title: Screening for caspase-3 inhibitors: effect of a reducing agent on identified hit chemotypes.
abstract: When studying cysteinyl proteases in general and caspases in particular, it is generally accepted that a reaction buffer must contain a reducing agent to prevent essential cysteinyl groups from spontaneous oxidation. Dithiothreitol (DTT) and beta-mercaptoethanol (beta-MCE) are 2 of the most broadly used reducing agents. While screening a library of small molecules against caspase-3, the authors have found that the nature of the reducing agent used, DTT or beta-MCE, dramatically affects screening results and leads to identification of nonoverlapping hits. Screening in DTT-containing buffer revealed few novel classes of small molecules that selectively and reversibly inhibit caspase-3 but failed to identify isatin sulfonamides recently found to be potent and selective caspase-3 inhibitors (false negatives). On the other hand, screening in the presence of beta-MCE failed to identify a series of hit compounds, 1,3-dioxo-2,3-dichloro-1H-pyrrolo[3,4-c]quinolines, discovered with DTT, whereas isatin sulphonamides in these conditions exhibited strong caspase-3 inhibition. In this work, the authors show that thiol-containing reducing agents can affect catalytic activity of caspase-3 and modify its thermostability in a redox-potential-independent manner. The authors speculate that the differential structural modifications of caspase-3 seen with different reducing agents represent structurally different caspase-3 conformations and are responsible for its differential sensitivity to small molecules of different chemotypes. Hence, selection of the reducing agent may dramatically affect the quality of high-throughput screening campaigns.
authors.affiliation: ChemDiv, Inc., San Diego, California 92121, USA. io@chemdiv.com
authors.forename: Ilya
authors.initials: I
authors.lastname: Okun
authors.forename: Sergei
authors.initials: S
authors.lastname: Malarchuk
authors.forename: Elena
authors.initials: E
authors.lastname: Dubrovskaya
authors.forename: Alexander
authors.initials: A
authors.lastname: Khvat
authors.forename: Sergey
authors.initials: S
authors.lastname: Tkachenko
authors.forename: Volodymyr
authors.initials: V
authors.lastname: Kysil
authors.forename: Dmitry
authors.initials: D
authors.lastname: Kravchenko
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ivachtchenko
mesh_terms: D053148:Caspase 3; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis* / Q000494:pharmacology; D004229:Dithiothreitol / Q000378:metabolism; D008623:Mercaptoethanol / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008623:Mercaptoethanol; C505407:CASP3 protein, human; D053148:Caspase 3; D004229:Dithiothreitol