Gene Therapy Fas Inhibitor(ONL)

Two multivariate studies, a PCA-SAR and a PLS-QSAR, of 3-aryl-4-hydroxyquinolin-2(1H)-one derivatives described as type I fatty acid synthase (FAS) inhibitors, are presented in this work. The variable selection was performed with the Fisher's weight and Ordered Predictors Selection (OPS) algorithm, respectively. In the PCA, a separation between active and inactive compounds was obtained by six descriptors (topological and geometrical). The PLS model presented five descriptors and two Latent Variables. Leave-N-out cross validation and y-randomization test showed that the model presented robustness and no chance correlation, respectively, and the descriptors indicated that the FAS inhibition depends on electronic distribution of the investigated compounds. The model obtained in this study may provide a guidance for proposition of new FAS inhibitors.

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.