[Description]:
YM-201636 is a potent and selective PIKfyve inhibitor with an IC50 of 33 nM. YM-201636 also inhibits p110α with IC50 of 3.3 μM.
[Related Catalog]:
[Target]
PIKfyve:33 nM (IC50)
p110α:3.3 μM (IC50)
Autophagy
[In Vitro]
Acute treatment of cells with YM-201636 shows that the PIKfyve pathway is involved in the sorting of endosomal transport, with inhibition leading to the accumulation of a late endosomal compartment and blockade of retroviral exit. The yeast orthologue of PIKfyve, Fab1, is found to be insensitive to YM-201636 (IC50>5 μM). YM-201636 does not inhibit a type IIγ PtdInsP kinase even at 10 μM and inhibits a mouse type Iα PtdInsP kinase with an IC50>2 μM[1]. YM-201636 almost completely inhibits basal and insulin-activated 2-deoxyglucose uptake at doses as low as 160 nM, with IC50=54 nM for the net insulin response. YM-201636 also completely inhibits insulin-dependent activation of class IA PI 3-kinase[2]. At low doses (10-25 nM), YM-201636 inhibits preferentially PtdIns5P rather than PtdIns(3,5)P2 production, whereas at higher doses, the two products are similarly inhibited. YM-201636 at 160 nM inhibits PtdIns5P synthesis twice more effectively compared with PtdIns(3,5)P2 synthesis[3]. MDCK cells treated with YM-201636 accumulate the tight junction protein claudin-1 intracellularly. YM-201636 treatment blocks the continuous recycling of claudin-1/claudin-2 and delays epithelial barrier formation[4].
[Kinase Assay]
Following 3T3L1 adipocyte serum-starvation and insulin stimulation, cell lysates containing protease inhibitors are clarified and then subjected to immunoprecipitation with anti-PIKfyve antibodies. Washed beads are mixed with 100 μM PtdIns and preincubated for 15 min with YM-201636 (100 nM) or vehicle in the assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EGTA and 10 mM MgCl2). The kinase assay (50 μL final volume) is carried out for 15 min at 37 °C with 15 μM ATP and [γ-32P]ATP (30 μCi). Lipids are extracted, spotted on TLC glass plates (250 μm), resolved by a chloroform/methanol/water/ammonia solvent system and detected by autoradiography[2].
[Cell Assay]
YM-201636 is dissolved in DMSO and diluted with DMEM and added to cells at a final concentration of 800 nM. Cells are treated with YM-201636 or a DMSO control for 2 h. For TER measurements cells are plated at confluency on Transwell permeable polyester filters (0.4 µm pore size) with surface area of 0.33 cm2. Media is changed ever 2-3 days and cells are grown for 7 days prior to TER measurements[4].
[References]
[1]. Jefferies HB, et al. A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep, 2008, 9(2), 164-170.
[2]. Ikonomov OC, et al. YM-201636, an inhibitor of retroviral budding and PIKfyve-catalyzed PtdIns(3,5)P2 synthesis, halts glucose entry by insulin in adipocytes. Biochem Biophys Res Commun. 2009 May 8;382(3):566-70.
[3]. Sbrissa D, et al. Functional dissociation between PIKfyve-synthesized PtdIns5P and PtdIns(3,5)P2 by means of the PIKfyve inhibitor YM-201636. Am J Physiol Cell Physiol. 2012 Aug 15;303(4):C436-46.
[4]. Dukes JD, et al. The PIKfyve inhibitor YM-201636 blocks the continuous recycling of the tight junction proteins claudin-1 and claudin-2 in MDCK cells. PLoS One. 2012;7(3):e28659.
[Related Small Molecules]