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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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Related in: MedlinePlus

Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).
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pone-0045948-g009: Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).

Mentions: Huh7 cells were incubated with spiroligomer 1 and Nutlin-3 in media at 2, 5, 10 and 20 µM for 17 hours at 37°C. Western blot analysis showed the surprising result that HDM2 levels in the cells treated with 1 increased more than 30-fold relative to control cells in a dose-dependent manner while p53Y220C levels and p21 levels (a protein that is transcriptionally activated by p53) did not change (Figure 9). As a control experiment Huh7 cells were treated with Nutlin-3 at the same concentrations and duration and Nutlin-3 shows a very weak stabilization effect on HDM2. No visible effect on the levels of p53Y220C was seen with either compound 1 or Nutlin-3 in Huh7 cells, suggesting that this temperature sensitive p53Y220C mutant is neither ubiquitinated nor negatively regulated by HDM2. Quantitative real-time PCR analysis of Huh7 cells treated with spiroligomer 1 showed no effect of 1 on the level of mRNA for HDM2 (see supporting information). Huh7 cells were then incubated with 1 at 15 µM and cells were harvested at 2, 4, 6, 8, 12 and 24 hours. Western blot analysis of these cells showed a large increase in HDM2 concentration with time and no change in p53Y220C levels with time (Figure 10, Figure 11A). When the same experiment was carried out using Nutlin-3 at 15 µM only very small changes in the levels of HDM2 relative to the β-actin loading control were seen with time. Our hypothesis is that compound 1 stabilizes HDM2 to proteolysis. Studies are underway to determine the mechanism of HDM2 stabilization by compound 1.


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)

Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0045948-g009: Western blot analysis of HDM2, p53 and p21 in Huh7 cells as a function of concentration of spiroligomer 1 (left) or Nutlin-3 (right).
Mentions: Huh7 cells were incubated with spiroligomer 1 and Nutlin-3 in media at 2, 5, 10 and 20 µM for 17 hours at 37°C. Western blot analysis showed the surprising result that HDM2 levels in the cells treated with 1 increased more than 30-fold relative to control cells in a dose-dependent manner while p53Y220C levels and p21 levels (a protein that is transcriptionally activated by p53) did not change (Figure 9). As a control experiment Huh7 cells were treated with Nutlin-3 at the same concentrations and duration and Nutlin-3 shows a very weak stabilization effect on HDM2. No visible effect on the levels of p53Y220C was seen with either compound 1 or Nutlin-3 in Huh7 cells, suggesting that this temperature sensitive p53Y220C mutant is neither ubiquitinated nor negatively regulated by HDM2. Quantitative real-time PCR analysis of Huh7 cells treated with spiroligomer 1 showed no effect of 1 on the level of mRNA for HDM2 (see supporting information). Huh7 cells were then incubated with 1 at 15 µM and cells were harvested at 2, 4, 6, 8, 12 and 24 hours. Western blot analysis of these cells showed a large increase in HDM2 concentration with time and no change in p53Y220C levels with time (Figure 10, Figure 11A). When the same experiment was carried out using Nutlin-3 at 15 µM only very small changes in the levels of HDM2 relative to the β-actin loading control were seen with time. Our hypothesis is that compound 1 stabilizes HDM2 to proteolysis. Studies are underway to determine the mechanism of HDM2 stabilization by compound 1.

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