The efficacy of the PDK1 inhibitor (PDKi) GSK2334470 is tested in newborn BrafV600E::Pten−/−mice subjected to systemic administration of 4-HT. Twice weekly administration of PDK1 results in marked inhibition of pigmented lesions and concomitant melanomagenesis, as well as significant inhibition of lung metastases, seen by H&E staining-based quantification (~80%), and lymph node metastases as by S100 immunostaining, similar to the phenotype seen upon genetic ablation of Pdk1[3].
In Vitro
Small molecule GSK2334470 inhibits PDK1 with an IC50 of ~10 nM, but does not suppress the activity of 93 other protein kinases including 13 AGC-kinases most related to PDK1 at 500-fold higher concentrations. Addition of GSK2334470 ablates T-loop residue phosphorylation and activation of SGK isoforms and S6K1 induced by serum or IGF-1 (insulin-like growth factor 1). GSK2334470 and AZD8055 effectively inhibite phosphorylation of PDK1 and mTOR, respectively, and induce higher G0–G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effecte on phosphorylation of GSK3β in some of resistant sublines[2].
Kinase Assay
Cell Assay
GSK2334470 is dissolved in DMSO and diluted with appropriate medium before use. To study the inhibitory effect of GSK2334470 on mTOR-S6K pathway, non-resistant cells and the resistant sublines are treated with GSK2334470 at 5 μM for 1.5 and 12 h in 10 % FBS medium with/without MK-2206 (5 μM)[2].
Animal Administration
Mice is dissolved in DMSO and then diluted with PBS or saline. BrafV600E::Pten−/− are generated as previously described. Cohorts of six animals per group are used in each experimental group. GSK2334470 is administered through IP injection (100 mg/kg) 3 times per week starting the same day of topical administration of 4-hydroxytamoxifen and ending at the time of mouse collection, based on earlier studies[3].
References
[1]. Najafov A, et al. Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1. Biochem J.?2011 Jan 15;433(2):357-69.
[2]. Qi L, et al. PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells. Cancer Cell Int.?2015 Sep 29;15:91.
[3]. Scortegagna M, et al. Genetic inactivation or pharmacological inhibition of Pdk1 delays development and inhibits metastasis of Braf(V600E)::Pten(-/-) melanoma. Oncogene. 2014 Aug 21;33(34):4330-9.
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