Scalable Synthesis of All Stereoisomers of 2-Aminocyclopentanecarboxylic Acid─A Toolbox for Peptide Foldamer Chemistry.

Although the construction of peptides with well-defined three-dimensional structures and predictable functions, including biological activity, using conformationally constrained ß-amino acids has been shown to be a very successful strategy, their broad application is limited by access to the appropriate building blocks. In particular, trans- and cis-stereoisomers of 2-aminocyclopentanecarboxylic acid (ACPC) are of high interest. The scalable synthesis of all four stereoisomers of Fmoc derivatives of ACPC is presented with NMR-based analysis methods for their enantiomeric purity.

Antiviral Protein-Protein Interaction Inhibitors.

Continually repeating outbreaks of pathogenic viruses necessitate the construction of effective antiviral strategies. Therefore, the development of new specific antiviral drugs in a well-established and efficient manner is crucial. Taking into account the strong ability of viruses to change, therapies with diversified molecular targets must be sought. In addition to the widely explored viral enzyme inhibitor approach, inhibition of protein-protein interactions is a very valuable strategy. In this Perspective, protein-protein interaction inhibitors targeting HIV, SARS-CoV-2, HCV, Ebola, Dengue, and Chikungunya viruses are reviewed and discussed. Antibodies, peptides/peptidomimetics, and small molecules constitute three classes of compounds that have been explored, and each of them has some advantages and disadvantages for drug development.

Amyloid engineering - how terminal capping modifies morphology and secondary structure of supramolecular peptide aggregates.

The effects of peptide N- and C-termini on aggregation behavior have been scarcely studied. Herein, we examine (105-115) peptide fragments of transthyretin (TTR) containing various functional groups at both termini and study their impact on the morphology and the secondary structure. We synthesized TTR(105-115) peptides functionalized with α-amino (H-), N-acetyl-α-amino (Ac-) or N,N-dimethyl-α-amino (DiMe-) groups at the N-terminus, and with amide (-NH2) or carboxyl (-OH) functions at the C-terminus. We also investigated quasi-racemic mixtures by mixing the L-enantiomers with the D-enantiomer capped by H- and -NH2 groups. We observed that fibril formation is promoted by the sufficient number of hydrogen bonds at peptides' termini. Moreover, the final morphology of the aggregates can be controlled by the functional groups at the N-terminus. Remarkably, all quasi-racemic mixtures resulted in the robust formation of fibrils. Overall, this work illustrates how modifications of peptide termini may help to engineer supramolecular aggregates with a predicted morphology.

Structural and biological characterization of pAC65, a macrocyclic peptide that blocks PD-L1 with equivalent potency to the FDA-approved antibodies.

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.

Peptide foldamer-based inhibitors of the SARS-CoV-2 S protein-human ACE2 interaction.

The entry of the SARS-CoV-2 virus into a human host cell begins with the interaction between the viral spike protein (S protein) and human angiotensin-converting enzyme 2 (hACE2). Therefore, a possible strategy for the treatment of this infection is based on inhibiting the interaction of the two abovementioned proteins. Compounds that bind to the SARS-CoV-2 S protein at the interface with the alpha-1/alpha-2 helices of ACE2 PD Subdomain I are of particular interest. We present a stepwise optimisation of helical peptide foldamers containing trans-2-aminocylopentanecarboxylic acid residues as the folding-inducing unit. Four rounds of optimisation led to the discovery of an 18-amino-acid peptide with high affinity for the SARS-CoV-2 S protein (Kd = 650 nM) that inhibits this protein-protein interaction with IC50 = 1.3 µM. Circular dichroism and nuclear magnetic resonance studies indicated the helical conformation of this peptide in solution.

Exploration of Thiourea-Based Scaffolds for the Construction of Bacterial Ureases Inhibitors.

A series of 32 thiourea-based urease inhibitors were synthesized and evaluated against native bacterial enzyme and whole cells of Sporosarcina pasteurii and Proteus mirabilis strains. The proposed inhibitors represented structurally diverse thiosemicarbazones and thiocarbohydrazones, benzyl-substituted thiazolyl thioureas, 1H-pyrazole-1-carbothioamides, and dihydropirimidine-2(1H)-thiones. Kinetic characteristics with purified S. pasteurii enzyme determined low micromolar inhibitors within each structural group. (E)-2-(1-Phenylethylidene)hydrazine-1-carbothioamide 19 (Ki = 0.39 ± 0.01 µM), (E)-2-(4-methylbenzylidene)hydrazine-1-carbothioamide 16 (Ki = 0.99 ± 0.04 µM), and N'-((1E,2E)-1,3-diphenylallylidene)hydrazinecarbothiohydrazide 29 (Ki = 2.23 ± 0.19 µM) were used in modeling studies that revealed sulfur ion coordination of the active site nickel ion and hydrogen bonds between the amide group and the side chain of Asp363 and Ala366 carbonyl moiety. Whole-cell studies proved the activity of compounds in Gram-positive and Gram-negative microorganisms. Ureolysis control observed in P. mirabilis PCM 543 (e.g., IC50 = 304 ± 14 µM for 1-benzyl-3-(4-(4-hydroxyphenyl)thiazol-2-yl)thiourea 52) is a valuable achievement, as urease is recognized as a major virulence factor of this urinary tract pathogen.

Inhibitory activity of catecholic phosphonic and phosphinic acids against Helicobacter pylori ureolysis.

Catechols have been reported to be potent covalent inhibitors of ureases, and they exhibit activity by modifying cysteine residues at the entrance to enzymatic active sites. Following these principles, we designed and synthesized novel catecholic derivatives that contained carboxylate and phosphonic/phosphinic functionalities and assumed expanded specific interactions. When studying the chemical stability of the molecules, we found that their intrinsic acidity catalyzes spontaneous esterification/hydrolysis reactions in methanol or water solutions, respectively. Regarding biological activity, the most promising compound, 2-(3,4-dihydroxyphenyl)-3-phosphonopropionic acid (15), exhibited significant anti-urease potential (Ki = 2.36 µM, Sporosarcinia pasteurii urease), which was reflected in the antiureolytic effect in live Helicobacter pylori cells at a submicromolar concentration (IC50 = 0.75 µM). As illustrated by molecular modeling, this compound was bound in the active site of urease through a set of concerted electrostatic and hydrogen bond interactions. The antiureolytic activity of catecholic phosphonic acids could be specific because these compounds were chemically inert and not cytotoxic to eukaryotic cells.

The application of the hierarchical approach for the construction of foldameric peptide self-assembled nanostructures.

In this paper, we show that a hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is a rational method for the design of novel self-assembled nanomaterials based on peptides. Incorporation of a trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue into the outer positions of the model coiled-coil peptide led to the formation of helical foldamers, which was determined by circular dichroism (CD) and vibrational spectroscopy. The oligomerization state of the obtained peptides in water was established by analytical ultracentrifugation (AUC). The thioflavin T assay and Congo red methods showed that the obtained α,ß-peptides possess a strong tendency to aggregate, leading to the formation of self-assembled nanostructures, which were assessed by microscopic techniques. The location of the ß-amino acid in the heptad repeat of the coiled-coil structure proved to have an influence on the secondary structure of the obtained peptides and on the morphology of the self-assembled nanostructures.

Optimized Ebselen-Based Inhibitors of Bacterial Ureases with Nontypical Mode of Action.

Screening of 25 analogs of Ebselen, diversified at the N-aromatic residue, led to the identification of the most potent inhibitors of Sporosarcina pasteurii urease reported to date. The presence of a dihalogenated phenyl ring caused exceptional activity of these 1,2-benzisoselenazol-3(2H)-ones, with Ki value in a low picomolar range (<20 pM). The affinity was attributed to the increased π-π and π-cation interactions of the dihalogenated phenyl ring with αHis323 and αArg339 during the initial step of binding. Complementary biological studies with selected compounds on the inhibition of ureolysis in whole Proteus mirabilis cells showed a very good potency (IC50 < 25 nM in phosphate-buffered saline (PBS) buffer and IC90 < 50 nM in a urine model) for monosubstituted N-phenyl derivatives. The crystal structure of S. pasteurii urease inhibited by one of the most active analogs revealed the recurrent selenation of the Cys322 thiolate, yielding an unprecedented Cys322-S-Se-Se chemical moiety.

Miniproteins in medicinal chemistry.

Miniproteins exhibit great potential as scaffolds for drug candidates because of their well-defined structure and good synthetic availability. Because of recently described methodologies for their de novo design, the field of miniproteins is emerging and can provide molecules that effectively bind to problematic targets, i.e., those that have been previously considered to be undruggable. This review describes methodologies for the development of miniprotein scaffolds and for the construction of biologically active miniproteins.

Rational Development of Bacterial Ureases Inhibitors.

Urease, an enzyme that catalyzes the hydrolysis of urea, is a virulence factor of various pathogenic bacteria. In particular, Helicobacter pylori, that colonizes the digestive tract and Proteus spp., that can infect the urinary tract, are related to urease activity. Therefore, urease inhibitors are considered as potential therapeutics against these infections. This review describes current knowledge of the structures, activity, and biological importance of bacterial ureases. Moreover, the structure-based design of several classes of bacterial urease inhibitors is presented and discussed. Phosphinic and phosphonic acids were applied as transition-state analogues, while Michael acceptors and ebselen derivatives were applied as covalent binders of cysteine residue. This review incorporates bacterial urease inhibitors from literature published between 2008 and 2021.

Controlling the conformational stability of coiled-coil peptides with a single stereogenic center of a peripheral ß-amino acid residue.

The key issue in the research on foldamers remains the understanding of the relationship between the monomers structure and conformational properties at the oligomer level. In peptidomimetic foldamers, the main goal of which is to mimic the structure of proteins, a main challenge is still better understanding of the folding of peptides and the factors that influence their conformational stability. We probed the impact of the modification of the peptide periphery with trans- and cis-2-aminocyclopentanecarboxylic acid (ACPC) on the structure and stability of the model coiled-coil using circular dichroism (CD), analytical ultracentrifugation (AUC) and two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). Although, trans-ACPC and cis-ACPC-containing mutants differ by only one peripheral stereogenic center, their conformational stability is strikingly different.

Design and Engineering of Miniproteins.

The potential of miniproteins in the biological and chemical sciences is constantly increasing. Significant progress in the design methodologies has been achieved over the last 30 years. Early approaches based on propensities of individual amino acid residues to form individual secondary structures were subsequently improved by structural analyses using NMR spectroscopy and crystallography. Consequently, computational algorithms were developed, which are now highly successful in designing structures with accuracy often close to atomic range. Further perspectives include construction of miniproteins incorporating non-native secondary structures derived from sequences with units other than α-amino acids. Noteworthy, miniproteins with extended structures, which are now feasibly accessible, are excellent scaffolds for construction of functional molecules.

A Conformationally Stable Acyclic ß-Hairpin Scaffold Tolerating the Incorporation of Poorly ß-Sheet-Prone Amino Acids.

The ß-hairpin is a structural element of native proteins, but it is also a useful artificial scaffold for finding lead compounds to convert into peptidomimetics or non-peptide structures for drug discovery. Since linear peptides are synthetically more easily accessible than cyclic ones, but are structurally less well-defined, we propose XWXWXpPXK(/R)X(R) as an acyclic but still rigid ß-hairpin scaffold that is robust enough to accommodate different types of side chains, regardless of the secondary-structure propensity of the X residues. The high conformational stability of the scaffold results from tight contacts between cross-strand cationic and aromatic side chains, combined with the strong tendency of the d-Pro-l-Pro dipeptide to induce a type II' ß-turn. To demonstrate the robustness of the scaffold, we elucidated the NMR structures and performed molecular dynamics (MD) simulations of a series of peptides displaying mainly non-ß-branched, poorly ß-sheet-prone residues at the X positions. Both the NMR and MD data confirm that our acyclic ß-hairpin scaffold is highly versatile as regards the amino-acid composition of the ß-sheet face opposite to the cationic-aromatic one.

Constrained beta-amino acid-containing miniproteins.

The construction of ß-amino acid-containing peptides that fold to tertiary structures in solution remains challenging. Two model miniproteins, namely, Trp-cage and FSD, were scanned using a constrained ß-amino acid in order to evaluate its impact on the folding process. Relationships between forces stabilizing the miniprotein structure and conformational stability of analogues were found. The possibility of a significant increase of the conformational stability of the studied miniproteins by substitution with the ß-amino acid at the terminus of a helix is shown. On the basis of these results, ß-amino acid containing-peptide analogs with helical fragments substantially altered by the incorporation of several constrained ß-amino acids were designed, synthesized and evaluated with respect to their structure and stability. The smallest known ß-amino acid-containing peptide with a well-defined tertiary structure is described.

A computationally designed ß-amino acid-containing miniprotein.

A new miniprotein built from three helices, including one structure based on the ααßαααß sequence pattern was developed. Its crystal structure revealed a compact conformation with a well-packed hydrophobic core of unprecedented structure. The miniprotein formed dimers that were stabilized by the interaction of their hydrophobic surfaces.

Autofluorescence of Amyloids Determined by Enantiomeric Composition of Peptides.

Amyloid fibrils are peptide or protein aggregates possessing a cross-ß-sheet structure. They possess intrinsic fluorescence property, which is still not fully understood. Herein, we compare structural and optical properties of fibrils formed from L- and D-enantiomers of the (105-115) fragment of transthyretin (TTR) and from their racemic mixture. Our results show that autofluorescence of fibrils obtained from enantiomers differs from that of fibrils from the racemic mixture. In order to elucidate the origin of observed differences, we analyzed the structure and morphology of fibrils and showed how variations in ß-sheet organization influence optical properties of fibrils. We clarified the contribution of aromatic rings and the amyloid backbone to the final blue-green emission of fibrils. This work demonstrates how enantiomeric composition of amino acids allows us to modulate the self-assembly and final morphology of well-defined fibrillar bionanostructures with optical properties controlled by supramolecular organization.

Towards Foldameric Miniproteins: A Helix-Turn-Helix Motif.

Numerous beta-amino acid containing peptides forming secondary structures have been already described, however the design of higher-order structures remains poorly explored. The methodology allowing construction of sequence patterns containing few rigid secondary element was proposed and experimentally validated. On the basis of 9/10/9/12-helix containing cis-2-aminocyclopentanecarboxylic acid (cis-ACPC) residues arranged in an ααßß sequence pattern, a conformationally stable helix-turn-helix structure was designed. The connection between two helices was also constructed using cis-ACPC residues. Five examples of designed peptides were obtained and analyzed using circular dichroism and nuclear magnetic resonance spectroscopy, which confirmed the assumed way of folding. The NMR structure was calculated for the peptide with the highest number of non-sequential contacts.

Hierarchical approach for the rational construction of helix-containing nanofibrils using α,ß-peptides.

The rational design of novel self-assembled nanomaterials based on peptides remains a great challenge in modern chemistry. A hierarchical approach for the construction of nanofibrils based on α,ß-peptide foldamers is proposed. The incorporation of a helix-promoting trans-(1S,2S)-2-aminocyclopentanecarboxylic acid residue in the outer positions of the model coiled-coil peptide led to its increased conformational stability, which was established consistently by the results of CD, NMR and FT-IR spectroscopy. The designed oligomerization state in the solution of the studied peptides was confirmed using analytical ultracentrifugation. Moreover, the cyclopentane side chain allowed additional interactions between coiled-coil-like structures to direct the self-assembly process towards the formation of well-defined nanofibrils, as observed using AFM and TEM techniques.

Covalent Inhibition of Bacterial Urease by Bifunctional Catechol-Based Phosphonates and Phosphinates.

In this study, a new class of bifunctional inhibitors of bacterial ureases, important molecular targets for antimicrobial therapies, was developed. The structures of the inhibitors consist of a combination of a phosphonate or (2-carboxyethyl)phosphinate functionality with a catechol-based fragment, which are designed for complexation of the catalytic nickel ions and covalent bonding with the thiol group of Cys322, respectively. Compounds with three types of frameworks, including ß-3,4-dihydroxyphenyl-, α-3,4-dihydroxybenzyl-, and α-3,4-dihydroxybenzylidene-substituted derivatives, exhibited complex and varying structure-dependent kinetics of inhibition. Among irreversible binders, methyl ß-(3,4-dihydroxyphenyl)-ß-(2-carboxyethyl)phosphorylpropionate was observed to be a remarkably reactive inhibitor of Sporosarcina pasteurii urease (kinact/KI = 10 420 s-1 M-1). The high potential of this group of compounds was also confirmed in Proteus mirabilis whole-cell-based inhibition assays. Some compounds followed slow-binding and reversible kinetics, e.g., methyl ß-(3,4-dihydroxyphenyl)-ß-phosphonopropionate, with Ki* = 0.13 µM, and an atypical low dissociation rate (residence time τ = 205 min).