Abstract 2837: Assay development for the discovery of semaphorin 3B-inducing agents from natural product sources

Abstract

Abstract The family of semaphorins has been found to regulate cell adhesion and cell motility, the immune response, angiogenesis, and tumor progression. The semaphorins are emerging as regulators of key processes of tumor progression. Semaphorin 3B, in particular, has been found to inhibit proliferation and promote apoptosis. Since there is yet no screening assay available to be used in drug discovery for this crucial target, the present study focuses on the development of a new absorbance-based bioassay to search for new semaphorin 3B inducers from natural sources. Among the parameters considered include the type of cancer cell line, the time of incubation, the type of lysis buffer, secondary effects that affect absorbance levels, and ways to minimize false positives. Vitamin D and actinomycin are being used as controls in this assay. Vitamin D acts as a positive control since it is a semaphorin 3B inducer. Comparison of the levels of induction of the active substances to that of the positive control provides an indication about the level of potential a hit will have if being developed as a drug. Actinomycin inhibits transcription by binding to DNA at the transcription initiation level. When using actinomycin in this assay, its effect is considered to be proportional to that of semaphorin 3B inhibitors. Hence, actinomycin serves as a negative control to discriminate inducers from inhibitors of semaphorin 3B. The use of both controls, therefore, offers the opportunity to discover selective and highly potent semaphorin 3B inducers of natural origin. The assay has been standardized in a 96-well plate format to optimize sensitivity, selectivity, accuracy, robustness, and cost effectiveness. A library of natural product compounds isolated from higher plants, filamentous fungi, and cyanobacteria is currently being screened using this new bioassay. A total of 50 samples have been tested already with two samples exhibiting low induction levels. The most active substances will be further tested using a series of immunoblot secondary assays to validate the potential hits. Additionally, the screening assay presented could be modified to discover new small molecules or drug-like scaffolds as inhibitors or inducers of other semaphorins. Thus, an assay to identify compounds that selectively and potently affect this pathway may lead to new ways of understanding the cell signaling mechanism of these agents, which could be beneficial for the discovery of new natural product leads for cancer treatment. Program project grant P01-CA125066-S1 funded by the National Cancer Institute, NIH. 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 2837. doi:1538-7445.AM2012-2837