A peptoid antagonist of VEGF receptor 2 recognizes a 'hotspot' in the extracellular domain distinct from the hormone-binding site

Antagonists of VEGF-mediated angiogenesis are of great interest clinically for the treatment of solid tumors and certain forms of macular degeneration. We recently described a novel peptoid antagonist of VEGF Receptor 2 (VEGFR2) that binds to the extracellular domain of the receptor and inhibits VEGF-mediated autophosphorylation and subsequent downstream signaling. Given the structural similarities between peptides and peptoids, an obvious model for the mode of action of the peptoid is that it competes with VEGF for binding to VEGFR2. However, we present evidence here that this is not the case and that VEGF and the peptoid antagonist recognize non-overlapping surfaces located within the first three immunoglobulin-like subdomains of the receptor. These data argue that the peptoid inhibits receptor-mediated autophosphorylation by a novel allosteric mechanism that may prevent the receptor from acquiring the conformation necessary to propagate downstream signals.     

Design and conformational analysis of peptoids containing N-hydroxy amides reveals a unique sheet-like secondary structure

N-hydroxy amides can be found in many naturally occurring and synthetic compounds and are known to act as both strong proton donors and chelators of metal cations. We have initiated studies of peptoids, or N-substituted glycines which contain N-hydroxy amide side chains to investigate the potential effects of these functional groups on peptoid backbone amide rotamer equilibria and local conformations. We reasoned that the propensity of these functional groups to participate in hydrogen bonding could be exploited to enforce intramolecular or intermolecular interactions that yield new peptoid structures. Here, we report the design, synthesis, and detailed conformational analysis of a series of model N-hydroxy peptoids. These peptoids were readily synthesized, and their structures were analyzed in solution by 1D and 2D NMR and in the solid-state by X-ray crystallography. The N-hydroxy amides were found to strongly favor trans conformations with respect to the peptoid backbone in chloroform. More notably, unique sheet-like structures held together via intermolecular hydrogen bonds were observed in the X-ray crystal structures of an N-hydroxy amide peptoid dimer, which to our knowledge represent the first structure of this type reported for peptoids. These results suggest that the N-hydroxy amide can be utilized to control both local backbone geometries and longer-range intermolecular interactions in peptoids, and represents a new functional group in the peptoid design toolbox.     

Osmoprotective polymer additives attenuate the membrane pore‐forming activity of antimicrobial peptoids

Antimicrobial peptides (AMPs) are critical components of the innate immune system and exhibit bactericidal activity against a broad spectrum of bacteria. We investigated the use of N‐substituted glycine peptoid oligomers as AMP mimics with potent antimicrobial activity. The antimicrobial mechanism of action varies among different AMPs, but many of these peptides can penetrate bacterial cell membranes, causing cell lysis. We previously hypothesized that amphiphilic cyclic peptoids may act through a similar pore formation mechanism against methicillin‐resistant Staphylococcus aureus (MRSA). Peptoid‐induced membrane disruption is observed by scanning electron microscopy and results in a loss of membrane integrity. We demonstrate that the antimicrobial activity of the peptoids is attenuated with the addition of polyethylene glycol osmoprotectants, signifying protection from a loss of osmotic balance. This decrease in antimicrobial activity is more significant with larger osmoprotectants, indicating that peptoids form pores with initial diameters of ～2.0–3.8 nm. The initial membrane pores formed by cyclic peptoid hexamers are comparable in diameter to those formed by larger and structurally distinct AMPs. After 24 h, the membrane pores expand to >200 nm in diameter. Together, these results indicate that cyclic peptoids exhibit a mechanism of action that includes effects manifested at the cell membrane of MRSA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 227–236, 2015.     

Unconstrained peptoid tetramer exhibits a predominant conformation in aqueous solution

Conformational control in peptoids, N-substituted glycines, is crucial for the design and synthesis of biologically-active compounds and atomically-defined nanomaterials. While there are a growing number of structural studies in solution, most have been performed with conformationally-constrained short sequences (e.g., sterically-hindered sidechains or macrocyclization). Thus, the inherent degree of heterogeneity of unconstrained peptoids in solution remains largely unstudied. Here, we explored the folding landscape of a series of simple peptoid tetramers in aqueous solution by NMR spectroscopy. By incorporating specific ¹³C-probes into the backbone using bromoacetic acid-2-¹³C as a submonomer, we developed a new technique for sequential backbone assignment of peptoids based on the 1,n-Adequate pulse sequence. Unexpectedly, two of the tetramers, containing an N-(2-aminoethyl)glycine residue (Nae), had preferred conformations. NMR and molecular dynamics studies on one of the tetramers showed that the preferred conformer (52%) had a trans-cis-trans configuration about the three amide bonds. Moreover, >80% of the ensemble contained a cis amide bond at the central amide. The backbone dihedral angles observed fall directly within the expected minima in the peptoid Ramachandran plot. Analysis of this compound against similar peptoid analogs suggests that the commonly used Nae monomer plays a key role in the stabilization of peptoid structure via a side-chain-to-main-chain interaction. This discovery may offer a simple, synthetically high-yielding approach to control peptoid structure, and suggests that peptoids have strong intrinsic conformational preferences in solution. These findings should facilitate the predictive design of folded peptoid structures, and accelerate application in areas ranging from drug discovery to biomimetic nanoscience.     

Extreme stability of helices formed by water-soluble poly-N-substituted glycines (polypeptoids) with alpha-chiral side chains.

Poly-N-substituted glycines or "peptoids" are protease-stable peptide mimics. Although the peptoid backbone is achiral and lacks hydrogen-bond donors, substitution with alpha-chiral side chains can drive the formation of stable helices that give rise to intense CD spectra. To systematically study the solution properties and stability of water-soluble peptoid helices with alpha-chiral side chains, we have synthesized and characterized an amphipathic, 36-residue N-substituted glycine oligomer. CD was used to investigate effects of concentration and solvent environment on this helical peptoid. We saw no significant dependence of helical structure on concentration. Intense, "alpha-helix-like" CD spectra were observed for the 36-mer in aqueous, 2,2,2-trifluorethanol (TFE), and methanol solution, proving a relative insensitivity of peptoid helical structure to solvent environment. While CD spectra taken in these different solvents were fundamentally similar in shape, we did observe some interesting differences in the intensities of particular CD bands in the various solvents. For example, the addition of TFE to an aqueous solvent increases the degree of peptoid helicity, as is observed for polypeptide alpha-helices. Moreover, the helical structure of peptoids appears to be virtually unaffected by heat, even in an aqueous buffer containing 8 M urea. The extraordinary resistance of these peptoid helices to denaturation is consistent with a dominant role of steric forces in their structural stabilization. The structured polypeptoids studied here may have potential as robust mimics of helical polypeptides of therapeutic interest.     

Peptoids advance multidisciplinary research and undergraduate education in parallel: Sequence effects on conformation and lipid interactions

Peptoids are versatile peptidomimetic molecules with wide-ranging applications from drug discovery to materials science. An understanding of peptoid sequence features that contribute to both their three-dimensional structures and their interactions with lipids will expand functions of peptoids in varied fields. Furthermore, these topics capture the enthusiasm of undergraduate students who prepare and study diverse peptoids in laboratory coursework and/or in faculty led research. Here, we present the synthesis and study of 21 peptoids with varied functionality, including 19 tripeptoids and 2 longer oligomers. We observed differences in fluorescence spectral features for 10 of the tripeptoids that correlated with peptoid flexibility and relative positioning of chromophores. Interactions of representative peptoids with sonicated glycerophospholipid vesicles were also evaluated using fluorescence spectroscopy. We observed evidence of conformational changes effected by lipids for select peptoids. We also summarize our experiences engaging students in peptoid-based projects to advance both research and undergraduate educational objectives in parallel.     

Solution effects on the self-association of a water-soluble peptoid

A desire to replicate the structural and functional complexity of proteins with structured, sequence-specific oligomers motivates study of the structural features of water-soluble peptoids (N-substituted glycine oligomers). Understanding the molecular-level details of peptoid self-assembly in water is essential to advance peptoids' application as novel materials. Peptoid 1 , an amphiphilic, putatively helical peptoid previously studied in our laboratory, shows evidence of self-association in aqueous solution. In this work, we evaluate how changes to aqueous solution conditions influence the self-association of 1 . We report that changes to pH influence the fluorescence and CD spectroscopic features as well as the peptoid's interaction with a solvatochromic fluorophore and its apparent size as estimated by size exclusion chromatography. Addition of guanidine hydrochloride and ammonium sulfate also modulate spectroscopic features of the peptoid, its interaction with a solvatochromic fluorophore, and its elution in size exclusion chromatography. These data suggest that the ordering of the self-assembly changes in response to pH and with solvent additives and is more ordered at higher pH and in the presence of guanidine hydrochloride. The deeper understanding of the self-association of 1 afforded by these studies informs the design of new stimuli-responsive peptoids with stable tertiary or quaternary structures.     

Oligo(N-alkoxy glycines): trans substantiating peptoid conformations

Peptoid oligomers possess many desirable attributes bioactive peptidomimetic agents, including their ease of synthesis, chemical diversity, and capability for molecular recognition. Ongoing efforts to develop functional peptoids will necessitate improved capability for control of peptoid structure, particularly of the backbone amide conformation. We introduce alkoxyamines as a new reagent for solid phase peptoid synthesis. Herein, we describe the synthesis of N-alkoxy peptoids, and present NMR data indicating that the oligomers adopt a single stable conformation featuring trans amide bonds. These findings, combined with results from computational modeling, suggest that N-alkoxy peptoid oligomers have a strong propensity to adopt a polyproline II type secondary structure.     

High-throughput evaluation of relative cell permeability between peptoids and peptides

Peptides are limited in their use as drugs due to low cell permeability and vulnerability to proteases. In contrast, peptoids are immune to enzymatic degradation and some peptoids have been shown to be relatively cell permeable. In order to facilitate future design of peptoid libraries for screening experiments, it would be useful to have a high-throughput method to estimate the cell permeability of peptoids containing different residues. In this paper, we report the strengths and limitations of a high-throughput cell-based permeability assay that registers the relative ability of steroid-conjugated peptides and peptoids to enter a cell. A comparative investigation of the physicochemical properties and side chain composition of peptoids and peptides is described to explain the observed higher cell permeability of peptoids over peptides. These data suggest that the conversion of the monomeric residues in peptides to an N-alkylglycine moiety in peptoids reduced the hydrogen-bonding potential of the molecules and is the main contributor to the observed permeability improvement.     

Peptoid drug discovery and optimization via surface X-ray scattering

Synthetic polymers mimicking antimicrobial peptides have drawn considerable interest as potential therapeutics. N-substituted glycines, or peptoids, are recognized by their in vivo stability and ease of synthesis. Peptoids are thought to act primarily on the negatively charged lipids that are abundant in bacterial cell membranes. A mechanistic understanding of lipid–peptoid interaction at the molecular level will provide insights for rational design and optimization of peptoids. Here, we highlight recent studies that utilize synchrotron liquid surface X-ray scattering to characterize the underlying peptoid interactions with bacterial and eukaryotic membranes. Cellular membranes are highly complex, and difficult to characterize at the molecular level. Model systems including Langmuir monolayers, are used in these studies to reduce system complexity. The general workflow of these systems and the corresponding data analysis techniques are presented alongside recent findings. These studies investigate the role of peptoid physicochemical characteristics on membrane activity. Specifically, the roles of cationic charge, conformational constraint via macrocyclization, and hydrophobicity are shown to correlate their membrane interactions to biological activities in vitro. These structure–activity relationships have led to new insights into the mechanism of action by peptoid antimicrobials, and suggest optimization strategies for future therapeutics based on peptoids.     

In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.     

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both α-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.     

Peptoid conformational free energy landscapes from implicit-solvent molecular simulations in AMBER

To test the accuracy of existing AMBER force field models in predicting peptoid conformation and dynamics, we simulated a set of model peptoid molecules recently examined by Butterfoss et al. (JACS 2009, 131, 16798-16807) using QM methods as well as three peptoid sequences with experimentally determined structures. We found that AMBER force fields, when used with a Generalized Born/Surface Area (GBSA) implicit solvation model, could accurately reproduce the peptoid torsional landscape as well as the major conformers of known peptoid structures. Enhanced sampling by replica exchange molecular dynamics (REMD) using temperatures from 300 to 800 K was used to sample over cis-trans isomerization barriers. Compared to (Nrch)5 and cyclo-octasarcosyl, the free energy of N-(2-nitro-3-hydroxyl phenyl)glycine-N-(phenyl)glycine has the most "foldable" free energy landscape, due to deep trans-amide minima dictated by N-aryl sidechains. For peptoids with (S)-N (1-phenylethyl) (Nspe) side chains, we observe a discrepancy in backbone dihedral propensities between molecular simulations and QM calculations, which may be due to force field effects or the inability to capture n --> n* interactions. For these residues, an empirical phi-angle biasing potential can "rescue" the backbone propensities seen in QM. This approach can serve as a general strategy for addressing force fields without resorting to a complete reparameterization. Overall, this study demonstrates the utility of implicit-solvent REMD simulations for efficient sampling to predict peptoid conformational landscapes, providing a potential tool for first-principles design of sequences with specific folding properties.     

Incorporation of chemoselective functionalities into peptoids via solid-phase submonomer synthesis

A simple route to the introduction of a number of chemoselective functional groups into peptoids (oligo(N-substituted glycines)) by an extension of the standard solid-phase submonomer method is reported. The following groups were introduced: aminooxyacetamide, N-(carbamoylmethyl)acetohydrazide, mercaptoacetamide, 2-pyridinesulfenylmercaptoacetamide, and aldehyde-terminated peptoids. The method uses commercially available reagents, is fully compatible with standard peptoid submonomer synthesis conditions, is easily automated, and generates the desired functionalized peptoid in high yield and purity. Peptoids with suitable pairs of chemoselective ligation groups were joined in high yield.     

Synthesis and structure–activity relationship studies of peptidomimetic PKB/Akt inhibitors: The significance of backbone interactions

Elevated levels of activated Protein Kinase B (PKB/Akt) have been detected in many types of human cancer. In contrast to ATP site inhibitors, substrate-based inhibitors are more likely to be selective because of extensive interactions with the specific substrate binding site. Unfortunately, peptide-based inhibitors lack important pharmacological properties that are required of drug candidates. Chemical modifications of potent peptide inhibitors, such as peptoids and N^α-methylated amino acids, may overcome these drawbacks, while maintaining potency. We present a structure-activity relationship study of a potent, peptide-based PKB/Akt inhibitor, PTR6154. The study was designed to evaluate backbone modifications on the inhibitory activity of PTR6154. Two peptidomimetic libraries, peptoid and N^α-methylation, based on PTR6154, were synthesized and evaluated for in vitro PKB/Akt inhibition efficiency. All the peptoid analogs reduced potency significantly, as well as most of the members of the N-methyl library, suggesting that the backbone conformation and/or hydrogen bond interactions of PTR6154 derivatives are essential for inhibition activity. Two N-terminal members of the N-methyl library did not decrease potency and can be used as future drug leads.