation is measured using the Cell Titer Glo Assay , as noted in figure legends. To generate dose–response curves, cell lines are seeded at a density of 5,000 cells per well in a 96-well plate. After 24 hours of plating, cells are dosed with varying concentrations of either OSI-027 or rapamycin. The signal for Cell Titer Glo Assay is determined 72 hours after dosing and normalized to that of vehicle-treated controls. Inhibition of proliferation, relative to vehicle-treated controls, is expressed as a fraction of 1 and graphed using PRISM software.

Animal Models	GEO colorectal cells are injected s.c. into the right flank of nu/nu CD-1 mice.
Formulation	Dissolved in DMSO and then diluted in water.
Dosages	≤65 mg/kg
Administration	Administered via gavage.
Solubility	1% DMSO/30% polyethylene glycol/1% Tween 80, 30 mg/mL
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Umrechnung verschiedener Modelltiere basierend auf BSA (Wert basierend auf Daten aus FDA Draft Guidelines)

Species	Baboon	Dog	Monkey	Rabbit	Guinea pig	Rat	Hamster	Mouse
Weight (kg)	12	10	3	1.8	0.4	0.15	0.08	0.02
Body Surface Area (m2)	0.6	0.5	0.24	0.15	0.05	0.025	0.02	0.007
Km factor	20	20	12	12	8	6	5	3

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

Um beispielsweise die Resveratrol-Dosis für eine Maus (22,4 mg/kg) auf eine Dosis basierend auf dem BSA für eine Ratte zu ändern, multiplizieren Sie 22,4 mg/kg mit dem Km-Faktor für eine Maus und dividieren Sie dann durch den Km-Faktor für eine Ratte. Diese Berechnung ergibt eine Ratten-Äquivalentdosis für Resveratrol von 11,2 mg/kg.

Rat dose (mg/kg) = mouse dose (22.4 mg/kg) ×	mouse Km(3)	= 11.2 mg/kg
	rat Km(6)

Chemische Informationen

Molecular Weight (MW)	406.44
Formula	C21H22N6O3
CAS No.	936890-98-1

Storage	3 years -20℃Powder
	6 months-80℃in solvent (DMSO, water, etc.)
Synonyms	ASP4786

Solubility (25°C) *	In vitro	DMSO	18 mg/mL (44.28 mM)
		Water	<1 mg/mL (
		Ethanol	<1 mg/mL (
	In vivo	1% DMSO/30% polyethylene glycol/1% Tween 80	30 mg/mL
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Name	(1r,4r)-4-(4-amino-5-(7-methoxy-1H-indol-2-yl)imidazo[1,5-f][1,2,4]triazin-7-yl)cyclohexanecarboxylic acid

Molaritätsrechner Verdünnungsrechner Molekulargewichtsrechner

Produktgruppe : PI3K/Akt/mTOR > mTOR-Inhibitor