title: Differential scanning fluorimetry as secondary screening platform for small molecule inhibitors of Bcl-XL.
abstract: Since apoptosis is impaired in malignant cells overexpressing prosurvival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Small molecule inhibitors of Bcl-X(L) function have been discovered from diverse structure classes using rational drug design as well as high-throughput screening (HTS) approaches. However, most of the BH3 mimetics that have been identified via screening based on fluorescence polarization displayed an affinity for their presumed protein targets that is far lower than that of BH3-only proteins. Therefore, it is important to establish a simple and inexpensive secondary platform for hit validation which is pertinent to current efforts for developing compounds that mimic the action of BH3-only proteins as novel anticancer agents. These considerations prompted us to explore the differential scanning fluorimetry (DSF) method that is based on energetic coupling between ligand binding and protein unfolding. We have systematically tested known Bcl-X(L)/Bcl-2 inhibitors using DSF and have revealed distinct subsets of inhibitors. More importantly, we report that some of these inhibitors interacted selectively with glutathione S-transferase tagged Bcl-X(L), whereas certain inhibitors exhibited marked selectivity towards native untagged Bcl-X(L). Therefore, we propose that the affinity tag may cause a significant conformational switch in the Bcl-X(L), which results in the selectivity for certain subsets of small molecule inhibitors. This finding also implies that the previous screens involving tagged proteins need to be carefully reexamined while further investigations must ensure that the right conformation of protein is used in future screens.
authors.affiliation: Experimental Therapeutics Center, A*STAR, Biopolis, Singapore.
authors.forename: Kah Fei
authors.initials: KF
authors.lastname: Wan
authors.forename: Sifang
authors.initials: S
authors.lastname: Wang
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Brown
authors.forename: Victor C
authors.initials: VC
authors.lastname: Yu
authors.forename: Michael
authors.initials: M
authors.lastname: Entzeroth
authors.forename: David P
authors.initials: DP
authors.lastname: Lane
authors.forename: May Ann
authors.initials: MA
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D015195:Drug Design; D005454:Fluorescence Polarization / Q000379:methods*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism; D057166:High-Throughput Screening Assays; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D054852:Small Molecule Libraries; D013696:Temperature; D051020:bcl-X Protein / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C474007:Bax protein (53-86); D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D054852:Small Molecule Libraries; D051020:bcl-X Protein; D005982:Glutathione Transferase