This phase 1 project has as its overall objectives: to design, engineer, optimize and implement a novel, genetically encoded, fluorescent drug sensor (FDS) based on Förster 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 96-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: 1. Convert the SA gene into Gateway-compatible format, insert into a suite of Gateway vectors (~60), transform E. coli and grow cultures, test responses in crude lysates to drugs, and monitor spectra of promising affinity-purified sensor proteins. 2. Obtain binding isotherms and determine affinities, compare to published data, for drug binding to SA constructs in comparison with standard SA-fluorescein. 3. Optimize signal to noise ratio by linker mutagenesis and create a series of specificity and affinity mutants. 4. Screen NCI Oncology Drug Set (124 compounds) to confirm sensor'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 Statement: 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).
Project Terms: Address; Affinity; Affinity Chromatography; Area; Bacteria; base; Binding (Molecular Function); Binding Sites; Biological; Biological Assay; Biosensor; c-myc Genes; Cells; Chimeric Proteins; Collaborations; cost; Data; design; design and construction; Development; Diffusion; Disease; drug candidate; drug development; Drug Interactions; Drug Monitoring; drug testing; Drug usage; Energy Transfer; Engineering; engineering design; Escherichia coli; Fluorescein; Fluorescence; fluorophore; Genes; Goals; Green Fluorescent Proteins; Human; improved; Industry; inhibitor/antagonist; Institution; International; Knowledge; Lead; Ligands; linker mutagenesis; Lipids; Liposomes; Measures; member; Membrane; Multi-Drug Resistance; mutant; Noise; novel; oncology; Open Reading Frames; Permeability; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Preclinical Drug Evaluation; Property; protein E; Proteins; public health relevance; Publishing; Resolution; response; Science; screening; sensor; Series; Serum Albumin; Signal Transduction; small molecule; Specificity; Structure; Subgroup; System; Techniques; Technology; Testing; Therapeutic; Universities; Variant; vector; wasting; Yeasts
