Eric Beausoleil

RAC1 Inhibition Targets Amyloid Precursor Protein Processing by γ-Secretase and Decreases Aβ Production in Vitro and in Vivo

β-Amyloid peptides (Aβ) that form the senile plaques of Alzheimer disease consist mainly of 40- and 42-amino acid (Aβ 40 and Aβ 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of Aβ involves β-secretase and γ-secretase activities and is regulated by membrane trafficking of the proteins involved in Aβ production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks Aβ 40 and Aβ 42 production but does not impact sAPPα levels and does not inhibit β-secretase. Rather, EHT 1864 modulates APP processing at the level of γ-secretase to prevent Aβ 40 and Aβ 42 generation. This effect does not result from a direct inhibition of the γ-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces Aβ 40 and Aβ 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting Aβ formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease.

Structure–activity relationship of isoform selective inhibitors of Rac1/1b GTPase nucleotide binding

The synthesis of a series of berberine, phenantridine and isoquinoline derivatives was realized to explore their Rho GTPase nucleotide inhibitory activity. The compounds were evaluated in a nucleotide binding competition assay against Rac1, Rac1b, Cdc42 and in a cellular Rac GTPase activation assay. The insertion of 19 AA in the splice variant Rac1b is shown to be sufficient to introduce a conformational difference that allows compounds 4, 21, 22, and 26 to exhibit selective inhibition of Rac 1b over Rac1.

Identification of aldehyde dehydrogenase 1A1 modulators using virtual screening

The highly similar aldehyde dehydrogenase isozymes (ALDH1A1 and ALDH2) have been implicated in the metabolism of toxic biogenic aldehydes such as 3,4-dihydroxyphenylacetaldehyde (DOPAL) and 4-hydroxy-2E-nonenal. We report the down-regulation of ALDH1A1 mRNA found in substantia nigra tissue of human Parkinson’s disease (PD) samples using the Genome-Wide SpliceArray™ (GWSA™) technology. Since DOPAL can rapidly inactivate ALDH1A1 in vitro, we set up a DOPAL-induced ALDH1A1 inactivation assay and used this assay to demonstrate that Alda-1, a compound originally identified as an activator of ALDH2, can also activate ALDH1A1. We carried out a virtual screening of 19,943 compounds and the top 21 hits from this screen were tested in the DOPAL inactivation assay with ALDH1A1 which led to identification of an activator as well as two inhibitors among these hits. These findings represent an attractive starting point for developing higher potency activator compounds that may have utility in restoring the metabolism of DOPAL in PD.

Vascular disrupting activity and the mechanism of action of EHT 6706, a novel anticancer tubulin polymerization inhibitor

SummaryTumor blood vessels are an important emerging target for anticancer therapy. Here, we characterize the in vitro antiproliferative and antiangiogenic properties of the synthetic small molecule, 7-ethoxy-4-(3,4,5-trimethoxybenzyl)isoquinolin-8-amine dihydrochloride, EHT 6706, a novel microtubule-disrupting agent that targets the colchicine-binding site to inhibit tubulin polymerization. At low nM concentrations, EHT 6706 exhibits highly potent antiproliferative activity on more than 60 human tumor cell lines, even those described as being drug resistant. EHT 6706 also shows strong efficacy as a vascular-disrupting agent, since it prevents endothelial cell tube formation and disrupts pre-established vessels, changes the permeability of endothelial cell monolayers and inhibits endothelial cell migration. Genome-wide transcriptomic analysis of EHT 6706 effects on human endothelial cells shows that the antiangiogenic activity elicits gene deregulations of antiangiogenic pathways. These findings indicate that EHT 6706 is a promising tubulin-binding compound with potentially broad clinical antitumor efficacy.

Abstract 2794: Cytotoxic and vascular disrupting activity of EHT 6706, a novel tubulin polymerization inhibitor

Among cytotoxics commonly used for cancer treatment, microtubule-targeting agents harbor a broad clinical anti-tumor efficacy. Microtubule-targeting agents act by their direct cytotoxic actions on tumor cells but also by their anti-angiogenic and vascular-disrupting actions. In contrast to classical antiangiogenic agents, vascular disrupting agents can, at the same time, block cell survival, prevent the formation of new vessels and damage the existing tumor vasculature. Here we describe EHT 6706, the current lead compound of a novel chemical class of tubulin targeting agents. EHT 6706 inhibits in a dose-dependent manner microtubule formation in an in vitro tubulin polymerization assay. EHT 6706 binds at or near the colchicine binding site of the tubulin-colchicine complex, hence displacing the [3] H-colchicine tracer. This binding competition is comparable to that of combretastain A-4, an established colchicine site binder with a higher affinity for tubulin than colchicine. At low concentrations, EHT 6706 decreases the amount of microtubular materials and induces alteration in tumor cell morphology. Perturbing microtubule dynamics with EHT 6706 halts the rapid cancer cells division by blocking cells in the metaphase/anaphase transition of the cell cycle and leads to apoptosis. EHT 6706 exhibits broad cytotoxicity in vitro with potency at sub-nanomolar/low nanomolar concentrations (0.3-10nM) in multiple cancer types (more than 60 cell lines, including the NCI60 panel) such as breast, melanoma, colorectal, renal, ovarian, non-small cell lung, leukaemia, as well as pancreatic cancer cells. EHT 6706 exerts this growth inhibition of human tumor cells independently of the type of main multidrug resistance mechanisms (ABC transporter MDR-1 (Pgp-1), MRP-1…). Furthermore, EHT 6706 retains cytotoxic activity against all of the resistant cell lines tested (combretastatin, vinblastin, imatinib), thus indicating the potential of this agent for the treatment of resistant tumors. EHT 6706 carries a potential for enhancing antitumor effects of radiation. In H460 and MiaPaca2 cell lines, indeed, EHT 6706 treatment inhibits clonogenic survival synergistically with ionizing radiation and exerts additive inhibition of proliferation when given in combination with conventional chemotherapeutic agents. Finally, in vitro experiments demonstrate that EHT 6706 has anti-angiogenic and vascular disrupting effects. Endothelial cell tube formation inhibition, pre-established vessels disruption, and vascular permeability increase were observed in a concentration- and time-dependent manner. These studies demonstrate that EHT 6706 has potent in vitro efficacy as a novel tubulin-targeted anti-proliferative and anti-vascular agent. EHT 6706 is a candidate for in vivo pharmacokinetic and efficacy studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2794. doi:1538-7445.AM2012-2794

Abstract C89: Rac1 splicing variant Rac1b: Towards isoform selective inhibitors for the treatment of cancer

The pivotal role of Rac1, a member of the Rho family of small GTPases, has been well characterized in the genesis of many cancers. The aberrant activation of Rac1 promotes uncontrolled proliferation, invasion and metastatic properties of human cancer cells. Via ExonHit Therapeutics9 alternative splicing discovery plateform DATAS™, we identified a repertoire of genes deregulated in cancer. Among them, Rac1b, an alternative splice variant of Rac1, was found deregulated and overexpressed in diverse cancer types, including breast and colon. Rac1b is constitutively active and transforming, and since it was shown to sustain cancer cell survival, such as for colorectal cancer cells, there is considerable interest in the development of small molecule inhibitors of Rac1/Rac1b. We previously synthesized and fully characterized EHT 1864 as a pharmacological tool to study Rac1 functions(Shutes A et al. (2007). J Biol Chem. 282(49):35666‐78). Then, using molecular docking algorithms and ExonHit9s screening platform for nucleotide binding inhibitors, novel compounds were synthesized and characterized for Rac1/Rac1b inhibition and Rac1 versus Rac1b selectivity. Here, we describe new Rac1/Rac1b inhibitors with better pharmacological profile than EHT 1864 and better potency in various in vitro cancer cell models. Some of these pharmacological tools are good candidates for subsequent in vivo cancer studies to study Rac1 and Rac1b mediated cellular functions and to modulate pathological conditions in which Rac1 or Rac1b deregulation may play a role. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C89.