Ispinesib (SB-715992)

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Biological Activity

Description	Ispinesib (SB-715992, CK0238273) is a potent, specific and reversible inhibitor of kinesin spindle protein (KSP) with Ki app of 1.7 nM, no inhibition to CENP-E, RabK6, MCAK, MKLP1, KHC or Kif1A. Phase 1/2.
Targets	KSP (HsEg5)
IC50	1.7 nM (Ki app) [1]
In vitro	Ispinesib is a potent, allosteric, reversible, and specific inhibitor of KSP, which changes the binding property of KSP to microtubules and disturbs its movement by inhibiting ADP release without altering the release of the KSP-ADP complex from the microtubule. [1] Ispinesib shows potent cytotoxic activity in a panel of tumor cell lines, including Colo205, Colo201, HT-29, M5076, Madison-109, and MX-1, with IC50 of 1.2 nM to 9.5 nM. [2] In PC-3 prostate cancer cells, Ispinesib (15 nM and 30 nM) blocks cell proliferation and induces apoptosis by regulating the expression levels of genes that controls apoptosis, cell proliferation, cell cycle, and cell signaling, such as EGFR, p27, p15, and IL-11. [3] In a panel of 53 breast cell lines, Ispinesib (7.4 nM–600 nM) demonstrates broad inhibitory activity. In BT-474 and MDA-MB-468 cells, Ispinesib (150 nM) induces apoptosis, as revealed by a higher proportion of apoptotic cells, lower antiapoptotic Bcl-XL level, and higher proapoptotic Bax and Bid levels. [4]
In vivo	Ispinesib (4.5 mg/kg–15 mg/kg) exhibits inhibitory effects against Colo205, Colo201, HT-29, but not MX-1 cells, in mouse xenograft models. SB-715992 (6 mg/kg–10 mg/kg ) also inhibits murine solid tumors, including Madison 109 lung carcinoma, M5076 sarcoma, as well as L1210 and P388 leukemias. [2] In mice xenograft models of breast cancer cells MCF-7, HCC1954, MDA-MB-468, and KPL4, Ispinesib (8 mg/kg–10 mg/kg) inhibits tumor growth. [4]
Features	An allosteric, potent, specific, and reversible inhibitor of the mitotic kinesin spindle protein (KSP) (HsEg5).

Protocol(Only for Reference)

Kinase Assay: ^[1]

Steady-State Kinetic Analysis of Human KSP ATPase Activity and Inhibition by Ispinesib

Kinesin specificity analysis is carried out using a pyruvate kinase-lactate dehydrogenase detection system that couples the production of ADP to oxidation of NADH. Absorbance changes are monitored at 340 nm. Steady-state studies using nanomolar concentrations of KSP are performed using a sensitive fluorescence-based assay utilizing a pyruvate kinase, pyruvate oxidase, and horseradish peroxidase (HRP) coupled detection system that couples the generation of ADP to oxidation of Amplex Red to fluorescent resorufin. Generation of resorufin is monitored by fluorescence (λexcitation = 520 nm and λemission = 580 nm). Steady-state biochemical experiments are performed in PEM25 buffer [25 mM Pipes-K+ (pH 6.8), 2 mM MgCl2, 1 mM EGTA] supplemented with 10 µM paclitaxel for experiments involving microtubules. The IC50 for steady-state inhibition is determined at 500 µM ATP, 5 µM Microtubules, and 1 nM KSP in PEM25 buffer. Ki app (apparent inhibitor dissociation constant) values of Ispinesib are extracted from the dose-response curves, with explicit correction for enzyme concentration by using the Morrison equation. Inhibitor modality (e.g., competitive, noncompetitive, uncompetitive, or mixed) under steady-state conditions is determined by measuring the effect of inhibitor concentration on initial velocity as a function of substrate concentrations. Data are fit using equations in GraFit to velocity equations for the various modes of inhibition.

Cell Assay: ^[4]

Cell lines	Breast cancer cells, including MCF-7, HCC1954, MDA-MB-468, and KPL4
Concentrations	0.085 nM–33 µM
Incubation Time	72 hours
Method	Cells are plated in log phase of growth in 96-well plates and treated with Ispinesib for 72 hours. Then, cell growth is measured using CellTiter-Glo, and luminescence is detected using BioTek FLx800. Data are analyzed and the IC50 value, defined as the drug concentration that results in 50% growth inhibition relative to control, is calculated.

Animal Study: ^[4]

Animal Models	Nude (nu/nu) mice models of MCF7, KPL4, and HCC1954 cells; severe combined immunodeficient (SCID) mice model of MDA-MB-468 cells;
Dosages	10 mg/kg for nude mice or 8 mg/kg for SCID mice
Administration	Intraperitoneal injection on a q4d× schedule (3 doses, every 4 day

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00095628	Completed	Metastatic Squamous Neck Cancer With Occult Primary Squamous Cell Carcinoma|Recurrent Metastatic Squamous Neck Cancer With Occult Primary|Recurrent Salivary Gland Cancer etc.	National Cancer Institute (NCI)	2005-01	Phase 2
NCT00096499	Completed	Adenocarcinoma of the Prostate|Recurrent Prostate Cancer|Stage IV Prostate Cancer	National Cancer Institute (NCI)	2005-04	Phase 2
NCT00354250	Completed	Recurrent Renal Cell Cancer|Stage III Renal Cell Cancer|Stage IV Renal Cell Cancer	National Cancer Institute (NCI)	2006-05	Phase 2
NCT00363272	Completed	Childhood Burkitt Lymphoma|Childhood Central Nervous System Germ Cell Tumor|Childhood Choroid Plexus Tumor|Childhood Craniopharyngioma etc.	National Cancer Institute (NCI)	2006-06	Phase 1
NCT00607841	Completed	Breast Neoplasms	Cytokinetics	2007-12	Phase 1|Phase 2

Chemical Information

Download Ispinesib (SB-715992) SDF

Chemical Name	(R)-N-(3-aminopropyl)-N-(1-(3-benzyl-7-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-2-methylpropyl)-4-methylbenzamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.51706	5.1706	10.3412	15.5118

Customer Reviews (2)

Click to enlarge	Rating
	Source	PLoS One. 2013 Apr 10;8(4):e60334 . Ispinesib (SB-715992) purchased from Selleck
	Method	Cell imaging-based efflux assay
	Cell Lines	KB-V1 cells
	Concentrations	0-20 uM
	Incubation Time	24 h
	Results	Ispinesib inhibited ABCB1-medicated calcein AM efflux in the flow cytometry assay and displayed dose-dependent inhibition of ABCB1-mediated efflux in our cell imaging-based efflux assay.

Product Citations (1)

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  Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference. [Ansbro MR, et al. PLoS One 2013;8(4):e60334]