Enhanced Hydrogen Bonding by Urea Functionalization Tunes the Stability and Biological Properties of Peptide Amphiphiles.

Self-assembled nanostructures such as those formed by peptide amphiphiles (PAs) are of great interest in biological and pharmacological applications. Herein, a simple and widely applicable chemical modification, a urea motif, was included in the PA's molecular structure to stabilize the nanostructures by virtue of intermolecular hydrogen bonds. Since the amino acid residue nearest to the lipid tail is the most relevant for stability, we decided to include the urea modification at that position. We prepared four groups of molecules (13 PAs in all), with varying levels of intermolecular cohesion, using amino acids with distinct ß-sheet promoting potential and/or containing hydrophobic tails of distinct lengths. Each subset contained one urea-modified PA and nonmodified PAs, all with the same peptide sequence. The varied responses of these PAs to variations in pH, temperature, counterions, and biologically related proteins were examined using microscopic, X-ray, spectrometric techniques, and molecular simulations. We found that the urea group contributes to the stabilization of the morphology and internal arrangement of the assemblies against environmental stimuli for all peptide sequences. In addition, microbiological and biological studies were performed with the cationic PAs. These assays reveal that the addition of urea linkages affects the PA-cell membrane interaction, showing the potential to increase the selectivity toward bacteria. Our data indicate that the urea motif can be used to tune the stability of a wide range of PA nanostructures, allowing flexibility on the biomaterial's design and opening a myriad of options for clinical therapies.

Engineering a nanoantibiotic system displaying dual mechanism of action.

In recent decades, peptide amphiphiles (PAs) have established themselves as promising self-assembling bioinspired materials in a wide range of medical fields. Herein, we report a dual-therapeutic system constituted by an antimicrobial PA and a cylindrical protease inhibitor (LJC) to achieve broad antimicrobial spectrum and to enhance therapeutic efficacy. We studied two strategies: PA-LJC nanostructures (Encapsulation) and PA nanostructures + free LJC (Combination). Computational modeling using a molecular theory for amphiphile self-assembly captures and explains the morphology of PA-LJC nanostructures and the location of encapsulated LJC in agreement with transmission electron microscopy and two-dimensional (2D) NMR observations. The morphology and release profile of PA-LJC assemblies are strongly correlated to the PA:LJC ratio: high LJC loading induces an initial burst release. We then evaluated the antimicrobial activity of our nanosystems toward gram-positive and gram-negative bacteria. We found that the Combination broadens the spectrum of LJC, reduces the therapeutic concentrations of both agents, and is not impacted by the inoculum effect. Further, the Encapsulation provides additional benefits including bypassing water solubility limitations of LJC and modulating the release of this molecule. The different properties of PA-LJC nanostructures results in different killing profiles, and reduced cytotoxicity and hemolytic activity. Meanwhile, details in membrane alterations caused by each strategy were revealed by various microscopy and fluorescent techniques. Last, in vivo studies in larvae treated by the Encapsulation strategy showed better antimicrobial efficacy than polymyxin B. Collectively, this study established a multifunctional platform using a versatile PA to act as an antibiotic, membrane-penetrating assistant, and slow-release delivery vehicle.

Structure-Activity Relationship Study to Develop Peptide Amphiphiles as Species-Specific Antimicrobials.

Antimicrobial peptide amphiphiles (PAs) are a promising class of molecules that can disrupt the bacterial membrane or act as drug nanocarriers. In this study, we prepared 33 PAs to establish supramolecular structure-activity relationships. We studied the morphology and activity of the nanostructures against different Gram-positive and Gram-negative bacterial strains (such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii). Next, we used principal component analysis (PCA) to determine the key contributors to activity. We found that for S. aureus, the zeta potential was the major contributor to the activity while Gram-negative bacteria were more influenced by the partition coefficient (LogP) with the following order P. aeruginosa>E. coli>A. baumannii. We also performed a study of the mechanism of action of selected PAs on the bacterial membrane assessing the membrane permeability and depolarization, changes in zeta potential and overall integrity. We studied the toxicity of the nanostructures against mammalian cells. Finally, we performed an in vivo study using the wax moth larvae to determine the therapeutic efficacy of the active PAs. This study shows cationic PA nanostructures can be an intriguing platform for the development of nanoantibacterials.

Designing peptide amphiphiles as novel antibacterials and antibiotic adjuvants against gram-negative bacteria.

Gram-negative strains are intrinsically resistant to most antibiotics due to the robust and impermeable characteristic of their outer membrane. Self-assembling cationic peptide amphiphiles (PAs) have the ability to disrupt bacteria membranes, constituting an excellent antibacterial alternative to small molecule drugs that can be used alone or as antibiotic adjuvants to overcome bacteria resistance. PA1 (C16KHKHK), self-assembled into micelles, which exhibited low antibacterial activity against all strains tested, and showed strong synergistic antibacterial activity in combination with Vancomycin with a Fractional Inhibitory Concentration index (FICi) of 0.15 against E. coli. The molecules, PA2 (C16KRKR) and PA3 (C16AAAKRKR), also self-assembled into micelles, displayed a broad-spectrum antibacterial activity against all strains tested, and low susceptibility to resistance development over 21 days. Finally, PA1, PA 2 and PA3 displayed low cytotoxicity against mammalian cells, and PA2 showed a potent antibacterial activity and low toxicity in preliminary in vivo models using G. mellonella. The results show that PAs are a great platform for the future development of effective antibiotics to slow down the antibiotic resistance and can act as antibiotic adjuvants with synergistic mechanism of action, which can be repurposed for use with existing antibiotics commonly used to treat gram-positive bacteria to treat infections caused by gram-negative bacteria.

De novo design of a stapled peptide targeting SARS-CoV-2 spike protein receptor-binding domain.

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

Synthesis and biological evaluation of sulfonylpyridine derivatives as potential anti-chlamydia agents.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the United States and the world. This pathogen can cause health problems ranging from trachoma (blindness) to damage of the fallopian tubes or ectopic pregnancy, which can be life-threatening if not treated properly. To this day, there is no chlamydia-specific drug on the market. Previously, we reported the activity and basic structure-activity relationships (SAR) of sulfonylpyridine molecules that possess antichlamydial action. Based on those results, we prepared a new series of derivatives. Our data indicate the new analogs can halt the growth of C. trachomatis. The lead compound, 22, was more active than our previous molecules and did not affect the growth of S. aureus and E. coli, suggesting bacterial selectivity. We performed docking studies on the presumed target, the cylindrical protease of Chlamydia. The in-silico studies partially explained the in vitro biological result as well as predicted a possible binding pose in the binding pocket. The top compound displayed a good cytotoxicity profile towards mammalian cell lines and was stable in both serum and stimulated gastric fluid. The presented data suggests the sulfonylpyridines are promising and selective anti-chlamydial compounds that merit further structural optimization.

Design, Biological Evaluation, and Computer-Aided Analysis of Dihydrothiazepines as Selective Antichlamydial Agents.

Chlamydia trachomatis (CT) causes the most prevalent sexually transmitted bacterial disease in the United States. The lack of drug selectivity is one of the main challenges of the current antichlamydial pharmacotherapy. The metabolic needs of CT are controlled, among others, by cylindrical proteases and their chaperones (e.g., ClpX). It has been shown that dihydrothiazepines can disrupt CT-ClpXP. Based on this precedent, we synthesized a dihydrothiazepine library and characterized its antichlamydial activity using a modified semi-high-throughput screening assay. Then, we demonstrated their ability to inhibit ClpX ATPase activity in vitro, supporting ClpX as a target. Further, our lead compound displayed a promising selectivity profile against CT, acceptable cytotoxicity, no mutagenic potential, and good in vitro stability. A two-dimensional quantitative structure-activity relationship (2D QSAR) model was generated as a support tool in the identification of more potent antichlamydial molecules. This study suggests dihydrothiazepines are a promising starting point for the development of new and selective antichlamydial drugs.

Pharmacophore modeling, molecular docking, and molecular dynamics studies to identify new 5-HT2AR antagonists with the potential for design of new atypical antipsychotics.

Some important atypical antipsychotic drugs target the serotonergic receptor 2A (5-HT2AR). Currently, new therapeutic strategies are needed to offer faster onset of action with fewer side effects and, therefore, greater efficacy in a substantial proportion of patients with neuropsychological disorders such as Autism and Parkinson. The main objective of this work was to use SBDD methods to identify new hit compounds potentially useful as precursors of novel and selective 5-HT2AR antagonists. A structure-based pharmacophore screening study based on a selective antagonist was carried out in ten databases. The set obtained was refined using molecular docking, and the five most promising compounds were subjected to molecular dynamics simulations. The most stable and promising hit occupied a side pocket present in the 5-HT2AR, a site that can be explored to obtain selective ligands. Simulations against 5-HT2CR and D2R showed that the best hit could not form stable complexes with these targets, strengthening the hypothesis that the hit presents selective binding by the receptor of interest. The selected hits showed some predicted toxicity risk or violated some drug-likeness property. However, it can be concluded that the identified hits are the most promising for performing in vitro assays. Once the presence of activity is confirmed, they could become precursors of optimized and selective antagonists of 5-HT2AR. An SBDD study was carried out to identify new selective 5-HT2AR ligands potentially useful for designing selective atypical antipsychotics.

The structure of caseinolytic protease subunit ClpP2 reveals a functional model of the caseinolytic protease system from Chlamydia trachomatis.

Chlamydia trachomatis (ct) is the most reported bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Caseinolytic proteases (ClpP) from pathogenic bacteria are attractive antibiotic targets, particularly for bacterial species that form persister colonies with phenotypic resistance against common antibiotics. ClpP functions as a multisubunit proteolytic complex, and bacteria are eradicated when ClpP is disrupted. Although crucial for chlamydial development and the design of agents to treat chlamydia, the structures of ctClpP1 and ctClpP2 have yet to be solved. Here, we report the first crystal structure of full-length ClpP2 as an inactive homotetradecamer in a complex with a candidate antibiotic at 2.66 Å resolution. The structure details the functional domains of the ClpP2 protein subunit and includes the handle domain, which is integral to proteolytic activation. In addition, hydrogen-deuterium exchange mass spectroscopy probed the dynamics of ClpP2, and molecular modeling of ClpP1 predicted an assembly with ClpP2. By leveraging previous enzymatic experiments, we constructed a model of ClpP2 activation and its interaction with the protease subunits ClpP1 and ClpX. The structural information presented will be relevant for future rational drug design against these targets and will lead to a better understanding of ClpP complex formation and activation within this important human pathogen.

Urea-Modified Self-Assembling Peptide Amphiphiles That Form Well-Defined Nanostructures and Hydrogels for Biomedical Applications.

Hydrogen bonding plays a critical role in the self-assembly of peptide amphiphiles (PAs). Herein, we studied the effect of replacing the amide linkage between the peptide and lipid portions of the PA with a urea group, which possesses an additional hydrogen bond donor. We prepared three PAs with the peptide sequence Phe-Phe-Glu-Glu (FFEE): two are amide-linked with hydrophobic tails of different lengths and the other possesses an alkylated urea group. The differences in the self-assembled structures formed by these PAs were assessed using diverse microscopies, nuclear magnetic resonance (NMR), and dichroism techniques. We found that the urea group influences the morphology and internal arrangement of the assemblies. Molecular dynamics simulations suggest that there are about 50% more hydrogen bonds in nanostructures assembled from the urea-PA than those assembled from the other PAs. Furthermore, in silico studies suggest the presence of urea-π stacking interactions with the phenyl group of Phe, which results in distinct peptide conformations in comparison to the amide-linked PAs. We then studied the effect of the urea modification on the mechanical properties of PA hydrogels. We found that the hydrogel made of the urea-PA exhibits increased stability and self-healing ability. In addition, it allows cell adhesion, spreading, and growth as a matrix. This study reveals that the inclusion of urea bonds might be useful in controlling the morphology, mechanical, and biological properties of self-assembled nanostructures and hydrogels formed by the PAs.

In Vitro and In Vivo Activity of (Trifluoromethyl)pyridines as Anti-Chlamydia trachomatis Agents.

Chlamydia trachomatis is the leading pathogen in sexually transmitted bacterial infections across the globe. The development of a selective treatment against this pathogen could be an attractive therapeutic option that will reduce the overuse of broad-spectrum antibiotics. Previously, we reported some sulfonylpyridine-based compounds that showed selectivity against C. trachomatis. Here, we describe a set of related compounds that display enhanced anti-chlamydial potency when compared to our early leads. We found that the active molecules are bactericidal and have no impact on Staphylococcus aureus or Escherichia coli strains. Importantly, the molecules were not toxic to mammalian cells. Furthermore, a combination of molecule 20 (the most active molecule) and azithromycin at subinhibitory concentrations acted synergistically to inhibit chlamydial growth. Molecule 20 also eradicated Chlamydia in a 3D infection model and accelerated the recovery of Chlamydia-infected mice. This work presents compounds that could be further developed to be used alone or in combination with existing treatment regimens against chlamydial infections.

Control of Peptide Amphiphile Supramolecular Nanostructures by Isosteric Replacements.

Supramolecular nanostructures with tunable properties can have applications in medicine, pharmacy, and biotechnology. In this work, we show that the self-assembly behavior of peptide amphiphiles (PAs) can be effectively tuned by replacing the carboxylic acids exposed to the aqueous media with isosteres, functionalities that share key physical or chemical properties with another chemical group. Transmission electron microscopy, atomic force microscopy, and small-angle X-ray scattering studies indicated that the nanostructure's morphologies are responsive to the ionization states of the side chains, which are related to their pKa values. Circular dichroism studies revealed the effect of the isosteres on the internal arrangement of the nanostructures. The interactions between diverse surfaces and the nanostructures and the effect of salt concentration and temperature were assessed to further understand the properties of these self-assembled systems. These results indicate that isosteric replacements allow the pH control of supramolecular morphology by manipulating the pKa of the charged groups located on the nanostructure's surface. Theoretical studies were performed to understand the morphological transitions that the nanostructures underwent in response to pH changes, suggesting that the transitions result from alterations in the Coulomb forces between PA molecules. This work provides a strategy for designing biomaterials that can maintain or change behaviors based on the pH differences found within cells and tissues.

Scission energies of surfactant wormlike micelles loaded with nonpolar additives.

HYPOTHESIS: The previously observed effects of nonpolar additives on the scission energy and rheological properties of surfactant wormlike micelles can be explained in terms of the spatial distribution of the additive within the micelles. The dependence of the scission energy with the molecular organization of the system can be analyzed with a molecular theory capable of describing the thermodynamics and structure of the micelles. THEORY: A new theoretical method to determine the scission energy of surfactant wormlike micelles is introduced. This methodology is based on a molecular theory that explicitly considers molecular details of all components of the micelles, and their inter- and intramolecular interactions without the use of fitting and/or empirical macroscopic parameters. FINDINGS: The predicted effects of the concentration, molecular structure and hydrophobicity of the additive on the scission energy of cetyltrimethylammonium bromide (CTAB) wormlike micelles are found to be in qualitative agreement with previous experimental observations. In particular, our theory captures the decrease of micellar length with increasing content of highly hydrophobic additives and the non-monotonic dependence of the viscosity with additive hydrophobicity. The latter effect arises because highly and mildly hydrophobic additives affect the scission energy of wormlike micelles via markedly different molecular mechanisms.

Targeting SARS-CoV-2 Receptor Binding Domain with Stapled Peptides: An In Silico Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into a pandemic of unprecedented scale. This coronavirus enters cells by the interaction of the receptor binding domain (RBD) with the human angiotensin-converting enzyme 2 receptor (hACE2). In this study, we employed a rational structure-based design to propose 22-mer stapled peptides using the structure of the hACE2 α1 helix as a template. These peptides were designed to retain the α-helical character of the natural structure, to enhance binding affinity, and to display a better solubility profile compared to other designed peptides available in the literature. We employed different docking strategies (PATCHDOCK and ZDOCK) followed by a double-step refinement process (FIBERDOCK) to rank our peptides, followed by stability analysis/evaluation of the interaction profile of the best docking predictions using a 500 ns molecular dynamics (MD) simulation, and a further binding affinity analysis by molecular mechanics with generalized Born and surface area (MM/GBSA) method. Our most promising stapled peptides presented a stable profile and could retain important interactions with the RBD in the presence of the E484K RBD mutation. We predict that these peptides can bind to the viral RBD with similar potency to the control NYBSP-4 (a 30-mer experimentally proven peptide inhibitor). Furthermore, our study provides valuable information for the rational design of double-stapled peptide as inhibitors of SARS-CoV-2 infection.

Molecular Basis for the Morphological Transitions of Surfactant Wormlike Micelles Triggered by Encapsulated Nonpolar Molecules.

Surfactant wormlike micelles are prone to experience morphological changes, including the transition to spherical micelles, upon the addition of nonpolar additives. These morphological transitions have profound implications in diverse technological areas, such as the oil and personal-care industries. In this work, additive-induced morphological transitions in wormlike micelles were studied using a molecular theory that predicts the equilibrium morphology and internal molecular organization of the micelles as a function of their composition and the molecular properties of their components. The model successfully captures the transition from wormlike to spherical micelles upon the addition of a nonpolar molecule. Moreover, the predicted effects of the concentration, molecular structure, and degree of hydrophobicity of the nonpolar additive on the wormlike-to-sphere transition are shown to be in good agreement with experimental trends in the literature. The theory predicts that the location of the additive in the micelle (core or hydrophobic-hydrophilic interface) depends on the additive hydrophobicity and content, and the morphology of the micelles. Based on the results of our model, simple molecular mechanisms were proposed to explain the morphological transitions of wormlike micelles upon the addition of nonpolar molecules of different polarities.

The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development.

Chlamydia trachomatis is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, the elementary body (EB) and reticulate body (RB), each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiations require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system relies on a serine protease subunit, ClpP, and an AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, ClpX is encoded within an operon 3' to clpP2 We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability with observable defects in EB morphology noted. Conversely, overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2 Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation.IMPORTANCEChlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.

Synthesis and Antichlamydial Activity of Molecules Based on Dysregulators of Cylindrical Proteases.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease globally and the leading cause of infertility and preventable infectious blindness (trachoma) in the world. Unfortunately, there is no FDA-approved treatment specific for chlamydial infections. We recently reported two sulfonylpyridines that halt the growth of the pathogen. Herein, we present a SAR of the sulfonylpyridine molecule by introducing substituents on the aromatic regions. Biological evaluation studies showed that several analogues can impair the growth of C. trachomatis without affecting host cell viability. The compounds did not kill other bacteria, indicating selectivity for Chlamydia. The compounds presented mild toxicity toward mammalian cell lines. The compounds were found to be nonmutagenic in a Drosophila melanogaster assay and exhibited a promising stability in both plasma and gastric fluid. The presented results indicate this scaffold is a promising starting point for the development of selective antichlamydial drugs.

Twisting of Charged Nanoribbons to Helicoids Driven by Electrostatics.

Charged amphiphiles in solution usually self-assemble into flat nanoribbons that spontaneously twist into different shapes. The role of electrostatics in this process is still under strong debate. This work studies the electrostatic free energy of twisting a nanoribbon at the level of the nonlinear Poisson-Boltzmann approximation. It is shown that helicoid-shaped ribbons are more stable than flat ribbons, while other shapes under consideration (cylindrical helixes and bent ribbons) are always less stable than the flat ribbon. The unexpected electrostatics-driven twisting of the ribbon into a helicoid is ascribed to the increase in its perimeter with increasing degree of twisting, as charges near the edge of the ribbon are electrostatically more stable than those near its center. This argument successfully explains the effects of salt concentration and the width of the ribbon on the optimal twisting period and allows us to approximately describe the problem of ribbon twisting in terms of two dimensionless variables that combine the helicoid twisting period, the Debye length of the solution, and the width of the ribbon. The magnitude of the electrostatic twisting energy predicted by our calculations is comparable to that of restoring elastic forces for typical ribbons of self-assembled amphiphiles, which indicates that electrostatics plays an important role in determining the equilibrium shape of charged nanoribbons.

Protecting Groups in Peptide Synthesis.

The protection of amino acid reactive functionalities including the α-amino group, the side chain (amines, carboxylic acids, alcohols, and thiols), or the carboxylic acid terminus is an essential strategy in peptide chemistry. This is mandatory to prevent polymerization of the amino acids and to minimize undesirable side reactions during the synthetic process. Proper protecting group manipulation strategies can maximize the yield of the desired product or allow the construction of complex peptide-based structures. Thus, the compatibility and orthogonality of each protecting group are key to achieve the proper control of molecular structure. Herein, we describe some common protecting groups and their general unmasking methods, in order to mask and expose amine, carboxylic acid, alcohol, and thiol functionalities to achieve the synthesis of peptides and related molecules.