Enzymatic synthesis of a skin active ingredient - glochidone by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans.

In this study, we applied AcmB2, sourced from Sterolibacterium denitrificans, to catalyze the oxidative dehydrogenation of 3-ketolupeol (lupenone), a derivative of lupeol, triterpene obtained from birch bark. This enzymatic Δ1-dehydrogenation catalyzed by AcmB2 yielded glochidone, a bioactive compound frequently obtained from medicinal plants like Salvia trichoclada and Maytenus boria. Glochidone is known for its broad biological activities, including antibacterial, antifungal, anti-inflammatory, anticancer, antidiabetic as well as acetylcholinesterase inhibition. Our research demonstrates >99% conversion efficiency with 100% regioselectivity of the reaction. The effective conversion to glochidone employed an electron acceptor e.g., potassium hexacyanoferrate III, in mild, environmentally friendly conditions: 8-16% 2-hydroxypropyl-ß-cyclodextrin, and 2-3% 2-methoxyethanol. AcmB2 reaction optimum was determined at pH 8.0 and 30 °C. Enzyme's biochemical attributes such as electron acceptor type, concentration and steroid substrate specificity were investigated. Among 4-, 5- and 6-ring steroid derivatives androst-4-en-3,17-dione and testosterone propionate were determined as the best substrates of AcmB2. Δ1-Dehydrogenation of substrates such as lupenone, diosgenone and 3-ketopetromyzonol was confirmed. We have assessed the antioxidant and rejuvenating characteristics of glochidone as an active component in formulations, considering its precursors, lupeol, and lupenone as well. Glochidone exhibited limited antioxidant and chelating capabilities compared to lupeol and reference compounds. However, it demonstrated robust rejuvenating properties, with a sirtuin induction level of 61.5 ± 1.87%, notably surpassing that of the reference substance, E-resveratrol (45.15 ± 0.09%). Additionally, glochidone displayed 26.5±0.67 and 19.41±0.76% inhibition of elastase and collagenase, respectively. The safety of all studied triterpenes was confirmed on skin reconstructed human Epidermis model. These findings provide valuable insights into the potential applications of glochidone in formulations aimed at addressing skin health concerns. This research presents the first example of an enzyme in the 3-ketosteroid dehydrogenase (KstD) family catalyzing the Δ1-dehydrogenation of a pentacyclic triterpene. We also explored structural differences between AcmB, AcmB2, and related KstDs pointing to G52 and P532 as potentially responsible for the unique substrate specificity of AcmB2. Our findings not only highlight the enzyme's capabilities but also present novel enzymatic pathways for bioactive compound synthesis.

Virtual screening and in vitro experimental verification of LuxS inhibitors from natural products for Lactobacillus reuteri.

The rapid proliferation and colonization of probiotics in the intestines are essential for human health. Quorum sensing (QS) is a communication mechanism among bacteria, which can regulate various bacterial crowd behavior. This study aimed to enhance the viability of Lactobacillus reuteri 1-12 by regulating QS. Herein, we built a database containing 72 natural products (previously reported) that can improve intestinal flora. Virtual screening (VS) was subsequently conducted to screen four potential active compounds. After that, molecular docking was conducted to analyze the binding mode of the four natural products to S-Ribosylhomocysteinase (LuxS). The results showed that norathyriol, mangiferin, baicalein, and kaempferol had good binding ability to LuxS. The validation experiment showed that norathyriol, mangiferin, baicalein, and kaempferol could inhibit the production of autoinducer-2 (AI-2). Moreover, mangiferin significantly increased L. reuteri 1-12 biomass and promoted L. reuteri 1-12 biofilm formation and structure. Besides, only mangiferin inhibited luxS expression, thus increasing L. reuteri 1-12 biomass. This research indicated that mangiferin may be a potential inhibitor of LuxS, promoting the probiotic properties of L. reuteri and human health.

Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Ursolic acid as a potential inhibitor of Mycobacterium tuberculosis cytochrome bc1 oxidase-a molecular modelling perspective.

The escalating burden of tuberculosis disease and drastic effects of current medicine has stimulated a search for alternative drugs. A medicinal plant Warburgia salutaris has been reported to possess inhibitory properties against M. tuberculosis. In this study, we apply computational methods to investigate the probability of W. salutaris compounds as potential inhibitors of M. tuberculosis QcrB protein. We performed molecular docking, molecular dynamics simulations, radius of gyration, principal component analysis (PCA), and molecular mechanics-generalized born surface area (MM-GBSA) binding-free energy calculations in explicit solvent to achieve our objective. The results suggested that ursolic acid (UA) and ursolic acid acetate (UAA) could serve as preferred potential inhibitors of mycobacterial QcrB compared to lansoprazole sulphide (LSPZ) and telacebec (Q203)-UA and UAA have a higher binding affinity to QcrB compared to LSPZ and Q203 drugs. UA binding affinity is attributed to hydrogen bond formation with Val120, Arg364 and Arg366, and largely resonated from van der Waals forces resulting from UA interactions with hydrophobic amino acids in its vicinity. UAA binds to the porphyrin ring binding site with higher binding affinity compared to LSPZ. The binding affinity results primarily from van der Waals forces between UAA and hydrophobic residues of QcrB in the porphyrin ring binding site where UAA binds competitively. UA and UAA formed stable complexes with the protein with reduced overall residue mobility, consequently supporting the magnitude of binding affinity of the respective ligands. UAA could potentially compete with the porphyrin ring for the binding site and deprive the mycobacterial cell from oxygen, consequently disturbing mycobacterial oxygen-dependent metabolic processes. Therefore, discovery of a compound that competes with porphyrin ring for the binding site may be useful in QcrB pharmocological studies. UA proved to be a superior compound, although its estimated toxicity profile revealed UA to be hepatotoxic within acceptable parameters. Although preliminary findings of this report still warrant experimental validation, they could serve as a baseline for the development of new anti-tubercular drugs from natural resources that target QcrB.

Discovery of Novel Inhibitors of Uridine Diphosphate-N-Acetylenolpyruvylglucosamine Reductase (MurB) from Pseudomonas aeruginosa, an Opportunistic Infectious Agent Causing Death in Cystic Fibrosis Patients.

Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 µM (LE 0.35).

Combination of bactericidal antibiotics and inhibitors of Universal stress protein A (UspA): a potential therapeutic alternative against multidrug resistant Escherichia coli in urinary tract infections.

The increasing incidence of multidrug resistant uropathogenic E. coli (MDR-UPEC), the most common opportunistic pathogen in urinary tract infections (UTI) pose a global health problem and demands searching for alternative therapeutics. Antibiotics generate oxidative stress in bacteria which results in overexpression of the universal stress protein, UspA that helps in bacterial survival. An in silico study showed that two compounds ZINC000104153710, and ZINC000000217308 effectively bound bacterial UspA. This study aimed to determine the activity of ZINC000104153710, and ZINC000000217308 against bacterial UspA function in MDR-UPEC in vitro. Twenty-five highly MDR-UPEC were screened against ZINC000104153710, and, ZINC000000217308 either alone or in combination with the bactericidal antibiotics; ciprofloxacin (CIP), ceftazidime(CAZ), gentamicin(GEN) respectively by determining minimum inhibitory concentrations (MICs) using a broth microdilution assay. Additionally, the effect of ZINC000104153710, and ZINC000000217308 in the absence and presence of antibiotics on the bacteria was monitored by bacterial growth curve assays, ROS production, structure of the organism by scanning electron microscopy (FESEM) and quantitating UspA using a western blot technique. A 2-8 fold reduction in MIC values against ZINC000104153710, and ZINC000000217308 was observed against all 25 MDR-UPEC isolates in the presence of antibiotics with no alteration in intracellular ROS production. Discrete changes in cell morphology was evident in bacteria treated with ZINC000104153710 or ZINC000000217308 and antibiotics individually by FESEM compared with untreated control. Reduction in the level of UspA protein in bacteria treated with combination of ZINC000104153710 or ZINC000000217308 with individual antibiotics established their ability to inhibit UspA whose expression was elevated in presence of antibiotics alone. Therefore this study validated ZINC000104153710, and ZINC000000217308 as potent inhibitors of bacterial UspA function and indicated their potential as alternative therapeutics to combat the MDR-UPEC.

Virtual high-throughput screening and simulation studies of compounds from selected traditionally important medicinal plants for the identification of potential inhibitors of AcrB.

One of the significant human health concerns today is the emergence of drug-resistant bacteria and their propagation worldwide, rendering all available treatment options useless. One of the molecular mechanisms behind the evolution and dissemination of multidrug-resistant species is the overexpression of efflux pumps. AcrB is a major component of the AcrAB-TolC efflux pump belonging to the RND division and responsible for the extrusion of antibiotics from the bacterial cell leading to resistance. In this study, we applied the reverse chemogenomics technique to find inhibitors of AcrB to combat the issue of drug resistance. A total of 102 compounds from five different plants having medicinal properties were passed through Lipinski filter and ADMET screening was done to check their drug likeliness before docking against the AcrB protein and based on the -cDocker energy scores and toxicity analysis report, the compounds with best values were analyzed. A comparison was made between them and known inhibitors as well as antibiotics. Heat maps, frequency histograms, 2 D diagrams were generated, and the molecules were simulated for 60 ns using GROMACS. From the study, we have found Dihydrocapsaicin and Garcinexanthone-A to be a potential efflux pump inhibitors having all the characters of a promising drug candidate.Communicated by Ramaswamy H. Sarma.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Suppressing Alpha-Hemolysin as Potential Target to Screen of Flavonoids to Combat Bacterial Coinfection.

The rapid emergence of bacterial coinfection caused by cytosolic bacteria has become a huge threat to public health worldwide. Past efforts have been devoted to discover the broad-spectrum antibiotics, while the emergence of antibiotic resistance encourages the development of antibacterial agents. In essence, bacterial virulence is a factor in antibiotic tolerance. However, the discovery and development of new antibacterial drugs and special antitoxin drugs is much more difficult in the antibiotic resistance era. Herein, we hypothesize that antitoxin hemolytic activity can serve as a screening principle to select antibacterial drugs to combat coinfection from natural products. Being the most abundant natural drug of plant origins, flavonoids were selected to assess the ability of antibacterial coinfections in this paper. Firstly, we note that four flavonoids, namely, baicalin, catechin, kaempferol, and quercetin, have previously exhibited antibacterial abilities. Then, we found that baicalin, kaempferol, and quercetin have better inhibitions of hemolytic activity of Hla than catechin. In addition, kaempferol and quercetin, have therapeutic effectivity for the coinfections of Staphylococcus aureus and Pseudomonas aeruginosa in vitro and in vivo. Finally, our results indicated that kaempferol and quercetin therapied the bacterial coinfection by inhibiting S. aureus α-hemolysin (Hla) and reduced the host inflammatory response. These results suggest that antitoxins may play a promising role as a potential target for screening flavonoids to combat bacterial coinfection.

Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis.

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 µM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

Molecular Insights into Binding Mode and Interactions of Structure-Based Virtually Screened Inhibitors for Pseudomonas aeruginosa Multiple Virulence Factor Regulator (MvfR).

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa, uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

Evaluation of small molecule kinase inhibitors as novel antimicrobial and antibiofilm agents.

Antibiotic resistance is a global and pressing concern. Our current therapeutic arsenal is increasingly limited as bacteria are developing resistance at a rate that far outpaces our ability to create new treatments. Novel approaches to treating and curing bacterial infections are urgently needed. Bacterial kinases have been increasingly explored as novel drug targets and are poised for development into novel therapeutic agents to combat bacterial infections. This review describes several general classes of bacterial kinases that play important roles in bacterial growth, antibiotic resistance, and biofilm formation. General features of these kinase classes are discussed and areas of particular interest for the development of inhibitors will be highlighted. Small molecule kinase inhibitors are described and organized by phenotypic effect, spotlighting particularly interesting inhibitors with novel functions and potential therapeutic benefit. Finally, we provide our perspective on the future of bacterial kinase inhibition as a viable strategy to combat bacterial infections and overcome the pressures of increasing antibiotic resistance.

Orientin mediates protection against MRSA-induced pneumonia by inhibiting Sortase A.

Drug-resistant pathogenic Staphylococcus aureus (S. aureus) has severely threatened human health and arouses widespread concern. Sortase A (SrtA) is an essential virulence factor of S. aureus, which is responsible for the covalent anchoring of a variety of virulence-related proteins to the cell wall. SrtA has always been regarded as an ideal pharmacological target against S. aureus infections. In this research, we have determined that orientin, a natural compound isolated from various medicinal plants, can effectively inhibit the activity of SrtA with an IC50 of 50.44 ± 0.51 µM. We further demonstrated that orientin inhibited the binding of S. aureus to fibrinogen and diminished biofilm formation and the attaching of Staphylococcal protein A (SpA) to the cell wall in vitro. Using the fluorescence quenching assay, we demonstrated a direct interaction between orientin and SrtA. Further mechanistic studies revealed that the residues Glu-105, Thr-93, and Cys-184 were the key sites for the binding of SrtA to orientin. Importantly, we demonstrated that treatment with orientin attenuated S. aureus virulence of in vivo and protected mice against S. aureus-induced lethal pneumonia. These findings indicate that orientin is a potential drug to counter S. aureus infections and limit the development of drug resistance.

Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus.

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase.

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

Discovery of novel SecA inhibitors against "Candidatus Liberibacter asiaticus" through virtual screening and biological evaluation.

"Candidatus Liberibacter asiaticus" (Ca. L. asiaticus) is the causal agent of Huanglongbing disease of citrus and current study focuses on the discovery of novel small-molecule inhibitors against SecA protein of Ca. L. asiaticus. In this study, homologous modeling was used to construct the three-dimensional structure of SecA. Then, molecular docking-based virtual screening and two rounds of in vitro bacteriostatic experiments were utilized to identify novel small-molecule inhibitors of SecA. Encouragingly, 93 compounds were obtained and two of them (P684-2850, P684-3808) showed strong antimicrobial activities against Liberibacter crescens BT-1 in bacteriostatic experiments. Finally, molecular dynamics simulations were employed to explore the binding modes of the receptor-ligand complexes. Results in MD simulations showed that compound P684-3808 was relatively stable during simulation, while compound P684-2850 left the binding pocket. Compound P684-3808 might be suitable as a lead compound for further development of antimicrobial compounds against SecA of Ca. L. asiaticus.

Discovery of novel Staphylococcus aureus penicillin binding protein 2a inhibitors by multistep virtual screening and biological evaluation.

Penicillin-binding protein 2a (PBP2a) is an essential protein involved in the resistance to ß-lactam antibiotics acquired by methicillin-resistant Staphylococcus aureus (MRSA) and is a potential antibacterial target. In the current study, we employed a strategy that combined virtual screening with biological evaluation to discover novel inhibitors of PBP2a. In this investigation, a hybrid virtual screening method, consisting of drug-likeness evaluation (Lipinski's Rule of Five and ADMET) and rigid (LibDock) and semi-flexible (CDOCKER) docking-based virtual screenings, was used for retrieving novel PBP2a inhibitors from commercially available chemical databases. 11 compounds were selected from the final hits and subsequently shifted to experimental studies. Among them, Hit 2, Hit 3, and Hit 10 exhibited excellent anti-MRSA ATCC 33591 activity and weak toxicity in vitro. The affinity of the three compounds to bind to PBP2a was further confirmed by surface plasmon resonance (SPR) experiments and molecular dynamics (MD) simulation. An inter-complex interaction study showed that all hit compounds adapted well to the allosteric site of the PBP2a protein. In addition, Hit 2 (with best binding affinity to PBP2a, KD = 1.29 × 10-7 M) significantly inhibits proliferation of MRSA clinical isolates. Together, the 3 hit compounds, especially Hit 2, may be potential non-ß-lactam antibiotics against MRSA and the work will provide clues for the future development of specific compounds that block the interaction of PBP2a with their targets.

Isolation, Structure Elucidation and In Silico Prediction of Potential Drug-Like Flavonoids from Onosma chitralicum Targeted towards Functionally Important Proteins of Drug-Resistant Bad Bugs.

Admittedly, the disastrous emergence of drug resistance in prokaryotic and eukaryotic human pathogens has created an urgent need to develop novel chemotherapeutic agents. Onosma chitralicum is a source of traditional medicine with cooling, laxative, and anthelmintic effects. The objective of the current research was to analyze the biological potential of Onosma chitralicum, and to isolate and characterize the chemical constituents of the plant. The crude extracts of the plant prepared with different solvents, such as aqueous, hexane, chloroform, ethyl acetate, and butanol, were subjected to antimicrobial activities. Results corroborate that crude (methanol), EtoAc, and n-C6H14 fractions were more active against bacterial strains. Among these fractions, the EtoAc fraction was found more potent. The EtoAc fraction was the most active against the selected microbes, which was subjected to successive column chromatography, and the resultant compounds 1 to 7 were isolated. Different techniques, such as UV, IR, and NMR, were used to characterize the structures of the isolated compounds 1-7. All the isolated pure compounds (1-7) were tested for their antimicrobial potential. Compounds 1 (4',8-dimethoxy-7-hydroxyisoflavone), 6 (5,3',3-trihydroxy-7,4'-dimethoxyflavanone), and 7 (5',7,8-trihydroxy-6,3',4'-trimethoxyflavanone) were found to be more active against Staphylococcus aureus and Salmonella Typhi. Compound 1 inhibited S. typhi and S. aureus to 10 ± 0.21 mm and 10 ± 0.45 mm, whereas compound 6 showed inhibition to 10 ± 0.77 mm and 9 ± 0.20 mm, respectively. Compound 7 inhibited S. aureus to 6 ± 0.36 mm. Compounds 6 and 7 showed significant antibacterial potential, and the structure-activity relationship also justifies their binding to the bacterial enzymes, i.e., beta-hydroxyacyl dehydratase (HadAB complex) and tyrosyl-tRNA synthetase. Both bacterial enzymes are potential drug targets. Further, the isolated compounds were found to be active against the tested fungal strains. Whereas docking identified compound 7, the best binder to the lanosterol 14α-demethylase (an essential fungal cell membrane synthesizing enzyme), reported as an antifungal fluconazole binding enzyme. Based on our isolation-linked preliminary structure-activity relationship (SAR) data, we conclude that O. chitralicum can be a good source of natural compounds for drug development against some potential enzyme targets.

The TCM prescription Ma-xing-shi-gan-tang inhibits Streptococcus pneumoniae pathogenesis by targeting pneumolysin.

ETHNOPHARMACOLOGICAL RELEVANCE: Ma-xing-shi-gan-tang (MXSGT), which is documented in the Treatise on Febrile Diseases and is a therapeutic drug, is a well-known classic prescription in China and has been widely studied. Previous studies have shown that MXSGT has various pharmacological activities, including anti-influenza virus activity, and ameliorates microvascular hyperpermeability and inflammatory reactions. However, no study has reported the effect of MXSGT in the treatment of bacterial pneumonia. AIM OF THE STUDY: In this study, the potential inhibition of MXSGT against the virulence of S. pneumoniae by targeting PLY was investigated. MATERIALS AND METHODS: First, HPLC analysis was used to determine the main components of MXSGT. Then PLY protein was constructed and used for hemolysis assay and western blot to test the ability of MXSGT to inhibit PLY activity, production and widowed characteristics. The growth curve of S. pneumoniae was drawled with or without MXSGT treatment. In addition, the inhibition of MXSGT against PLY-mediated A549 cell death was examined by cytotoxicity assay. Finally, the mouse experiment was used to verify the effect of MXSGT on mouse lungs. RESULTS: This work has discovered that MXSGT, a TCM prescription, is an effective inhibitor of PLY, an important virulence factor that is essential for S. pneumoniae pathogenicity. MXSGT inhibits the oligomerization of PLY without affecting S. pneumoniae growth and PLY production. In addition, experimental MXSGT treatment was effective against S. pneumoniae infection both in vitro and in vivo. CONCLUSION: These findings directly demonstrate the potential mechanism of the Chinese herbal formula MXSGT in the treatment of pneumococcal disease and provide additional evidence for promotion of the wide use of MXSGT in the clinic.

Tannic Acid-Stabilized Silver Nanoparticles Used in Biomedical Application as an Effective Antimelioidosis and Prolonged Efflux Pump Inhibitor against Melioidosis Causative Pathogen.

Burkholderia pseudomallei is the causative pathogen of melioidosis and this bacterium is resistant to several antibiotics. Silver nanoparticles (AgNPs) are an interesting agent to develop to solve this bacterial resistance. Here, we characterize and assess the antimelioidosis activity of AgNPs against these pathogenic bacteria. AgNPs were characterized and displayed a maximum absorption band at 420 nm with a spherical shape, being well-monodispersed and having high stability in solution. The average size of AgNPs is 7.99 ± 1.46 nm. The antibacterial efficacy of AgNPs was evaluated by broth microdilution. The bactericidal effect of AgNPs was further assessed by time-kill kinetics assay. Moreover, the effect of AgNPs on the inhibition of the established biofilm was investigated by the crystal violet method. In parallel, a study of the resistance induction development of B. pseudomallei towards AgNPs with efflux pump inhibiting effect was performed. We first found that AgNPs had strong antibacterial activity against both susceptible and ceftazidime-resistant (CAZ-resistant) strains, as well as being efficiently active against B. pseudomallei CAZ-resistant strains with a fast-killing mode via a bactericidal effect within 30 min. These AgNPs did not only kill planktonic bacteria in broth conditions, but also in established biofilm. Our findings first documented that the resistance development was not induced in B. pseudomallei toward AgNPs in the 30th passage. We found that AgNPs still showed an effective efflux pump inhibiting effect against these bacteria after prolonged exposure to AgNPs at sublethal concentrations. Thus, AgNPs have valuable properties for being a potent antimicrobial agent to solve the antibiotic resistance problem in pathogens.