title: Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.
abstract: Because of the intimate role of caspase-8 in apoptosis signaling pathways from FAS, TNFR1, and other death receptors, the enzyme is a potentially important therapeutic target. We have generated an Escherichia coli expression construct for caspase-8 in which a His-tag sequence is inserted ahead of codon 217 of caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein could be purified to 98% purity. Hydroxyapatite resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 units additional mass. The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins could be activated by autoproteolysis to the appropriate 19- plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4-5 units/mg against 200 microM Ac-IETD-pNA at 25 degrees C. The fully active protein was used in a high-throughput screen for inhibitors of caspase-8. A preliminary robustness screen demonstrated that caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. A new class of caspase-8 inhibitors, steroid-derived diacids, with affinity in the low micromolar range were discovered in the refined screen. Structure--activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity.
authors.affiliation: GlaxoSmithKline Research, 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA. gks39645@gsk.com
authors.forename: Gary K
authors.initials: GK
authors.lastname: Smith
authors.forename: David G
authors.initials: DG
authors.lastname: Barrett
authors.forename: Kevin
authors.initials: K
authors.lastname: Blackburn
authors.forename: Michael
authors.initials: M
authors.lastname: Cory
authors.forename: Walter S
authors.initials: WS
authors.lastname: Dallas
authors.forename: Roderick
authors.initials: R
authors.lastname: Davis
authors.forename: Daniel
authors.initials: D
authors.lastname: Hassler
authors.forename: Randy
authors.initials: R
authors.lastname: McConnell
authors.forename: Mary
authors.initials: M
authors.lastname: Moyer
authors.forename: Kurt
authors.initials: K
authors.lastname: Weaver
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002021:Buffers; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013256:Steroids / Q000737:chemistry / Q000494:pharmacology*; D013379:Substrate Specificity; D014162:Transfection
chemical_list: D002021:Buffers; D061945:Caspase Inhibitors; D013256:Steroids; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases