Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains.

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.

Versatile Peptide Macrocyclization with Diels-Alder Cycloadditions.

Macrocyclization can improve bioactive peptide ligands through preorganization of molecular topology, leading to improvement of pharmacologic properties like binding affinity, cell permeability, and metabolic stability. Here we demonstrate that Diels-Alder [4 + 2] cycloadditions can be harnessed for peptide macrocyclization and stabilization within a range of peptide scaffolds and chemical environments. Diels-Alder cyclization of diverse diene-dienophile reactive pairs proceeds rapidly, in high yield and with tunable stereochemical preferences on solid-phase or in aqueous solution. This reaction can be applied alone or in concert with other stabilization chemistries, such as ring-closing olefin metathesis, to stabilize loop, turn, and α-helical secondary structural motifs. NMR and molecular dynamics studies of model loop peptides confirmed preferential formation of endo cycloadduct stereochemistry, imparting significant structural rigidity to the peptide backbone that resulted in augmented protease resistance and increased biological activity of a Diels-Alder cyclized (DAC) RGD peptide. Separately, we demonstrated the stabilization of DAC α-helical peptides derived from the ERα-binding protein SRC2. We solved a 2.25 Å cocrystal structure of one DAC helical peptide bound to ERα, which unequivocally corroborated endo stereochemistry of the resulting Diels-Alder adduct, and confirmed that the unique architecture of stabilizing motifs formed with this chemistry can directly contribute to target binding. These data establish Diels-Alder cyclization as a versatile approach to stabilize diverse protein structural motifs under a range of chemical environments.

Steroid receptor/coactivator binding inhibitors: An update.

The purpose of this review is to highlight recent developments in small molecules and peptides that block the binding of coactivators to steroid receptors. These coactivator binding inhibitors bind at the coregulator binding groove, also known as Activation Function-2, rather than at the ligand-binding site of steroid receptors. Steroid receptors that have been targeted with coactivator binding inhibitors include the androgen receptor, estrogen receptor and progesterone receptor. Coactivator binding inhibitors may be useful in some cases of resistance to currently prescribed therapeutics. The scope of the review includes small-molecule and peptide coactivator binding inhibitors for steroid receptors, with a particular focus on recent compounds that have been assayed in cell-based models.

Recent structural advances in constrained helical peptides.

Given the ubiquity of the ⍺-helix in the proteome, there has been much research in developing mimics of ⍺-helices, and most of this study has been toward developing protein-protein interaction inhibitors. A common strategy for mimicking ⍺-helices has been through the use of constrained, helical peptides. The addition of a constraint typically provides for conformational and proteolytic stability and, in some cases, cell permeability. Some of the most well-known strategies included are lactam formation and hydrocarbon "stapling." Beyond those strategies, there have been many recent advances in developing constrained peptides. The purpose of this review is to highlight recent advances in the development of new helix-stabilizing technologies, constraint diversification strategies, tether diversification strategies, and combination strategies that create new bicyclic helical peptides.

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules.

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules in vivo and in vitro, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an ex vivo method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after in vitro incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides.

A "cross-stitched" peptide with improved helicity and proteolytic stability.

A new computational approach to obtain quantitative energy profiles for helix folding was used in the design of orthogonal hydrocarbon and lactam bicyclic peptides. The proteolytically stable, "cross-stitched" peptide SRC2-BCP1 shows nanomolar affinity for estrogen receptor α and X-ray crystallography confirms a helical binding pose.

A Cell-Permeable Stapled Peptide Inhibitor of the Estrogen Receptor/Coactivator Interaction.

We and others have proposed that coactivator binding inhibitors, which block the interaction of estrogen receptor and steroid receptor coactivators, may represent a potential class of new breast cancer therapeutics. The development of coactivator binding inhibitors has been limited, however, because many of the current molecules which are active in in vitro and biochemical assays are not active in cell-based assays. Our goal in this work was to prepare a coactivator binding inhibitor active in cellular models of breast cancer. To accomplish this, we used molecular dynamics simulations to convert a high-affinity stapled peptide with poor cell permeability into R4K1, a cell-penetrating stapled peptide. R4K1 displays high binding affinity for estrogen receptor α, inhibits the formation of estrogen receptor/coactivator complexes, and distributes throughout the cell with a high percentage of nuclear localization. R4K1 represses native gene transcription mediated by estrogen receptor α and inhibits proliferation of estradiol-stimulated MCF-7 cells. Using RNA-Seq, we demonstrate that almost all of the effects of R4K1 on global gene transcription are estrogen-receptor-associated. This chemical probe provides a significant proof-of-concept for preparing cell-permeable stapled peptide inhibitors of the estrogen receptor/coactivator interaction.

Stapled Peptides with γ-Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction.

"Stapled" peptides are typically designed to replace two non-interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ-position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC50 =89 nm) replaces isoleucine 689 with an S-γ-methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm). Through X-ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S-γ-methyl peptide minimizes the syn-pentane interactions between the α- and γ-methyl groups.