Isoquinoline-1,3,4-trione derivatives inactivate caspase-3 by generation of reactive oxygen species.
Caspase-3 is an attractive therapeutic target for treatment of diseases involving disregulated apoptosis. We report here the mechanism of caspase-3 inactivation by isoquinoline-1,3,4-trione derivatives. Kinetic analysis indicates the compounds can irreversibly inactivate caspase-3 in a 1,4-dithiothreitol (DTT)- and oxygen-dependent manner, implying that a redox cycle might take place in the inactivation process. Reactive oxygen species detection experiments using a chemical indicator, together with electron spin resonance measurement, suggest that ROS can be generated by reaction of isoquinoline-1,3,4-trione derivatives with DTT. Oxygen-free radical scavenger catalase and superoxide dismutase eliciting the inactivation of caspase-3 by the inhibitors confirm that ROS mediates the inactivation process. Crystal structures of caspase-3 in complexes with isoquinoline-1,3,4-trione derivatives show that the catalytic cysteine is oxidized to sulfonic acid (-SO(3)H) and isoquinoline-1,3,4-trione derivatives are bound at the dimer interface of caspase-3. Further mutagenesis study shows that the binding of the inhibitors with caspase-3 appears to be nonspecific. Isoquinoline-1,3,4-trione derivative-catalyzed caspase-3 inactivation could also be observed when DTT is substituted with dihydrolipoic acid, which exists widely in cells and might play an important role in the in vivo inactivation process in which the inhibitors inactivate caspase-3 in cells and then prevent the cells from apoptosis. These results provide valuable information for further development of small molecular inhibitors against caspase-3 or other oxidation-sensitive proteins.
Du
Jun-Qing
JQ
Chinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 189 Guo Shou Jing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China.
Wu
Jian
J
Zhang
Hua-Jie
HJ
Zhang
Ya-Hui
YH
Qiu
Bei-Ying
BY
Wu
Fang
F
Chen
Yi-Hua
YH
Li
Jing-Ya
JY
Nan
Fa-Jun
FJ
Ding
Jian-Ping
JP
Li
Jia
J