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 Frster 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

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).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Small Business Technology Transfer (STTR) Grants - Phase I (R41)
Project #
1R41GM110813-01A1
Application #
8826873
Study Section
Special Emphasis Panel (ZRG1-IMST-L (11))
Program Officer
Cole, Alison E
Project Start
2015-01-15
Project End
2016-01-14
Budget Start
2015-01-15
Budget End
2016-01-14
Support Year
1
Fiscal Year
2015
Total Cost
$320,466
Indirect Cost
Name
Glsynthesis, Inc.
Department
Type
DUNS #
003231854
City
Worcester
State
MA
Country
United States
Zip Code
01605