Synthesis Of Peptide Libraries Research Articles

Solid-phase peptide synthesis in 384-well plates.

Newer solid-phase peptide synthesis and release strategies enable the production of short peptides with high purity, allowing direct screening for desired bioactivity without prior chromatographic purification. However, the maximum number of peptides that can currently be synthesized per microplate reactor is 96, allowing the parallel synthesis of 384 peptides in modern devices that have space for 4 microplate reactors. To synthesize larger numbers of peptides, we modified a commercially available peptide synthesizer to enable the production of peptides in 384-well plates, which allows the synthesis of 1,536 peptides in one run (4× 384 peptides). We report new hardware components and customized software that allowed for the synthesis of 1,536 short peptides in good quantity (average > 0.5μmol), at high concentration (average > 10 mM), and decent purity without purification (average > 80%). The high-throughput peptide synthesis, which we developed with peptide drug development in mind, may be widely used for peptide library synthesis and screening, antibody epitope scanning, epitope mimetic development, or protease/kinase substrate screening.

Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability.

Membrane permeability is an important factor that determines the virtue of peptides targeting intracellular molecules. By introducing a membrane penetration motif, some peptides exhibit better membrane permeabilities. Previous choices for such motifs have usually been polycationic sequences, but their protease vulnerabilities and modest endosome escapability remain challenging. Here, we report a strategy for macrocyclization of peptides closed by a hydrophobic bipyridyl (BPy) unit, which grants an improvement of their membrane permeability and proteolytic stability compared with the conventional polycationic peptides. We chemically prepared model macrocyclic peptides closed by a thioether-BPy unit and determined their cell membrane permeability, giving 200 nM CP50 (an indicative value of membrane permeability), which is 40-fold better than that of the ordinary thioether macrocycle consisting of the same sequence composition. To discover potent target binders consisting of the BPy unit, we reprogrammed the initiator with chloromethyl-BPy (ClMeBPy) for the peptide library synthesis with a downstream Cys residue(s) and executed RaPID (Random nonstandard Peptide Integrated Discovery) against the bromodomains of BRD4. One of the obtained sequences exhibited a single-digit nanomolar dissociation constant against BRD4 in vitro and showed approximately 2-fold and 10-fold better membrane permeability than positive controls, R9 and Tat peptides, respectively. Moreover, we observed an intracellular activity of the BPy macrocycle tagged with a proteasome target peptide motif (RRRG), resulting in modest but detectable degradation of BRD4. The present demonstration indicates that the combination of the RaPID system with an appropriate hydrophobic unit, such as BPy, would provide a potential approach for devising cell penetrating macrocycles targeting various intracellular proteins.

Development of Bioactive Foldamers Using Ribosomally Synthesized Nonstandard Peptide Libraries

Abstract Genetic code manipulation enables the ribosomal synthesis of peptide libraries bearing diverse nonproteinogenic amino acids, which can be applied to the discovery of bioactive peptides in combination with screening methodologies, such as mRNA display. Despite a tremendous number of successes in incorporation of l-α-amino acids with non-proteinogenic sidechains and N-methyl-l-α-amino acids into nascent peptide chains, d-, β-, and γ-amino acids have suffered from low translation efficiency. This obstacle has been hindering their integration into such peptide libraries. However, the use of engineered tRNAs, which can effectively recruit EF-Tu or/and EF-P, has recently made possible significant improvement of their incorporation efficiency into nascent peptides. This article comprehensively summarizes advances in such methodology and applications to the discovery of peptide ligands against target proteins of interest.

Post-translational formation of strained cyclophanes in bacteria.

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence-function space.

PTMiner: Localization and Quality Control of Protein Modifications Detected in an Open Search and Its Application to Comprehensive Post-translational Modification Characterization in Human Proteome*[S

The open (mass tolerant) search of tandem mass spectra of peptides shows great potential in the comprehensive detection of post-translational modifications (PTMs) in shotgun proteomics. However, this search strategy has not been widely used by the community, and one bottleneck of it is the lack of appropriate algorithms for automated and reliable post-processing of the coarse and error-prone search results. Here we present PTMiner, a software tool for confident filtering and localization of modifications (mass shifts) detected in an open search. After mass-shift-grouped false discovery rate (FDR) control of peptide-spectrum matches (PSMs), PTMiner uses an empirical Bayesian method to localize modifications through iterative learning of the prior probabilities of each type of modification occurring on different amino acids. The performance of PTMiner was evaluated on three data sets, including simulated data, chemically synthesized peptide library data and modified-peptide spiked-in proteome data. The results showed that PTMiner can effectively control the PSM FDR and accurately localize the modification sites. At 1% real false localization rate (FLR), PTMiner localized 93%, 84 and 83% of the modification sites in the three data sets, respectively, far higher than two open search engines we used and an extended version of the Ascore localization algorithm. We then used PTMiner to analyze a draft map of human proteome containing 25 million spectra from 30 tissues, and confidently identified over 1.7 million modified PSMs at 1% FDR and 1% FLR, which provided a system-wide view of both known and unknown PTMs in the human proteome.

Use of Peptide Libraries for Identification and Optimization of Novel Antimicrobial Peptides.

The increasing rates of resistance among bacteria and to a lesser extent fungi have resulted in an urgent need to find new molecules that hold therapeutic promise against multidrug-resistant strains. Antimicrobial peptides have proven very effective against a variety of multidrug-resistant bacteria. Additionally, the low levels of resistance reported towards these molecules are an attractive feature for antimicrobial drug development. Here we summarise information on diverse peptide libraries used to discover or to optimize antimicrobial peptides. Chemical synthesized peptide libraries, for example split and mix method, tea bag method, multi-pin method and cellulose spot method are discussed. In addition biological peptide library screening methods are summarized, like phage display, bacterial display, mRNA-display and ribosomal display. A few examples are given for small peptide libraries, which almost exclusively follow a rational design of peptides of interest rather than a combinatorial approach.

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Rapid Optimization of Mcl-1 Inhibitors using Stapled Peptide Libraries Including Non-Natural Side Chains.

Alpha helices form a critical part of the binding interface for many protein-protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.

A Chemically Synthesized Capture Agent Enables the Selective, Sensitive, and Robust Electrochemical Detection of Anthrax Protective Antigen

We report on a robust and sensitive approach for detecting protective antigen (PA) exotoxin from Bacillus anthracis in complex media. A peptide-based capture agent against PA was developed by improving a bacteria display-developed peptide into a highly selective biligand through in situ click screening against a large, chemically synthesized peptide library. This biligand was coupled with an electrochemical enzyme-linked immunosorbent assay utilizing nanostructured gold electrodes. The resultant assay yielded a limit of detection of PA of 170 pg/mL (2.1 pM) in buffer, with minimal sensitivity reduction in 1% serum. The powdered capture agent could be stably stored for several days at 65 °C, and the full electrochemical biosensor showed no loss of performance after extended storage at 40 °C. The engineered stability and specificity of this assay should be extendable to other cases in which biomolecular detection in demanding environments is required.

HomoSAR: Bridging comparative protein modeling with quantitative structural activity relationship to design new peptides

Peptides play significant roles in the biological world. To optimize activity for a specific therapeutic target, peptide library synthesis is inevitable; which is a time consuming and expensive. Computational approaches provide a promising way to simply elucidate the structural basis in the design of new peptides. Earlier, we proposed a novel methodology termed HomoSAR to gain insight into the structure activity relationships underlying peptides. Based on an integrated approach, HomoSAR uses the principles of homology modeling in conjunction with the quantitative structural activity relationship formalism to predict and design new peptide sequences with the optimum activity. In the present study, we establish that the HomoSAR methodology can be universally applied to all classes of peptides irrespective of sequence length by studying HomoSAR on three peptide datasets viz., angiotensin-converting enzyme inhibitory peptides, CAMEL-s antibiotic peptides, and hAmphiphysin-1 SH3 domain binding peptides, using a set of descriptors related to the hydrophobic, steric, and electronic properties of the 20 natural amino acids. Models generated for all three datasets have statistically significant correlation coefficients (r(2)) and predictive r2 (r(pred)2) and cross validated coefficient ( q(LOO)2). The daintiness of this technique lies in its simplicity and ability to extract all the information contained in the peptides to elucidate the underlying structure activity relationships. The difficulties of correlating both sequence diversity and variation in length of the peptides with their biological activity can be addressed. The study has been able to identify the preferred or detrimental nature of amino acids at specific positions in the peptide sequences.

Determination of CK2 Specificity and Substrates by Proteome-Derived Peptide Libraries

Understanding the specificity of kinases enables prediction of their substrates and uncovering kinase functions in signaling pathways. Traditionally synthesized peptide libraries are used to determine the kinase specificity. In this study, a proteomics-based method was developed to determine the specificity of kinase by taking the advantages of proteome-derived peptide libraries and quantitative proteomics. Proteome-derived peptide libraries were constructed by digesting proteins in total cell lysate followed with dephosphorylation of the resulting peptides. After incubating the peptide libraries with/without CK2 for in vitro kinase assay, stable isotopic labeling based quantitative phosphoproteomics was applied to distinguish the in vitro phosphosites generated by CK2. By using the above approach, 404 CK2 in vitro phosphosites were identified by 1D LC-MS/MS. Those sites allowed the statistic determination of the CK2 specificity. In addition to the easy construction of the proteome-derived peptide library, another significant advantage of this method over the method with synthesized peptide libraries is that the identified phosphosites could be directly mapped to proteins for the screening of putative kinase substrates. It was found that the confidence for substrate identification could be significantly improved by comparing the in vitro CK2 sites with the in vivo sites identified by phosphoproteomics analysis of the same cell lines. By applying this integrated strategy, 138 phosphosites from 105 putative CK2 substrates of high confidence were determined.

New method of peptide cleavage based on Edman degradation

A straightforward cleavage method for N- acylated peptides based on the phenylthiohydantoin (PTH) formation is presented. The procedure could be applied to acid-stable resins, such as TentaGel HL–NH_{2}. We designed a cleavable linker that consists of a lysine residue with the upalpha -amino group blocked by Boc, whereas the upvarepsilon -amino group is used for peptide synthesis. After the peptide assembly is completed, the protecting groups in peptide side chains are removed using trifluoroacetic acid, thus liberating also the upalpha -amino group of the lysine in the linker. Then the reaction with phenyl isothiocyanate followed by acidolysis causes an efficient peptide release from the resin as a stable PTH derivative. Furthermore, the application of a fixed charge tag in the form of 2-(4-aza-1-azoniabicyclo[2.2.2]octylammonium)acetyl group increases ionization efficiency and reduces the detection limit, allowing ESI-MS/MS sequencing of peptides in the subfemtomolar range. The proposed strategy is compatible with standard conditions during one-bead-one-compound peptide library synthesis. The applicability of the developed strategy in combinatorial chemistry was confirmed using a small training library of upalpha -chymotrypsin substrates.Electronic supplementary materialThe online version of this article (doi:10.1007/s11030-013-9453-y) contains supplementary material, which is available to authorized users.

Thermodynamic Additivity of Sequence Variations: An Algorithm for Creating High Affinity Peptides Without Large Libraries or Structural Information

BackgroundThere is a significant need for affinity reagents with high target affinity/specificity that can be developed rapidly and inexpensively. Existing affinity reagent development approaches, including protein mutagenesis, directed evolution, and fragment-based design utilize large libraries and/or require structural information thereby adding time and expense. Until now, no systematic approach to affinity reagent development existed that could produce nanomolar affinity from small chemically synthesized peptide libraries without the aid of structural information.Methodology/Principal FindingsBased on the principle of additivity, we have developed an algorithm for generating high affinity peptide ligands. In this algorithm, point-variations in a lead sequence are screened and combined in a systematic manner to achieve additive binding energies. To demonstrate this approach, low-affinity lead peptides for multiple protein targets were identified from sparse random sequence space and optimized to high affinity in just two chemical steps. In one example, a TNF-α binding peptide with Kd = 90 nM and high target specificity was generated. The changes in binding energy associated with each variation were generally additive upon combining variations, validating the basis of the algorithm. Interestingly, cooperativity between point-variations was not observed, and in a few specific cases, combinations were less than energetically additive.Conclusions/SignificanceBy using this additivity algorithm, peptide ligands with high affinity for protein targets were generated. With this algorithm, one of the highest affinity TNF-α binding peptides reported to date was produced. Most importantly, high affinity was achieved from small, chemically-synthesized libraries without the need for structural information at any time during the process. This is significantly different than protein mutagenesis, directed evolution, or fragment-based design approaches, which rely on large libraries and/or structural guidance. With this algorithm, high affinity/specificity peptide ligands can be developed rapidly, inexpensively, and in an entirely chemical manner.

Solid‐Phase Synthesis of Peptide Libraries Combining α‐Amino Acids with Inorganic and Organic Chromophores

The synthesis of two series of peptidic chains composed of bis(terpyridine)ruthenium(II) acceptor units and organic chromophores (coumarin, naphthalene, anthracene, fluorene) by stepwise solid-phase peptide synthesis (SPPS) techniques is described. The first series of dyads comprises directly amide linked chromophores, while the second one possesses a glycine spacer between the two chromophores. All dyads were studied by UV/Vis and NMR spectroscopy, steady-state luminescence, luminescence decay and electrochemistry, as well as by DFT calculations. The results of these studies indicate weak electronic coupling of the chromophores in the ground state. Absorption spectra of all dyads are dominated by metal-to-ligand charge-transfer (MLCT) bands around 500 nm. The bichromophoric systems, especially with coumarin as organic chromophore, display additional strong absorptions in the visible spectral region. All complexes are luminescent at room temperature ((3)MLCT). Efficient quenching of the fluorescence of the organic chromophore by the attached ruthenium complex is observed in all dyads. Excitation spectra indicate energy transfer from the organic dye to the ruthenium chromophore.

N-Methylation of Peptides: A New Perspective in Medicinal Chemistry

The potential of peptides as drug candidates is limited by their poor pharmacokinetic properties. Many peptides have a short half-life in vivo and a lack of oral availability. Inspired by the excellent pharmacokinetic profile of cyclosporine, a natural, multiply N-methylated cyclic peptide, we envisioned multiple N-methylation as a promising way to rationally improve key pharmacokinetic characteristics. In this Account, we summarize our efforts toward modulating the properties of peptides by multiple N-methylation. As a first step, we simplified the synthesis of N-methylated amino acids in solution, by employing very mild conditions that could be tolerated by the diverse protecting groups required when working with naturally occurring amino acids. We also report the rapid and inexpensive syntheses of N-methylated peptides on a solid support; this facilitated the N-methyl scanning of bioactive peptides. Because of a lack of information regarding the conformational behavior of multiply N-methylated peptides, a complete library of N-methylated cyclic alanine pentapeptides was synthesized. The library provided valuable insight into the conformational modulation of cyclic peptides by N-methylation. This information is extremely valuable for the design of bioactive peptides and spatial screening of cyclic N-methylated peptides. To demonstrate the applicability of N-methylation to highly active but poorly bioavailable peptides, we performed a full N-methyl scan of the cyclopeptidic somatostatin analog cyclo(-PFwKTF-), known as the Veber-Hirschmann peptide. We show here for the first time that the simple approach of multiple N-methylation can drastically improve the metabolic stability and intestinal permeability of peptides, for example, resulting in 10% oral bioavailability for a tri-N-methylated Veber-Hirschmann peptide analog. In addition, we also describe a designed approach to N-methylated peptide library synthesis, which can accelerate the screening of N-methylated bioactive peptides. Finally, we find that multiple N-methylation of a cyclic hexapeptide integrin antagonist of GPIIb-IIIa (alphaIIb beta3 integrin), cyclo(-GRGDfL-), increases the selectivity of this peptide toward different integrin subtypes. This result demonstrates the utility of multiple N-methylation in elucidating the bioactive conformation of peptides.

Parallel synthesis of peptide libraries using microwave irradiation

The application of microwave irradiation to solid-phase peptide synthesis increases product purity and reduces reaction time. Parallel synthesis in 96-well polypropylene filter plates with microwave irradiation is an efficient method for the rapid generation of combinatorial peptide libraries in sufficient purity to assay the products directly for biological activity without HPLC purification. In this protocol, the solid-phase support is arrayed into each well of a 96-well plate, reagents are delivered using a multichannel pipette and a microwave reactor is used to complete peptide coupling reactions in 6 min and Fmoc-removal reactions in 4 min under temperature-controlled conditions. The microwave-assisted parallel peptide synthesis protocol has been used to generate a library of difficult hexa-beta-peptides in 61% average initial purity (50% yield) and has been applied to the preparation of longer alpha- and beta-peptides. Using this protocol, a library of 96 different hexapeptides can be synthesized in 24 h (excluding characterization).

Combinatorial Solid‐Phase Peptide Synthesis and Bioassays

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Tailoring Surface Properties To Build Colloidal Diagnostic Devices: Controlling Interparticle Associations

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized. The interparticle association must withstand a harsh solvent environment, multiple synthetic and washing procedures, and biological screening buffers. The encoded support beads were exposed to different solvents used for peptide synthesis, and different solutions used for biological screening including phosphate buffered saline (PBS), 2-[N-morpholino]ethane sulfonic acid (MES) and a mixture of MES and N-(3-dimethyl-aminopropyl)-N'-ethylcarbodiimide (EDC). The number of reporters remaining adhered to the support bead was quantified after each step. The nature of the associations were explored and tested to optimize the efficiency of these phenomena. Results presented illustrate the influence of the surface functionality and polyelectrolyte modification of the reporters. These parameters were investigated through zeta potential and X-ray photoelectron spectroscopy.

Combinatorial Solid Phase Peptide Synthesis and Bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.

Development of a Genetic Algorithm to Design and Identify Peptides that can Cross the Blood‐Brain Barrier

AbstractThe design of peptide drugs to treat central nervous system (CNS) diseases is hampered by our ignorance of the factors that determine whether a given peptide can cross the blood‐brain‐barrier (BBB). We are developing an approach to this problem that combines computer‐aided ligand design, parallel synthesis of peptide libraries, and biological evaluation using in vitro BBB models. We present a genetic algorithm (GA) to search for peptides that can cross the BBB. In the design and optimization of this GA we used a genetic meta‐algorithm to optimize the GA parameters. The GA is validated in silico by virtual screening of a peptide library of more than 1015 molecules. We used a virtual fitness function dervied from statistical analysis of the few experimental data on peptide‐BBB permeability available.