title: A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution.
abstract: We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quantitative high-throughput screening (qHTS) was used to determine IC(50)s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC(50) < 10 microM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.
authors.affiliation: NIH Chemical Genomics Center, National Institutes of Health, Bethesda, Maryland 20892-3370, USA. dauld@mail.nih.gov
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Auld
authors.forename: Ya-Qin
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Noel T
authors.initials: NT
authors.lastname: Southall
authors.forename: Ganesha
authors.initials: G
authors.lastname: Rai
authors.forename: Marc
authors.initials: M
authors.lastname: Landsman
authors.forename: Jennifer
authors.initials: J
authors.lastname: MacLure
authors.forename: Daniel
authors.initials: D
authors.lastname: Langevin
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Thomas
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: James
authors.initials: J
authors.lastname: Inglese
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000737:chemistry*; D048888:Fireflies / Q000201:enzymology*; D049454:Firefly Luciferin / Q000737:chemistry; D057166:High-Throughput Screening Assays; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008163:Luminescent Measurements; D008958:Models, Molecular; D010069:Oxadiazoles / Q000737:chemistry; D021281:Quantitative Structure-Activity Relationship
chemical_list: D052160:Benzothiazoles; D004791:Enzyme Inhibitors; D010069:Oxadiazoles; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly