Minimalist and universal peptidomimetics

Many "new generation" peptidomimetics are designed to present amino acid side chains only; they do not have structural features that resemble peptide main chains. These types of molecules have frequently been presented in the literature as mimics of specific secondary structures. However, many "side-chain only" peptidomimetics do not rest in single conformational states, but exist in a limited number of freely interconverting forms. These different conformations may resemble different secondary structures, so referring to them as, for instance, turn- or helical-mimics understates the ways they could adapt to various binding situations. Sets of scaffolds that can be used to mimic aspects of nearly every secondary structure, i.e. universal peptidomimetics, can be constructed. These may assume a privileged place in library design, particularly in high throughput screening for pharmacological probes for which binding conformations, or even the target itself, is unknown at the time the library is designed (critical review, 101 references).     

Universal peptidomimetics

This paper concerns peptidomimetic scaffolds that can present side chains in conformations resembling those of amino acids in secondary structures without incurring excessive entropic or enthalpic penalties. Compounds of this type are referred to here as minimalist mimics. The core hypothesis of this paper is that small sets of such scaffolds can be designed to analogue local pairs of amino acids (including noncontiguous ones) in any secondary structure; i.e., they are universal peptidomimetics. To illustrate this concept, we designed a set of four peptidomimetic scaffolds. Libraries based on them were made bearing side chains corresponding to many of the protein-derived amino acids. Modeling experiments were performed to give an indication of kinetic and thermodynamic accessibilities of conformations that can mimic secondary structures. Together, peptidomimetics based on these four scaffolds can adopt conformations that resemble almost any combination of local amino acid side chains in any secondary structure. Universal peptidomimetics of this kind are likely to be most useful in the design of libraries for high-throughput screening against diverse targets. Consequently, data arising from submission of these molecules to the NIH Molecular Libraries Small Molecule Repository (MLSMR) are outlined.     

Topochemical exploration of potent compounds using retro-enantiomer libraries of cyclic pentapeptides

Cyclic pentapeptides have been adopted as conformationally restricted peptide templates to dispose pharmacophores of bioactive peptides. In our recent study, use of two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries containing critical residues of a bioactive peptide led to the discovery of potent downsized peptides that possess activity comparable to that of the parent peptide. The present study demonstrates that a third library consisting of retro-enantiomers (retro-inverso peptides) that possess not only all residues with the opposite configuration to those in the corresponding original peptide but also amino acid sequences with reversed arrangement, is important as an alternative library for rationally finding active compounds.     

Synthesis and screening of libraries of synthetic tripodal receptor molecules with three different amino acid or peptide arms: identification of iron binders

A novel selectively deprotectable triazacyclophane scaffold was used for the design and split-mix synthesis of two libraries of solid-phase bound tripodal synthetic receptors possessing three different amino acid or peptidic arms. In the synthesis of the first library, the two outer arms consisted of amino acid Ala, Arg, Asp, Gln, Gly, Lys, Phe, Ser, Tyr, or Val and the middle arm consisted of amino acid Asn, Glu, His, Leu, or Pro. The second library contained amino acid and/or (di)peptide arms. The arms were different in all library members. The first outer arm consisted of amino acid(s) Ala, Arg, Gln, Phe, or Ser, the second outer arm consisted of amino acid(s) Asp, Gly, Lys, Tyr, or Val, and the middle arm consisted of amino acid(s) Asn, Glu, His, Leu, or Pro, leading to a 27 000 member library of synthetic tripodal receptor molecules. In on-bead screening experiments, a remarkable selectivity of some library members for Fe(3+) was observed and decoding of their structures by Edman degradation revealed consensus sequences with structural resemblance to non-heme iron proteins.     

A general method for designing combinatorial peptide libraries decodable by amino acid analysis

Herein we describe an algorithm for designing combinatorial peptide libraries for split-and-mix synthesis on solid support that are decodable by amino acid analysis (AAA) of the beads. AAA is a standard service analysis available in most biochemical laboratories, and it allows one to control the quality of the peptide on each bead, an important feature that is missing from most library decoding protocols. In the algorithm, each AA is assigned to two variable positions in the sequence grouped in a "unique pair". This arrangement limits sequence design because both the number of unique pairs U (setting the maximum number of variable AA) and the maximum number S of different AA per variable position depend on the peptide length N (U=N(N-1)/2), S=N-1). The method is therefore only suitable for focused libraries. An application example is shown for the selection of peptides with N-terminal proline or hydroxyproline catalyzing an aldol reaction from a combinatorial library of 65536 octapeptides. A simple enumeration program is available to help design combinatorial libraries decodable by amino acid analysis. The method applies to linear and cyclic peptides, can be used for nonnatural building blocks, including beta-amino acids, and should help to explore the vast chemistry of linear and cyclic peptide for catalysis and bioactivity.     

Intracellular protein scaffold-mediated display of random peptide libraries for phenotypic screens in mammalian cells

BACKGROUND: Mammalian cell screens of peptide libraries for changes in cellular phenotype may identify novel functional peptides and their cognate binding partners, and allow identification of signal transduction network members or proteins important in disease processes. RESULTS: Green fluorescent protein (GFP) peptide libraries with different structural biases were tested by retroviral expression in A549 carcinoma cells, HUVEC and other cell types. Three different loop replacement libraries, containing 12 or 18 random residues, were compatible with enhanced GFP (EGFP) folding, as was a C-terminally fused random 20-mer library. Library concentrations in A549 cells ranged from ca. 1 to 54 microM. Replacement of loop 3 with known nuclear localization sequence (NLS) peptides, but not with inactive mutants, directed EGFP to the nucleus. Microscopy-based screens of three different libraries for non-uniform localization revealed novel NLS peptides, novel variants of a peroxisomal localization motif, a variety of partial NLS peptides, peptides localized to the nucleolus, and nuclear-excluded peptides. CONCLUSIONS: Peptides can be presented by EGFP in conformations that can functionally interact with cellular constituents in mammalian cells. A phenotypic screen resulting in the discovery of novel localization peptides that were not cell type-specific suggests that this methodology may be applied to other screens in cells derived from diseased organisms, and illustrates the use of intracellular combinatorial peptide chemistry in mammalian cells.     

Microwave-assisted solid-phase aza-peptide synthesis: aza scan of a PKB/Akt inhibitor using aza-arginine and aza-proline precursors

Aza-peptides are peptidomimetics in which one or more of the α-carbons, bearing the side-chain residues, has been replaced by a nitrogen. These peptidomimetics have been shown to be promising for the generation of drug leads and for structure-activity relationship studies. Aza-scan is the systematic replacement of amino acid residues in a given peptide with their aza counterparts. We report here an aza-scan of a potent, peptide-based PKB/Akt inhibitor, PTR6154. Procedures for microwave-assisted, Fmoc/t-Bu chemistry, solid-phase aza-peptide synthesis were developed which significantly reduce standard reaction time and are suitable for automation. Novel substituted hydrazines have been prepared for the straightforward incorporation of aza-arginine and aza-proline residues. This work will enable aza-scan to become a more common and standard method for structure-activity relationship studies of peptides.     

Importance of chirality and reduced flexibility of protein side chains: a study with square and tetrahedral lattice models

Side chains of amino acid residues are the determining factor that distinguishes proteins from other unstable chain polymers. In simple models they are often represented implicitly (e.g., by spin states) or simplified as one atom. Here we study side chain effects using two-dimensional square lattice and three-dimensional tetrahedral lattice models, with explicitly constructed side chains formed by two atoms of different chirality and flexibility. We distinguish effects due to chirality and effects due to side chain flexibilities, since residues in proteins are L residues, and their side chains adopt different rotameric states. For short chains, we enumerate exhaustively all possible conformations. For long chains, we sample effectively rare events such as compact conformations and obtain complete pictures of ensemble properties of conformations of these models at all compactness region. This is made possible by using sequential Monte Carlo techniques based on chain growth method. Our results show that both chirality and reduced side chain flexibility lower the folding entropy significantly for globally compact conformations, suggesting that they are important properties of residues to ensure fast folding and stable native structure. This corresponds well with our finding that natural amino acid residues have reduced effective flexibility, as evidenced by statistical analysis of rotamer libraries and side chain rotatable bonds. We further develop a method calculating the exact side chain entropy for a given backbone structure. We show that simple rotamer counting underestimates side chain entropy significantly for both extended and near maximally compact conformations. We find that side chain entropy does not always correlate well with main chain packing. With explicit side chains, extended backbones do not have the largest side chain entropy. Among compact backbones with maximum side chain entropy, helical structures emerge as the dominating configurations. Our results suggest that side chain entropy may be an important factor contributing to the formation of alpha helices for compact conformations.     

Use of phage display methods to identify heptapeptide sequences for use as affinity purification 'tags' with novel chelating ligands in immobilized metal ion affinity chromatography

This study describes the screening of a peptide phage display library for amino acid sequences that bind with different affinities to a novel class of chelating ligands complexed with Ni2+ ions. These chelating ligands are based on the 1,4,7-triazacyclononane (TACN) structure and have been chosen to allow enhanced efficiency in protein capture and decreased propensity for metal ion leakage in the immobilized metal ion affinity chromatographic (IMAC) purification of recombinant proteins. Utilising high stringency screening conditions, various peptide sequences containing multiple histidine, tryptophan, and/or tyrosine residues were identified amongst the different phage peptide sequences isolated. The structures, and particularly the conserved locations of these key amino acid residues within the selected heptapeptides, form a basis to design specific peptide tags for use with these novel TACN ligands as a new mode of IMAC purification of recombinant proteins.     

A novel strategy for in vitro selection of peptide-drug conjugates

The chemical diversity of peptide and protein libraries generated from biological display systems is typically confined to the 20 naturally occurring amino acids. Here, we have developed a general strategy to introduce non-natural side chains into mRNA-display libraries via specific chemical derivatization. We constructed a mRNA-display library containing 3 x 10(12) different peptides bearing a pendant penicillin moiety in a fixed position. In vitro selection using this hybrid peptide-drug library resulted in novel inhibitors of the Staphylococcus aureus penicillin binding protein 2a (PBP2a). This strategy resulted in a penicillin-peptide conjugate that has at least 100-fold higher activity than the parent penicillin itself. Our approach provides a convenient way to enhance the efficacy of known drugs and facilitates the discovery of powerful new hybrid ligands with functionalities beyond those provided by the 20 naturally occurring residues.     

Stereoselective synthesis of 2,4-methanoproline homologues

The stereoselective synthesis of 2-azabicyclo[2.2.1]heptane-1-carboxylic acid and 6-azabicyclo[3.2.1]octane-5-carboxylic acid, novel rigid bicyclic proline analogues, is reported. The synthesis was performed in five steps from the corresponding 2-cycloalken-1-ones. A known approach to 2,4-methanoproline is improved. The three amino acids constitute a library of conformationally constrained proline analogues, which can be used for the design of peptidomimetics and peptide models.     

Aza-amino acid scanning of secondary structure suited for solid-phase peptide synthesis with fmoc chemistry and aza-amino acids with heteroatomic side chains

Aza-peptides, peptide analogues in which the alpha-carbon of one or more of the amino acid residues is replaced with a nitrogen atom, exhibit a propensity for adopting beta-turn conformations. A general Fmoc-protection protocol for the stepwise solid-phase synthesis of aza-peptides has now been developed based on the activation of N'-alkyl fluoren-9-ylmethyl carbazates with phosgene for coupling the aza-amino acid residues. This method has proven effective for introducing aza-amino acid residues with aliphatic (Ala, Leu, Val, and Gly) and aromatic (Phe, Tyr, and Trp) side chains. Acid promoted loss of aromatic side chains was noted with aza-Trp and aza-Tyr residues during peptide cleavage and suppressed by temperature control in the case of the latter. In addition, aza-peptides with heteroatomic side chain residues (Lys, Orn, Arg, and Asp) were conveniently synthesized using this protocol. Partial aza-amino acid scans were performed on three biologically active peptides: the potent tetrapeptide melanocortin receptor agonist, Ac-His-d-Phe-Arg-Trp-NH2; the growth hormone secretagogue hexapeptide, GHRP-6, His-d-Trp-Ala-Trp-d-Phe-Lys-NH2; and the human calcitonin gene-related peptide (hCGRP) antagonist, FVPTDVGPFAF-NH2. This practical procedure for aza-amino acid scanning using Fmoc-based solid-phase synthesis should find general utility for probing the existence and importance of beta-turn conformations in bioactive peptides.     

TAC-scaffolded tripeptides as artificial hydrolytic receptors: a combinatorial approach toward esterase mimics

In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.     

Cyclic Peptidomimetics Derived from the Apical Membrane Antigen I of Plasmodium falciparum and Their Use in Malaria Vaccine Design

A library of 35 cyclic peptidomimetics has been prepared, by a combination of solid‐ and solution‐phase methods, which, together, scan amino acid residues 444–489 in a protruding loop in the subdomain‐III of the apical membrane antigen‐I (AMA‐I), an integral membrane protein found on the surface of Plasmodium falciparum merozoites. The mimetics each contain twelve residues from AMA‐I linked through the N‐ and C‐termini to a β‐hairpin‐inducing template, comprising the dipeptide D ‐proline‐L ‐Apro (Apro=cis‐4‐amino‐L ‐proline). These peptidomimetics were each coupled via the 4‐amino group to a phospholipid, which allows their incorporation into reconstituted influenza virus‐like particles (virosomes) for immunization of mice, as well as their use in ELISA to characterize epitopes recognized by anti‐AMA‐I growth‐inhibitory monoclonal antibodies.     

Conformational analysis of furanoid epsilon-sugar amino acid containing cyclic peptides by NMR spectroscopy,molecular dynamics simulation,and X-ray crystallography: evidence for a novel turn structure

Sugar amino acids (SAAs) are useful building blocks for the design of peptidomimetics and peptide scaffolds. The three-dimensional structures of cyclic hybrid molecules containing the furanoid epsilon-SAA III and several amino acids were elucidated to study the preferred conformation of such an epsilon-SAA and its conformational influence on the backbone of cyclic peptides. NMR-based molecular dynamics simulations and empirical calculations of the cyclic tetramer 1, consisting of two copies of the SAA residue and two amino acids, revealed that it is conformationally restrained. The two SAA residues adopt different conformations. One of them forms an unusual turn, stabilized by an intraresidue nine-member hydrogen bond. The methylene functionalities of the other SAA residue are positioned in such a way that an intraresidue H bond is not possible. The X-ray crystal structure of 1 strongly resembles the solution conformation. Molecular dynamics calculations in combination with NMR analysis were also performed for compounds 2 and 3, which contain the RGD (Arg-Gly-Asp) consensus sequence and were previously shown to inhibit alpha(IIb)beta(3)-receptor-mediated platelet aggregation. The biologically most active compound 2 adopts a preferred conformation with the single SAA residue folded into the nine-member H bond-containing turn. Compound 3, containing an additional valine residue, as compared with compound 2, is conformational flexible. Our studies demonstrate that the furanoid epsilon-SAA III is able to introduce an unusual intraresidue hydrogen bond-stabilized beta-turn-like conformation in two of the three cyclic structures.     

cis-Apa: a practical linker for the microwave-assisted preparation of cyclic pseudopeptides via RCM cyclative cleavage

A new linker cis-5-aminopent-3-enoic acid (cis-Apa) was prepared for the synthesis of cyclic pseudopeptides by cyclization-cleavage by using ring-closing methatesis (RCM). We developed a new synthetic pathway for the preparation of the cis-Apa linker that was tested in the cyclization-cleavage process of different RGD peptide sequences. Different macrocyclic peptidomimetics were prepared by using this integrated microwave-assisted method, showing that the readily available cis-Apa amino acid is well adapted as a linker in the cyclization-cleavage process.     

Controlled peptide solvation in portion-mixing libraries of FRET peptides: improved specificity determination for Dengue 2 virus NS2B-NS3 protease and human cathepsin S

The solubility of peptides in aqueous buffers used for the enzyme assays is a common limitation for all peptide libraries. In principle, the more water-soluble peptides are, the more susceptible they will be to peptidase hydrolysis. We have demonstrated that this bias can be circumvented in a portion-mixing fluorescence resonance energy transfer (FRET) peptide library by introducing k (lysine in the D-form) in both termini of the peptides. This more solvated library and another one without the k were assayed using trypsin and chymotrypsin as standard peptidases with high selectivity for R and K and for hydrophobic F and Y, respectively. Significantly improved consistency of the information on substrate profiles was obtained from the solvated library. The influence of improved solvation on substrate specificity determination was successfully demonstrated by the difference in specificity observed between the two libraries employing the human cathepsin S (accepts acidic, basic, or neutral amino acids at P1 position) and Dengue 2 virus NS2B-NS3 protease (high specificity to the pair of basic amino acids K-R, R-R, or Q-R/K at P2-P1 positions). In conclusion, hydration of the peptides has a major influence on protease processing, and this bias can be reduced in bound peptide libraries, improving reliability.     

Design,structure-based focusing and in silico screening of combinatorial library of peptidomimetic inhibitors of Dengue virus NS2B-NS3 protease

Abstract  

Serine protease activity of the NS3 protein of Dengue virus is an important target of antiviral agents that interfere with the viral polyprotein precursor processing catalyzed by the NS3 protease (NS3pro), which is important for the viral replication and maturation. Recent studies showed that substrate-based peptidomimetics carrying an electrophilic warhead inhibit the NS2B-NS3pro cofactor-protease complex with inhibition constants in the low micromolar concentration range when basic amino acid residues occupy P₁ and P₂ positions of the inhibitor, and an aldehyde warhead is attached to the P₁. We have used computer-assisted combinatorial techniques to design, focus using the NS2B-NS3pro receptor 3D structure, and in silico screen a virtual library of more than 9,200 peptidomimetic analogs targeted around the template inhibitor Bz-Nle-Lys-Arg-Arg-H (Bz—benzoyl) that are composed mainly of unusual amino acid residues in all positions P₁–P₄. The most promising virtual hits were analyzed in terms of computed enzyme-inhibitor interactions and Adsorption, Distribution, Metabolism and Excretion (ADME) related physico-chemical properties. Our study can direct the interest of medicinal chemists working on a next generation of antiviral chemotherapeutics against the Dengue Fever towards the explored subset of the chemical space that is predicted to contain peptide aldehydes with NS3pro inhibition potencies in nanomolar range which display ADME-related properties comparable to the training set inhibitors.     

Role of the site of protonation in the low-energy decompositions of gas-phase peptide ions

The dissociation of singly or multiply protonated peptide ions by using low-energy collisional activation (CA) is highly dependent on the sites of protonation. The presence of strongly basic amino acid residues in the peptide primary structure dictates the sites of protonation, which generates a precursor ion population that is largely homogeneous with respect to charge sites. Attempts to dissociate this type of precursor ion population by low-energy CA result in poor fragmentation via few pathways. The work described here represents a systematic investigation of the effects of charge heterogeneity in the precursor ion population of a series of model peptides in low-energy CA experiments. Incorporation of acidic residues in the peptide RLC*IFSC*FR (where C* indicates a cysteic acid residue), for example, balances the charge on the basic arginine residues, which enables the ionizing protons to reside on a number of less basic sites along the peptide backbone. This results in a precursor ion population that is heterogeneous with respect to charge site. Low-energy CA of these ions results in diverse and efficient fragmentation. Molecular modeling has been utilized to demonstrate that energetically preferred conformations incorporate an intraionic interaction between arginine and cysteic acid residues.     

Preferred 3D‐Structure of Peptides Rich in a Severely Conformationally Restricted Cyclopropane Analogue of Phenylalanine

Terminally blocked, homo‐peptide amides of (R,R)‐1‐amino‐2,3‐diphenylcyclopropane‐1‐carboxylic acid (c₃diPhe), a chiral member of the family of C^α‐tetrasubstituted α‐amino acids, from the dimer to the tetramer, and diastereomeric co‐oligopeptides of (R,R)‐ or (S,S)‐c₃diPhe with (S)‐alanine residues to the trimer level were prepared in solution and fully characterized. The synthetic effort was extended to terminally protected co‐oligopeptide esters to the hexamer, where c₃diPhe residues are combined with achiral α‐aminoisobutyric acid residues. The preferred conformations of the peptides were assessed in solution by FT‐IR absorption, NMR, and CD techniques, and for seven oligomers in the crystal state (by X‐ray diffraction) as well. This study clearly indicates that c₃diPhe, a sterically demanding cyclopropane analogue of phenylalanine, tends to fold peptides into β‐turn and 3₁₀‐helix conformations. However, when c₃diPhe is in combination with other chiral residues, the conformation preferred by the resulting peptides is also dictated by the chiral sequence of the amino acid building blocks. The (S,S)‐enantiomer of this α‐amino acid, unusually lacking asymmetry in the main chain, strongly favors the left‐handedness of the turn/helical peptides formed.