The purpose of this R03 application is to describe our proposed drug design effort designed to potentiate TRAIL-induced apoptosis in cancer cells. Apoptosis plays an essential role in the regulation of cell number in many physiological and pathological settings. Apoptosis mediates therapy-induced cytotoxicity in chemotherapy, ?-irradiation and immunotherapy. Many tumors, however, have proved to be drug- and treatment-resistant. TRAIL is a highly promising cancer therapeutic because it can induce apoptosis in a broad spectrum of cancer cell types but not in normal cells. Unfortunately, certain tumor cell lines have already acquired resistance to TRAIL. There are chemotherapeutic agents that augment TRAIL-mediated apoptosis when co-administered with TRAIL. These existing combined therapies, however, have demonstrated unacceptable side-effects when therapy affects both tumor and normal cells. We believe that the most realistic and practical means to employ TRAIL-induced apoptosis in cancer treatment is to identify chemical leads that specifically potentiate TRAIL-mediated anti-tumor cytotoxicity. We fully expect to identify these leads by using HTS techniques to screen comprehensive libraries of chemicals. Hypothesis/Aims. Because it is known that TRAIL induces apoptosis in a broad spectrum of cancer cells but not in normal cells and that available drugs used in combined therapy targeting the apoptotic pathways have serious deleterious side-effects, we hypothesize: (1) that it is of the utmost importance to discover and identify chemicals that can be developed into drugs that specifically augment TRAIL-mediated anti-tumor toxicity, and (2) that the use of HTS techniques within the MLSCN is the most efficient manner to accomplish this task. Our approach is based on the sequential treatment of human fibrosarcoma HT1080 cells with chemical library compounds followed by treatment with the trimeric, yeast-derived, TRAIL-LZ construct with a known low hepatotoxicity. The initial hits that augment TRAIL-induced apoptosis will be validated in an additional assay to select chemicals that are non-toxic to normal human hepatocytes.
Our Specific Aims are: (1) To identify low molecular weight chemicals that can efficiently enhance TRAIL-mediated apoptosis in human fibrosarcoma HT1080 cells. (2) To identify, among selected hits, compounds that are non-toxic to normal cells in an additional cell-based assay. (3) To determine which of the hits specifically sensitize tumor cells to TRAIL (and other apoptosis inducing TNF family cytokines) without impacting the sensitivity of cells to other types of apoptosis pathways. The resulting compounds will be specific sensitizers of the """"""""extrinsic"""""""" pathway and consequently will serve as valuable research tools for improving our understanding of the mechanisms of TRAIL resistance. In addition, these compounds can provide a starting point for the development of a novel class of less toxic and more powerful anticancer drugs. One liter of Pichia conditioned medium provides us with 5 mg of purified TRAIL-LZ. This amount is sufficient for the analysis of 500,000 individual chemicals. Future Plans include: (1)To identify the putative targets and to determine the efficiency and selectivity of the apoptosis activation mechanism exerted by initial hits in cell-based assays; (2) Optimize the structure of the selected hits to improve their properties such as potency and ADMET (absorption, distribution, metabolism, excretion, and toxicity) attributes; (3) Confirm the selectivity and efficiency of the derivatized compounds to potentiate TRAIL-mediated apoptosis employing multiple cancer cell types; (4) Confirm the efficiency and safety of the derivatized hits in vivo in tumor xenograft models. Assays. We propose to use the cell-based assays employing HT1080 cells (primary screen) and human hepatocytes (secondary screen). The cells will be incubated with chemicals followed by TRAIL-LZ treatment. This assay with a Z'-factor equal to 0.6-0.9 is readily adaptable to automation to fit 384-well or 1536-well plates. We are confident that reproducibility between plates and day-to-day experiments will be greater than 95% and the coefficient of variation (CV) will not exceed 5%. To select compounds non-toxic to normal cells, we will evaluate the toxicity of the identified hits in human primary hepatocytes. The normal human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center. The main technical parameters of the primary assay are as follows: assay, CellTiter-Glo Luminescent Cell Viability Assay (Promega); assay volume, 0.1 ml; number of cells/well, 50,000; plates, 96-well transparent, flat bottom; temperature, ambient; cost, unless a bulk purchase is arranged, the $2,159.0 costs of the commercially available CellTiter-Glo Cell Viability Assay will be sufficient for the analysis of 10,000 compounds in a 96-well plate format. The cost of all other assay reagents is minimal. Stability of the cells and the substrate: DMSO up to a 1% concentration does not affect the viability of HT1080 target cells. The HT1080 cells, human primary hepatocytes, TRAIL-LZ, and the detailed experimental protocol will be provided to the screening center. ? ? ?
| Rozanov, Dmitri; Cheltsov, Anton; Sergienko, Eduard et al. (2015) TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response. PLoS One 10:e0129566 |
