De novo design of potential peptide analogs against the main protease of Omicron variant using in silico studies.

SARS-CoV-2 and its variants are crossing the immunity barrier induced through vaccination. Recent Omicron sub-variants are highly transmissible and have a low mortality rate. Despite the low severity of Omicron variants, these new variants are known to cause acute post-infectious syndromes. Nowadays, novel strategies to develop new potential inhibitors for SARS-CoV-2 and other Omicron variants have gained prominence. For viral replication and survival the main protease of SARS-CoV-2 plays a vital role. Peptide-like inhibitors that mimic the substrate peptide have already proved to be effective in inhibiting the Mpro of SARS-CoV-2 variants. Our systematic canonical amino acid point mutation analysis on the native peptide has revealed various ways to improve the native peptide of the main protease. Multi mutation analysis has led us to identify and design potent peptide-analog inhibitors that act against the Mpro of the Omicron sub-variants. Our in-depth analysis of all-atom molecular dynamics studies has paved the way to characterize the atomistic behavior of Mpro in Omicron variants. Our goal is to develop potent peptide-analogs that could be therapeutically effective against Omicron and its sub-variants.

Light-Fueled Primitive Replication and Selection in Biomimetic Chemical Systems.

The concept of chemically evolvable replicators is central to abiogenesis. Chemical evolvability requires three essential components: energy-harvesting mechanisms for nonequilibrium dissipation, kinetically asymmetric replication and decomposition pathways, and structure-dependent selective templating in the autocatalytic cycles. We observed a UVA light-fueled chemical system displaying sequence-dependent replication and replicator decomposition. The system was constructed with primitive peptidic foldamer components. The photocatalytic formation-recombination cycle of thiyl radicals was coupled with the molecular recognition steps in the replication cycles. Thiyl radical-mediated chain reaction was responsible for the replicator death mechanism. The competing and kinetically asymmetric replication and decomposition processes led to light intensity-dependent selection far from equilibrium. Here, we show that this system can dynamically adapt to energy influx and seeding. The results highlight that mimicking chemical evolution is feasible with primitive building blocks and simple chemical reactions.

Tilted State Population of Antimicrobial Peptide PGLa Is Coupled to the Transmembrane Potential.

Cationic antimicrobial peptide PGLa gets into close contact with the anionic bacterial cell membrane, facilitating cross-membrane transport phenomena and membrane disruption depending on the concentration. The mechanisms of action are closely associated with the tilted insertion geometry of PGLa. Therefore, we aimed to understand the interaction between the transmembrane potential (TMP) and the orientation of the membrane-bound PGLa helix. Molecular dynamics simulations were performed with TMP, and we found that the PGLa tilt angle relative to the membrane is coupled with the TMP. Elevated TMP increases the population of the tilted state. We observed positive feedback between the tilt angle and the TMP, which occurs due to the electrostatic interaction between the peptidic helix and the Na+ cations at the membrane-water interface. These TMP coupled phenomena can contribute to understanding the direct antimicrobial and adjuvant effects of PGLa in combination with regular antibiotics.

DPP-4 Cleaves α/ß-Peptide Bonds: Substrate Specificity and Half-Lives.

The incorporation of ß-amino acids into a peptide sequence has gained particular attention as ß- and α/ß-peptides have shown remarkable proteolytic stability, even after a single homologation at the scissile bond. Several peptidases have been shown to cleave such bonds with high specificity but at a much slower rate compared to α-peptide bonds. In this study, a series of analogs of dipeptidyl peptidase-4 (DPP-4) substrate inhibitors were synthesized in order to investigate whether ß-amino acid homologation at the scissile bond could be a valid approach to improving peptide stability towards DPP-4 degradation. DPP-4 cleaved the α/ß-peptide bond after the N-terminal penultimate Pro with a broad specificity and retained full activity regardless of the ß3 -amino acid side chain and peptide length. Significantly improved half-lives were observed for ß3 Ile-containing peptides. Replacing the penultimate Pro with a conformationally constrained Pro mimetic led to proteolytic resistance. DPP-4 cleavage of α/ß-peptide bonds with a broad promiscuity represents a new insight into the stability of peptide analogs containing ß-amino acids as such analogs were thought to be stable towards enzymatic degradation.

Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations.

Multitargeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets, are generally considered to be a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multitargeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here, we propose that such individual stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an >2,000-fold reduction in susceptibility, while individually, none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, coselected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones toward resistance against antimicrobial compounds still under development. Our research indicates that even balanced multitargeting antibiotics are prone to resistance evolution.

Peripheral cyclic ß-amino acids balance the stability and edge-protection of ß-sandwiches.

Engineering water-soluble stand-alone ß-sandwich mimetics is a current challenge because of the difficulties associated with tailoring long-range interactions. In this work, single cis-(1R,2S)-2-aminocyclohexanecarboxylic acid mutations were introduced into the edge strands of the eight-stranded ß-sandwich mimetic structures from the betabellin family. Temperature-dependent NMR and CD measurements, together with thermodynamic analyses, demonstrated that the modified peripheral strands exhibited an irregular and partially disordered structure but were able to exert sufficient shielding on the hydrophobic core to retain the predominantly ß-sandwich structure. Although the frustrated interactions decreased the free energy of unfolding, the temperature of the maximum stabilities increased to or remained at physiologically relevant temperatures. We found that the irregular peripheral strands were able to prevent edge-to-edge association and fibril formation in the aggregation-prone model. These findings establish a ß-sandwich stabilization and aggregation inhibition approach, which does not interfere with the pillars of the peptide bond or change the net charge of the peptide.

Structural Optimization of Foldamer-Dendrimer Conjugates as Multivalent Agents against the Toxic Effects of Amyloid Beta Oligomers.

Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.

Target-specific NMR detection of protein-ligand interactions with antibody-relayed 15N-group selective STD.

Fragment-based drug design has been successfully applied to challenging targets where the detection of the weak protein-ligand interactions is a key element. 1H saturation transfer difference (STD) NMR spectroscopy is a powerful technique for this work but it requires pure homogeneous proteins as targets. Monoclonal antibody (mAb)-relayed 15N-GS STD spectroscopy has been developed to resolve the problem of protein mixtures and impure proteins. A 15N-labelled target-specific mAb is selectively irradiated and the saturation is relayed through the target to the ligand. Tests on the anti-Gal-1 mAb/Gal-1/lactose system showed that the approach is experimentally feasible in a reasonable time frame. This method allows detection and identification of binding molecules directly from a protein mixture in a multicomponent system.

Induced folding of protein-sized foldameric ß-sandwich models with core ß-amino acid residues.

The mimicry of protein-sized ß-sheet structures with unnatural peptidic sequences (foldamers) is a considerable challenge. In this work, the de novo designed betabellin-14 ß-sheet has been used as a template, and αâ†’ß residue mutations were carried out in the hydrophobic core (positions 12 and 19). ß-Residues with diverse structural properties were utilized: Homologous ß(3) -amino acids, (1R,2S)-2-aminocyclopentanecarboxylic acid (ACPC), (1R,2S)-2-aminocyclohexanecarboxylic acid (ACHC), (1R,2S)-2-aminocyclohex-3-enecarboxylic acid (ACEC), and (1S,2S,3R,5S)-2-amino-6,6-dimethylbicyclo[3.1.1]heptane-3-carboxylic acid (ABHC). Six α/ß-peptidic chains were constructed in both monomeric and disulfide-linked dimeric forms. Structural studies based on circular dichroism spectroscopy, the analysis of NMR chemical shifts, and molecular dynamics simulations revealed that dimerization induced ß-sheet formation in the 64-residue foldameric systems. Core replacement with (1R,2S)-ACHC was found to be unique among the ß-amino acid building blocks studied because it was simultaneously able to maintain the interstrand hydrogen-bonding network and to fit sterically into the hydrophobic interior of the ß-sandwich. The novel ß-sandwich model containing 25 % unnatural building blocks afforded protein-like thermal denaturation behavior.

Predicting order and disorder for ß-peptide foldamers in water.

Following a quantitative validation approach, we tested the AMBER ff03 and GAFF force fields with the TIP3P explicit water model in molecular dynamic simulations of ß-peptide foldamers. The test sequences were selected to represent a wide range of folding behavior in water: compact helix, strand mimetic geometry, and the state of disorder. The combination AMBER ff03-TIP3P successfully predicted the experimentally observed conformational properties and reproduced the NOE distances and backbone (3)J coupling data at a good level. GAFF was unable to produce folded structures correctly due to its biased torsion potentials. We can recommend AMBER ff03-TIP3P for simulations involving ß-peptide sequences in aqueous media including ordered and disordered structures.

Foldameric α/ß-peptide analogs of the ß-sheet-forming antiangiogenic anginex: structure and bioactivity.

The principles of ß-sheet folding and design for α-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of ß-amino-acid-containing foldamers, we followed a top-down approach to study a series of α/ß-peptidic analogs of anginex, a ß-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic α → ß(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the ß-sheet tendency, though with a decreased folding propensity. ß-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the α → ß(3) exchange was located in the ß-sheet core. Analysis of the ß-sheet stability versus substitution pattern and the local conformational bias of the bulky ß(3)V and ß(3)I residues revealed that a mismatch between the H-bonding preferences of the α- and ß-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the α/ß-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The α → ß(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive ß-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties.

Peptidic foldamers: ramping up diversity.

Non-natural folded polymers (foldamers) display considerable versatility, and the design of such molecules is of great current interest. In this respect, peptidic foldamers are perhaps the best-characterized systems, as they populate a number of residue-controlled secondary structures, which have found various biological applications and have also led to the creation of nanostructured materials. This critical review covers recent developments related to diverse building blocks and modern foldamer design principles, such as the stereochemical patterning methods. The recent achievements concerning tertiary/quaternary structures and the self-assembling foldameric nanostructures are also addressed (176 references).

Improved Metal-Free Approach for the Synthesis of Protected Thiol Containing Thymidine Nucleoside Phosphoramidite and Its Application for the Synthesis of Ligatable Oligonucleotide Conjugates.

Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5' or 3' end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation.

Structural Adaptation of the Single-Stranded DNA-Binding Protein C-Terminal to DNA Metabolizing Partners Guides Inhibitor Design.

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide-protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy-entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.

Self-association-driven transition of the ß-peptidic H12 helix to the H18 helix.

Various patterns of foldameric oligomers formed by trans-ABHC ((1S,2S,3S,5S)-2-amino-6,6-dimethylbicyclo[3.3.1]heptane-3-carboxylic acid) and ß(3)-hSer residues were studied. NMR, ECD and molecular modelling demonstrated that octameric and nonameric sequences with multiple i-i+3 ABHC pair repulsions attain the ß-H18 helix in CD(3)OH. As a close relative of the α-helix, this helix type is stabilized by i-i+4 backbone H-bond interactions. The formation of the ß-H18 helix was found to be solvent- and concentration-dependent. Upon dilution, the ß-H18 → ß-H12 helix transition was revealed by concentration-dependent ECD, DOSY-NMR and TEM measurements.

Promiscuity mapping of the S100 protein family using a high-throughput holdup assay.

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally occurring S100 partners in the human proteome. Such information will be precious for future drug design to interfere with S100 related pathologies.

Rationally designed foldameric adjuvants enhance antibiotic efficacy via promoting membrane hyperpolarization.

The negative membrane potential of bacterial cells influences crucial cellular processes. Inspired by the molecular scaffold of the antimicrobial peptide PGLa, we have developed antimicrobial foldamers with a computer-guided design strategy. The novel PGLa analogues induce sustained membrane hyperpolarization. When co-administered as an adjuvant, the resulting compounds - PGLb1 and PGLb2 - have substantially reduced the level of antibiotic resistance of multi-drug resistant Escherichia coli, Klebsiella pneumoniae and Shigella flexneri clinical isolates. The observed antibiotic potentiation was mediated by hyperpolarization of the bacterial membrane caused by the alteration of cellular ion transport. Specifically, PGLb1 and PGLb2 are selective ionophores that enhance the Goldman-Hodgkin-Katz potential across the bacterial membrane. These findings indicate that manipulating bacterial membrane electrophysiology could be a valuable tool to overcome antimicrobial resistance.

α/ß-Peptides as Nanomolar Triggers of Lipid Raft-Mediated Endocytosis through GM1 Ganglioside Recognition.

Cell delivery of therapeutic macromolecules and nanoparticles is a critical drug development challenge. Translocation through lipid raft-mediated endocytic mechanisms is being sought, as it can avoid rapid lysosomal degradation. Here, we present a set of short α/ß-peptide tags with high affinity to the lipid raft-associated ganglioside GM1. These sequences induce effective internalization of the attached immunoglobulin cargo. The structural requirements of the GM1-peptide interaction are presented, and the importance of the membrane components are shown. The results contribute to the development of a receptor-based cell delivery platform.

(15)N and (13)C group-selective techniques extend the scope of STD NMR detection of weak host-guest interactions and ligand screening.

Saturation transfer difference (STD) is a valuable tool for studying the binding of small molecules to large biomolecules and for obtaining detailed information on the binding epitopes. Here, we demonstrate that the proposed (15)N/(13)C variants of group-selective, "GS-STD" experiments provide a powerful approach to mapping the binding epitope of a ligand even in the absence of efficient spin diffusion within the target protein. Therefore, these experimental variants broaden the scope of STD studies to smaller and/or more-dynamic targets. The STD spectra obtained in four different experimental setups (selective (1)H STD, (15)N GS-STD, (13)C(Ar) and (13)C(aliphatic) GS-STD approaches) revealed that the signal-intensity pattern of the difference spectra is affected by both the type and the spatial distribution of the excited "transmitter" atoms, as well as by the efficiency of the spin-diffusion-mediated magnetization transfer. The performance of the experiments is demonstrated on a system by using the lectin, galectin-1 and its carbohydrate ligand, lactose.

Galectin-1-asialofetuin interaction is inhibited by peptides containing the tyr-xxx-tyr motif acting on the glycoprotein.

Galectin-1 (Gal-1), a ubiquitous beta-galactoside-binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal-1 depend on its affinity for beta-galactoside-containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr-Xxx-Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal-1-asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr-Xxx-Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr-Xxx-Tyr peptides studied do not bind to Gal-1, whereas their binding to ASF is clearly detected. (15)N,(1)H HSQC titrations with (15)N-labeled Gal-1 confirm the absence of any peptide-Gal-1 interaction. These data indicate that the Tyr-Xxx-Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.