Cyclic Peptide Linker Design and Optimization by Molecular Dynamics Simulations.

Cyclic peptides are an emerging therapeutic modality that can target protein-protein interaction sites with high affinity and selectivity. A common medicinal chemistry strategy for the optimization of peptide hits is conformational stabilization through macrocyclization. We present a method based on explicit solvent enhanced sampling molecular dynamics simulations for estimating the impact of varying linker lengths and chemistry on the conformational stability of a peptide. The method is demonstrated on three cyclic peptide series that bind to proteins PCSK9, trypsin, and MDM2 adopting loop, ß-sheet, and helical secondary structures. In general, the simulations show greater solution stability of the receptor-bound conformation for the higher-affinity peptides, consistent with the idea that preorganizing a ligand for binding can enhance binding affinity. The impact of the force field and sampling is discussed for one series that does not follow this trend. We have successfully applied this method to internal discovery programs to design peptides with increased potency and chemical stability.

Macropinocytosis as a Key Determinant of Peptidomimetic Uptake in Cancer Cells.

Peptides and peptidomimetics represent the middle space between small molecules and large proteins-they retain the relatively small size and synthetic accessibility of small molecules while providing high binding specificity for biomolecular partners typically observed with proteins. During the course of our efforts to target intracellular protein-protein interactions in cancer, we observed that the cellular uptake of peptides is critically determined by the cell line-specifically, we noted that peptides show better uptake in cancer cells with enhanced macropinocytic indices. Here, we describe the results of our analysis of cellular penetration by different classes of conformationally stabilized peptides. We tested the uptake of linear peptides, peptide macrocycles, stabilized helices, ß-hairpin peptides, and cross-linked helix dimers in 11 different cell lines. Efficient uptake of these conformationally defined constructs directly correlated with the macropinocytic activity of each cell line: high uptake of compounds was observed in cells with mutations in certain signaling pathways. Significantly, the study shows that constrained peptides follow the same uptake mechanism as proteins in macropinocytic cells, but unlike proteins, peptide mimics can be readily designed to resist denaturation and proteolytic degradation. Our findings expand the current understanding of cellular uptake in cancer cells by designed peptidomimetics and suggest that cancer cells with certain mutations are suitable mediums for the study of biological pathways with peptide leads.

Bridgehead-Substituted Triptycenes for Discovery of Nucleic Acid Junction Binders.

Recently, the utility of triptycene as a scaffold for targeting nucleic acid three-way junctions was demonstrated. A rapid, efficient route for the synthesis of bridgehead-substituted triptycenes is reported, in addition to solid-phase diversification to a new class of triptycene peptides. The triptycene peptides were evaluated for binding to a d(CAG)·(CTG) repeat DNA junction exhibiting potent affinities. The bridgehead-substituted triptycenes provide new building blocks for rapid access to diverse triptycene ligands with novel architectures.

Modulation of the E.†coli rpoH Temperature Sensor with Triptycene-Based Small Molecules.

Regulation of the heat shock response (HSR) is essential in all living systems. In E. coli, the HSR is regulated by an alternative σ factor, σ(32) , which is encoded by the rpoH gene. The mRNA of rpoH adopts a complex secondary structure that is critical for the proper translation of the σ(32) protein. At low temperatures, the rpoH gene transcript forms a highly structured mRNA containing several three-way junctions, including a rare perfectly paired three-way junction (3WJ). This complex secondary structure serves as a primitive but highly effective strategy for the thermal control of gene expression. In this work, the first small-molecule modulators of the E. coli σ(32) mRNA temperature sensor are reported.

Synthesis of 9-Substituted Triptycene Building Blocks for Solid-Phase Diversification and Nucleic Acid Junction Targeting.

Triptycenes have been shown to bind nucleic acid three-way junctions, but rapid and efficient methods to diversify the triptycene core are lacking. An efficient synthesis of a 9-substituted triptycene scaffold is reported that can be used as a building block for solid-phase peptide synthesis and rapid diversification. The triptycene building block was diversified to produce a new class of tripeptide-triptycenes, and their binding abilities toward d(CAG)·(CTG) repeat junctions were investigated.

Triptycene-based small molecules modulate (CAG)·(CTG) repeat junctions.

Nucleic acid three-way junctions (3WJs) play key roles in biological processes such as nucleic acid replication in addition to being implicated as dynamic transient intermediates in trinucleotide repeat sequences. Structural modulation of specific nucleic acid junctions could allow for control of biological processes and disease states at the nucleic acid level. Trinucleotide repeat expansions are associated with several neurodegenerative diseases where dynamic slippage is thought to occur during replication, forming transient 3WJ intermediates with the complementary strand. Here, we report triptycene-based molecules that bind to a d(CAG)·(CTG) repeat using a gel shift assay, fluorescence-quenching and circular dichroism.

Triple Aryne-Tetrazine Reaction Enabling Rapid Access to a New Class of Polyaromatic Heterocycles.

One of the most challenging goals of modern synthetic chemistry is to develop multi-step reactions for rapid and efficient access to complex molecules. We report a triple aryne-tetrazine reaction that enables rapid access to a new class of polyaromatic heterocycles. This new reaction, which couples diverse reactivity modes between simple aryne and tetrazine starting materials, proceeds in a single operation and takes less than 5 minutes in air with no metal catalyst.

Recognition of nucleic acid junctions using triptycene-based molecules.

The modulation of nucleic acids by small molecules is an essential process across the kingdoms of life. Targeting nucleic acids with small molecules represents a significant challenge at the forefront of chemical biology. Nucleic acid junctions are ubiquitous structural motifs in nature and in designed materials. Herein, we describe a new class of structure-specific nucleic acid junction stabilizers based on a triptycene scaffold. Triptycenes provide significant stabilization of DNA and RNA three-way junctions, providing a new scaffold for the development of nucleic acid junction binders with enhanced recognition properties. Additionally, we report cytotoxicity and cell uptake data in two human ovarian carcinoma cell lines.