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.

Isoquinoline-1,3,4-trione Derivatives Inactivate Caspase-3 by Generation of Reactive Oxygen Species

“…Previously Okun et al (32) found that in the presence of ␤-mercaptoethanol the inhibitory activity of 1,3-dioxo-2,3-dichloro-1H-pyrrolo [3,4-c]quinolines, which were identified as selective and reversible inhibitors of caspase-3 from screening in a DTT-containing buffer could not be measured. It was proposed that DTT and ␤-mercaptoethanol differentially affect the enzymatic parameters and the thermoinactivation of caspase-3 and thus define its sensitivity to different chemo types of small molecule inhibitors.…”

“…However, these agents had only weak effects on the inactivation by compound III (even in 100 M) ( Table 2). Lipoic acid and some other dithiol-containing agents (such as 1,2-butanedithiol, 1,4-butanedithiol, 1,2-benzenedithiol, and 4,4Ј-thiobisbenzenthiol) have been reported to be able to facilitate 1,3-dioxo-2,3-dihydro-1H-pyrrolo [3,4-c]quinolines to produce the same potent inhibition of caspase-3 as DTT (32). However, our results show that dihydrolipoic acid (reduced lipoic acid) but not lipoic acid has a great effect on the sensitivity of caspase-3 to isoquinoline-1,3,4-trione derivatives (Table 2).…”

“…A series of isoquinoline-1,3,4-trione derivatives developed through structural modification exhibit nanomolar potency against caspase-3 in vitro and are validated as selective, irreversible, slow-binding and pancaspase inhibitors. Furthermore, we have found that these inhibitors could attenuate apoptosis induced by ␤-amyloid (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) in PC12 cells and primary neuronal cells (23) and decrease in infarct volume in the transient middle cerebral artery occlusion stroke model (22). These caspase-3 inhibitors with novel structures have provided a new therapeutic strategy directed against diseases involving abnormally up-regulated apoptosis.…”

Enzymatic Characterisation of USP7 Deubiquitinating activity and Inhibition

“…As a consequence of both our findings and previously reported effects on the ability of reductants to alter Caspase-3 catalytic activity and inhibition [17], the effects of varying reductants on the enzymatic activity of USP7 were examined. Activity measurements were therefore taken immediately after incubation of the enzyme in buffer containing no additional reductant (other than remaining TCEP from the storage buffer), 1 mM DTT, 1 mM Cysteine, 1 mM glutathione, or 1 mM TCEP.…”

“…This is in contrast to some other cysteine proteases, such as caspase-3, where the presence of different reductants not only affects the catalytic activity, and potentially structural modification of the enzyme, but also the enzyme's sensitivity to small molecules of different chemotypes [17].…”