title: A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs.
abstract: Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand-p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.
authors.affiliation: Bioscience Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK. Electronic address: m.redhead@sygnaturediscovery.com.
authors.forename: M
authors.initials: M
authors.lastname: Redhead
authors.affiliation: Bioscience Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK.
authors.forename: R
authors.initials: R
authors.lastname: Satchell
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania; Department of Neurobiology and Biophysics, Faculty of Natural Sciences, Vilnius University, Vilnius 03101, Lithuania.
authors.forename: V
authors.initials: V
authors.lastname: Morkūnaitė
authors.affiliation: Bioscience Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK.
authors.forename: D
authors.initials: D
authors.lastname: Swift
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania.
authors.forename: V
authors.initials: V
authors.lastname: Petrauskas
authors.affiliation: Bioscience Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK.
authors.forename: E
authors.initials: E
authors.lastname: Golding
authors.affiliation: Chemistry Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK.
authors.forename: S
authors.initials: S
authors.lastname: Onions
authors.affiliation: Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, Vilnius LT-02241, Lithuania. Electronic address: daumantas.matulis@bti.vu.lt.
authors.forename: D
authors.initials: D
authors.lastname: Matulis
authors.affiliation: Bioscience Department, Sygnature Discovery, BioCity, Nottingham NG1 1GF, UK. Electronic address: j.unitt@sygnaturediscovery.com.
authors.forename: J
authors.initials: J
authors.lastname: Unitt
mesh_terms: D055808:Drug Discovery / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008024:Ligands; D048308:Mitogen-Activated Protein Kinase 14 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D008024:Ligands; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D048308:Mitogen-Activated Protein Kinase 14
keywords: Assay interference; Biophysics; Drug discovery; FTSA; SPR; Screening