A novel drug detection assay using fluorescent biosensor technology

DESCRIPTION provided by applicant This phase project has as its overall objectives to design engineer optimize and implement a novel genetically encoded fluorescent drug sensor FDS based on F rster Resonance Energy Transfer FRET by exploiting conformational rearrangements induced by drug interactions with human serum albumin SA SA is uniquely suited for a general drug sensor because it possesses multiple binding sites for small molecules We have acquired the ORF open reading frame encoding human SA and will insert this ORF into Gateway vectors purify the chimeric proteins from E coli and analyze fluorescence responses to drugs using a well microplate spectrofluorimeter During the first year we will focus on design and construction of sensors once a successful sensor has been obtained we will optimize and fine tune the sensor to different ligand groups The following specific aims are proposed to achieve the goals of phase I of this project Convert the SA gene into Gateway compatible format insert into a suite of Gateway vectors transform E coli and grow cultures test responses in crude lysates to drugs and monitor spectra of promising affinity purified sensor proteins Obtain binding isotherms and determine affinities compare to published data for drug binding to SA constructs in comparison with standard SA fluorescein Optimize signal to noise ratio by linker mutagenesis and create a series of specificity and affinity mutants Screen NCI Oncology Drug Set compounds to confirm sensorandapos s general sensing capacity Development of a successful new drug requires identifying those that show maximal ability to reach target cells permeability while having minimal effects on drug transporters drug drug interactions For this reason the Fluorosome division of GLSynthesis Inc has established collaboration with Prof W Frommer at the Carnegie Institution for Science Stanford University to create genetically encoded FRET drug sensors FDS from SA This novel drug sensor technology is expected to enhance the commercial use of Fluorosome r based assays and lead to the development of novel assay systems PUBLIC HEALTH RELEVANCE We will engineer optimize and implement a novel genetically encoded fluorescent drug sensor that will assist in identifying drug candidates that show maximal ability to reach target cells permeability while having minimal effects on drug transporters drug drug interactions