Moguntinones--new selective inhibitors for the treatment of human colorectal cancer.

3-Indolyl and 3-azaindolyl-4-aryl maleimide derivatives, called moguntinones (MOG), have been selected for their ability to inhibit protein kinases associated with angiogenesis and induce apoptosis. Here, we characterize their mode of action and their potential clinical value in human colorectal cancer in vitro and in vivo. MOG-19 and MOG-13 were characterized in vitro using kinase, viability, and apoptosis assays in different human colon cancer (HT-29, HCT-116, Caco-2, and SW480) and normal colon cell lines (CCD-18Co, FHC, and HCoEpiC) alone or in combination with topoisomerase I inhibitors. Intracellular signaling pathways were analyzed by Western blotting. To determine their potential to inhibit tumor growth in vivo, the human HT-29 tumor xenograft model was used. Moguntinones prominently inhibit several protein kinases associated with tumor growth and metastasis. Specific signaling pathways such as GSK3ß and mTOR downstream targets were inhibited with IC(50) values in the nanomolar range. GSK3ß signaling inhibition was independent of KRAS, BRAF, and PI3KCA mutation status. While moguntinones alone induced apoptosis only in concentrations >10 µmol/L, MOG-19 in combination with topoisomerase I inhibitors induced apoptosis synergistically at lower concentrations. Consistent with in vitro data, MOG-19 significantly reduced tumor volume and weight in combination with a topoisomerase I inhibitor in vivo. Our in vitro and in vivo data present significant proapoptotic, antiangiogenic, and antiproliferative effects of MOG-19 in different human colon cancer cells. Combination with clinically relevant topoisomerase I inhibitors in vitro and xenograft mouse model demonstrate a high potency of moguntinones to complement and improve standard chemotherapy options in human colorectal cancer.

Novel 3-Azaindolyl-4-arylmaleimides exhibiting potent antiangiogenic efficacy, protein kinase inhibition, and antiproliferative activity.

Tumor growth and metastasis are highly associated with the overexpression of protein kinases (PKs) regulating cell growth, apoptosis resistance, and prolonged cell survival. This study describes novel azaindolyl-maleimides with significant inhibition of PKs, such as VEGFR, FLT-3, and GSK-3ß which are related to carcinogenesis. Furthermore, these compounds exhibit high kinase selectivity and potent inhibition of angiogenesis and cell proliferation, offering versatile options in cancer treatment strategies.

3,4-Diarylmaleimides-a novel class of kinase inhibitors-effectively induce apoptosis in FLT3-ITD-dependent cells.

FLT3 kinase has become an attractive drug target in AML with up to 30% of cases harboring internal-tandem-duplication (ITD) mutations. For these, conferring a worse prognosis and decreased overall survival, several FLT3 tyrosine kinase inhibitors (TKIs) are currently being tested in clinical trials. However, when using these drugs as monotherapy, the problem of short duration of remissions and high incidence of TKI resistance has emerged. Here, we investigated two members of a novel class of tyrosine kinase inhibitors, 3,4-diarylmaleimides, for their efficacy on mutated FLT3 kinase. These compounds inhibit FLT3 kinase in an ATP-competitive manner and effectively inhibit phosphorylation of downstream targets. 3,4-Diarylmaleimides (DHF125 and 150) induce apoptosis in FLT3-ITD-dependent cells lines and patient blasts at low micromolar concentrations. They are retained in the cytoplasm of exposed cells for more than 24 h and synergize with chemotherapy and midostaurin. Both 3,4-diarylmaleimides show inhbition of FLT3-ITD-related kinase autophosphorylation at distinct tyrosine residues when compared to midostaurin. In conclusion, this novel group of compounds shows differential inhibition patterns with regard to FLT3 kinase and displays a promising profile for further clinical development. Currently, experiments evaluating toxicity in murine models and unraveling the exact binding mechanism are under way to facilitate a potential clinical application.

Vanillin suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo.

Vanillin, a food flavoring agent, has been shown to suppress cancer cell migration and metastasis in a mouse model, but its mechanism of action is unknown. In this report, we have examined the antimetastatic potential of vanillin and its structurally related compounds, vanillic acid, vanillyl alcohol, and apocynin on hepatocyte growth factor (HGF)-induced migration of human lung cancer cells by the Transwell assay. Vanillin and apocynin could inhibit cell migration, and both compounds selectively inhibited Akt phosphorylation of HGF signaling, without affecting phosphorylation of Met and Erk. Vanillin and apocynin could inhibit the enzymatic activity of phosphoinositide 3-kinase (PI3K), as revealed by an in vitro lipid kinase assay, suggesting that inhibition of PI3K activity was a mechanism underlying the inhibitory effect on cancer cell migration, and the presence of an aldehyde or ketone group in the vanillin structure was important for this inhibition. Vanillin and apocynin also inhibited angiogenesis, determined by the chick chorioallantoic membrane assay.

Juice of eclipta prostrata inhibits cell migration in vitro and exhibits anti-angiogenic activity in vivo.

BACKGROUND: The invasion of cancer cells is critical for metastasis. The effects of Eclipta prostrata, a Thai medicinal plant, on invasion, migration and adhesion of cancer cells were investigated and the anti-angiogenic activity in vivo was evaluated. MATERIALS AND METHODS: In vitro invasion and migration assays were performed in modified Boyden chambers. In vivo anti-angiogenic activity was determined using the chick chorioallantoic membrane (CAM) assay. RESULTS: E. prostrata juice inhibited cancer invasion and migration, without affecting cell adhesion. Cell migration was inhibited in a variety of cancer cell types and in endothelial cells, with IC50 values of 31-70 microg/ml, much lower than the IC50 values for cytotoxicity of 203-1,217 microg/ml for cancer cells and >4,000 microg/ml for endothelial cells. Fifty percent inhibition of angiogenesis by E. prostrata juice was observed at 200 microg/egg. CONCLUSION: E. prostrata juice inhibited cancer and endothelial cell migration in vitro and also showed in vivo anti-angiogenic activity.

Synthesis of GABAA receptor agonists and evaluation of their alpha-subunit selectivity and orientation in the GABA binding site.

Drugs used to treat various disorders target GABA A receptors. To develop alpha subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at alpha 2-, alpha 3-, and alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in alpha 2-, alpha 4-, and alpha 6-containing receptors and potentiated alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants alpha 1F64C and alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing alpha subtype selective GABA mimetic drugs.

Design, synthesis, and biological evaluation of 3,4-diarylmaleimides as angiogenesis inhibitors.

The new analogue 2 of combretastatin A-4 was discovered to be an inhibitor of tubulin polymerization with an IC50 of 7.6 microM and reduced angiogenesis in the in vivo chick embryo model. Interestingly, in a series of 2,3-diarylmaleimides closely related to this lead, no other compound was found to be active in the tubulin polymerization assay. However, by screening in the in vivo chick embryo assay 10 was identified as a potent angiogenesis inhibitor indicating an alternative target. Indeed, molecular modeling studies suggest a reasonable binding mode of 10 at the ATP-binding site of the model kinase CDK2. Motivated by these results, analogues of 10 were screened for inhibitory activity in a panel of 12 selected protein kinases and a high affinity of 10 to VEGF-R2 was found showing an IC50 of 2.5 nM. Structure-activity relationships (SAR) for this compound series with the isolated enzyme and equivalent antiangiogenic activity in the chick embryo assay are presented herein.

A novel quantitative chick embryo assay as an angiogenesis model using digital image analysis.

A number of pathological events in humans such as solid tumour growth and metastasis have been found to be associated with angiogenesis, the formation of new blood vessels front pre-existing vasculature. A novel approach in cancer treatment consists of blocking or delaying the progression of neovascularisation to dysplastic cells in order to stop delivery of nutrients and oxygen. Thus, agents with antiangiogenic activity may be useful to treat cancer with potentially less systemic toxicity than conventional cytotoxic therapeutics. In this study we report the development and validation of a modified quantitative and objective in vivo chick embryo assay with Digital Image Analysis to screen compounds for antiangiogenic activity. The evaluation of vascular responses was optimised to less than one minute per sample in order to make this automatic method suitable for larger experimental series. Significant activity of the known antiangiogenic compounds thalidomide and captopril was determined, which resulted in approximately 50% inhibition of the vessel area compared to the control. The stability and release of thalidomide and captopril within 48 h after application to the extraembryonic vessel system was monitored by HPLC analysis. In conclusion, this multiple target test system will be complementary to existing angiogenesis assays and may be useful to quantify the antiangiogenic activity of compounds.

Convergent synthesis and preliminary biological evaluations of the stilbenolignan (+/-)-aiphanol and various congeners.

Treatment of an equimolar mixture of stilbene 7 and cinnamyl alcohol 8 with silver carbonate in acetone-benzene afforded a ca. 2:1:2:1 mixture of the stilbenolignan (+/-)-aiphanol (1) and congeners 2-4 each of which show significant anti-angiogenic and COX-2 inhibitory properties.

Synthesis and biological evaluation of cycloalkylidene carboxylic acids as novel effectors of Ras/Raf interaction.

The protooncogenes Ras and Raf play important roles in signal transduction pathways regulated by mitogen-activated protein kinases. Mutations of Ras that arrest the protein in its active state are frequently implicated in tumor formation. We used Ras and Raf proteins in the yeast two-hybrid system to search for natural or synthesized substances capable of modulating Ras/Raf interaction by specifically binding to one of the interacting partners. We found that cycloalkylidene carboxylic acids enhanced Ras/Raf interaction by acting on the cysteine-rich domain of Raf. Several analogues of the active substance 2-cyclohexylidene propanoic acid were synthesized and the importance of the semicyclic double bond in the stabilization of Ras/Raf interaction was demonstrated. Variation of the size and the substituents of the cyclic system as well as the length of the carboxylic acid resulted in enhanced Ras/Raf interaction.