Pierre Falardeau

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.

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.

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.