Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition.

Rapamycin, a natural product inhibitor of the Raptor-mammalian target of rapamycin complex (mTORC1), is known to induce Protein kinase B (Akt/PKB) Ser-473 phosphorylation in a subset of human cancer cell lines through inactivation of S6K1, stabilization of insulin receptor substrate (IRS)-1, and increased signaling through the insulin/insulin-like growth factor-I/phosphatidylinositol 3-kinase (PI3K) axis. We report that A-443654, a potent small-molecule inhibitor of Akt serine/threonine kinases, induces Akt Ser-473 phosphorylation in all human cancer cell lines tested, including PTEN- and TSC2-deficient lines. This phenomenon is dose-dependent, manifests coincident with Akt inhibition and likely represents an alternative, rapid-feedback pathway that can be functionally dissociated from mTORC1 inhibition. Experiments performed in TSC2-/- cells indicate that TSC2 and IRS-1 cooperate with, but are dispensable for, A-443654-mediated Akt phosphorylation. This feedback event does require PI3K activity, however, as it can be inhibited by LY294002 or wortmannin. Small interfering RNA-mediated knockdown of mTOR or Rictor, components of the rapamycin-insensitive mTORC2 complex, but not the mTORC1 component Raptor, also inhibited Akt Ser-473 phosphorylation induced by A-443654. Our data thus indicate that Akt phosphorylation and activity are coupled in a manner not previously appreciated and provide a novel mode of Akt regulation that is distinct from the previously described rapamycin-induced IRS-1 stabilization mechanism.

RNAi-based screening of the human kinome identifies Akt-cooperating kinases: a new approach to designing efficacious multitargeted kinase inhibitors.

Tumors comprise genetically heterogeneous cell populations, whose growth and survival depend on multiple signaling pathways. This has spurred the development of multitargeted therapies, including small molecules that can inhibit multiple kinases. A major challenge in designing such molecules is to determine which kinases to inhibit in each cancer to maximize efficacy and therapeutic index. We describe an approach to this problem implementing RNA interference technology. In order to identify Akt-cooperating kinases, we screened a library of kinase-directed small interfering RNAs (siRNAs) for enhanced cancer cell killing in the presence of Akt inhibitor A-443654. siRNAs targeting casein kinase I gamma 3 (CSNK1G3) or the inositol polyphosphate multikinase (IPMK) significantly enhanced A-443654-mediated cell killing, and caused decreases in Akt Ser-473 and ribosomal protein S6 phosphorylation. Small molecules targeting CSNK1G3 and/or IPMK in addition to Akt may thus exhibit increased efficacy and have the potential for improved therapeutic index.

Heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids as leukotriene biosynthesis inhibitors.

A novel series of heteroarylmethoxyphenylalkoxyiminoalkylcarboxylic acids was studied as leukotriene biosynthesis inhibitors. A hypothesis of structure-activity optimization by insertion of an oxime moiety was investigated using REV-5901 as a starting point. A systematic structure-activity optimization showed that the spatial arrangement and stereochemistry of the oxime insertion unit proved to be important for inhibitory activity. The promising lead, S-(E)-11, inhibited LTB(4) biosynthesis in the intact human neutrophil with IC(50) of 8 nM and had superior oral activity in vivo, in a rat pleurisy model (ED(50) = 0.14 mg/kg) and rat anaphylaxis model (ED(50) = 0.13 mg/kg). In a model of lung inflammation, S-(E)-11 blocked LTE(4) biosynthesis (ED(50) of 0.1 mg/kg) and eosinophil influx (ED(50) of 0.2 mg/kg). S-(E)-11 (A-93178) was selected for further preclinical evaluation.

Studies directed toward the design of orally active renin inhibitors. 1. Some factors influencing the absorption of small peptides.

A systematic evaluation of structure-absorption relationships using a high throughput intraduodenal rat screening model has led to the delineation of a set of structural parameters that appear to govern bioavailability in a series of peptide-based renin inhibitors. Optimum structures, exemplified by 25 and 41, incorporated a single, solubilizing substituent at the C- or N-terminus combined with a lipophilic P2-site residue. Both inhibitors gave unprecedented plasma drug levels upon intraduodenal administration to monkeys, and the calculated bioavailability for 41 (14 +/- 4%) is the highest reported for any peptidic renin inhibitor.