This paper describes the implementation of a biochemical and biophysical screening strategy to identify and optimize small molecule Akt1 inhibitors that act through a mechanism distinct from that observed for kinase domain ATP-competitive inhibitors. With the aid of an unphosphorylated Akt1 cocrystal structure of 12j solved at 2.25 Å, it was possible to confirm that as a consequence of binding these novel inhibitors, the ATP binding cleft contained a number of hydrophobic residues that occlude ATP binding as expected. These Akt inhibitors potently inhibit intracellular Akt activation and its downstream target (PRAS40) in vitro. In vivo pharmacodynamic and pharmacokinetic studies with two examples, 12e and 12j, showed the series to be similarly effective at inhibiting the activation of Akt and an additional downstream effector (p70S6) following oral dosing in mice.

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148 -7154), we showed that overlapping peptides, N20K (Asn 564 -Lys 583 ) and E20K (Glu 574 -Lys 593 ), from the catalytic domain of phospholipase C (PLC) ␤2 block G␤␥-dependent activation of PLC ␤2. The peptides could also be directly cross-linked to ␤␥ subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to G␤ 1 was inhibited by PLC ␤2 but not by ␣ i1 (GDP), indicating that the peptide-binding site on ␤ 1 represents a binding site for PLC ␤2 that does not overlap with the ␣ i1 -binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC ␤2 interaction with ␤ subunits. Each of the 14 cysteine residues in ␤ 1 were altered to alanine. The ability of the PLC ␤2-derived peptide to cross-link to each ␤␥ mutant was then analyzed to identify the reactive sulfhydryl moiety on the ␤ subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of ␤ 1 which is located in the amino-terminal coiled-coil region of ␤ 1 and identifies a PLC-binding site distinct from the ␣ subunit interaction site.Guanine nucleotide-binding proteins (G proteins) 1 are a large group of structurally similar proteins consisting of three subunits (␣, ␤, and ␥) that are central molecules coupling seven-transmembrane domain-spanning receptors to downstream effector molecules. Activation of G proteins begins with a ligand-induced conformational change of the receptor which catalyzes the release of GDP from the ␣ subunit in exchange for GTP (1, 2). In the GDP-bound heterotrimeric state, ␣(GDP)⅐␤␥, neither ␣(GDP) nor ␤␥ can regulate effector activity. Upon receptor-catalyzed G protein activation, the heterotrimer dissociates into free ␣(GTP) and free ␤␥ subunits. It is well understood that both ␣(GTP) and ␤␥ subunits can interact with a variety of downstream effector molecules including enzymes and ion channels. GTP is hydrolyzed to GDP, and reassociation of ␣(GDP) with ␤␥ results in deactivation of ␤␥-dependent signaling. Despite detailed knowledge of ␣-and ␤␥ subunit functions, the mechanism for how ␤␥ subunits activate its variety of effectors is not entirely understood.Effector-binding sites on the surface of ␤␥ are beginning to be mapped. The putative competition between ␣(GDP) and effectors for ␤␥ forms the premise for recent studies to map effectorbinding sites at the ␣ subunit-binding interface on ␤. The three-dimensional structure of the G protein heterotrimer reveals that the ␤ subunit is a ␤-propeller with seven "blades" and an amino-terminal ␣-helix (3, 4). The ␣ subunit binds to a portion of the top of the ␤-propeller and along side one of the blades of the propeller. Two groups have shown that alanine substitution of ␣-contacting residues on the top surface...

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Introduction: ARQ 092 is a potent and selective pan-AKT inhibitor currently in Phase 1 clinical studies for the treatment of advanced solid tumors. Here, we present preclinical in vitro drug-drug interaction, metabolism, disposition, and pharmacokinetic studies with ARQ 092. Methods: ARQ 092 was assessed in vitro for cross-species metabolic stability; CYP450 (cytochrome P450) and UGT (UDP-glucuronosyltransferase) reaction phenotyping; CYP450 inhibition/induction; Caco-2 absorption and efflux; and P-gp (P-glycoprotein), BCRP (Breast Cancer Resistance Protein), and MRP2 (Multidrug Resistance-associated Protein 2) mediated ATPase stimulation. Distribution of ARQ 092 to various tissues was measured after single and repeat dosing to mice as well as pharmacokinetics in mice, rats, and monkeys. Results: Cross-species NADPH-dependent metabolism studies revealed that ARQ 092 metabolism was highly species dependent with ARQ 092 being most stable in rat and human liver microsomes (t1/2 > 52 min) and least stable in mouse, dog, and monkey liver microsomes (t1/2 > 13-33 min). Reaction phenotyping with CYP450 and UGT isozymes and CYP450 inhibitors/antibodies indicated that CYP2D6, CYP3A, UGT1A4, and possibly CYP2C9 are involved in ARQ 092 metabolism. ARQ 092 inhibited CYP2D6, 2C9, and 2C19 in HLM with IC50 values of 10.2, 3.0, and 4.0 µM, respectively, but had little activity against other isozymes tested; and no induction of CYP3A, 2B6, or 1A2 was observed. Based on these in vitro results, interactions with CYP3A4, 2D6, CYP2C9, or CYP2C19 substrates and/or inhibitors in the clinic are possible. Caco-2 and ATPase transporter studies showed that ARQ 092 was highly permeable with little efflux in Caco-2 monolayers but may be an inhibitor/substrate of P-gp and mutant BCRP. Oral bioavailability values of 23, 62, and 50% were determined in mice, rats, and monkeys, respectively, with half-lives ranging widely from 1.6 to 13.6 hrs with iv dosing and 4.3 to 16.6 hrs with po dosing. In mice, ARQ 092 was highly distributed to tissues with liver, lung, and kidney having the highest concentrations. This result correlates with the high volume of distribution of the compound in mice of 24.5 L/kg. In toxicokinetic studies, increases in Cmax and AUC were generally greater than dose proportional with repeat dosing and accumulation was observed in rats but not monkeys. Conclusion: Collectively, these data indicate that ARQ 092 has sufficient oral bioavailability to advance into clinical testing. However, the potential for drug-drug interactions with ARQ 092 in the clinic will need to be investigated further. Additionally, careful selection of dosing regimens for ARQ 092 may be crucial based on the high volume of distribution and accumulation observed preclinically. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C83. Citation Format: Laurie P. Volak, Karen R. Bresciano, Terence Hall, David Vensel, Jean-Marc Lapierre, Inese Smukste, David K. McKearn, Ronald E. Savage. Nonclinical in vitro ADME, disposition, and pharmacokinetic assessment of ARQ 092, a selective AKT inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C83.

ARQ 621 is a potent and selective allosteric inhibitor of Eg5, a microtubule-based ATPase motor protein involved in cell division. Eg5 inhibition is recognized as a potential therapeutic strategy in cancer, supported by the observation that over-expression of Eg5 causes genomic instability and tumor formation in mice. ARQ 621 is currently being tested in a Phase I clinical trial in cancer patients. Prior to its entry into the clinic, the in vitro ADME properties of ARQ 621 were studied. Data from these studies showed that ARQ 621 had a t1/2 of 53 min in human liver microsomes. The t1/2 of ARQ 621 in male and female mouse, rat, dog and monkey liver microsomes was similar to that of human liver microsomes with t1/2 values of 43, 53, 56, 53, 47, 44, 36, and 32 minutes, respectively. Consistent with the microsomal stability data, in vitro metabolic studies conducted with individual human CYP P450 isoforms indicated that ARQ 621 was relatively stable with t1/2 values all greater than 27 min. IC50 values of ARQ 621 were measured for CYP 1A2, 2C9, 2D6, 3A4, 2C19, and 2C8 and determined to be >20, >20, >20, 4.1, 4.0, and 15 µM, respectively. ARQ 621 was found to modestly induce 1A2 but not 2A6 or 3A4. Data from in vitro Caco 2 studies demonstrated that ARQ 621 has poor GI absorption potential with a Papp value of 0.69 × 10−6 cm/s. Additionally, Caco 2 bi-directional experiments suggested that ARQ 621 may be a P-glycoprotein substrate with an efflux ratio of 45. These data are consistent with rat in vivo oral bioavailability studies in which the bioavailability of ARQ 621 was determined to be approximately 9%. Hence, ARQ 621 was subsequently developed for IV administration only. Protein binding studies conducted in human plasma showed that ARQ 621 is highly bound (∼96.4-99.2%) to plasma proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5783.