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ATP alters IKK‐2 inhibitor affinity for IKK2 enzyme and reveals non‐equivalent inhibitor binding sites
Author(s) -
Liu Jia Yeu,
Stevens Kirk,
Mourey Robert
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.963.7
Subject(s) - ic50 , chemistry , potency , enzyme , enzyme assay , biochemistry , in vitro
Method Inhibitor off‐rate was measured by two methods: (1) pre‐binding the enzyme with inhibitor followed by measuring the recovery of enzyme activity upon a 200‐fold dilution of the enzyme‐inhibitor complex in the presence of 1mM ATP and (2) measuring the shift in the compound potency (IC50) curves obtained by prebinding the enzyme and inhibitor in the absence of ATP followed by addition of either 1μM or 1mM ATP. Results Three literature inhibitors with potency (IC50) ranging from <1 to 35 nM were used for this study: PHA‐408 (Pfizer), GSK‐082 (GSK), and AVE‐002 (Aventis). The off‐rates (T1/2) measured by the dilution method are 124 and <15 min for PHA‐408 and GSK‐082, respectively. The off‐rate for AVE‐002 was biphasic with half of the enzyme‐inhibitor complex dissociating immediately (T1/2 < 2 min) and the other half bound tightly (T1/2 54 min). The IC50 curves at 1μM and 1mM ATP for PHA‐408 showed no shift using a short 2 min assay and is consistent with the slow off‐rate. GSK‐082 showed a > 10 fold loss of potency with 1mM ATP, as expected for a fast off‐rate inhibitor. AVE‐002 IC50 curve with 1μM ATP was monophasic but displayed a biphasic response with 1mM ATP. Conclusion ATP can change the off‐rate of IKK‐2 inhibitor bound to the enzyme. Additionally, high ATP revealed that AVE‐002 bound in the two inhibitor binding sites of the IKK‐2 homodimer non‐equivalently.