Henriette Gourdeau

Discovery of 4-aryl-4H-chromenes as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. 4. Structure-activity relationships of N-alkyl substituted pyrrole fused at the 7,8-positions.

In our continuing effort to discover and develop apoptosis inducing 4-aryl-4H-chromenes as novel anticancer agents, we explored the structure-activity relationship (SAR) of alkyl substituted pyrrole fused at the 7,8-positions. A methyl group substituted at the nitrogen in the 7-position of the pyrrole ring led to a series of potent apoptosis inducers with potency in the low nanomolar range. These compounds were also found to be low nanomolar or subnanomolar inhibitors of cell growth, and they inhibited tubulin polymerization, indicating that methylation of the 7-position nitrogen does not change the mechanism of action of these chromenes. Compound 2d was identified as a highly potent apoptosis inducer with an EC50 value of 2 nM and a highly potent inhibitor of cell growth with a GI50 value of 0.3 nM in T47D cells.

Identification, characterization and potent antitumor activity of ECO-4601, a novel peripheral benzodiazepine receptor ligand

PurposeECO-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER® technology, Thallion’s proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity in the low micromolar range when tested in the NCI 60 cell line panel. In the work presented here, ECO-4601 was further evaluated against brain tumor cell lines. Preliminary mechanistic studies as well as in vivo antitumor evaluation were performed.MethodsSince ECO-4601 has a benzodiazepinone moiety, we first investigated if it binds the central and/or peripheral benzodiazepine receptors. ECO-4601 was tested in radioligand binding assays on benzodiazepine receptors obtained from rat hearts. The ability of ECO-4601 to inhibit the growth of CNS cancers was evaluated on a panel of mouse, rat and human glioma cell lines using a standard MTT assay. Antitumor efficacy studies were performed on gliomas (rat and human), human breast and human prostate mouse tumor xenografts. Antitumor activity and pharmacokinetic analysis of ECO-4601 was evaluated following intravenous (IV), subcutaneous (SC), and intraperitoneal (IP) bolus administrations.ResultsECO-4601 was shown to bind the peripheral but not the central benzodiazepine receptor and inhibited the growth of CNS tumor cell lines. Bolus SC and IP administration gave rise to low but sustained drug exposure, and resulted in moderate to significant antitumor activity at doses that were well tolerated. In a rat glioma (C6) xenograft model, ECO-4601 produced up to 70% tumor growth inhibition (TGI) while in a human glioma (U-87MG) xenograft, TGI was 34%. Antitumor activity was highly significant in both human hormone-independent breast (MDA-MB-231) and prostate (PC-3) xenografts, resulting in TGI of 72 and 100%, respectively. On the other hand, IV dosing was followed by rapid elimination of the drug and was ineffective.ConclusionsAntitumor efficacy of ECO-4601 appears to be associated with the exposure parameter AUC and/or sustained drug levels rather than Cmax. These in vivo data constitute a rationale for clinical studies testing prolonged continuous administration of ECO-4601.

Oncodevelopmental and hormonal regulation of α1-fetoprotein gene expression

The main features of the oncodevelopmental biology of α1-fetoprotein (AFP) are reviewed. Progress made in the molecular biology of AFP gene regulation is discussed and we present our recent data on the mechanisms of AFP suppression by glucocorticoid hormones. The relationship between AFP gene transcription and cell replication is examined, and it is suggested that the degree of methylation of the AFP gene (or of co-methylated regulatory DNA sequences) conditions its response to hormones.

TLN-4601 suppresses growth and induces apoptosis of pancreatic carcinoma cells through inhibition of Ras-ERK MAPK signaling

Background TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered using Thallions proprietary DECIPHER® technology, a genomics and bioinformatics platform that predicts the chemical structures of secondary metabolites based on gene sequences obtained by scanning bacterial genomes. Our recent studies suggest that TLN-4601 inhibits the Ras-ERK MAPK pathway post Ras prenylation and prior to MEK activation. The Ras-ERK MAPK signaling pathway is a well-validated oncogenic cascade based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumors. Furthermore, RAS isoforms are the most frequently mutated oncogenes, occurring in approximately 30% of all human cancers, and KRAS is the most commonly mutated RAS gene, with a greater than 90% incidence of mutation in pancreatic cancer. Results To evaluate whether TLN-4601 interferes with K-Ras signaling, we utilized human pancreatic epithelial cells and demonstrate that TLN-4601 treatment resulted in a dose- and time-dependent inhibition of Ras-ERK MAPK signaling. The compound also reduced Ras-GTP levels and induced apoptosis. Finally, treatment of MIA PaCa-2 tumor-bearing mice with TLN-4601 resulted in antitumor activity and decreased tumor Raf-1 protein levels. Conclusion These data, together with phase I/II clinical data showing tolerability of TLN-4601, support conducting a clinical trial in advanced pancreatic cancer patients.

Tln-4601, a novel anticancer agent, inhibits Ras signaling post Ras prenylation and before Mek activation

TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER, Thallions proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity (low μmol/l) when tested in the NCI 60 tumor cell line panel and has shown in-vivo antitumor activity in several xenograft models. Related to its farnesylated moiety, the effect of TLN-4601 on Ras mitogen-activated protein kinase signaling was assessed. Downstream Ras signaling events, Raf-1, MEK, and ERK1/2 phosphorylation in MCF7 cells were evaluated by western blot analysis. TLN-4601 prevented epidermal growth factor-induced phosphorylation of Raf-1, MEK, and ERK1/2. This effect was time-dependent and dose-dependent with complete inhibition of protein phosphorylation within 4–6 h at 10 μmol/l. The inhibition of Ras signaling was not mediated by the inhibition of protein prenylation, documented by the lack of effect TLN-4601 on the prenylation of HDJ2 (specific substrate of farnesyltransferase), RAP1A (specific substrate of geranylgeranyl transferase-1), or Ras. As TLN-4601 did not inhibit EGFR, Raf-1, MEK or ERK1/2 kinase activities, the inhibitory effect of TLN-4601 on Ras signaling is not mediated by direct kinase inhibition. Using an Elk-1 trans-activation reporter assay, we found that TLN-4601 inhibits the MEK/ERK pathway at the level of Raf-1. Interestingly, TLN-4601 induces Raf-1 proteasomal-dependent degradation. These data indicate that TLN-4601 may inhibit the Ras-mitogen-activated protein kinase-signaling pathway by depleting the Raf-1 protein.

Species differences in Troxacitabine pharmacokinetics and pharmacodynamics: Implications for clinical development

Purpose: Troxacitabine is the first unnatural l-nucleoside analog to show potent preclinical antitumor activity and is currently under clinical investigation. Significant differences in troxacitabine toxicity between mice, rats, monkeys, and humans were observed during preclinical and clinical evaluations. To better understand the different toxicity and efficacy results observed between the human xenograft mouse tumor models used for preclinical assessment and the clinical study results, the pharmacodynamics and pharmacokinetics of troxacitabine were reassessed in murine and human models. Experimental Design: Clonal and thymidine incorporation assays were used to investigate the in vitro antiproliferative activity of troxacitabine on a selected panel of mouse and human tumor cell lines and normal hemapoietic cells. Analysis of the intracellular metabolites of [14C]troxacitabine was determined in mouse and human T-lymphocytes obtained from peripheral blood. The antitumor efficacy of troxacitabine administered either as single or repeated high-dose bolus administrations or as low-dose continuous infusions was evaluated in the human colon HT-29 xenograft model. We also determined plasma concentrations of troxacitabine using the different administration schedules. Results: Five to nine hundred-fold lower concentrations of troxacitabine were required to inhibit cell growth in human compared with murine tumor and normal hemapoietic cell lines. Furthermore, the sensitivity of cells of both species to troxacitabine was strongly time dependent, requiring >24 hours exposure for maximum activity. Analysis of the intracellular metabolites of [14C]troxacitabine in T-lymphocytes obtained from peripheral blood revealed subsequently higher levels of mono-, di-, and triphosphates in human compared with mouse. Antitumor efficacy studies revealed that prolonged exposure schedules (up to 6 days) showed equivalent efficacy to repeated high-dose bolus administrations. Five-day continuous infusion of 20 mg/mL troxacitabine via subcutaneous implanted mini-osmotic pump maintained systemic concentrations of 262 ng/mL (1.2 μmol/L) for the duration of administration, which are clinically achievable plasma concentrations, and led to significant antitumor activity [treated versus control (T/C) of 27% and tumor regression during treatment]. Conclusions: These studies support the hypothesis that troxacitabine infusions might be the administration regimen with the greatest likelihood of fully exploiting clinically the potent preclinical antitumor activity of troxacitabine.

TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins(*).

The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1H and 13C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity.

Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography

A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.

Abstract C4: TLN‐4601 is a novel tumor selective pro‐apoptotic agent acting independently of Bax, Bak or Bcl‐2 expression

Background: TLN‐4601 is a structurally novel farnesylated dibenzodiazepinone discovered through Thallion9s proprietary DECIPHER® technology platform. The compound has demonstrated broad in vitro cytotoxicity and in vivo tumor growth inhibition. Phase I clinical testing has shown the compound to be safe and well tolerated, with early signs of anti‐tumor activity. Although considerable research efforts have focused on the ability of TLN‐4601 to inhibit the Ras/MAPK signalling pathway, the compound has also been shown to induce apoptosis through caspase activation. The objectives of the present work were to further elucidate the molecular events, pathways and temporal patterns associated with TLN‐4601‐induced apoptosis. Material and Methods: Protein extracts from Jurkat cells (a human T cell lymphoblast‐like cell line), Jurkat cells with caspase 8 knocked‐down, Jurkat cells over‐expressing Bcl‐2, mouse embryonic fibroblasts isolated from Bax/Bak double knock‐out mice, and both quiescent and PHA‐L stimulated human peripheral blood lymphocytes (PBLs) treated with TLN‐4601 over different exposure times were analyzed by Western blot to evaluate the induction of apoptotic markers, including cytochrome c release, caspase activation and PARP cleavage. Apoptosis was assessed by DNA laddering and sub‐G1 DNA content. The effect of TLN‐4601 on cell viability were measured using commercial Caspase‐3/7 activity and ATP assays. Results: TLN‐4601 caused rapid apoptotic cell death in Jurkat cells as characterized by mitochondrial cytochrome c release, caspase activation, loss of cellular ATP and DNA laddering. Equal sensitivity of the wild‐type and caspase 8 knocked‐down Jurkat cells to TLN‐4601 suggests that the extrinsic apoptotic pathway was not involved in mediating this activity. Interestingly, TLN‐4601 exposure resulted in cytochrome c release independently of the apoptosis‐inducing proteins Bax and Bak. Furthermore, the over‐expression of the anti‐apoptotic protein Bcl‐2 did not affect the ability of TLN‐4601 to induce cell death. Finally, a short exposure of TLN‐4601 exhibited a preferential selectivity for triggering apoptosis in Jurkat cells as compared to either quiescent and PHA‐L stimulated PBLs. Conclusions: TLN‐4601 mediates rapid cell death via the intrinsic apoptotic pathway independent of Bax, Bak or Bcl‐2 expression. Moreover, the differential sensitivity to TLN‐4601 observed between Jurkat cells and PBLs highlights the potential for a usable therapeutic margin between cancer and normal cells. Taken together, these properties represent a very promising profile for an anti‐cancer agent and suggest that further investigation of TLN‐4601 in hematological malignancies is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C4.