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A spiroligomer α-helix mimic that binds HDM2, penetrates human cells and stabilizes HDM2 in cell culture.

Brown ZZ, Akula K, Arzumanyan A, Alleva J, Jackson M, Bichenkov E, Sheffield JB, Feitelson MA, Schafmeister CE - PLoS ONE (2012)

Bottom Line: Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM.This is a biological effect that is not seen with the HDM2 ligand nutlin-3a.We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

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Fluorescence polarization measurement of Flp4 peptide (Kd of 0.16 µM with HDM2) being displaced by spiroligomer 2.The fit curve is consistent with a Ki value of 5 µM.
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pone-0045948-g005: Fluorescence polarization measurement of Flp4 peptide (Kd of 0.16 µM with HDM2) being displaced by spiroligomer 2.The fit curve is consistent with a Ki value of 5 µM.

Mentions: To test whether spiroligomers were binding into the hydrophobic cleft of HDM2, we undertook competition experiments with a low nanomolar binding fluoresceinated mutant of the p53 peptide called Flp4 (Kd is 0.16 µM for HDM2) [20]. Incubating Flp4 at 10 nM with 2 µM HDM2 and a series of concentrations of spiroligomer 2 which lacked the fluorescein group gave a Ki of 5 µM (Figure 5). These results suggest that the spiroligomer 2 lacking a fluorescein was able to compete with a fluorescein labeled p53-analog that is known to bind to the hydrophobic groove of HDM2. To demonstrate the selectivity of spiroligomer 1, we undertook binding experiments with HDMX, a homolog of HDM2. Compound 1 had a measured Kd of 19 µM to HDMX, demonstrating that 1 is selective for HDM2 (data not shown).


A spiroligomer α-helix mimic that binds HDM2, penetrates human cells and stabilizes HDM2 in cell culture.

Brown ZZ, Akula K, Arzumanyan A, Alleva J, Jackson M, Bichenkov E, Sheffield JB, Feitelson MA, Schafmeister CE - PLoS ONE (2012)

Fluorescence polarization measurement of Flp4 peptide (Kd of 0.16 µM with HDM2) being displaced by spiroligomer 2.The fit curve is consistent with a Ki value of 5 µM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475717&req=5

pone-0045948-g005: Fluorescence polarization measurement of Flp4 peptide (Kd of 0.16 µM with HDM2) being displaced by spiroligomer 2.The fit curve is consistent with a Ki value of 5 µM.
Mentions: To test whether spiroligomers were binding into the hydrophobic cleft of HDM2, we undertook competition experiments with a low nanomolar binding fluoresceinated mutant of the p53 peptide called Flp4 (Kd is 0.16 µM for HDM2) [20]. Incubating Flp4 at 10 nM with 2 µM HDM2 and a series of concentrations of spiroligomer 2 which lacked the fluorescein group gave a Ki of 5 µM (Figure 5). These results suggest that the spiroligomer 2 lacking a fluorescein was able to compete with a fluorescein labeled p53-analog that is known to bind to the hydrophobic groove of HDM2. To demonstrate the selectivity of spiroligomer 1, we undertook binding experiments with HDMX, a homolog of HDM2. Compound 1 had a measured Kd of 19 µM to HDMX, demonstrating that 1 is selective for HDM2 (data not shown).

Bottom Line: Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM.This is a biological effect that is not seen with the HDM2 ligand nutlin-3a.We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Princeton University, Princeton, New Jersey, United States of America.

ABSTRACT
We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound 1, that binds HDM2 with a Kd value of 400 nM. The spiroligomer 1 penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner increases the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound 1 modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.

Show MeSH
Related in: MedlinePlus