Inhibition of neuronal apoptosis by the cyclin‐dependent kinase inhibitor GW8510: Identification of 3′ substituted indolones as a scaffold for the development of neuroprotective drugs

Abstract

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3¢ substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3b and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3¢ substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3¢ substituted indolone) as a neuroprotective compound, our findings identify the 3¢ substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.

Cyclin-Dependent Kinase 5 Inhibitors: Inhibition of Dopamine Transporter Activity

“…1C). GW8510, a structurally dissimilar inhibitor compared with roscovitine or olomoucine that displays selectivity toward Cdk5 in neuronal tissues (Johnson et al, 2005), also reduced specific […”

“…Roscovitine and olomoucine, two commonly used Cdk5 inhibitors, decrease purified Cdk5 activity, with IC 50 values of 0.16 and 3 M, respectively (Meijer et al, 1997), whereas the structurally dissimilar inhibitor GW8510 reduces neuronal apoptosis, with a predicted IC 50 value Ͻ1 M (Johnson et al, 2005). Here, roscovitine, olomoucine, and GW8510 all reduced specific [ 3 H]DA uptake into dSTR synaptosomes in a rapid, concentration-dependent manner, but with somewhat lower potencies than reported for Cdk5 inhibition (IC 50 values of 31, 37, and 7 M, respectively).…”

“…We used rat dSTR synaptosomes and slices containing dSTR to measure the effects of several Cdk5 inhibitors on 1) specific [ 3 H]DA uptake, 2) cell-surface DAT levels, and 3) [ 3 H]DA efflux/release. Cdk5 inhibitors used here included the widely used inhibitor roscovitine and the less selective inhibitor olomoucine (Meijer et al, 1997); isoolomoucine, an inactive congener of olomoucine (Veselý et al, 1994); and the structurally dissimilar inhibitor GW8510, which displays selectivity toward Cdk5 in neuronal tissues (Johnson et al, 2005). Porcine aortic endothelial (PAE) cells stably expressing yellow fluorescent protein (YFP)-hemagglutinin epitope (HA)-human (h)DATs (Sorkina et al, 2006) were used to explore further the findings from rat brain tissue and to test, by using Cdk5-siRNA, the involvement of Cdk5 in the pharmacological effects of roscovitine.…”

Tau-Based Treatment Strategies in Neurodegenerative Diseases

“…110,111 Cdk5 inhibition can be achieved by active site-directed inhibitors, by 2,6,9-trisubstituted purines and aloisines, which are small-molecule inhibitors that interfere with the cdk5/p25 complex formation (Cdk5 inhibitory peptide [CIP]), and by calpain inhibitors, which prevent p25 generation. 101,112,113 The use of active site inhibitors is hampered by their lack of selectivity toward cdk5. Indirubins, which belong to the chemical class indolinones, also inhibit GSK-3.…”

Regulation of S6 Kinase 1 Activation by Phosphorylation at Ser-411

“…GW8510 is a recently developed indolone derivative that specifically inhibits Cdk5 with low cytotoxicity toward cultured neurons (33). The GW8510 treatment of cortical neurons led to a dramatic decrease of Ser-411 and Thr-389 phosphorylation, while Thr-421/Ser-424 phosphorylation remained unchanged (Fig.…”