title: Fragment-based screening by biochemical assays: Systematic feasibility studies with trypsin and MMP12.
abstract: Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.
authors.affiliation: Novartis Institutes for BioMedical Research, Expertise Platform Proteases, Novartis Pharma AG, Basel, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Boettcher
authors.forename: Simon
authors.initials: S
authors.lastname: Ruedisser
authors.forename: Paulus
authors.initials: P
authors.lastname: Erbel
authors.forename: Daniela
authors.initials: D
authors.lastname: Vinzenz
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Schiering
authors.forename: Ulrich
authors.initials: U
authors.lastname: Hassiepen
authors.forename: Pascal
authors.initials: P
authors.lastname: Rigollier
authors.forename: Lorenz M
authors.initials: LM
authors.lastname: Mayr
authors.forename: Julian
authors.initials: J
authors.lastname: Woelcke
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D002853:Chromatography, Liquid; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005188:False Negative Reactions; D005189:False Positive Reactions; D005240:Feasibility Studies; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D053508:Matrix Metalloproteinase 12 / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D012542:Scattering, Radiation; D012995:Solubility; D020349:Surface Plasmon Resonance; D014357:Trypsin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D014357:Trypsin; D053508:Matrix Metalloproteinase 12