The PI3K-Akt pathway is dysregulated in the majority of solid tumors. Pharmacological inhibition of Akt is a promising strategy for treating tumors resistant to growth factor receptor antagonists due to mutations in PI3K or PTEN. We have developed allosteric, isozyme-specific inhibitors of Akt activity and activation, as well as ex vivo kinase assays to measure inhibition of individual Akt isozymes in tissues. Here we describe the relationship between PK, Akt inhibition, hyperglycemia and tumor efficacy for a selective inhibitor of Akt1 and Akt2 (AKTi). In nude mice, AKTi treatment caused transient insulin resistance and reversible, dose-dependent hyperglycemia and hyperinsulinemia. Akt1 and Akt2 phosphorylation was inhibited in mouse lung with EC50 values of 1.6 and 7 μM, respectively, and with similar potency in other tissues and xenograft tumors. Weekly subcutaneous dosing of AKTi resulted in dose-dependent inhibition of LNCaP prostate cancer xenografts, an AR-dependent tumor with PTEN deletion and constitutively activated Akt. Complete tumor growth inhibition was achieved at 200 mpk, a dose that maintained inhibition of Akt1 and Akt2 of greater than 80% and 50%, respectively, for at least 12 hours in xenograft tumor and mouse lung. Hyperglycemia could be controlled by reducing Cmax, while maintaining efficacy in the LNCaP model, but not by insulin administration. AKTi treatment was well tolerated, without weight loss or gross toxicities. These studies supported the rationale for clinical development of allosteric Akt inhibitors and provide the basis for further refining of pharmacokinetic properties and dosing regimens of this class of inhibitors.

We have identified and characterized potent and specific inhibitors of geranylgeranyl-protein transferase type I (GGPTase I), as well as dual inhibitors of GGPTase I and farnesyl-protein transferase. Many of these inhibitors require the presence of phosphate anions for maximum activity against GGPTase I in vitro. Inhibitors with a strong anion dependence were competitive with geranylgeranyl pyrophosphate (GGPP), rather than with the peptide substrate, which had served as the original template for inhibitor design. One of the most effective anions was ATP, which at low millimolar concentrations increased the potency of GGPTase I inhibitors up to several hundred-fold. In the case of clinical candidate L-778,123, this increase in potency was shown to result from two major interactions: competitive binding of inhibitor and GGPP, and competitive binding of ATP and GGPP. At 5 mM, ATP caused an increase in the apparent K d for the GGPP-GGPTase I interaction from 20 pM to 4 nM, resulting in correspondingly tighter inhibitor binding. A subset of very potent GGPP-competitive inhibitors displayed slow tight binding to GGPTase I with apparent on and off rates on the order of 10 6 M ؊1 s ؊1 and 10 ؊3 s ؊1 , respectively. Slow binding and the anion requirement suggest that these inhibitors may act as transition state analogs. After accounting for anion requirement, slow binding, and mechanism of competition, the structure-activity relationship determined in vitro correlated well with the inhibition of processing of GGPTase I substrate Rap1a in vivo.Inhibition of prenylation of Ras oncoproteins has long been considered an attractive approach to anti-cancer therapy of Ras-dependent tumors. Four isoforms of Ras are found in mammalian cells, Kirsten-Ras (Ki-Ras) 4A and 4B, Harvey-Ras (Ha-Ras), and N-Ras. Ki4B-Ras is the most frequently mutated isozyme in human cancer, ranging from 20% up to 90% in pancreatic cancers, whereas Ha-Ras mutations are found in a small percentage of bladder cancers. Most oncogenic mutations in Ras result in the elimination of the GTPase activity, locking Ras in its active, GTP-bound conformation. All Ras proteins undergo multiple post-translational modifications on their carboxyl termini that are required for membrane localization and biological activity. The COOH-terminal CAAX motif (where C is cysteine, A stands for aliphatic, and X for any amino acid) is recognized by farnesyl-protein transferase (FPTase), 1 which transfers a farnesyl group from farnesyl pyrophosphate (FPP) to the cysteine thiol. This prenylation step is followed by proteolytic clipping of the three terminal amino acids by a farnesyl-CAAX specific protease and subsequent methylation of the cysteine residue by a carboxymethyl transferase. Ha-Ras and N-Ras are further modified by palmitoylation which provides additional membrane interactions. A polylysine stretch in KiRas near the COOH terminus serves the same purpose. However, the prenylation step has been shown to be the single most critical modification for function of all Ras prote...

An attempt was made to determine if the P3-J line of Burkitt's lymphoma cells could synthesize specific antibody in response after exposure to bacteriophage as reported by Kamei and Moore (1, 2). The cells were exposed to various concentrations of phages MS-2 or T2 and cultured for up to 10 days, but we were unable to detect significant neutralizing activity in any experimental situation. We also attempted to induce a BALB/c myeloma tumor with specific antibody activity for phage MS-2. While two in vivo tumors had characteristics of producing neutralizing antibody for MS-2 on adaptation of one of these tumors to growth in cell culture, no activity could be detected. Further, exposure of the cultured tumor cells to MS-2 did not result in the synthesis of neutralizing antibody.

Cell-free extracts of bacteria, plants and animals have been utilized to investigate the mechanism of protein synthesis. The ability of polyuridylic acid (poly U) and other synthetic polyribonucleotides to direct specific polypeptide synthesis by these cell-free extracts has provided a tool not only for investigating the nature of the genetic code but also for elucidating the overall mechanism of protein synthesis. However, the regulation of protein synthesis at the transcriptional and translational levels has been studied in relatively few mammalian tissues (1)(2)(3)(4)(5)(6)(7)(8). In this report we describe a subcellular protein-synthesizing system that utilizes components derived from rabbit liver tissue which is capable of incorporating amino acids into protein at levels greater than that described for other mammalian cell-free systems.Materials and Methods. Materials. Foiyuridylic acid (poly U), with 250/260 and 280/260 optical density ratios of 0.78 and 0.3 5, respectively, was purchased from Calbiochem (Los Angeles, CA). Uniformly labeled L-[ 14C] -phenylalanine (4 14 mCi/ mmole) was purchased from New England Nuclear Corp. (Waltham, MA). Crystalline phosphoenolpyruvate kinase, potassium phosphoenolpyruvate, and sodium ATP and GTP were obtained from Sigma Chemical Corp. (St. Louis, MO). Nonidet P-40 was the gift of the Shell Chemical Co. (New York) . Methods. Preparation of ribosomes. San Juan rabbits were exsanguinated, their livers were removed and rinsed 3 times in cold buffer A (0.025 M KCl, 0.005 M MgC12, 0.006 M 2-mercaptoethanol, and 0.05 M Tris, pH 7.6).The tissue was resuspended in buffer A containing 0.2 5 M sucrose (homogenizing buffer), homogenized with a motor driven loose-fitting Teflon pestle and rehomogenized by 6 strokes each in a loose-and a tight-fitting Dounce homogenizer. The homogenizations and all subsequent preparative steps were carried out a t 4' . The homogenate was centrifuged at 15,000g for 10 min and the supernate was centrifuged at 105,OOOg for 2 hr in a type 50.1 rotor on a Spinco L2-65B ultracentrifuge. The resulting supernate (S-105) was used to prepare pH 5 enzyme and transfer factors (see below). The microsomal pellet was resuspended in buffer A containing 0.5% Nonidet P-40, carefully layered over an equal volume of 0.5 M sucrose in buffer A and centrifuged at lOS,OOOg for 2.5 hr. The ribosomal pellet was resuspended in buffer A, dispensed into aliquots, rapidly frozen and stored a t -70". Ribosomal RNA (rRNA) was assayed by a modification of the procedure of Fleck and Munro (9).Preparation of pH 5 enzyme. The S-105 supernatant was diluted with 2 vol of cold, sterile glass-distilled water containing 0.006 M 2-mercaptoethanol, made pH 5.1 with 1 M acetic acid and incubated at 4' for 15 min. The preparation was centrifuged at 10, OOOg for 20 min and the clear supernatant was used for the preparation of transfer facnis remwh was supported by u.s. PubEc tors ( 1 1). The pellet was resuspended in buffer B (0.07 M KCI, 0.006 M 2-mercapto-MgC12, 0.35 k ! SWrOSe and 0.05 & i ? ...