Identification of a Water-Soluble Indirubin Derivative as Potent Inhibitor of Insulin-like Growth Factor 1 Receptor through Structural Modification of the Parent Natural Molecule.

Indirubins have been identified as potent ATP-competitive protein kinase inhibitors. Structural modifications in the 5- and 3'-position have been extensively investigated, but the impact of substituents in 5'-position is not equally well-studied. Here, we report the synthesis of new indirubin 3'- and 5'-derivatives in the search of water-soluble indirubins by introducing basic centers. Antiproliferative activity of all compounds in tumor cells was evaluated along with kinase inhibition of selected compounds. The results show the 3'-position to tolerate large substituents without compromising activity, whereas bulk and rigid substituents in 5'-position appear unfavorable. Screening molecular targets of water-soluble 3'-oxime ethers revealed 6ha as preferential inhibitor of insulin-like growth factor 1 receptor (IGF-1R) in a panel of 22 protein kinases and in cells. Consistently, 6ha inhibited tumor cell growth in the NCI 60 cell line panel and induced apoptosis. The results indicate that the 5'-position provides limited space for chemical modifications and identify 6ha as a potent water-soluble indirubin-based IGF-1R inhibitor.

7,7'-Diazaindirubin--a small molecule inhibitor of casein kinase 2 in vitro and in cells.

Aza- and diaza-bisindoles were synthesized by coupling of 7-azaisatin, 7-azaoxindol, 7-azaindoxyl acetate, and their non-aza counterparts, respectively. Whereas 7,7'-diazaindigo (10) and 7,7'-diazaisoindigo (11) did not show antiproliferative activity in several human tumor cell lines up to 100 µM, 7-azaindirubin (12) and 7'-azaindirubin (13) were more active than the parent molecule, indirubin, in LXFL529L cells (human large cell lung tumor xenograft), and 7,7'-diazaindirubin (14) was exhibiting substantially enhanced growth inhibitory activity in these cells. In the NCI 60 cell line panel, 14 displayed antiproliferative activity preferentially in certain melanoma and non-small cell lung cancer cells. In contrast to the potent serine/threonine/tyrosine kinase inhibition observed for indirubins, kinase inhibition profiling of 14 in 220 kinases revealed largely a loss of kinase inhibitory activity towards most kinases, with retained inhibitory activity for just a few kinases. At 1 µM concentration, especially casein kinases CK1γ3, CK2α, CK2α2, and SIK were inhibited by more than 50%. In cell-based assays, 14 markedly affected CK2-mediated signaling in various human tumor cells. In MCF7 cells, 14 induced cell cycle arrest at G1 and G2/M and apoptosis, whereas CK2-deficient MCF7 cells were resistant. These findings reveal a novel key mechanism of action for 14, suggesting primarily CK2 inhibition to be causally related to growth inhibition of human tumor cells.

Synthesis and cytotoxicity of novel indirubin-5-carboxamides.

Indirubins have been reported to act as potent inhibitors of protein kinases relevant to tumorigenesis and of tumor cell growth, but their development to antitumor drugs suffer from their poor water solubility. We synthesized a novel class of indirubin derivatives, indirubin-5-carboxamides, carrying amide substituents with basic centers. Quaternization or protonation of these alkylamino substituents provided indirubins with significantly improved solubility without loss of bioactivity.

Limited stability in cell culture medium and hydrogen peroxide formation affect the growth inhibitory properties of delphinidin and its degradation product gallic acid.

In the present study we investigated the stability of anthocyanidins under cell culture conditions and addressed the question whether degradation products might contribute to the cellular effects assigned to the parent compounds. Substantial degradation was found already after 30 min, measured by HPLC/DAD. However, the decrease of detectable anthocyanidins exceeded by far the formation of the respective phenolic acids. From the formed phenolic acids only gallic acid (GA) exhibited growth inhibitory properties. However, also GA was found to be degraded rapidly. Furthermore, the incubation with delphinidin (DEL) or GA resulted in a substantial formation of hydrogen peroxide. The suppression of hydrogen peroxide accumulation by catalase modified significantly the growth inhibitory effects of DEL and GA, indicating that hydrogen peroxide formation might generate experimental artefacts. In summary, the results show that the phenolic acids formed by the degradation of cyanidin (CY), pelargonidin (PG), peonidin (PN) and malvidin (MV) do not contribute to the growth inhibitory effect of the parent compound. The degradation of DEL generates a phenolic acid with substantial growth inhibitory properties (GA). However, taken into account the small proportion of generated GA and its lacking stability, the contribution of GA to the growth inhibitory properties of DEL might be limited.

Biological activities of malvidin, a red wine anthocyanidin.

Malvidin (mv) has been identified as a potential inhibitor of 3',5'-cyclic adenosine monophosphate (cAMP) phosphodiesterases (PDE). This study was to investigate if, as a possible consequence of intracellular PDE inhibition, the activity of the mitogen-activated protein kinase (MAPK) cascade is affected by mv treatment. At a concentration of 5 microM of mv a significant decrease of phosphorylated ERK1 and ERK2 (ERK, extracellular regulated kinase) in HT29 cells was observed. However, an increase in substance concentration led to a substantial recurrence of the phosphorylated enzymes. Cell cycle analysis underlined that indeed G(1)-relevant targets are only marginally affected by mv. The recurrence of phosphorylated ERK1/2 and the lack of effectiveness on the G(1)-passage up to 100 microM indicated that the inhibition of cAMP-specific PDEs is of minor relevance for the growth-inhibitory properties of mv in HT29 cells. In contrast, the release of cells, synchronised in the G(2)/M-phase of the cell cycle by nocodazole treatment, was effectively blocked in the presence of 1 microM mv. These results suggest that mv interferes with cellular targets relevant for G(2)/M-progression which have not been identified so far.

Differential phosphodiesterase expression and cytosolic Ca2+ in human CNS tumour cells and in non-malignant and malignant cells of rat origin.

A promising attempt in the field of tumour therapy is the modulation of intracellular, proliferation-associated signalling pathways. The role of cyclic nucleotide phosphodiesterases (PDEs), key enzymes in cAMP/cGMP signal transduction, was investigated in two human CNS tumour cell lines as well as in the rat glioblastoma cell line C6 in comparison with rat cerebellar astrocytes with the emphasis on target evaluation. We found differential PDE expression patterns in human CNS tumour cell lines as well as in CNS cells of rat origin. In human glioblastoma cells, intracellular cAMP and Ca(2+) levels correlated well with the PDE expression pattern. There were, however, marked differences in PDE expression and Ca(2+) kinetics between the human glioblastoma cell lines. In contrast to human epithelial tumour cells, shown earlier by us to express significantly enhanced cAMP-specific PDE activity, this was not the case in rat glioblastoma cells compared with non-malignant rat astrocytes. Despite different levels of PDE1 and PDE4 expression and activity, cyclic nucleotide and Ca(2+) levels in non-malignant and malignant rat CNS cells were similar. These in vitro data do not support the concept of PDE1C representing a target exploitable for drug treatment of malignant CNS tumours.

Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells.

Stat3 protein has an important role in oncogenesis and is a promising anticancer target. Indirubin, the active component of a traditional Chinese herbal medicine, has been shown previously to inhibit cyclin-dependent kinases, resulting in cell cycle arrest. Here, we show that the indirubin derivatives E564, E728, and E804 potently block constitutive Stat3 signaling in human breast and prostate cancer cells. In addition, E804 directly inhibits Src kinase activity (IC(50) = 0.43 microM) in an in vitro kinase assay. Levels of tyrosyl phosphorylation of c-Src are also reduced in cultured cells 30 min after E804 treatment. Tyrosyl phosphorylation of Stat3, which is known to be phosphorylated by c-Src, was decreased, and constitutive Stat3 DNA binding-activity was suppressed in cells 30 min after E804 treatment. The antiapoptotic proteins Mcl-1 and Survivin, which are encoded in target genes of Stat3, were down-regulated by indirubin derivatives, followed by induction of apoptosis. These results demonstrate that E804 directly blocks the Src-Stat3 signaling pathway, suggesting that the antitumor activity of indirubin compounds is at least partially due to inhibition of this pathway.