Abstract
The phosphoinositide 3-kinase/3-phosphoinositide-dependent kinase 1 (PDK1)/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis and represents a promising target for anticancer drugs. Here, we describe three potent PDK1 inhibitors, BX-795, BX-912, and BX-320 (IC(50) = 11-30 nm) and their initial biological characterization. The inhibitors blocked PDK1/Akt signaling in tumor cells and inhibited the anchorage-dependent growth of a variety of tumor cell lines in culture or induced apoptosis. A number of cancer cell lines with elevated Akt activity were >30-fold more sensitive to growth inhibition by PDK1 inhibitors in soft agar than on tissue culture plastic, consistent with the cell survival function of the PDK1/Akt signaling pathway, which is particularly important for unattached cells. BX-320 inhibited the growth of LOX melanoma tumors in the lungs of nude mice after injection of tumor cells into the tail vein. The effect of BX-320 on cancer cell growth in vitro and in vivo indicates that PDK1 inhibitors may have clinical utility as anticancer agents.
Highlights
3-Phosphoinositide-dependent kinase 1 (PDK1)1 is a Ser/Thr protein kinase that can phosphorylate and activate a number of kinases in the AGC kinase superfamily, including Akt/protein kinase B, protein kinase C (PKC), PKC-related kinases (PRK1 and PRK2), p70 ribobsomal S6-kinase (S6K1), and serum and glucocorticoid-regulated kinase (SGK) (1)
Phosphoinositides produced by PI 3-kinase bind directly to the regulatory pleckstrin homology (PH) domain of Akt, driving a conformational change in the molecule which enables the activation loop of AKT1 to be phosphorylated by phosphoinositide-dependent kinase 1 (PDK1) at Thr308 (Thr309 for AKT2 and Thr305 for AKT3) (9, 10)
We found that the compounds potently inhibited PDK1 enzyme activity in a direct kinase assay format (IC50 values for BX-795, BX-912, and BX-320 were 11, 26, and 30 nM, respectively), they failed to block preactivated AKT2 activity (IC50 Ͼ 10 M)
Summary
Materials—Polyclonal antibodies against phospho-Thr308-Akt, phospho-Ser473-Akt, Akt, phospho-Thr389-S6K1, S6K1, phospho-Ser241PDK1, PDK1, phospho-Thr505-PKC␦, PKC␦, and phospho-Ser21/9GSK3, were obtained from Cell Signaling Technologies (Beverly, MA). To measure PDK1 activity directly, the final assay mixture (60 l) contained 50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, 0.1 mM EDTA, 0.1% -mercaptoethanol, 1 mg/ml bovine serum albumin, 10 mM MgOAc, 10 M ATP, 0.2 Ci of [␥-33P]ATP, 7.5 M substrate peptide (H2N-ARRRGVTTKTFCGT), and 60 ng of purified recombinant human PDK1. Trypsinized cells, dispersed into single cells using a 21-gauge needle, were seeded in a 1.5-ml volume of 0.3% agar in growth medium (42 °C temperature) added over the bottom agar layer and allowed to incubate overnight. Quantification of Tumor Burden by Quantitative Real Time PCR—We determined the tumor burden in the mouse lungs by extracting total DNA, measuring the amount of the human CCR5 gene present by quantitative real time PCR. Data Curve Fitting—Data were curve fit to a four-parameter logistic equation using Kaleidagraph software, version 3.51 (Synergy Software, Reading, PA)
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