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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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(A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells.(B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.
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pone-0045948-g006: (A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells.(B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.

Mentions: With the sub-micromolar HDM2 binding spiroligomer 1 in hand we sought to evaluate its effect in cell-culture. We first examined the cell-penetrating capability of two of the functionalized spiroligomers. Compound 3 was incubated with Huh7 cells (human hepatoma cells, mutant-p53 with a p53-Y220C/del genotype, purchased from ATCC (Manassas, VA)) for 24 hours at a concentration of 2 µM and observed with fluorescence microscopy (including confocal imaging), after washing away the media containing the fluorescein labeled spiroligomer using phosphate buffered saline (PBS). Significant uptake of 3 was seen as judged by the amount and fluorescent intensity of the cells, including distribution throughout the cytoplasm and nucleus as seen with confocal microscopy (Figure 6A). We then incubated spiroligomer 1, with both Huh7 cells (not shown) and HepG2 (human liver hepatoblastoma cells, purchased from ATCC) [26] and imaged them (Figure 6B). Compound 1 at concentrations ranging from 2 µM to 40 µM had no visible effect on cell morphology or viability with Huh7, HepG2 or primary human myocytes (data not shown) and no visible cytotoxicity as observed using fluorescence microscopy after exposures of up to 72 hours. Confocal fluorescence images verified the distribution of compound 1 throughout the cytoplasm and nucleus (Figure 7). When the cells were incubated with fluorescein for similar times, no fluorescence uptake was observed (not shown). With this evidence of cellular uptake of fluorescently labeled spiroligomers we sought to determine if the uptake was through active transport or passive diffusion. HepG2 cells were pre-cooled for 30 min at 4°C in 475 µL of media and then 25 µL of spiroligomer 1 in phosphate buffered saline (pH 7.4) was added to the media to achieve a concentration of 5 µM of compound 1. The cells were then incubated at 4°C for 3.75 hours and then the media with 1 was removed. Fluorescence imaging showed substantial uptake of fluorescence that was indistinguishable from control cells treated with 1 at 37°C (Figure 8). In further experiments, HepG2 cells at 37°C were pre-treated with 10 mM sodium azide together with 50 mM deoxy-D-glucose (60 minutes pre-treatment) followed by addition of compound 1. These cells also showed uptake of fluorescence at levels that were indistinguishable from control cells treated with 1 at 37°C (Figure 8G&I). Incubation of both Huh7 and HepG2 cell lines with fluorescein at 37°C as a control produced no visible uptake of fluorescence. Despite having molecular weights in excess of 1,300 Daltons, both compound 1 and 3 penetrate human cells and compound 1 penetrates cells when active transport is inhibited suggesting that these compounds penetrate cells by passive diffusion. The lack of flexibility and the fact that five of the eight amides of these compounds are N-alkylated may contribute to their good bioavailability despite their large size [33].


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)

(A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells.(B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475717&req=5

pone-0045948-g006: (A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells.(B) HepG2 cells that were incubated with spiroligomer 1 (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.
Mentions: With the sub-micromolar HDM2 binding spiroligomer 1 in hand we sought to evaluate its effect in cell-culture. We first examined the cell-penetrating capability of two of the functionalized spiroligomers. Compound 3 was incubated with Huh7 cells (human hepatoma cells, mutant-p53 with a p53-Y220C/del genotype, purchased from ATCC (Manassas, VA)) for 24 hours at a concentration of 2 µM and observed with fluorescence microscopy (including confocal imaging), after washing away the media containing the fluorescein labeled spiroligomer using phosphate buffered saline (PBS). Significant uptake of 3 was seen as judged by the amount and fluorescent intensity of the cells, including distribution throughout the cytoplasm and nucleus as seen with confocal microscopy (Figure 6A). We then incubated spiroligomer 1, with both Huh7 cells (not shown) and HepG2 (human liver hepatoblastoma cells, purchased from ATCC) [26] and imaged them (Figure 6B). Compound 1 at concentrations ranging from 2 µM to 40 µM had no visible effect on cell morphology or viability with Huh7, HepG2 or primary human myocytes (data not shown) and no visible cytotoxicity as observed using fluorescence microscopy after exposures of up to 72 hours. Confocal fluorescence images verified the distribution of compound 1 throughout the cytoplasm and nucleus (Figure 7). When the cells were incubated with fluorescein for similar times, no fluorescence uptake was observed (not shown). With this evidence of cellular uptake of fluorescently labeled spiroligomers we sought to determine if the uptake was through active transport or passive diffusion. HepG2 cells were pre-cooled for 30 min at 4°C in 475 µL of media and then 25 µL of spiroligomer 1 in phosphate buffered saline (pH 7.4) was added to the media to achieve a concentration of 5 µM of compound 1. The cells were then incubated at 4°C for 3.75 hours and then the media with 1 was removed. Fluorescence imaging showed substantial uptake of fluorescence that was indistinguishable from control cells treated with 1 at 37°C (Figure 8). In further experiments, HepG2 cells at 37°C were pre-treated with 10 mM sodium azide together with 50 mM deoxy-D-glucose (60 minutes pre-treatment) followed by addition of compound 1. These cells also showed uptake of fluorescence at levels that were indistinguishable from control cells treated with 1 at 37°C (Figure 8G&I). Incubation of both Huh7 and HepG2 cell lines with fluorescein at 37°C as a control produced no visible uptake of fluorescence. Despite having molecular weights in excess of 1,300 Daltons, both compound 1 and 3 penetrate human cells and compound 1 penetrates cells when active transport is inhibited suggesting that these compounds penetrate cells by passive diffusion. The lack of flexibility and the fact that five of the eight amides of these compounds are N-alkylated may contribute to their good bioavailability despite their large size [33].

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