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On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

Bista M, Smithson D, Pecak A, Salinas G, Pustelny K, Min J, Pirog A, Finch K, Zdzalik M, Waddell B, Wladyka B, Kedracka-Krok S, Dyer MA, Dubin G, Guy RK - PLoS ONE (2012)

Bottom Line: Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53.The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein.This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Biochemistry, Martinsried, Germany.

ABSTRACT
SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

Show MeSH
Inhibition of MDMX-p53 peptide binding by compound 1 (IC50 = 3 µM), compound 2 (IC50>100 uM).
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pone-0037518-g003: Inhibition of MDMX-p53 peptide binding by compound 1 (IC50 = 3 µM), compound 2 (IC50>100 uM).

Mentions: Careful consideration of these results suggests that compound 1 exerts its effect upon MDMX binding p53 by forming a covalent complex with MDMX through reaction with the cysteine residue in the binding domain but that the resulting adduct exists in dynamic equilibria rather than being irreversible. If this were the case, then the activity would be dependent upon the presence of the electrophilic group within the chemotype and upon the presence of the cysteine. To test this hypothesis, an experiment was carried out to compare inhibition of MDMX-p53 peptide binding with compound 1 and compound 2, which lacks the required electrophilic center (Figure 3). Saturation of the ene-amide group reduced the inhibitory potency by at roughly 30-fold, consistent with covalent bond formation being important to the mechanism of action. A similar result could be obtained by allowing compound 1 to react with glutathione in situ, reducing potency by 10-fold but not completely removing efficacy (Figure S5). These experiments suggest that the binding of compound 1 to MDMX utilizes both covalent and non-covalent interactions. Most likely this takes the form of pre-organization of the compound with the “active site” cysteine, followed by formation of a covalent adduct, which we have previously reported with other cysteine reactive electrophilic inhibitors [19].


On the mechanism of action of SJ-172550 in inhibiting the interaction of MDM4 and p53.

Bista M, Smithson D, Pecak A, Salinas G, Pustelny K, Min J, Pirog A, Finch K, Zdzalik M, Waddell B, Wladyka B, Kedracka-Krok S, Dyer MA, Dubin G, Guy RK - PLoS ONE (2012)

Inhibition of MDMX-p53 peptide binding by compound 1 (IC50 = 3 µM), compound 2 (IC50>100 uM).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366986&req=5

pone-0037518-g003: Inhibition of MDMX-p53 peptide binding by compound 1 (IC50 = 3 µM), compound 2 (IC50>100 uM).
Mentions: Careful consideration of these results suggests that compound 1 exerts its effect upon MDMX binding p53 by forming a covalent complex with MDMX through reaction with the cysteine residue in the binding domain but that the resulting adduct exists in dynamic equilibria rather than being irreversible. If this were the case, then the activity would be dependent upon the presence of the electrophilic group within the chemotype and upon the presence of the cysteine. To test this hypothesis, an experiment was carried out to compare inhibition of MDMX-p53 peptide binding with compound 1 and compound 2, which lacks the required electrophilic center (Figure 3). Saturation of the ene-amide group reduced the inhibitory potency by at roughly 30-fold, consistent with covalent bond formation being important to the mechanism of action. A similar result could be obtained by allowing compound 1 to react with glutathione in situ, reducing potency by 10-fold but not completely removing efficacy (Figure S5). These experiments suggest that the binding of compound 1 to MDMX utilizes both covalent and non-covalent interactions. Most likely this takes the form of pre-organization of the compound with the “active site” cysteine, followed by formation of a covalent adduct, which we have previously reported with other cysteine reactive electrophilic inhibitors [19].

Bottom Line: Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53.The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein.This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Biochemistry, Martinsried, Germany.

ABSTRACT
SJ-172550 (1) was previously discovered in a biochemical high throughput screen for inhibitors of the interaction of MDMX and p53 and characterized as a reversible inhibitor (J. Biol. Chem. 2010; 285:10786). Further study of the biochemical mode of action of 1 has shown that it acts through a complicated mechanism in which the compound forms a covalent but reversible complex with MDMX and locks MDMX into a conformation that is unable to bind p53. The relative stability of this complex is influenced by many factors including the reducing potential of the media, the presence of aggregates, and other factors that influence the conformational stability of the protein. This complex mechanism of action hinders the further development of compound 1 as a selective MDMX inhibitor.

Show MeSH