Structure-Based Design and Synthesis of Covalent Inhibitors for Deubiquitinase and Acetyltransferase ChlaDUB1 of Chlamydia trachomatis.

Upon infection by an intracellular pathogen, host cells activate apoptotic pathways to limit pathogen replication. Consequently, efficient proliferation of the obligate intracellular pathogen Chlamydia trachomatis, a major cause of trachoma and sexually transmitted diseases, depends on the suppression of host cell apoptosis. C. trachomatis secretes deubiquitinase ChlaDUB1 into the host cell, leading among other interactions to the stabilization of antiapoptotic proteins and, thus, suppression of host cell apoptosis. Targeting the bacterial effector protein may, therefore, lead to new therapeutic possibilities. To explore the active site of ChlaDUB1, an iterative cycle of computational docking, synthesis, and enzymatic screening was applied with the aim of lead structure development. Hereby, covalent inhibitors were developed, which show enhanced inhibition with a 22-fold increase in IC50 values compared to previous work. Comprehensive insights into the binding prerequisites to ChlaDUB1 are provided, establishing the foundation for an additional specific antichlamydial therapy by small molecules.

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.

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.

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.

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.

Effects of Kinesio Taping® on pulmonary function of individuals with COPD: A systematic review and meta-analysis.

BACKGROUND: Currently, there is no consensus regarding indication and benefits of Kinesio Taping® on pulmonary function of individuals with chronic obstructive pulmonary disease (COPD). OBJECTIVES: To identify the effects of Kinesio Taping® on pulmonary function of individuals with COPD through a systematic review and meta-analysis. METHODS: Systematic review including experimental or quasi-experimental studies in English, Portuguese, or Spanish. Searches were conducted on LILACS, Scielo, Medline, Web of Science, and Scopus databases. Two reviewers independently conducted study selection, data extraction, and analysis. Methodological quality was assessed using PEDro scale, while meta-analyses were conducted using RevMan software. This review was registered (PROSPERO CRD42020223752). RESULTS: Five studies were included. Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and peak expiratory flow were not different between Kinesio Taping® group and control group. CONCLUSION: Results suggest that Kinesio Taping® does not improve pulmonary function of individuals with COPD.

Design, synthesis and in vivo evaluation of 1,4-dioxo-2-butenyl aryl amine derivatives as a promising anti-inflammatory drug prototype.

Inflammation is a natural response of the organism to an infection, trauma, or cellular stress. Pain is the first symptom of acute and chronic inflammation. The standard class of medication to treat inflammatory pain is the nonsteroidal anti-inflammatory drug (NSAID). These drugs are associated with severe side effects such as gastric ulcers, gastritis, or internal bleeding. One of NSAIDs, Dipyrone® (metamizole) is largely used in many European and South American countries despite its dubious effectivity and its withdrawal from the market of several countries. Here, aiming to identify a new anti-inflammatory drug prototype based on Dipyrone® structure, a set of 27 molecules were virtually screened, and 4 compounds containing the active metabolite 4-aminoantipyrine and 1,4-dioxo-2-butenyl fragment were selected for docking, synthesis, and biological evaluation. The selection was based on the number of H-bonds and π- π stacking interactions between the inhibitor and the amino acids within the binding site of the enzyme. Carrageenan-induced paw edema, acetic acid-induced writhing, and formalin assays were used to evaluate inflammation and pain response. The selected compounds 1-4 inhibited the involvement of biogenic amines in the formation of paw edema. Compounds 1-4 also reduced pain in the inflammatory response phase. It has to be noted that 4-AA may cause agranulocytosis, which should be borne in mind when developing drug candidates of similar structure. Our new drug prototypes based on 4-aminoantipyrine and 1,4-dioxo-2-butenyl moieties open a gate for developing a prototype of nonsteroidal anti-inflammatory drugs.

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.

Low-power laser irradiation decreases lipid droplet accumulation in the parotid glands of diabetic rats.

Lipid droplet accumulation has been related to salivary gland hypofunction in diabetes. In this study, the effect of laser irradiation on the parotid glands (PGs) of diabetic rats was analyzed with regard to its effect on lipid droplet accumulation, intracellular calcium concentration and calmodulin expression. The animals were distributed into 6 groups: D0, D5, D20 and C0, C5, C20, for diabetic (D) and control animals (C), respectively. Twenty-nine days following diabetes induction, PGs of groups D5 and C5; D20 and C20 were irradiated with 5 and 20 J/cm2 of a red diode laser at 100 mW, respectively. After 24 hours, PGs were removed for histological, biochemical, and western blotting analysis. The diabetic animals showed lipid droplet accumulation, which was decreased after irradiation. Ultrastructurally, the droplets were nonmembrane bound and appeared irregularly located in the cytoplasm. Moreover, diabetic animals showed an increased intracellular calcium concentration. In contrast, after laser irradiation a progressive decrease in the concentration of this ion was observed, which would be in agreement with the results found in the increased expression of calmodulin in D20. These data are promising for using laser to decrease lipid droplet accumulation in PGs, however, more studies are necessary to better understand its mechanisms. Micrographs showing decreased lipid accumulation after laser irradiation in light micrographs (LM), and morphology of lipid droplet in transmission electron microscopic (TEM). LM: (A) PGs from nondiabetic rats that did not receive Laser irradiation (LI), (B) PGs from nondiabetic rats that received a dose of 20 J/cm2 , (C) lipid accumulation (arrows) in the secretory cells from diabetic rats that did not receive irradiation, (D) reduction of lipid accumulation in the secretory cells from diabetic rats that received a dose of 20 J/cm2 and TEM: (E) scale bar = 5 µm, (F) scale bar = 1 µm, and (G) scale bar = 0.5 µm.

Modeling structure-activity relationships of prodiginines with antimalarial activity using GA/MLR and OPS/PLS.

In the present study, we performed a multivariate quantitative structure-activity relationship (QSAR) analysis of 52 prodiginines with antimalarial activity. Variable selection was based on the genetic algorithm (GA) and ordered predictor selection (OPS) approaches, and the models were built using the multiple linear regression (MLR) and partial least squares (PLS) regression methods. The leave-N-out crossvalidation and y-randomization tests showed that the models were robust and free from chance correlation. The mechanistic interpretation of the results was supported by earlier findings. In addition, the comparison of our models with those previously described indicated that the OPS/PLS-based model had a higher quality of external prediction. Thus, this study provides a comprehensive approach to the evaluation of the antimalarial activity of prodiginines, which may be used as a support tool in designing new therapeutic agents for malaria.

Laser phototherapy as topical prophylaxis against radiation-induced xerostomia.

The common consequences of radiotherapy (RT) to the head and neck are oral mucositis, xerostomia, and severe pain. The aim of this study was to verify how laser phototherapy (LPT) used for oral mucositis could influence xerostomia symptoms and hyposalivation of patients undergoing RT. Patients were divided into two groups: 12 individuals receiving three laser irradiations per week (G1) and 10 patients receiving one laser irradiation per week (G2). A diode laser (660 nm, 6 J/cm(2), 0.24 J, 40 mW) was used until completely healing of the lesions or the end of the RT. At the first and last laser sessions, whole resting and stimulated saliva were collected, and questionnaires were administered. According to Wilcoxon and Student statistical test, xerostomia for G1 was lower than for G2 (p < 0.05), and salivary flow rate was no different before and after RT, except for stimulated collection of G2, which was lower (p < 0.05). Our results suggest that LPT can be beneficial as an auxiliary therapy for hypofunction of salivary glands.