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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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Thermal stability equilibria of MDMX.Panel a. Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound 1. The panel shows individual data sampling points from 3 independent experiments from each condition. Panel b. Dose dependency and time dependency of the effect showing an apparent EC50 of roughly 1 µM and minimal time dependency. Panel c. Dose dependent reversal of the effects of compound 1 by TCEP. Panel d. Dose dependent reversal of the effects of compound 1 by DTT.
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pone-0037518-g005: Thermal stability equilibria of MDMX.Panel a. Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound 1. The panel shows individual data sampling points from 3 independent experiments from each condition. Panel b. Dose dependency and time dependency of the effect showing an apparent EC50 of roughly 1 µM and minimal time dependency. Panel c. Dose dependent reversal of the effects of compound 1 by TCEP. Panel d. Dose dependent reversal of the effects of compound 1 by DTT.

Mentions: Next, the effects of buffer condition changes and exposure to compound 1 upon the conformational equilibria of MDMX were examined by a different technique – thermal stability as measured by hydrophobic dye binding (Figure 5) [20]. Initially, MDMX was allowed to interact with varying concentrations of 1 for 1 h. Then the dye binding capacity of the protein was assessed across a temperature range in order to induce a phase transition from low to high dye binding – normally interpreted as the “melting point” of the protein – the point at which the conformational flexibility of the protein cooperatively opens to many confirmations (Panel a)[9]. In this case, compound 1 increases the temperature required to reach a phase transition, which would normally be interpreted as increasing stability. Our prior work has shown that similar covalent inhibitors of protein interactions often show slow on rates, relative to non-covalent inhibitors, and will show time dependencies in their behaviors. [21]–[23] In order to assess if the shift in MDMX melting point was time dependent the experiment was carried out with long (1 h) and short (5 min) incubation times; no change was observed in the phase transition temperature. Next, the effects of reducing agents were examined. For both TCEP and DTT, addition of the reducing agent to the preformed mixture of MDMX and compound 1 (at apparent EC50 from the first experiment) reversed the stabilization of the protein caused by compound 1. When used alone, TCEP actually destabilized the protein at high concentrations while DTT had no apparent effect. This study strongly suggests that the binding of 1 to MDMX is reversible and that its effect is suppressed by reducing agents, whether or not they contain a nucleophilic thiol.


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)

Thermal stability equilibria of MDMX.Panel a. Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound 1. The panel shows individual data sampling points from 3 independent experiments from each condition. Panel b. Dose dependency and time dependency of the effect showing an apparent EC50 of roughly 1 µM and minimal time dependency. Panel c. Dose dependent reversal of the effects of compound 1 by TCEP. Panel d. Dose dependent reversal of the effects of compound 1 by DTT.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037518-g005: Thermal stability equilibria of MDMX.Panel a. Thermal shift data for MDMX (23–111) showing a 7 degree stabilization of the protein’s melting point by addition of compound 1. The panel shows individual data sampling points from 3 independent experiments from each condition. Panel b. Dose dependency and time dependency of the effect showing an apparent EC50 of roughly 1 µM and minimal time dependency. Panel c. Dose dependent reversal of the effects of compound 1 by TCEP. Panel d. Dose dependent reversal of the effects of compound 1 by DTT.
Mentions: Next, the effects of buffer condition changes and exposure to compound 1 upon the conformational equilibria of MDMX were examined by a different technique – thermal stability as measured by hydrophobic dye binding (Figure 5) [20]. Initially, MDMX was allowed to interact with varying concentrations of 1 for 1 h. Then the dye binding capacity of the protein was assessed across a temperature range in order to induce a phase transition from low to high dye binding – normally interpreted as the “melting point” of the protein – the point at which the conformational flexibility of the protein cooperatively opens to many confirmations (Panel a)[9]. In this case, compound 1 increases the temperature required to reach a phase transition, which would normally be interpreted as increasing stability. Our prior work has shown that similar covalent inhibitors of protein interactions often show slow on rates, relative to non-covalent inhibitors, and will show time dependencies in their behaviors. [21]–[23] In order to assess if the shift in MDMX melting point was time dependent the experiment was carried out with long (1 h) and short (5 min) incubation times; no change was observed in the phase transition temperature. Next, the effects of reducing agents were examined. For both TCEP and DTT, addition of the reducing agent to the preformed mixture of MDMX and compound 1 (at apparent EC50 from the first experiment) reversed the stabilization of the protein caused by compound 1. When used alone, TCEP actually destabilized the protein at high concentrations while DTT had no apparent effect. This study strongly suggests that the binding of 1 to MDMX is reversible and that its effect is suppressed by reducing agents, whether or not they contain a nucleophilic thiol.

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