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

Abstract

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.

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“…182,183 Time-dependent activity or unusual Hill coefficients can usefully point to general assay interference/protein-reactive compounds . 92,184,185 Increasing Prevalence of PAINS in the Screening Literature. A simple literature searching exercise reveals the influence that rhodanine-based PAINS are having on the scientific literature.…”

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy

“…Unexpectedly, the combination between Smac mimetics and PDT caused a weaker caspase-3 cleavage compared to Smac mimetic treatment alone, although it somehow stimulated caspase-3 processing after PDT treatment. This suggests that, beside displaying intrinsic defects in the apoptotic machinery, PDT by itself may negatively interfere with caspase signaling in these cells, probably through a ROS-mediated inhibition of caspases, as already reported [39]. In this case, cells would preferentially undergo necrosis in response to PDT because cells in which caspases cannot be efficiently activated often A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 undergo necrosis in response to apoptotic stimuli [40].…”

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“…Mitochondrial membrane potential assays were based on a previous report [27] . Briefly, HeLa cells were treated with P1 for 40 min before being stained with 0.1% JC-1 for another 20 min.…”