Akt Pathway Inhibitors.

Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the identification of new targets, the war on cancer was launched, in 1972. The P13K/Akt pathway is a growth-regulating cellular signaling pathway, which in many human cancers is over-activated. Studies have demonstrated that a decrease in Akt activity by Akt inhibitors is associated with a reduction in tumor cell proliferation. There have been several promising drug candidates that have been studied, including but not limited to ipatasertib (RG7440), 1; afuresertib (GSK2110183), 2; uprosertib (GSK2141795), 3; capivasertib (AZD5363), 4; which reportedly bind to the ATP active site and inhibit Akt activity, thus exerting cytotoxic and antiproliferative activities against human cancer cells. For most of the compounds discussed in this review, data from preclinical studies in various cancers suggest a mechanistic basis involving hyperactivated Akt signaling. Allosteric inhibitors are also known to alter the activity of kinases. Perifosine (KRX- 0401), 5, an alkylphospholipid, is known as the first allosteric Akt inhibitor to enter clinical development and is mechanistically characterized as a PH-domain dependent inhibitor, non-competitive with ATP. This results in a reduction in Akt enzymatic and cellular activities. Other small molecule (MK- 2206, 6, PHT-427, Akti-1/2) inhibitors with a similar mechanism of action, alter Akt activity through the suppression of cell growth mediated by the inhibition of Akt membrane localization and subsequent activation. The natural product solenopsin has been identified as an inhibitor of Akt. A few promising solenopsin derivatives have emerged through pharmacophore modeling, energy-based calculations, and property predictions.

Akt Pathway Inhibition of the Solenopsin Analog, 2-Dodecylsulfanyl-1,-4,-5,-6-tetrahydropyrimidine.

BACKGROUND/AIM: The P13K/Akt signaling pathway is a growth-regulating cellular pathway that is constitutively activated in a variety of human cancers. In previous studies, we reported that a solenopsin analog, compound B (MU-06-SC-608-7), shows inhibitory effects on Akt phosphorylation at a key activation site, as well as on proliferation of tumorigenic cells at sub-micromolar concentrations. The purpose of this study was to evaluate the effect of compound B on downstream effectors of Akt kinase, phosphorylation of Akt at a second activation site, Akt kinase activity in vitro, tumorigenic cell viability and other signaling pathways. MATERIALS AND METHODS: Western blot analyses were performed using WBras1 epithelial and H2009 human carcinoma cells and cell viability assays were performed on H2009 cells. In vitro Akt kinase assays were performed using a commercially available kit. RESULTS: Compound B decreased the phosphorylation of Akt at the Thr308 activation site and key downstream effectors of Akt kinase, but did not directly inhibit Akt kinase. Substantial decreases in cell viability were observed at concentrations above 5 µM. No effect was seen on ERK or JNK pathways. CONCLUSION: The results earmark this compound for further studies as a potential targeted cancer therapy.

Modulator of the PI3K/Akt oncogenic pathway affects mTOR complex 2 in human adenocarcinoma cells.

Chaetoglobosin K (ChK) is a natural product that has been shown to promote F-actin capping, inhibit growth, arrest cell cycle G2 phase, and induce apoptosis. ChK also has been shown to downregulate two important kinases involved in oncogenic pathways, Akt and JNK. This report investigates how ChK is involved in the receptor tyrosine kinase pathway (RTK/PI3K/mTORC2/Akt) to the centrally located protein kinase, Akt. Studies have reported that ChK does not inhibit PI3K comparable to wortmannin and does not affect PDK1 activation. PDK1 is responsible for phosphorylation on Akt T308, while mTORC2 phosphorylates Akt S473. Yet, Akt's two activation sites, T308 and S473, are known to be affected by ChK treatment. It was our hypothesis that ChK acts on the mTORC2 complex to inhibit the phosphorylation seen at Akt S473. This inhibition at mTORC2 should decrease phosphorylation at both these proteins, Akt and mTORC2 complex, compared to a known mTOR specific inhibitor, Torin1. Human lung adenocarcinoma H1299 and H2009 cells were treated with IGF-1 or calyculin A to increase phosphorylation at complex mTORC2 and Akt. Pretreatment with ChK was able to significantly decrease phosphorylation at Akt S473 similarly to Torin1 with either IGF-1 or calyculin A treatment. Moreover, the autophosphorylation site on complex mTORC2, S2481, was also significantly reduced with ChK pretreatment, similar to Torin1. This is the first report to illustrate that ChK has a significant effect at mTORC2 S2481 and Akt S473 comparable to Torin1, indicating that it may be a mTOR inhibitor.

Discovery and biological activity of computer-assisted drug designed Akt pathway inhibitors.

The P13K/Akt pathway is a growth-regulating cellular signaling pathway that is over-activated in numerous human cancers. A novel series of Akt pathway inhibitors were identified using iterative pharmacophore modeling, energy-based calculations, and property predictions of known Akt inhibitors. Inhibitory effects on activation of Akt and growth of human neoplastic cells are reported. Results show variable inhibitory effects of three selected compounds on Akt phosphorylation at a key activation site, and on proliferation of tumorigenic cells. We identify one lead compound with potent inhibitory activity on both human carcinoma cell proliferation and Akt activation.

Amidation inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester are novel HDAC inhibitors with anti-tumorigenic properties.

4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.

Comparative effects of 4-phenyl-3-butenoic acid and vorinostat on cell growth and signaling.

BACKGROUND/AIM: 4-phenyl-3-butenoic acid (PBA) is a small-molecule anti-inflammatory agent, which has been shown to inhibit growth, increase gap junction intercellular communication and modulate activation of p38 mitogen-activated protein kinase (p38 MAPK) and c-jun n-terminal kinase (JNK) in tumorigenic cells at concentrations that do not similarly affect non-tumorigenic cells. Vorinostat is an anticancer agent structurally similar to PBA. The purpose of this study was to compare the effects of these two agents on JNK and p38 activation, cell growth and gap junction intercellular communication (GJIC). MATERIALS AND METHODS: Cell growth, GJIC and western blot analyses were performed utilizing tumorigenic WBras1 and H2009 human carcinoma cells, and non-tumorigenic WBneo3 and human bronchial epithelial (HBE) cells. RESULTS: Both compounds significantly inhibited WBras1 and H2009 tumorigenic cell growth and increased GJIC in WBras1 cells, as previously reported for PBA. Under similar conditions, both compounds increased phosphorylation of p38 MAPK in tumorigenic but not in non-tumorigenic cells and decreased phosphorylation of JNK in tumorigenic cells. However, a decrease in phosphorylation of JNK occurred in non-tumorigenic WBras1 cells following vorinostat treatment but not PBA treatment. Both compounds showed a selective growth inhibition of H2009 human carcinoma over normal HBE lung cells but, unlike PBA, vorinostat significantly decreased cell growth in WBneo3 cells. CONCLUSION: Overall, PBA exhibited similar effects to vorinostat in tumorigenic cells, while also showing reduced effects on JNK phosphorylation and growth in non-tumorigenic cells compared to ras-transformed cells.

Inhibition of JNK and p38 MAPK phosphorylation by 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester and 4-phenyl-butenoic acid decreases substance P-induced TNF-α upregulation in macrophages.

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the undecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4-phenyl-3-butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP-stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA-Me and PBA both inhibit SP-stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA-Me and PBA dock at the ATP binding site of apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKs upstream of both JNK and p38 MAPK, with predicted interaction energies of -7.0 kcal/mol and -5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings suggest that AOPHA-Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition of JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule.

Dual modulation of JNK and Akt signaling pathways by chaetoglobosin K in human lung carcinoma and ras-transformed epithelial cells.

Chaetoglobosin K (ChK) is a natural product that inhibits anchorage-dependent and anchorage-independent growth of ras-transformed cells, prevents tumor-promoter disruption of cell-cell communication, and reduces Akt activation in tumorigenic cells. This study demonstrates how ChK modulates the JNK pathway in ras-transformed and human lung carcinoma cells and investigates regulatory mechanisms controlling ChK's effect on the Akt and JNK signaling pathways. Human lung carcinoma and ras-transformed epithelial cell lines treated with ChK or vehicle for varying times were assayed for cell growth or extracted for total proteins for western blot analysis using phosphorylation site-specific antibodies to monitor changes in activation of JNK, Akt, and other signaling enzymes. Results show that ChK inhibited both Akt and JNK phosphorylation at key activation sites in ras-transformed cells as well as human lung carcinoma cells. Downstream effectors of both kinases were accordingly affected. Direct upstream kinases of JNK were not affected by ChK. Wortmannin and LY294002, two PI3 kinase inhibitors, inhibited Akt but not JNK phosphorylation in ras-transformed cells. This report establishes the dual inhibitory effect of ChK on both the Akt and JNK signaling pathways in ras-transformed epithelial and human carcinoma cells. The unique effect of ChK on these two key pathways involved in carcinogenesis earmarks ChK for further studies to determine its molecular target(s) and in vivo anti-tumor potential.

A Cell-Cell Communication Marker for Identifying Targeted Tumor Therapies.

Cell-cell communication through gap junctions is aberrant or absent in a majority of human cancer cells, compared to cells in corresponding normal tissues. This and other evidence has led to the hypothesis that gap junction channels, comprised of connexin proteins, are important in growth control and cancer progression. The major goal of this ongoing study was to identify bioactive compounds that specifically upregulate gap junction channel-mediated cell-cell communication as potential anti-tumor therapies. Control of cell-cell communication is linked to growth regulatory intracellular signaling pathways; we therefore further aimed to identify signaling pathways modulated by these compounds in order to assess their potential as targeted anti-tumor therapies. Compounds were screened for their ability to upregulate gap junction-mediated cell-cell communication by using a fluorescent dye transfer assay to measure cell-cell communication between tumor promoter-treated astroglial cells or ras-transformed epithelial cells. Western blotting using connexin-specific and phosphorylation site-specific antibodies was used to monitor phosphorylation changes in signaling pathway proteins. Our results identified three compounds that upregulate gap junction-mediated cell-cell communication in our screening assays, chaetoglobosin K(ChK), 4-phenyl-3-butenoic acid (PBA) and the methyl ester of PBA (PBA-Me). Further analyses demonstrated that in tumorigenic cells, ChK downregulates phosphorylation of Akt kinase, an enzyme in the PI3-kinase signaling pathway that is found to be upregulated in a number of human cancers, on a key activation site. However, ChK did not inhibit PI-3 kinase in vitro as did the classic PI-3 kinase inhibitor, Wortmannin. PBA and PBA-Me were found to upregulate phosphorylation of p38 MAPK on a key activation site in tumorigenic cells, which is downregulated in several human cancer cell types. ChK and PBA also decreased activation of SAPK/JNK, another kinase found to be upregulated in a number of human cancers. These studies highlight the potential of monitoring gap junction intercellular communication for identifying experimental anti-tumor compounds.

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid.

Human lung neoplasms frequently express mutations that down-regulate expression of various tumor suppressor molecules, including mitogen-activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti-tumor therapies. We now report that 4-phenyl-3-butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non-cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell-cell communication. H2009 human lung carcinoma cells and ras-transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras-transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell-cell communication and connexin 43 phosphorylation in ras-transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up-regulation of p38 MAPK activity, altered connexin 43 expression, and down-regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down-regulated p38 MAPK activity and/or activated JNK and altered cell-cell communication.

Protective effect of the natural product, chaetoglobosin K, on lindane- and dieldrin-induced changes in astroglia: identification of activated signaling pathways.

PURPOSE: The purpose of the present study was to identify the biochemical mechanism(s) of the preventative and reversal effects of Chaetoglobosin K (ChK), a bioactive natural product, on inhibition of gap junction-mediated communication and connexin phosphorylation by the tumor promoting organochlorine compounds, lindane, and dieldrin. MATERIALS AND METHODS: A fluorescent dye transfer assay was used to quantify gap junction-mediated communication and sensitivity to lindane and dieldrin. Analyses of connexin 43, PKC, ERK, GSK-3beta, Raf, and Akt kinase phosphorylation were performed by Western blotting. RESULTS: Pre-incubation of astroglial cells with 10 microM ChK prevented inhibition of dye transfer by lindane and dieldrin, which correlates with stabilization of the connexin 43 P2 isoform, and addition of ChK after lindane or dieldrin reversed the inhibitory effect, which correlated with re-appearance of the P2 isoform. Using phosphorylation site-specific antibodies, we demonstrated that lindane, dieldrin, and ChK all activated p44/42 ERK, but only ChK activated Akt kinase. ChK also activated a downstream effector of Akt, GSK-3beta, and activation of both kinases was inhibited by Wortmannin. Wortmannin also blocked ChK's ability to prevent dieldrin-induced inhibition of gap junction-mediated communication between RG-2 cells. CONCLUSION: ChK's protective effects, both preventative and reversal, on lindane and dieldrin inhibition of gap junction-mediated communication are associated with stabilization and reappearance of the connexin 43 P2 phosphoform and may be mediated by the Akt pathway.

Inhibition of cytokinesis and akt phosphorylation by chaetoglobosin K in ras-transformed epithelial cells.

PURPOSE: Chaetoglobosin K (ChK), a bioactive natural product previously shown to have anti-tumor promoting activity in glial cells and growth inhibitory effects in ras-transformed fibroblasts, inhibited anchorage-dependent and anchorage-independent growth in ras-transformed liver epithelial cells. The purpose of this study was to identify cellular targets of ChK that mediate its anti-tumor effects. METHODS: Anchorage-independent cell growth assays, using soft agar-coated dishes, and anchorage-dependent growth assays were performed on transformed WB- ras1 cells. Phase/contrast and fluorescent microscopy were used to visualize cell morphological changes and DAPI-stained nuclei. Analyses of p21 Ras membrane versus cytosolic forms, p44/42 mitogen-activated protein kinase (MAPK) phosphorylation, Akt kinase phosphorylation and connexin 43 phosphorylation were performed by Western blotting. Gap junction-mediated cellular communication was measured by fluorescent dye transfer. RESULTS: Treatment of WB- ras1 cells with a non-cytotoxic dose of ChK inhibited both anchorage-dependent and anchorage-independent growth. Inhibited cells were generally larger and less spindle-shaped in morphology than vehicle-treated cells, many of which were multinucleate. Removal of ChK induced cytokinesis and a return to predominantly single-nucleate cells, suggesting that ChK inhibits cytokinesis. The proportion of membrane-associated versus cytosolic forms of p21 Ras was unchanged by ChK treatment, suggesting that ChK does not act as a farnesylation inhibitor. ChK treatment decreased the level of phosphorylation of Akt kinase, a key signal transducer of the Phosphatidylinositol 3-kinase pathway. In contrast, ChK had no effect on phosphorylation of p44/42 MAPK, which mediates the MAPK/ERK Ras effector pathway. Phosphorylation of the gap junction protein, connexin 43, shown previously to increase following treatment with other anti-Ras compounds, was also not altered by ChK, which correlated with its lack of effect on gap junction-mediated cellular communication. CONCLUSIONS: Our results demonstrate that ChK inhibits Akt kinase phosphorylation and cytokinesis in ras-transformed cells, which likely contribute to its ability to inhibit tumorigenic growth.

Reversal of the transformed phenotype and inhibition of peptidylglycine alpha-monooxygenase in Ras-transformed cells by 4-phenyl-3-butenoic acid.

Recent studies have shown that the proliferation of some tumor cells is dependent on autocrine growth loops that require amidated autocrine growth factors. Peptidylglycine alpha-monooxygenase (PAM) is required for amidation of these growth factors and, therefore, this enzyme is an attractive target for anti-tumor compounds. 4-Phenyl-3-butenoic acid (PBA) is an irreversible turnover-dependent inhibitor of PAM in vitro and has been shown to decrease lung cancer cell proliferation by inhibiting the synthesis of amidated growth factors. We show here that PBA (0.1 mg/mL) inhibits the growth of Ras-transformed epithelial cells (WB-Ras) but has little effect on the proliferation of normal epithelial cells (WB-Neo). The methyl ester derivative of PBA (PBA-Me) at 10-fold lower concentration also exhibits a selective inhibition of Ras-transformed cell growth compared to normal epithelial cell growth. In addition, PBA produces a significant upregulation of gap junctional communication between WB-Ras cells following 2-5 day treatments, with a corresponding increase in the degree of connexin 43 phosphorylation and an increase in the number of connexin 43-containing plasma membrane gap junction plaques. Western blot analyses indicate no effect of PBA on the proportion of p21 Ras in the membrane versus cytosolic fractions or on p44/42 MAP kinase phosphorylation. Furthermore, the cell morphology of PBA-treated WB-Ras cells is altered, so as to more closely resemble that of non-transformed WB-Neo cells. PAM activity was assayed in both WB-Ras and WB-Neo cells, and we demonstrate that PBA at long treatment times (4 days) inhibits PAM activity in both cell types at concentrations that produce selective growth inhibition of WB-Ras cells. Shorter PBA treatment times (24 h), however, inhibit PAM activity in WB-Ras but not WB-Neo cells, an effect that was mimicked by PBA-Me. Taken together, these results clearly demonstrate that PBA returns Ras-transformed cells to a more normal phenotype, a finding consistent with the known increased dominance of the Ras signaling pathway in transformed epithelial cells.

Connexin 36 in bovine retina: lack of phosphorylation but evidence for association with phosphorylated proteins.

In vertebrate retina interneuronal communication through gap junctions is involved in light adaptation and in the transfer of visual information from the rod pathway to the cone pathway. Reports over the last two decades have indicated that these gap junctions are regulated by cyclic nucleotide-dependent protein kinases suggesting that the gap junction proteins, connexins, are phosphorylated. Though all the connexins involved in light adaptation and information transfer from rod to cone pathway are not yet known, connexin 36 has been shown to be definitively involved in the latter process. We have therefore attempted to investigate the cyclic nucleotide-dependent phosphorylation of this connexin in bovine retina. We found several soluble and membrane proteins in bovine retina whose phosphorylation was regulated by cyclic nucleotides. However, no protein of about 36 kDa with cyclic nucleotide-regulated phosphorylation was found in gap junction-enriched membrane preparations. A 36-kDa phosphorylated protein was found in gap junction-enriched membranes phosphorylated in the presence of calcium. However, this protein was not immunoprecipitated by anti-connexin 36 antibodies indicating that it was not connexin 36 in spite of its similarity in molecular weight. Immunoprecipitation did reveal phosphorylated proteins coimmunoprecipitated with connexin 36. Two of these proteins were identified as beta and alpha tubulin subunits. Though cyclic GMP and calcium did not greatly influence the association of these proteins with connexin 36, the results suggest the possibility of connexin 36 associating with other proteins. Together, these observations indicate that interneuronal communication at gap junctions made by connexin 36 may not be regulated by direct phosphorylation of connexin 36, but possibly by phosphorylation of associated proteins.