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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.

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Variation in function of MDMX depending upon buffer conditions.Panela. SPR binding of hMDMX(23–111) to p53 peptide under non-reducing conditions. Note poor response indicating many of the protein molecules are not “active.” Panelb. The p53 binding curve generated in non-reducing conditions showing a Kd of 940 nM. Panelc. SPR binding of hMDMX(23–111) to p53 in the presence of 1 mM TCEP is much improved, indicating that more of the protein molecules are “active.” Paneld. The p53 binding curve generated under reducing conditions – the Kd is the same as that determined under non-reducing conditions.
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pone-0037518-g004: Variation in function of MDMX depending upon buffer conditions.Panela. SPR binding of hMDMX(23–111) to p53 peptide under non-reducing conditions. Note poor response indicating many of the protein molecules are not “active.” Panelb. The p53 binding curve generated in non-reducing conditions showing a Kd of 940 nM. Panelc. SPR binding of hMDMX(23–111) to p53 in the presence of 1 mM TCEP is much improved, indicating that more of the protein molecules are “active.” Paneld. The p53 binding curve generated under reducing conditions – the Kd is the same as that determined under non-reducing conditions.

Mentions: The results of the SPR study are shown in Figure 4. In these experiments, hMDMX (23–111) was immobilized to the SPR chips using a biotin tag. Synthetic p53 peptide, in the presence or absence of reducing agent, was then flowed across the SPR chip and binding measured. When the experiment is carried out under non-reducing conditions (Figure 4, Panels a and b; mimicking the conditions of the original assays), the p53 peptide does bind, but magnitude of the response is small (Panel a), the data quite noisy, and the resulting binding isotherm not well defined (Panel b). On the other hand, when the same protein on the same chip is reduced in situ (1 mM TCEP) and probed with p53 peptide, the response is robust (Panel c), the data clean, and the resulting binding isotherm well defined (Panel d). Of note is the fact that the measured Kd remains the same for both assays.


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)

Variation in function of MDMX depending upon buffer conditions.Panela. SPR binding of hMDMX(23–111) to p53 peptide under non-reducing conditions. Note poor response indicating many of the protein molecules are not “active.” Panelb. The p53 binding curve generated in non-reducing conditions showing a Kd of 940 nM. Panelc. SPR binding of hMDMX(23–111) to p53 in the presence of 1 mM TCEP is much improved, indicating that more of the protein molecules are “active.” Paneld. The p53 binding curve generated under reducing conditions – the Kd is the same as that determined under non-reducing conditions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037518-g004: Variation in function of MDMX depending upon buffer conditions.Panela. SPR binding of hMDMX(23–111) to p53 peptide under non-reducing conditions. Note poor response indicating many of the protein molecules are not “active.” Panelb. The p53 binding curve generated in non-reducing conditions showing a Kd of 940 nM. Panelc. SPR binding of hMDMX(23–111) to p53 in the presence of 1 mM TCEP is much improved, indicating that more of the protein molecules are “active.” Paneld. The p53 binding curve generated under reducing conditions – the Kd is the same as that determined under non-reducing conditions.
Mentions: The results of the SPR study are shown in Figure 4. In these experiments, hMDMX (23–111) was immobilized to the SPR chips using a biotin tag. Synthetic p53 peptide, in the presence or absence of reducing agent, was then flowed across the SPR chip and binding measured. When the experiment is carried out under non-reducing conditions (Figure 4, Panels a and b; mimicking the conditions of the original assays), the p53 peptide does bind, but magnitude of the response is small (Panel a), the data quite noisy, and the resulting binding isotherm not well defined (Panel b). On the other hand, when the same protein on the same chip is reduced in situ (1 mM TCEP) and probed with p53 peptide, the response is robust (Panel c), the data clean, and the resulting binding isotherm well defined (Panel d). Of note is the fact that the measured Kd remains the same for both assays.

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