Zosuquidar (LY335979) 3HCl

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Biological Activity

Description	Zosuquidar (LY335979) is a potent modulator of P-glycoprotein-mediated multi-drug resistance with Ki of 60 nM. Phase 3.
Targets	P-glycoprotein
IC50	60 nM (Ki) [1]
In vitro	LY335979 competitively inhibits equilibrium binding of [3H]vinblastine to Pgp by blocking [3H]azidopine photoaffinity labeling of the Pgp in CEM/VLB100 plasma membranes. [1] LY335979 alone shows the cytotoxicity to drug-sensitive and MDR cell lines with IC50 ranging from 6 μM-16 μM and produces its ability to completely reverse the resistance of the oncolytics (vinblastine, doxorubicin, or etoposide) to the MDR cell lines P388/ADR, MCF7/ADR, 2780AD, or UCLA-P3.003VLB at concentration of 0.1 and 0.5 μM. [1] LY335979 significantly restores drug sensitivity in P-gp-expressing leukemia cell lines including K562/HHT40, K562/HHT90, K562/DOX and HL60/DNR, and enhances the cytotoxicity of anthracyclines (daunorubicin, idarubicin, mitoxantrone) and gemtuzumab ozogamicin (Mylotarg) in primary AML blasts with active P-gp. [2] A latest paper indicates that LY335979 completely inhibits apically directed transport of (Z)-endoxifen in the ABCB1-transduced cells. [3]
In vivo
Features

Protocol(Only for Reference)

Kinase Assay: [1]

ATPase Assay

P-Glycoprotein ATPase activity is measured by the liberation of inorganic phosphate from ATP. The assay is measured in a 96-well plate for 90 min at 37 °C. Membranes (8 μg-10 μg protein) are incubated in a total volume of 100 μL of buffer A containing 5 mM sodium azide, 1 mM ouabain, 1 mM EGTA, 3 mM ATP, an ATP regenerating system composed of 5 mM phosphoenolpyruvate, and 3.6 units/mL pyruvate kinase in the presence and absence of 1 mM sodium vanadate. Pgp-ATPase activity is defined as the vanadate-sensitive portion of the total ATPase activity. Plates are read 3 minutes after the addition of the detection solution. The absorbance is measured at 690 nm by a microtiter dish reader. A phosphate standard curve is used to calculate the μmol of phosphate formed. Samples are measured in triplicate.

Cell Assay: [1]

Cell lines	CEM/VLB100, P388/ADR, MCF7/ADR, 2780AD, and UCLA-P3.OO3VLB cells
Concentrations	0.05 μM to 5 μM
Incubation Time	72 hours
Method	Cell viability is determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction method. Cells are harvested during logarithmic growth phase, and seeded in 96-well plates. The cells are then cultured for 72 hours in the presence of oncolytics with or without modulators. MCF-7 and MCF-7/ADR cells are incubated 24 hours before the addition of the drug with and without the LY335979. LY335979 is prepared as 2 nM DMSO stocks and added to wells to give final concentrations ranging from 0.05 to 5 μM. After 72 hours, 20 μL of freshly prepared 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL in Dulbecco’s PBS) is added to each well and incubated for 4 hours in a 37 °C incubator containing 5% CO2. Cells are pelleted in a Sorvall RT6000B centrifuge, 70 μL of medium is carefully removed from each well, and 100 μL of 2-propanol/0.04 N HC1 is added. Cells are resuspended 5-10 times with a Multipipettor or until no particulate matter is visible. Plates are immediately read on a Titertek Multiskan MCC/340 microplate reader Flow Laboratories with a test wavelength of 570 nm and a reference wavelength of 630 nm. Controls are measured in quadruplicate and modulators are measured in duplicate. Cytotoxicity analyses are also performed using the CeliTiter 96 AQueous assay kit.

Animal Study: [1]

Animal Models	P388 or P388/ADR cells are implanted by i.p. injection into female BDF1 mice.
Dosages	≤30 mg/kg
Administration	Administered via i.p. and i.v.
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 30 mg/mL

1

References

Chemical Information

Download Zosuquidar (LY335979) 3HCl SDF

Chemical Name	1-Piperazineethanol, 4-[(1aα,6α,10bα)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-α-[(5-quinolinyloxy)methyl]-,trihydrochloride

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.63699	6.3699	12.7398	19.1097

Customer Reviews (2)

Click to enlarge	Rating
	Source	Pharmacol Res, 2013, 67(1):79-83. Zosuquidar (LY335979) 3HCl purchased from Selleck
	Method	flow cytometry
	Cell Lines	K562, K562/DoxDR1, K562/DoxDR2, K562/DoxDR3, K562/Dox
	Concentrations
	Incubation Time
	Results	Application of P-gp inhibitor Zosuquidar(ZSQ) reversed the resistance to DNR.

Product Citations (2)

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  Can P-glycoprotein mediate resistance to nilotinib in human leukemia cells? [Kosztyu P, et al. Pharmacol Res 2013;67(1):79-83]

  
  


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  PARP Inhibitors as P-glyoprotein Substrates. [Lawlor D J Pharm Sci 2014;10.1002/jps.23952]