We propose to develop and validate a high throughput assay for cost/time-efficient screening and characterizing of compounds transported by human MDR1 P-glycoprotein (Pgp). The test (referred to as "the UIC2 Shift Assay") is based on a new principle: increased immunoreactivity of an anti-Pgp monoclonal antibody, UIC2, in the presence of MDR1 substrates. Our preliminary data shows the potential of the test: several new Pgp substrates have been identified using the flow cytometry version of the assay. The UIC2 Shift Assay is strictly MDR1-specific and allows for the dissection of multifactorial drug response in human malignancies. In order to develop a commercially viable, high throughput test, we designed and tested in preliminary experiments a 96-well version of the UIC2 Shift Assay. In Phase I, the design of the 96-well platform for the UIC2 Shift Assay will be optimized and validated using existing technologies (flow cytometry-based inhibition of the DiOC2 efflux test). A pilot screening will be performed on 250 well-characterized compounds (generic drugs and biochemical standards) from a commercial library. Screening results will be statistically evaluated, and the performance characteristics of the test will be determined. In Phase II, the optimized test will be validated on a large (several thousand) compound library, and a commercial kit for screening of new and characterizing of existing Pgp substrates will be developed and validated in collaboration with Beckman-Coulter, Inc. (BCI), and its subsidiary Immunotech. The technology will be compatible with BCI automated instrumentation. The project will provide a more accurate, specific and cost/time efficient assay for Pgp substrate specificity than currently available technologies. It will be used by pharmaceutical companies and academic institutions to identify new MDR1 modulators and eliminate undesired drugs - Pgp substrates from drug development programs. The potential economic effects can reach an estimated $2-3 billion annually. PROPOSED COMMERCIAL APPLICATION: We propose to develop a high throughput assay for cost/time-efficient screening and characterizing of compounds transported by human MDR1 P-glycoprotein. The automated version of this assay will be used by pharmaceutical companies and academic institutions to identify new MDR1 modulators and eliminate undesired drugs - Pgp substrates from drug development programs. The potential economic effects can reach an estimated $ 2-3 billion annually.
