A high-resolution structural characterization and physicochemical study of how a peptoid binds to an oncoprotein MDM2.

Peptoids are a promising drug modality targeting disease-related proteins, but how a peptoid engages in protein binding is poorly understood. This is primarily due to a lack of high-resolution peptoid-protein complex structures and systematic physicochemical studies. Here, we present the first crystal structure of a peptoid bound to a protein, providing high-resolution structural information about how a peptoid binds to a protein. We previously reported a rigid peptoid, oligo(N-substituted alanine) (oligo-NSA), and developed an oligo-NSA-type peptoid that binds to MDM2. X-ray crystallographic analysis of the peptoid bound to MDM2 showed that the peptoid recognizes the MDM2 surface predominantly through the interaction of the N-substituents, while the main chain acts as a scaffold. Additionally, conformational, thermodynamic, and kinetic analysis of the peptoid and its derivatives with a less rigid main chain revealed that rigidification of the peptoid main chain contributes to improving the protein binding affinity. This improvement is thermodynamically attributed to an increased magnitude of the binding enthalpy change, and kinetically to an increased association rate and decreased dissociation rate. This study provides invaluable insights into the design of protein-targeting peptoids.

Comprehensive Application of XFEL Microcrystallography for Challenging Targets in Various Organic Compounds.

There is a growing demand for structure determination from small crystals, and the three-dimensional electron diffraction (3D ED) technique can be employed for this purpose. However, 3D ED has certain limitations related to the crystal thickness and data quality. We here present the application of serial X-ray crystallography (SX) with X-ray free electron lasers (XFELs) to small (a few µm or less) and thin (a few hundred nm or less) crystals of novel compounds dispersed on a substrate. For XFEL exposures, two-dimensional (2D) scanning of the substrate coupled with rotation enables highly efficient data collection. The recorded patterns can be successfully indexed using lattice parameters obtained through 3D ED. This approach is especially effective for challenging targets, including pharmaceuticals and organic materials that form preferentially oriented flat crystals in low-symmetry space groups. Some of these crystals have been difficult to solve or have yielded incomplete solutions using 3D ED. Our extensive analyses confirmed the superior quality of the SX data regardless of crystal orientations. Additionally, 2D scanning with XFEL pulses gives an overall distribution of the samples on the substrate, which can be useful for evaluating the properties of crystal grains and the quality of layered crystals. Therefore, this study demonstrates that XFEL crystallography has become a powerful tool for conducting structure studies of small crystals of organic compounds.

Synthesis of Short Peptides with Perfluoroalkyl Side Chains and Evaluation of Their Cellular Uptake Efficiency.

With an increasing demand for macromolecular biotherapeutics, the issue of their poor cell-penetrating abilities requires viable and relevant solutions. Herein, we report tripeptides bearing an amino acid with a perfluoroalkyl (RF ) group adjacent to the α-carbon. RF -containing tripeptides were synthesized and evaluated for their ability to transport a conjugated hydrophilic dye (Alexa Fluor 647) into the cells. RF -containing tripeptides with the fluorophore showed high cellular uptake efficiency and none of them were cytotoxic. Interestingly, we demonstrated that the absolute configuration of perfluoroalkylated amino acids (RF -AAs) affects not only nanoparticle formation but also the cell permeability of the tripeptides. These novel RF -containing tripeptides are potentially useful as short and noncationic cell-penetrating peptides (CPPs).

Amide-to-ester substitution as a stable alternative to N-methylation for increasing membrane permeability in cyclic peptides.

Naturally occurring peptides with high membrane permeability often have ester bonds on their backbones. However, the impact of amide-to-ester substitutions on the membrane permeability of peptides has not been directly evaluated. Here we report the effect of amide-to-ester substitutions on the membrane permeability and conformational ensemble of cyclic peptides related to membrane permeation. Amide-to-ester substitutions are shown to improve the membrane permeability of dipeptides and a model cyclic hexapeptide. NMR-based conformational analysis and enhanced sampling molecular dynamics simulations suggest that the conformational transition of the cyclic hexapeptide upon membrane permeation is differently influenced by an amide-to-ester substitution and an amide N-methylation. The effect of amide-to-ester substitution on membrane permeability of other cyclic hexapeptides, cyclic octapeptides, and a cyclic nonapeptide is also investigated to examine the scope of the substitution. Appropriate utilization of amide-to-ester substitution based on our results will facilitate the development of membrane-permeable peptides.

Each side chain of cyclosporin A is not essential for high passive permeability across lipid bilayers.

We compared the passive permeability of cyclosporin A (CsA) derivatives with side chain deletions across lipid bilayers. CsA maintained passive permeability after losing any one of the side chains, which suggests that the propensity of the backbone of CsA is an important component for high passive permeability.

Label-free quantification of passive membrane permeability of cyclic peptides across lipid bilayers: penetration speed of cyclosporin A across lipid bilayers.

Cyclic peptides that passively penetrate cell membranes are under active investigation in drug discovery research. PAMPA (Parallel Artificial Membrane Permeability Assay) and Caco-2 assay are mainly used for permeability measurements in these studies. However, permeability rates across the artificial membrane and the cell monolayer used for these assays are intrinsically different from the ones across pure lipid bilayers. There are also membrane permeability assays for peptides using reconstructed lipid bilayers, but they require labeling for detection, and the absolute membrane permeability of the natural peptides themselves could not be determined. Here, we constructed a lipid bilayer permeability assay and realized the first label-free measurements of the lipid bilayer permeability of cyclic peptides. Quantitative permeability values across lipid bilayers were determined for model cyclic hexapeptides and an important natural product, cyclosporin A (CsA). The obtained quantitative permeability values will provide new and advanced knowledge about the passive permeability of cyclic peptides.

Residue-based program of a ß-peptoid twisted strand shape via a cyclopentane constraint.

N-Substituted peptides, such as peptoids and ß-peptoids, have been reported to have unique structures with diverse functions, like catalysis and manipulation of biomolecular functions. Recently, the preorganization of monomer shape by restricting bond rotations about all backbone dihedral angles has been demonstrated to be useful for de novo design of peptoid structures. Such design strategies are hitherto unexplored for ß-peptoids; to date, no preorganized ß-peptoid monomers have been reported. Here, we report the first design strategy for ß-peptoids, in which all four backbone dihedral angles (ω, ϕ, θ, ψ) are rotationally restricted on a per-residue basis. The introduction of a cyclopentane constraint realized the preorganized monomer structure and led to a ß-peptoid with a stable twisted strand shape.

Oligo(N-methylalanine) as a Peptide-Based Molecular Scaffold with a Minimal Structure and High Density of Functionalizable Sites.

Functionalizable synthetic molecules with nanometer sizes and defined shapes in water are useful as molecular scaffolds to mimic the functions of biomacromolecules and develop chemical tools for manipulating biomacromolecules. Herein, we propose oligo(N-methylalanine) (oligo-NMA) as a peptide-based molecular scaffold with a minimal structure and a high density of functionalizable sites. Oligo-NMA forms a defined shape in water without hydrogen-bonding networks or ring constraints, which enables the molecule to act as a scaffold with minimal atomic composition. Furthermore, functional groups can be readily introduced on the nitrogens and α-carbons of oligo-NMA. Computational and NMR spectroscopic analysis suggested that the backbone structure of oligo-NMA is not largely affected by functionalization. Moreover, the usefulness of oligo-NMA was demonstrated by the design of protein ligands. The ease of synthesis, minimal structure, and high functionalization flexibility makes oligo-NMA a useful scaffold for chemical and biological applications.

Peptoid-based reprogrammable template for cell-permeable inhibitors of protein-protein interactions.

The development of inhibitors of intracellular protein-protein interactions (PPIs) is of great significance for drug discovery, but the generation of a cell-permeable molecule with high affinity to protein is challenging. Oligo(N-substituted glycines) (oligo-NSGs), referred to as peptoids, are attractive as potential intracellular PPI inhibitors owing to their high membrane permeability. However, their intrinsically flexible backbones make the rational design of inhibitors difficult. Here, we propose a peptoid-based rational approach to develop cell-permeable PPI inhibitors using oligo(N-substituted alanines) (oligo-NSAs). The rigid structures of oligo-NSAs enable independent optimization of each N-substituent to improve binding affinity and membrane permeability, while preserving the backbone shape. A molecule with optimized N-substituents inhibited a target PPI in cells, which demonstrated the utility of oligo-NSA as a reprogrammable template to develop intracellular PPI inhibitors.

A comprehensive study on the effect of backbone stereochemistry of a cyclic hexapeptide on membrane permeability and microsomal stability.

Backbone stereochemistry of cyclic peptides has been reported to have a great influence on microsomal stability and membrane permeability, two important factors that determine oral bioavailability. Here, we comprehensively investigated the correlation between the backbone stereochemistry of cyclic hexapeptide stereoisomers and their stability in liver microsomes, as well as passive membrane permeability.

Methyl to trifluoromethyl substitution as a strategy to increase the membrane permeability of short peptides.

Here, we investigated the effect of CH3 to CF3 substitution on the membrane permeability of peptides. We synthesized a series of peptides with CF3 groups and corresponding nonfluorinated peptides and measured the membrane permeability of the peptides. As a result, we demonstrated that CH3 to CF3 substitution is useful for increasing the membrane permeability of di-/tri-peptides.

Per-Residue Program of Multiple Backbone Dihedral Angles of ß-Peptoids via Backbone Substitutions.

Unique folded structures of natural and synthetic oligomers are the most fundamental basis for their unique functions. N-Substituted ß-peptides, or ß-peptoids, are synthetic oligomers with great potential to fold into diverse three-dimensional structures because of the existence of four rotatable bonds in a monomer with highly modular synthetic accessibility. However, the existence of the four rotatable bonds poses a challenge for conformational control of ß-peptoids. Here, we report a strategy for per-residue programming of two dihedral angles of ß-peptoids, which is useful for restricting the conformational space of the oligomers. The oligomer was found to form a unique loop conformation that is stabilized by the backbone rotational restrictions. Circular dichroism and NMR spectroscopic analyses and X-ray crystallographic analysis of the oligomer are presented. The strategy would significantly facilitate the discovery of many more unique folded structures of ß-peptoids.

A Peptoid with Extended Shape in Water.

The term "peptoids" was introduced decades ago to describe peptide analogues that exhibit better physicochemical and pharmacokinetic properties than peptides. Oligo(N-substituted glycine) (oligo-NSG) was previously proposed as a peptoid due to its high proteolytic resistance and membrane permeability. However, oligo-NSG is conformationally flexible, and ensuring a defined shape in water is difficult. This conformational flexibility severely limits the biological application of oligo-NSG. Here, we propose oligo(N-substituted alanine) (oligo-NSA) as a peptoid that forms a defined shape in water. The synthetic method established in this study enabled the first isolation and conformational study of optically pure oligo-NSA. Computational simulations, crystallographic studies, and spectroscopic analysis demonstrated the well-defined extended shape of oligo-NSA realized by backbone steric effects. This new class of peptoid achieves the constrained conformation without any assistance of N-substituents and serves as a scaffold for displaying functional groups in well-defined three-dimensional space in water, which leads to effective biomolecular recognition.

A parallel permeability assay of peptides across artificial membranes and cell monolayers using a fluorogenic reaction.

Here, we report a facile permeability assay to quantitatively evaluate the membrane permeability of multiple peptides in parallel. With a fluorogenic click reaction between azidocoumarin and a terminal alkyne tag introduced on a peptide, the peptide that crossed an artificial membrane or a cell monolayer was quantitatively detected. The method allows a rapid measurement of the permeability of multiple compounds on a plate reader even in the presence of a complex mixture of biological molecules.

Isolation of a peptide containing d-amino acid residues that inhibits the α-helix-mediated p53-MDM2 interaction from a one-bead one-compound library.

α-Helix-mediated protein-protein interactions (PPIs) are important targets in biological research and drug development. Peptides containing d-amino acid residues are attractive molecules for inhibiting α-helix-mediated PPIs because of their wide surface area and high protease resistance. In this study, a peptide library was constructed using a one-bead one-compound format designed to isolate left-handed α-helical peptides, which are promising molecules as inhibitors of α-helix-mediated PPIs. Screening of the library against an α-helix-mediated PPI between MDM2 and p53 yielded an inhibitor of the PPI. Design and screening of the library, and biochemical and spectroscopic studies of the discovered peptide are presented.

Solid-Phase Synthesis of ß-Peptoids with Chiral Backbone Substituents Using Reductive Amination.

A new submonomeric synthetic method of ß-peptoids that allows introduction of chiral backbone substituents is established. The synthesis of ß-peptoids with various backbone substituents on ß-carbons and spectroscopic studies of synthesized oligomers are described.

Discovery of Phosphorylated Peripherin as a Major Humoral Autoantigen in Type 1 Diabetes Mellitus.

A major goal in understanding autoimmune diseases is to define the antigens that elicit a self-destructive immune response, but this is a difficult endeavor. In an effort to discover autoantigens associated with type 1 diabetes (T1D), we used epitope surrogate technology that screens combinatorial libraries of synthetic molecules for compounds that could recognize disease-linked autoantibodies and enrich them from serum. Autoantibodies from one patient revealed a highly phosphorylated form of peripherin, a neuroendocrine filament protein, as a candidate T1D antigen. Peripherin antibodies were detected in 72% of donor patient sera. Further analysis revealed that the T1D-associated antibodies only recognized a dimeric conformation of peripherin. These data explain why peripherin was dismissed as an important T1D antigen previously. The discovery of this novel autoantigen would not have been possible using standard methods, such as hybridizing serum antibodies to recombinant protein arrays, highlighting the power of epitope surrogate technology for probing the mechanism of autoimmune diseases.

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset.

Reliable diagnosis of murine type 1 diabetes using a panel of autoantigens and "antigen surrogates" mounted onto a liquid array.

Autoantibodies raised against ß cell antigens are the most reliable preclinical biomarkers for predicting the imminent onset of type 1 diabetes mellitus (T1DM). The most current detection platforms are technically challenging or are run on clinically esoteric equipment. Here, we present a straightforward approach to detect autoantibody biomarkers that employs highly PEGylated microspheres onto which are mounted various capture agents that include affinity-tagged antigens or small molecule "antigen surrogates." After incubation with small quantities of serum, the bound autoantibodies can be measured using a standard flow cytometer. By multiplexing this assay, we show that a panel of antigen and antigen surrogates reliably predicts hyperglycemia in a mouse model of diabetes without false positives.

Synthesis of a large library of macrocyclic peptides containing multiple and diverse N-alkylated residues.

Large combinatorial libraries of macrocyclic peptides are a useful source of bioactive compounds. However, peptides are not generally cell permeable, so there is great interest in the development of methods to create large libraries of modified peptides. In particular, N-alkylation of peptides is known to improve their bioavailability significantly. Incorporation of some level of N-methylated amino acids into peptide libraries has been accomplished with ribosome display or related methods, but the modest efficiency and the inability to employ more diverse N-alkylated amino acids in this type of system argue for the development of synthetic libraries. Here we present optimized procedures for synthesizing macrocyclic peptides containing multiple N-alkylated units and show that this chemistry is efficient enough for the creation of high quality combinatorial libraries by split and pool solid-phase synthesis.