Screening for caspase-3 inhibitors: effect of a reducing agent on identified hit chemotypes.
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.
Okun
Ilya
I
ChemDiv, Inc., San Diego, California 92121, USA. io@chemdiv.com
Malarchuk
Sergei
S
Dubrovskaya
Elena
E
Khvat
Alexander
A
Tkachenko
Sergey
S
Kysil
Volodymyr
V
Kravchenko
Dmitry
D
Ivachtchenko
Alexandre
A
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
D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D008623:Mercaptoethanol; C505407:CASP3 protein, human; D053148:Caspase 3; D004229:Dithiothreitol