Carbohydrates in peptide and protein design

Monosaccharides and amino acids are fundamental building blocks in the assembly of nature's polymers. They have different structural aspects and, to a significant extent, different functional groups. Oligomerization gives rise to oligosaccharides and peptides, respectively. While carbohydrates and peptides can be found conjoined in nature, e.g., in glycopeptides, the aim of this review is the radical redesign of peptide structures using carbohydrates, particularly monosaccharides and cyclic oligosaccharides, to produce novel peptides, peptidomimetics, and abiotic proteins. These hybrid molecules, chimeras, have properties arising largely from the combination of structural characteristics of carbohydrates with the functional group diversity of peptides. This field includes de novo designed synthetic glycopeptides, sugar (carbohydrate) amino acids, carbohydrate scaffolds for nonpeptidal peptidomimetics of cyclic peptides, cyclodextrin functionalized peptides, and carboproteins, i.e., carbohydrate-based proteinmimetics. These successful applications demonstrate the general utility of carbohydrates in peptide and protein architecture.     

An Index for Characterization of Natural and Non-Natural Amino Acids for Peptidomimetics

Bioactive peptides and peptidomimetics play a pivotal role in the regulation of many biological processes such as cellular apoptosis, host defense, and biomineralization. In this work, we develop a novel structural matrix, Index of Natural and Non-natural Amino Acids (NNAAIndex), to systematically characterize a total of 155 physiochemical properties of 22 natural and 593 non-natural amino acids, followed by clustering the structural matrix into 6 representative property patterns including geometric characteristics, H-bond, connectivity, accessible surface area, integy moments index, and volume and shape. As a proof-of-principle, the NNAAIndex, combined with partial least squares regression or linear discriminant analysis, is used to develop different QSAR models for the design of new peptidomimetics using three different peptide datasets, i.e., 48 bitter-tasting dipeptides, 58 angiotensin-converting enzyme inhibitors, and 20 inorganic-binding peptides. A comparative analysis with other QSAR techniques demonstrates that the NNAAIndex method offers a stable and predictive modeling technique for in silico large-scale design of natural and non-natural peptides with desirable bioactivities for a wide range of applications.     

Exploration of the P3 region of PEXEL peptidomimetics leads to a potent inhibitor of the Plasmodium protease,plasmepsin V

The use of arginine isosteres is a known strategy to overcome poor membrane permeability commonly associated with peptides or peptidomimetics that possess this highly polar amino acid. Here, we apply this strategy to peptidomimetics that are potent inhibitors of the malarial protease, plasmepsin V, with the aim of enhancing their activity against Plasmodium parasites, and exploring the structure–activity relationship of the P₃ arginine within the S₃ pocket of plasmepsin V. Of the arginine isosteres trialled in the P₃ position, we discovered that canavanine was the ideal and that this peptidomimetic potently inhibits plasmepsin V, efficiently blocks protein export and inhibits parasite growth. Structure studies of the peptidomimetics bound to plasmepsin V provided insight into the structural basis for the enzyme activity observed in vitro and provides further evidence why plasmepsin V is highly sensitive to substrate modification.     

Convergent synthesis of aminomethylene peptidomimetics

This protocol describes a convergent synthesis of reduced amide bond peptidomimetics using thioacid-terminated peptides and aziridine-containing peptide conjugates. This approach could be used to produce peptides and proteins with modified backbones. The peptide conjugates are made using readily available aziridine aldehydes. The convergent synthesis of peptidomimetics is demonstrated through the preparation of long and short peptide fragments with an aminomethylene group incorporated within them. This transformation is amenable to the synthesis of peptides with reduced amide bonds at cysteine and alanine. The procedure describes the preparation of each component used and highlights the ease of synthesis of aminomethylene peptidomimetics, and takes about 3 d to complete.     

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.     

The effect of substitution patterns on the release rates of opioid peptides DADLE and [Leu(5)]-enkephalin from coumarin prodrug moieties

A coumarin-based prodrug system has been developed in our laboratory for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. The drug release rates from this prodrug system were found to be dependent on the structural features of the drug moiety. The effect of the phenyl ring substitutions on the release kinetics of such prodrugs of model amines was examined recently and it was found that appropriately positioned alkyl substituents on the phenyl ring could help to facilitate the release. Aimed at further understanding the structure-release rate relationship of the coumarin-based cyclic prodrugs, we synthesized and examined a series of substituted coumarinic acid derivatives of opioid peptides, DADLE, and [Leu(5)]-enkephalin.     

Challenges in the design of insulin and relaxin/insulin-like peptide mimetics

Peptidomimetics are designed to overcome the poor pharmacokinetics and pharmacodynamics associated with the native peptide or protein on which they are based. The design of peptidomimetics starts from developing structure-activity relationships of the native ligand-target pair that identify the key residues that are responsible for the biological effect of the native peptide or protein. Then minimization of the structure and introduction of constraints are applied to create the core active site that can interact with the target with high affinity and selectivity. Developing peptidomimetics is not trivial and often challenging, particularly when peptides’ interaction mechanism with their target is complex. This review will discuss the challenges of developing peptidomimetics of therapeutically important insulin superfamily peptides, particularly those which have two chains (A and B) and three disulfide bonds and whose receptors are known, namely insulin, H2 relaxin, H3 relaxin, INSL3 and INSL5.     

Molecular dynamics simulations of conformational behavior of linear RGD peptidomimetics and cyclic prodrugs in aqueous and octane solutions

Conformations available to a class of cyclic prodrugs and corresponding linear RGD peptidomimetics were explored using 1 ns length molecular dynamics simulations performed with the program CHARMM. Water and octane, modeled explicitly, were used as solvents to mimic the change of the environment experienced by the solutes upon partition from water to membrane in the trans-cellular transport process. In water, the linear peptidomimetics tended to populate extended-like structures, characterized by strong favorable interactions with solvent and low intrinsic stability. In these extended conformations the charged termini are able to assume large distances, above 15 A for the longest systems. These linear peptidomimetics have been found to exhibit the highest potency in experimental studies, in accord with the trends experimentally observed for RGD peptides. In contrast, in octane compact conformers of the linear peptidomimetics were favored, with all charged groups aggregated and shielded from solvent, exhibiting high intrinsic stability and weak solute-solvent interactions. Our calculations predict a large unfavorable energy change for transferring the linear systems from water to octane, in agreement with experimental findings that these compounds are not transported via the trans-cellular pathway. The cyclic prodrugs did not exhibit major structural differences between the simulations in water and octane, adopting turn-like conformations in both solvents. The limited response of the cyclic structures to changes in the environment leads to energies of transfer from water to octane that are also unfavorable, but much less so than for the linear molecules. This effect is in accord with the observed enhanced passive trans-cellular transport of the cyclic prodrugs.     

Peptide Scaffolds: Flexible Molecular Structures With Diverse Therapeutic Potentials

Peptide scaffolds are diverse chemical structures providing a major base for drug development. Nature modifies a premature peptide with respect to a basic scaffold structure to create a mature and active peptide. Mimicking the natural scaffolds with desirable modifications i.e., scaffold-hopping will decrease the enormous efforts of chemical syntheses and testing for drug development. We have surveyed the scaffold-based compounds being used for anticancer, antiinfective, antiinflammatory and antidiabetic activities. Synthetic peptidomimetics like aptamers, dendrimers, arylamide foldamers, β peptides, d peptides etc. provide an anticipative picture for the therapeutic use of scaffold structures. Free energy based conformational analysis of peptidomimetics provides details of their structure–activity relationships. Diverse forms of such peptidomimetics with respect to their structure and applications are discussed alongwith the mimetics which reached clinical trials. The review gives an insight into the future panoramas of drug development and identifies few peptide scaffolds having diverse potential with chemical modifications.     

Molecular Dynamics Simulations of Conformational Behavior of Linear RGD Peptidomimetics and Cyclic Prodrugs in Aqueous and Octane Solutions

Abstract

Conformations available to a class of cyclic prodrugs and corresponding linear RGD peptidomimetics were explored using 1 ns length molecular dynamics simulations performed with the program CHARMM. Water and octane, modeled explicitly, were used as solvents to mimic the change of the environment experienced by the solutes upon partition from water to membrane in the trans-cellular transport process. In water, the linear peptidomimetics tended to populate extended-like structures, characterized by strong favorable interactions with solvent and low intrinsic stability. In these extended conformations the charged termini are able to assume large distances, above 15 Å for the longest systems. These linear peptidomimetics have been found to exhibit the highest potency in experimental studies, in accord with the trends experimentally observed for RGD peptides. In contrast, in octane compact conformers of the linear peptidomimetics were favored, with all charged groups aggregated and shielded from solvent, exhibiting high intrinsic stability and weak solute-solvent interactions. Our calculations predict a large unfavorable energy change for transferring the linear systems from water to octane, in agreement with experimental findings that these compounds are not transported via the trans-cellular pathway. The cyclic pro- drugs did not exhibit major structural differences between the simulations in water and octane, adopting turn-like conformations in both solvents. The limited response of the cyclic structures to changes in the environment leads to energies of transfer from water to octane that are also unfavorable, but much less so than for the linear molecules. This effect is in accord with the observed enhanced passive trans-cellular transport of the cyclic prodrugs.     

A Readily Applicable Strategy to Convert Peptides to Peptoid-based Therapeutics

Incorporation of unnatural amino acids and peptidomimetic residues into therapeutic peptides is highly efficacious and commonly employed, but generally requires laborious trial-and-error approaches. Previously, we demonstrated that C₂₀ peptide has the potential to be a potential antiviral agent. Herein we report our attempt to improve the biological properties of this peptide by introducing peptidomimetics. Through combined alanine, proline, and sarcosine scans coupled with a competitive fluorescence polarization assay developed for identifying antiviral peptides, we enabled to pinpoint peptoid-tolerant peptide residues within C₂₀ peptide. The synergistic benefits of combining these (and other) commonly employed methods could lead to a easily applicable strategy for designing and refining therapeutically-attractive peptidomimetics.     

Poly-lysine peptidomimetics having potent antimicrobial activity without hemolytic activity

Diversity of sequence and structure in naturally occurring antimicrobial peptides (AMPs) limits their intensive structure–activity relationship (SAR) study. In contrast, peptidomimetics have several advantages compared to naturally occurring peptide in terms of simple structure, convenient to analog synthesis, rapid elucidation of optimal physiochemical properties and low-cost synthesis. In search of short antimicrobial peptides using peptidomimetics, which provide facile access to identify the key factors involving in the destruction of pathogens through SAR study, a series of simple and short peptidomimetics consisting of multi-Lys residues and lipophilic moiety have been prepared and found to be active against several Gram-negative and Gram-positive bacteria containing methicillin-resistant Staphylococcus aureus (MRSA) without hemolytic activity. Based on the SAR studies, we found that hydrophobicity, +5 charges of multiple Lys residues, hydrocarbon tail lengths and cyclohexyl group were crucial for antimicrobial activity. Furthermore, membrane depolarization, dye leakage, inner membrane permeability and time-killing kinetics revealed that bacterial-killing mechanism of our peptidomimetics is different from the membrane-targeting AMPs (e. g. melittin and SMAP-29) and implied our peptidomimetics might kill bacteria via the intracellular-targeting mechanism as done by buforin-2.     

A new approach for modification of phenylalanine peptides by Suzuki-Miyaura coupling reaction

For the first time, we have modified phenylalanine peptides by the Suzuki-Miyaura coupling reaction which may be useful in developing combinatorial libraries of peptidomimetics.     

Peptide and peptidomimetic libraries

Various techniques for generation of peptide and peptidomimetic libraries are summarized in this article. Multipin, tea bag, and split-couple-mix techniques represent the major methods used to make peptides and peptidomimetics libraries. The synthesis of these libraries were made in either discrete or mixture format. Peptides and peptidomimetics combinatorial libraries were screened to discover leads against a variety of targets. These targets, including bacteria, fungus, virus, receptors, and enzymes were used in the screening of the libraries. Discovered leads can be further optimized by combinatorial approaches.     

VCD studies on cyclic peptides assembled from L‐α‐amino acids and a trans‐2‐aminocyclopentane‐ or trans‐2‐aminocyclohexane carboxylic acid

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans‐2‐aminocyclohexane carboxylic acid (Achc) or trans‐2‐aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three‐dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6‐31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR‐based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Role of azaamino acid residue in beta-turn formation and stability in designed peptide.

The structural perturbation induced by C(alpha)-->N(alpha) exchange in azaamino acid-containing peptides was predicted by ab initio calculation of the 6-31G* and 3-21G* levels. The global energy-minimum conformations for model compounds, For-azaXaa-NH2 (Xaa=Gly, Ala, Leu) appeared to be the beta-turn motif with a dihedral angle of phi= +/- 90 degrees, psi=0 degrees. This suggests that incorporation of the azaXaa residue into the i+2 position of designed peptides could stabilize the beta-turn structure. The model azaLeu-containing peptide, Boc-Phe-azaLeu-Ala-OMe, which is predicted to adopt a beta-turn conformation was designed and synthesized in order to experimentally elucidate the role of the azaamino acid residue. Its structural preference in organic solvents was investigated using 1H NMR, molecular modelling and IR spectroscopy. The temperature coefficients of amide protons, the characteristic NOE patterns, the restrained molecular dynamics simulation and IR spectroscopy defined the dihedral angles [ (phi i+1, psi i+1) (phi i+2, psi i+2)] of the Phe-azaLeu fragment in the model peptide, Boc-Phe-azaLeu-Ala-OMe, as [(-59 degrees, 127 degrees) (107 degrees, -4 degrees)]. This solution conformation supports a betaII-turn structural preference in azaLeu-containing peptides as predicted by the quantum chemical calculation. Therefore, intercalation of the azaamino acid residue into the i+2 position in synthetic peptides is expected to provide a stable beta-turn formation, and this could be utilized in the design of new peptidomimetics adopting a beta-turn scaffold.     

Membrane adsorption and binding, cellular uptake and cytotoxicity of cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone: effects of hydrogen bonding and α-chirality in the β-peptoid residues

Cell-penetrating peptides (CPPs) provide a promising approach for enhancing intracellular delivery of therapeutic biomacromolecules by increasing transport through membrane barriers. Here, proteolytically stable cell-penetrating peptidomimetics with α-peptide/β-peptoid backbone were studied to evaluate the effect of α-chirality in the β-peptoid residues and the presence of guanidinium groups in the α-amino acid residues on membrane interaction. The molecular properties of the peptidomimetics in solution (surface and intramolecular hydrogen bonding, aqueous diffusion rate and molecular size) were studied along with their adsorption to lipid bilayers, cellular uptake, and toxicity. The surface hydrogen bonding ability of the peptidomimetics reflected their adsorbed amounts onto lipid bilayers as well as with their cellular uptake, indicating the importance of hydrogen bonding for their membrane interaction and cellular uptake. Ellipsometry studies further demonstrated that the presence of chiral centers in the β-peptoid residues promotes a higher adsorption to anionic lipid bilayers, whereas circular dichroism results showed that α-chirality influences their overall mean residue ellipticity. The presence of guanidinium groups and α-chiral β-peptoid residues was also found to have a significant positive effect on uptake in living cells. Together, the findings provide an improved understanding on the behavior of cell-penetrating peptidomimetics in the presence of lipid bilayers and live cells.     

Grb2 SH2 domain-binding peptide analogs as potential anticancer agents

The growth factor receptor-bound protein 2 (Grb2) plays an important role in the Ras signaling pathway. Several proteins were found to be overexpressed by oncogenes in the Ras signaling pathway, rendering Grb2 a potential target for the design of antitumor agents. Blocking the interaction between the phosphotyrosine-containing activated receptor and the Src-homology 2 (SH2) domain of Grb2 thus constitutes an important strategy for the development of potential anticancer agents. X-ray, NMR structural investigations, and molecular modeling studies have provided the target structure of Grb2 SH2 domain-alone or complexed with a phosphotyrosine-containing peptide-which is useful for the structure-based design of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain. We review here the variety of approaches to Grb2 SH2 pepide inhibitors developed with the aim of interrupting Grb2 recognition. Inhibitory effects of peptide analogs on the Grb2 SH2 domain and their binding affinities for Grb2 SH2 were determined by ELISA, cell-based assays, or Surface Plasman Resonance (SPR) technology. Results of theses studies provide important information for further modifications of lead peptides, and should lead to the discovery of potent peptides as anticancer agents.     

Substituted coumarins as esterase-sensitive prodrug moieties with improved release rates.

Our laboratory has recently reported a coumarin-based prodrug system for the preparation of esterase-sensitive prodrugs of amines, peptides, and peptidomimetics. However, the release from this prodrug system was undesirably slow for some drug moieties. In this report, we describe the synthesis and evaluation of several substituted coumarin-based prodrugs of model amines with significantly increased release rates.