Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions.
We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.
Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*STAR , 61 Biopolis Drive, #03-12 Proteos, Singapore 138673.
Walter L
WL
Goh
Min Yen
MY
Lee
Thomas L
TL
Joseph
Soo Tng
ST
Quah
Christopher J
CJ
Brown
Chandra
C
Verma
Sydney
S
Brenner
Farid J
FJ
Ghadessy
Yin Nah
YN
Teo
D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009570:Nitriles / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011807:Quinolizines / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014783:Viscosity
D005456:Fluorescent Dyes; D009570:Nitriles; D010455:Peptides; D011807:Quinolizines; D016159:Tumor Suppressor Protein p53; C082390:9-(2-carboxy-2-cyanovinyl)julolidine; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2