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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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The solid-phase synthesis of spiroligomers 1 and 2 using the HMBA resin.Building blocks were successively coupled using either esterification conditions for residue 13 or conditions previously developed to achieve hindered active esters 14 or 15[16]. Further derivatization of the oligomers followed by cyclization of the terminal DKP released spiroligomers 1 and 2 from the solid support [16].
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pone-0045948-g002: The solid-phase synthesis of spiroligomers 1 and 2 using the HMBA resin.Building blocks were successively coupled using either esterification conditions for residue 13 or conditions previously developed to achieve hindered active esters 14 or 15[16]. Further derivatization of the oligomers followed by cyclization of the terminal DKP released spiroligomers 1 and 2 from the solid support [16].

Mentions: The synthesis of compounds 1 and 2 is shown as a representative example and other spiroligomers were synthesized using the same synthesis as compound 1 with different monomers (Figure 2). The synthesis is modular like peptide synthesis and can be extended to incorporate more monomers and increase the contact area of functionalized spiroligomers with MDM2 without increasing flexibility of the molecules at the same time. The synthesis utilized the acid-stable hydroxy-methyl benzamide (HMBA) resin in order to be able to remove the acid-labile protecting groups on solid support. First, Fmoc-protected building block 13 was immobilized on the resin using the esterification reagent 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) in the presence of methyl-imidazole. This and all subsequent couplings were repeated to ensure quantitative acylation. The tert-butyloxycarbonyl (Boc) and tert-butyl ester protecting groups were then simultaneously removed using 95% trifluoroacetic acid (TFA) in the presence of the cation scavenger triisopropylsilane (TIS) to expose the next resin-bound prolinyl amino acid. The next building block was then activated with 1 equivalent of diisopropylcarbodiimide (DIC) in the presence of 6-fold excess of 1-hydroxy-7-azabenzotriazole (HOAt) to give the hindered amino-OAt ester 14. These conditions have been shown to produce competent acylating species from hindered amino acid building blocks [15], [16]. The addition of activated building blocks such as 14 with prolinyl amino acids has been shown to produce mixtures of regioisomeric tertiary amides as well as the fully dehydrated diketopiperazine (DKP). The addition of another equivalent of DIC/HOAt to the resin converts all intermediate species into that of the desired DKP. The carbobenzyloxy (Cbz) protecting group and the tert-butyl ester protecting groups were then removed by treatment of the resin with 33% HBr in acetic acid to expose the next resin-bound prolinyl amino acid. The same procedure of acylation, DKP formation with DIC/HOAt, and then deprotection with HBr was then used to install the dichlorobenzyl residue 15. The next residue, Boc-homophenylalanine 16, was activated using HATU and 2 equivalents of diisopropylethylamine (DIPEA), followed by Boc removal with TFA and DKP formation using DIC/HOAt to yield resin-bound intermediate 17. The fluorenylmethyloxycarbonyl (Fmoc) group was then removed with piperidine, the resin was split into two portions and one portion was acylated with tert-butyl protected glutamic acid 18 to produce resin bound oligomer 20. The other portion was acylated with Fmoc protected lysine 19 followed by treatment with 20% piperidine to remove the Fmoc group and treatment of fluorescein isothiocyanate to yield resin-bound oligomer 21. The final Boc deprotection of both oligomer 20 and 21 was then accomplished by addition of TFA, and neutralization of the resin induced diketopiperazine formation with concomitant release of the oligomer from the solid support to yield fully rigidified spiroligomers 1 and 2.


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)

The solid-phase synthesis of spiroligomers 1 and 2 using the HMBA resin.Building blocks were successively coupled using either esterification conditions for residue 13 or conditions previously developed to achieve hindered active esters 14 or 15[16]. Further derivatization of the oligomers followed by cyclization of the terminal DKP released spiroligomers 1 and 2 from the solid support [16].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475717&req=5

pone-0045948-g002: The solid-phase synthesis of spiroligomers 1 and 2 using the HMBA resin.Building blocks were successively coupled using either esterification conditions for residue 13 or conditions previously developed to achieve hindered active esters 14 or 15[16]. Further derivatization of the oligomers followed by cyclization of the terminal DKP released spiroligomers 1 and 2 from the solid support [16].
Mentions: The synthesis of compounds 1 and 2 is shown as a representative example and other spiroligomers were synthesized using the same synthesis as compound 1 with different monomers (Figure 2). The synthesis is modular like peptide synthesis and can be extended to incorporate more monomers and increase the contact area of functionalized spiroligomers with MDM2 without increasing flexibility of the molecules at the same time. The synthesis utilized the acid-stable hydroxy-methyl benzamide (HMBA) resin in order to be able to remove the acid-labile protecting groups on solid support. First, Fmoc-protected building block 13 was immobilized on the resin using the esterification reagent 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT) in the presence of methyl-imidazole. This and all subsequent couplings were repeated to ensure quantitative acylation. The tert-butyloxycarbonyl (Boc) and tert-butyl ester protecting groups were then simultaneously removed using 95% trifluoroacetic acid (TFA) in the presence of the cation scavenger triisopropylsilane (TIS) to expose the next resin-bound prolinyl amino acid. The next building block was then activated with 1 equivalent of diisopropylcarbodiimide (DIC) in the presence of 6-fold excess of 1-hydroxy-7-azabenzotriazole (HOAt) to give the hindered amino-OAt ester 14. These conditions have been shown to produce competent acylating species from hindered amino acid building blocks [15], [16]. The addition of activated building blocks such as 14 with prolinyl amino acids has been shown to produce mixtures of regioisomeric tertiary amides as well as the fully dehydrated diketopiperazine (DKP). The addition of another equivalent of DIC/HOAt to the resin converts all intermediate species into that of the desired DKP. The carbobenzyloxy (Cbz) protecting group and the tert-butyl ester protecting groups were then removed by treatment of the resin with 33% HBr in acetic acid to expose the next resin-bound prolinyl amino acid. The same procedure of acylation, DKP formation with DIC/HOAt, and then deprotection with HBr was then used to install the dichlorobenzyl residue 15. The next residue, Boc-homophenylalanine 16, was activated using HATU and 2 equivalents of diisopropylethylamine (DIPEA), followed by Boc removal with TFA and DKP formation using DIC/HOAt to yield resin-bound intermediate 17. The fluorenylmethyloxycarbonyl (Fmoc) group was then removed with piperidine, the resin was split into two portions and one portion was acylated with tert-butyl protected glutamic acid 18 to produce resin bound oligomer 20. The other portion was acylated with Fmoc protected lysine 19 followed by treatment with 20% piperidine to remove the Fmoc group and treatment of fluorescein isothiocyanate to yield resin-bound oligomer 21. The final Boc deprotection of both oligomer 20 and 21 was then accomplished by addition of TFA, and neutralization of the resin induced diketopiperazine formation with concomitant release of the oligomer from the solid support to yield fully rigidified spiroligomers 1 and 2.

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