Potent Inhibitors of LXXLL‐Based Protein–Protein Interactions

Abstract

Protein-protein interactions between estrogen receptors, ERalpha and ERbeta, and their coactivators (CoAs) are an attractive target for drug intervention. This interaction is mediated by a small pentapeptide motif (LXXLL), termed the NR box. Based on this motif, a variety of cyclic and linear peptides were synthesized in order to gain a better understanding of the association of CoA proteins with the ER isoforms. Utilizing a time-resolved florescence-based coactivator interaction assay, we determined the abilities of these peptides to inhibit this interaction. Using molecular modeling and CD spectroscopy, we have examined the structural basis of their bioactivities with both hormone receptor isoforms. Either homocysteine or penicillamine was utilized as a substitute for cysteine in the disulfide-bridged peptides, while tertiary leucine and neopentyl glycine were used as the surrogates for the NR box leucines. The most potent disufide-bridged peptide (K(i)= 70 pM, with ERalpha) incorporates neopentyl glycine in the NR box, while the most active peptide in this series with ERbeta (K(i)=350 pM) incorporates tertiary leucine. Surprisingly, several linear peptides containing a single cysteine residue showed activities with low nanomolar K(i) values. Collectively, our results suggest a synthetic approach for designing potent and selective peptidomimetics for ERalpha and ERbeta interactions with CoA proteins effecting estrogen action.

International Union of Basic and Clinical Pharmacology. XC. Multisite Pharmacology: Recommendations for the Nomenclature of Receptor Allosterism and Allosteric Ligands

“…A range of peptides, peptidomimetics, and nonpeptide small molecules that inhibit the activity of a variety of NHRs even when agonists are present has been designed over the past decade (Chang et al, , 2005Norris et al, 1999;Hall et al, 2000;Nguyen et al, 2002;Kern and Zuiderweg, 2003;Leduc et al, 2003;Pike et al, 2003;Geistlinger et al, 2004;Arnold et al, 2005Arnold et al, , 2007Galande et al, 2005;Wang et al, 2006;Estebanez-Perpina et al, 2007a;Mettu et al, 2007;LaFrate et al, 2008;Parent et al, 2008); these are sometimes referred to as coactivator binding inhibitors. Coactivator binding inhibitors target the receptor at a site spatially distinct from the orthosteric site, leading to modulation of receptor activity.…”

Targeting the Nuclear Receptor–Cofactor Interaction

“…Based on this scaffold, LXXLL-containing disulfide-bridge nonapeptides were reported with high affinity and selectivity for ERa (2 and 3, Figure 2.3). Introduction of nonnatural amino acids, notably the neopentyl glycine amino acid, in the cyclic peptides and optimization of binding affinity even resulted in peptide binders with a K i of only 70 pM [61]. In a following detailed study it was shown that a high helical content of the peptide not always correlates with higher affinity to the hydrophobic ER LBD coactivator-binding groove [61,62].…”

“…Introduction of nonnatural amino acids, notably the neopentyl glycine amino acid, in the cyclic peptides and optimization of binding affinity even resulted in peptide binders with a K i of only 70 pM [61]. In a following detailed study it was shown that a high helical content of the peptide not always correlates with higher affinity to the hydrophobic ER LBD coactivator-binding groove [61,62]. Cyclic peptides with other thiol-containing amino acids, such as homocysteine and penicillamine, were also studied.…”

“…This resulted in a positive effect on the binding affinity but decreased the isoform, ERa and ERb, selectivity (4, Figure 2.3). On the other hand, the bulkiness introduced by the penicillamines provided additional constrain, resulting in a gain in selectivity (5, Figure 2.3) [61]. Studies on peptides that are not cyclic and feature only one Cys and thus a free sulfhydryl group have revealed a strong affinity, below 60 nM in every case, to ERs.…”

Bicyclo[2.2.2]octanes: Close structural mimics of the nuclear receptor-binding motif of steroid receptor coactivators

“…Biochemical studies using peptides and peptide mimics of the LXXLL motifs of the SRCs have demonstrated that this alternative approach is feasible. [13][14][15][16] If disruption of this helix-groove protein-protein interaction between the ERs and SRCs could be effected with small molecules, as is possible in other helix-groove interactions, 17-23 such molecules would be intriguing molecular probes for studying ER signaling. They might also afford an alternative strategy for blocking estrogen action in breast cancer less likely to be compromised by the cellular adaptations responsible for tamoxifen resistance.…”