Screening for specific proteasome inhibitors followed by the determination of proapoptotic and antitumoral properties in cell culture

• Grant number: 04/07636-9
• Support Opportunities: BIOTA-FAPESP Program - Regular Research Grants
• Start date: December 01, 2004
• End date: November 30, 2006
• Field of knowledge: Biological Sciences - Biochemistry
• Principal Investigator: Marilene Demasi
• Grantee: Marilene Demasi
• Host Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Proteasome inhibitors promote cell death in the cells of mammals by a process described, in many cases (depending on the cell lineage and dose), as apoptosis. This activity on the part of the specific inhibitors of protease has been explored as potentially beneficial in the chemotherapy of several tumors including solid and hematological ones. A highlight of recent scientific literature is the synthetic inhibitor known as bortezomib soon to be introduced into clinical medicine. Other evidence of the therapeutic potential of these inhibitors are the studies which have been published in recent years on the search for these compounds within products of natural origin, in addition to the chemical research of innumerable new synthetic compounds. The present study proposal has the objective of identifying among the natural products made available by the Biota-FAPESP Program, compounds that may be specific inhibitors of proteasome. The methodology to be used implies microtrials and envisages the monthly screening of hundreds of extracts and/or isolated derivatives. It will consist of the measurement of the activity of the proteasome in the presence of these products through the intermediary of trials on microplates, given that the proteasome will be isolated from a strain of S. cerevisiae where one of the subunits of the catalytic complex 20S is modified with a polyhistidine tag which enables the purification of the 20S complex in a few hours. The second phase of the project envisages the study of the specificity of the potential inhibitors found in the first stage. The specificity of the inhibitors will be evaluated by means of immunoprecipitation trials and enzymatic kinetics. After the identification and selection of the specific inhibitors of proteasome, evaluation will be undertaken of the pro-apoptotic activity of these compounds. The trials in this phase of the project will be undertaken in cultures of tumor cells of diverse strains in which the cell viability will be evaluated and the characterization of the apoptotic process undertaken by means of trials already consolidated in the literature. The stages of the project which involve the tests of the apoptotic effect on the culture of tumor cells and studies of cell viability could, as it is desirable for the program, be undertaken in collaboration with other groups within BIOprospecTA whose experimental objectives are similar. (AU)