Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors

Abstract

We developed a high-throughput HTRF (homogeneous time-resolved fluorescence) assay for Akt kinase activity and screened approx. 270000 compounds for their ability to inhibit the three isoforms of Akt. Two Akt inhibitors were identified that exhibited isoenzyme specificity. The first compound (Akt-I-1) inhibited only Akt1 (IC50 4.6 microM) while the second compound (Akt-I-1,2) inhibited both Akt1 and Akt2 with IC50 values of 2.7 and 21 microM respectively. Neither compound inhibited Akt3 nor mutants lacking the PH (pleckstrin homology) domain at concentrations up to 250 microM. These compounds were reversible inhibitors, and exhibited a linear mixed-type inhibition against ATP and peptide substrate. In addition to inhibiting kinase activity of individual Akt isoforms, both inhibitors blocked the phosphorylation and activation of the corresponding Akt isoforms by PDK1 (phosphoinositide-dependent kinase 1). A model is proposed in which these inhibitors bind to a site formed only in the presence of the PH domain. Binding of the inhibitor is postulated to promote the formation of an inactive conformation. In support of this model, antibodies to the Akt PH domain or hinge region blocked the inhibition of Akt by Akt-I-1 and Akt-I-1,2. These inhibitors were found to be cell-active and to block phosphorylation of Akt at Thr308 and Ser473, reduce the levels of active Akt in cells, block the phosphorylation of known Akt substrates and promote TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in LNCap prostate cancer cells.

Scheme 1

Figure 1. Inhibition of Akt phosphorylation in C33A cells

C33A cells were treated with Akt-I-1 (A) or Akt-I-1,2 (B) as described in the Experimental section, and the individual Akt isoforms were immunoprecipitated. The phosphorylation status of each isoform was determined by Western blot analysis using antibodies specific for phospho-Thr³⁰⁸ (P-T308) and phospho-Ser⁴⁷³ (P-S473). The phospho-Ser⁴⁷³ blots were stripped and probed for total Akt levels. Veh/V, DMSO vehicle; A, Akt-I-1; B, Akt-I-1,2. The numbers 1, 2 and 3 refer to the Akt isoenzymes.

Figure 2. Effects of inhibiting Akt1 and Akt2 on downstream signalling events

LnCaP cells were treated with vehicle (lane 1), 20 μM Akt-I-1,2 (lane 2) or 10 μM LY294002 (lane 3) for 18 h. Total cell lysates were prepared and Western blots were performed to determine the levels and migration patterns of Bad and p27^Kip1. Cytosolic extracts were prepared, and the relative amounts of p21^Cip1/Waf1 present following inhibitor treatment was determined by Western blot analysis.

Figure 3. Caspase 3 activity in LNCaP cells treated with Akt inhibitors or LY294002 in the presence or absence of TRAIL

LNCaP cells were treated with either vehicle, Akt-I-1 (A, 25 μM), Akt-I-1,2 (B, 25 μM) or LY294002 (LY, 15 μM) in the presence or absence of TRAIL. Each caspase 3 assay was performed in triplicate and results are means±S.D.

Figure 4. Model for Akt inhibition by PH-domain-dependent inhibitors

Inhibitors bind Akt outside of the active site and interact with the PH domain and/or hinge region. Inactive Akt (1) can be phosphorylated by PDK1 and activated (2) or bind inhibitor (3). Binding of inhibitor at (3) prevents phosphorylation of Thr³⁰⁸ by PDK1. Inhibitor can bind to activated Akt (4) and block phosphorylation of peptide substrates. Antibodies bound to the PH domain or hinge region (5) prevent inhibitor binding.