Androgen ablation augments human HLA2.1-restricted T cell responses to PSA self-antigen in transgenic mice.

BACKGROUND: In recent years, there has been an increasing interest in targeting human prostate tumor-associated antigens (TAAs) for prostate cancer immunotherapy as an alternative to other therapeutic modalities. However, immunologic tolerance to TAA poses a significant obstacle to effective, TAA-targeted immunotherapy. We sought to investigate whether androgen deprivation would result in circumventing immune tolerance to prostate TAA by impacting CD8 cell responses. METHODS: To this end, we generated a transgenic mouse that expresses the human prostate-specific antigen (PSA) specifically in the prostate, and crossed it to the HLA-A2.1 transgenic mouse to evaluate how androgen deprivation affects human HLA A2.1-resticted T cell responses following immunization of PSA-expressing mice by vaccinia-PSA (PROSTVAC). RESULTS: Our PSA transgenic mouse showed restricted expression of PSA in the prostate and detectable circulating PSA levels. Additionally, PSA expression was androgen-dependent with reduced PSA expression in the prostate within 1 week of castration, and undetectable PSA by day 42 after castration as evaluated by ELISA. Castration of the PSA/A2.1 hybrid mouse prior to immunization with a PSA-expressing recombinant vaccinia virus resulted in a significant augmentation of PSA-specific cytotoxic lymphocytes. CONCLUSIONS: This humanized hybrid mouse model provides a well-defined system to gain additional insight into the mechanisms of immune tolerance to PSA and to test novel strategies aiming at circumventing immune tolerance to PSA and other TAA for targeted prostate cancer immunotherapy.

An allosteric Akt inhibitor effectively blocks Akt signaling and tumor growth with only transient effects on glucose and insulin levels in vivo.

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 C(max), 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.

Allosteric inhibitors of Akt1 and Akt2: discovery of [1,2,4]triazolo[3,4-f][1,6]naphthyridines with potent and balanced activity.

A series of [1,2,4]triazolo[3,4-f][1,6]naphthyridine allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been shown to have potent dual Akt1 and 2 cell potency. The representative compound 13 provided potent inhibitory activity against Akt1 and 2 in vivo in a mouse model.

Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.

BACKGROUND: Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy. METHODOLOGY/PRINCIPAL FINDINGS: A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.

Discovery of potent and cell-active allosteric dual Akt 1 and 2 inhibitors.

This paper describes the improvement of cell potency in a class of allosteric Akt 1 and 2 inhibitors. Key discoveries include identifying the solvent exposed region of the molecule and appending basic amines to enhance the physiochemical properties of the molecules. Findings from the structure-activity relationships are discussed.

The design and synthesis of potent and cell-active allosteric dual Akt 1 and 2 inhibitors devoid of hERG activity.

This letter details the attenuation of hERG in a class of Akt inhibitors through heteroatom insertions into aromatic rings. The development of a cell-active dual Akt 1 and 2 inhibitors devoid of hERG activity is discussed using structure-activity relationships.

Allosteric inhibitors of Akt1 and Akt2: a naphthyridinone with efficacy in an A2780 tumor xenograft model.

A series of naphthyridine and naphthyridinone allosteric dual inhibitors of Akt1 and 2 have been developed. These compounds have been optimized to have potent dual activity against the activated kinase as well as the activation of Akt in cells. One molecule in particular, compound 17, has potent inhibitory activity against Akt1 and 2 in vivo in a mouse lung and efficacy in a tumor xenograft model.

Development of pyridopyrimidines as potent Akt1/2 inhibitors.

This communication reports a new synthetic route of pyridopyrimidines to facilitate their structural optimization in a library fashion and describes the development of pyridopyrimidines that have excellent enzymatic and cell potency against Akt1 and Akt2. This series also shows a high level of selectivity over other closely related kinases and significantly improved caspase-3 activity with the more optimized compounds.

Optimization of 2,3,5-trisubstituted pyridine derivatives as potent allosteric Akt1 and Akt2 inhibitors.

This letter shows inhibitor SAR on a pyridine series of allosteric Akt inhibitors to optimize enzymatic and cellular potency. We have optimized 2,3,5-trisubstituted pyridines to give potent Akt1 and Akt2 inhibitors in both enzyme and cell based assays. In addition, we will also highlight the pharmacokinetic profile of an optimized inhibitor that has low clearance and long half-life in dogs.

Rapid assembly of diverse and potent allosteric Akt inhibitors.

This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.

Development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved physical properties and cell activity.

This letter describes the development of potent, allosteric dual Akt1 and Akt2 inhibitors with improved aqueous solubility (approximately 18 mg/mL) that translates into enhanced cell activity and caspase-3 induction.

Discovery of 2,3,5-trisubstituted pyridine derivatives as potent Akt1 and Akt2 dual inhibitors.

This letter describes the discovery of a novel series of dual Akt1/Akt2 kinase inhibitors, based on a 2,3,5-trisubstituted pyridine scaffold. Compounds from this series, which contain a 5-tetrazolyl moiety, exhibit more potent inhibition of Akt2 than Akt1.

Tumor cell sensitization to apoptotic stimuli by selective inhibition of specific Akt/PKB family members.

Recent studies indicate that dysregulation of the Akt/PKB family of serine/threonine kinases is a prominent feature of many human cancers. The Akt/PKB family is composed of three members termed Akt1/PKBalpha, Akt2/PKBbeta, and Akt3/PKBgamma. It is currently not known to what extent there is functional overlap between these family members. We have recently identified small molecule inhibitors of Akt. These compounds have pleckstrin homology domain-dependent, isozyme-specific activity. In this report, we present data showing the relative contribution that inhibition of the different isozymes has on the apoptotic response of tumor cells to a variety of chemotherapies. In multiple cell backgrounds, maximal induction of caspase-3 activity is achieved when both Akt1 and Akt2 are inhibited. This induction is not reversed by overexpression of functionally active Akt3. The level of caspase-3 activation achieved under these conditions is equivalent to that observed with the phosphatidylinositol-3-kinase inhibitor LY294002. We also show that in different tumor cell backgrounds inhibition of mammalian target of rapamycin, a downstream substrate of Akt, is less effective in inducing caspase-3 activity than inhibition of Akt1 and Akt2. This shows that the survival phenotype conferred by Akt can be mediated by signaling pathways independent of mammalian target of rapamycin in some tumor cell backgrounds. Finally, we show that inhibition of both Akt1 and Akt2 selectively sensitizes tumor cells, but not normal cells, to apoptotic stimuli.

Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors.

This letter describes the development of two series of potent and selective allosteric Akt kinase inhibitors that display an unprecedented level of selectivity for either Akt1, Akt2 or both Akt1/Akt2. An iterative analog library synthesis approach quickly provided a highly selective Akt1/Akt2 inhibitor that induces apoptosis in tumor cells and inhibits Akt phosphorylation in vivo.

Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors.

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.

A prostate-specific antigen (PSA)-activated vinblastine prodrug selectively kills PSA-secreting cells in vivo.

Currently, there is no therapy for men with androgen-refractory prostate cancer that substantially extends survival. This report characterizes by in vitro and in vivo techniques a new chemotherapeutic that is composed of desacetyl-vinblastine covalently linked to a peptide that contains a peptide bond that can be hydrolyzed by prostate-specific antigen (PSA). This compound (referred to as vinblastine-conjugate) is minimally toxic to cells in culture which do not express PSA. In the presence of PSA, the peptide moiety is hydrolyzed, generating several highly toxic metabolites that contain vinblastine. Animals bearing PSA-positive human prostate tumors that were treated with the vinblastine-conjugate experienced a >99% reduction in PSA serum level. In contrast, animals bearing PSA-positive human prostate tumors treated with the cytotoxic metabolites derived from the PSA hydrolysis of the vinblastine-conjugate showed a nonsignificant change in both PSA and tumor weight values. The cell killing activity of the vinblastine-conjugate is PSA dependent because animals bearing non-PSA-producing human tumor xenografts had a nonsignificant increase in tumor weight after vinblastine-conjugate treatment. Exploratory efficacy/toxicity studies in LNCaP tumor-bearing nude mice were conducted with animals treated for 5 consecutive days with various doses of either the vinblastine-conjugate or a PSA-generated toxic metabolite (desacetyl-vinblastine). The desacetyl-vinblastine treatment resulted in 10-70% mortality with a very slight effect on tumor growth. In contrast, vinblastine-conjugate treatments resulted in no mortality, good to excellent antitumor efficacy, very slight to slight peripheral neuropathy and myelopathy, and slight to severe testicular degeneration. Similar treatment of beagle dogs with the vinblastine-conjugate showed even less toxicity. These data support the use of the PSA-hydrolyzable vinblastine-conjugate as an experimental therapy for prostate cancer in man.

Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties. Earlier work in these laboratories established that an appropriately engineered peptidyl pro-drug will release active cytotoxic agent strictly within the microenvironment of the tumor tissue (Garsky, V. M., et al. J. Med.Chem. 2001, 44, 4216-4224). Conjugate 5, which features an octapeptide segment attached by an ester linkage at the 4-position of vinblastine (1), undergoes rapid cleavage by PSA (T(1/2) = 12 min) between the Gln and Ser residues. In nude mouse xenograft studies, 5 reduced circulating PSA levels by 99% and tumor weight by 85% at a dose just below its MTD. By contrast, the putative end-point metabolite, the cytotoxic agent des-acetyl vinblastine (1b), was ineffective in reducing PSA levels and tumor burden at its maximum tolerated doses. Additional data from metabolism studies on 5 support the supervention of a novel in vivo processing mechanism, the spontaneous release of 1b from a dipeptidyl intermediate driven by favorable diketopiperazine formation.