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.

2). These enzymes catalyze the formation of thioether linkages between the C1 atom of farnesyl (15-carbon by FTase) or geranylgeranyl (20-carbon by GGTase-I and -II) isoprenoid lipids and cysteine residues at or near the C terminus of protein acceptors. Protein substrates of the prenyltransferases include Ras, Rho, Rab, other Ras-related small GTP-binding proteins, ␥ subunits of heterotrimeric G-proteins, nuclear lamins, centromeric proteins, and many proteins involved in visual signal transduction (2, 3). The attached lipid is required for proper functioning of the modified protein by mediating membrane associations and specific protein-protein interactions. FTase and GGTase-I, which are collectively known as the CaaX prenyltransferases, attach their respective isoprenoid to the cysteine residue of a C-terminal CaaX motif (C, cysteine; a, typically an aliphatic residue; X, C-terminal residue). GGTase-II attaches geranylgeranyl groups to two C-terminal cysteine residues in the Rab family of Rasrelated GTPases.Ras must be associated with the plasma membrane for proper functioning in the signal transduction pathway. Prenylation of Ras is required for this subcellular localization and is essential for the transforming activity of oncogenic variants of Ras (4-6). FTase is therefore a potential target for anticancer therapeutics. A critical advance in the development of FTase inhibitors was the finding that tetrapeptides that conformed to the CaaX sequence motif are competitive inhibitors (7). Surprisingly, a subset of these tetrapeptides (e.g., CVFM) are not farnesylated (8). Two features were identified as dominant determinants for the lack of farnesylation: a positively charged N terminus and an aromatic residue at the a 2 position (9). The distinction between competitive inhibitors that are competent substrates and non-substrate inhibitors is an important one, because farnesylation of the competitive inhibitor decreases their affinity for the enzyme, thereby reducing potency (10). These findings led to the design of several peptidomimetic compounds based on the CaaX motif (reviewed in ref. 11). The initial hurdles of low cell permeability and susceptibility to proteolytic degradation inherent to peptidebased compounds were overcome by the synthesis of ester prodrugs, such as L-744,832 (Fig. 1), which inhibited the growth of more than 70% of tumor cell lines (12) and caused tumor regression in H-ras transformed mice, without systemic toxicity (13). Numerous inhibitors of FTase are now in clinical trials for the treatment of human cancer (reviewed in ref. 14). L-744,832 is the isopropyl ester prodrug of L-739,750 ( Fig. 1; ref. 15), the peptidomimetic compound used in the structures presented in this paper, and was the first inhibitor of FTase to demonstrate tumor regression in animals (13).The three-dimensional structures of FTase-bound peptidomimetics were initially characterized by NMR spectroscopy. Twodimensional transferred nuclear Overhauser effect (TRNOE) experiments indicated that the peptide backbones of a...

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey. This is achieved by specific immunoprecipitation of the RISC from tissue lysates and quantification of small RNAs in the immunoprecipitates by stem-loop PCR. The method, expected to be independent of delivery vehicle and target, is label-free, and the throughput is acceptable for preclinical animal studies. We characterized a lipid-formulated siRNA by integrating these approaches and obtained a quantitative perspective on siRNA tissue accumulation, RISC loading, and gene silencing. The described methodologies have utility for the study of silencing mechanism, the development of siRNA therapeutics, and clinical trial design.

Deeper knowledge of pharmacokinetic and pharmacodynamic (PK/PD) concepts for RNA therapeutics is important to streamline the drug development process and for rigorous selection of best performing drug candidates. Here we characterized the PK/PD relationship for small interfering RNAs (siRNAs) targeting luciferase by examining siRNA concentration in plasma and liver, the temporal RNA-induced silencing complex binding profiles, mRNA reduction, and protein inhibition measured by noninvasive bioluminescent imaging. A dose-dependent and timerelated decrease in bioluminescence was detected over 25 days after a single treatment of a lipid nanoparticle-formulated siRNA targeting luciferase messenger RNA. A direct relationship was observed between the degree of in vivo mRNA and protein reduction and the Argonaute2 (Ago2)-bound siRNA fraction but not with the total amount of siRNA found in the liver, suggesting that the Ago2-siRNA complex is the key determinant of target inhibition. These observations were confirmed for an additional siRNA that targets endogenously expressed Sjö gren syndrome antigen B (Ssb) mRNA, indicating that our observations are not limited to a transgenic mouse system. Our data provide detailed information of the temporal regulation of siRNA liver delivery, Ago2 loading, mRNA reduction, and protein inhibition that are essential for the rapid and cost-effective clinical development of siRNAs therapeutics.