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.

Thiazole analogues of the NSAID indomethacin as selective COX-2 inhibitors.

The carboxyl group of the NSAID indomethacin was replaced with a variety of substituted thiazoles to obtain a series of potent, selective inhibitors of COX-2. Additional substitutions were made at the 1-position and 5-position of the indole of indomethacin.

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.

Water-soluble renin inhibitors: design of a subnanomolar inhibitor with a prolonged duration of action.

Incorporation of nonreactive polar functionalities at the C- and N-termini of renin inhibitors led to the development of a subnanomolar compound (21) with millimolar solubility. This inhibitor demonstrated excellent efficacy and a long duration of action upon intravenous administration to monkeys. While activity was also observed intraduodenally, a comparison of the blood pressure responses indicated low bioavailability. Subsequent experiments in rats showed that, although the compound was absorbed from the gastrointestinal tract, extensive liver extraction severely limited bioavailability.

Potent, low molecular weight renin inhibitors containing a C-terminal heterocycle: hydrogen bonding at the active site.

A series of low-nanomolar renin inhibitors containing novel C-terminal heterocycles has been designed by formally cyclizing the C-terminus of a glycol-based inhibitor to the second hydroxyl. Molecular modeling suggests that the heterocyclic oxygen hydrogen bonds as an acceptor to the flap region of renin and that the second hydroxyl in the glycol-based inhibitors behaves similarly.

Azido glycols: potent, low molecular weight renin inhibitors containing an unusual post scissile site residue.

Azidomethyl-substituted 1,2- and 1,3-diols were prepared from Boc-cyclohexylalanal and evaluated as transition state analogue renin inhibitors, leading to the development of a small (MW less than 600), nanomolar inhibitor. Remarkable aqueous solubility enhancement followed the incorporation of an N-terminal urea functionality. Evaluation of selected compounds both in vivo and in vitro demonstrated that while transport across the intestine occurred upon id administration, extensive liver extraction resulted in low systemic levels.

Novel renin inhibitors containing analogues of statine retro-inverted at the C-termini: specificity at the P2 histidine site.

Substituted 1,3- and 1,4-diamines were prepared from epoxides derived from Boc-leucine or Boc-cyclohexylalanine. These diamines were incorporated into renin inhibitors (IC50 = 4-1500 nM) replacing the Leu-Val scissile bond in small peptide analogues of angiotensinogen. Replacement of the P2 histidine imidazole with other heterocycles maintained or enhanced binding while changing the overall basicity of the inhibitor. Finally, substitution of O-methyltyrosine for the P3 phenylalanine suppressed chymotrypsin cleavage of the P3-P2 bond.