Antimicrobial Target Research Articles

Computational studies molecular structure investigation and experimental characterization of Bis(2-aminothiazolium): quantum chemical analysis, electronic transitions, interaction mechanisms, and molecular dynamics

Bis(2-aminothiazolium) succinate succinic acid (ATSA), was synthesized and evaluated using standard spectroscopic studies. Density Functional studies were employed for the theoretical investigation of the substance, aiding in the determination of the chemical molecule’s structure and form. The molecule’s structural stability and the flow of charges within and between molecules were explored using natural bond orbital calculations. The primary binding sites and subtle interactions of the molecule were identified through topological analyses analysis endorse the ultimate charge transfer (CT) within the molecule. In the calculation of a chemical’s potential as a therapeutic candidate, pharmacological research is undertaken. This assessment encompasses molecule’s drug-likeness, pharmaceutical profile, and environmentally friendly toxicity contemplations. A molecular docking technique was used toward ligand spanning different antimicrobial targets, and the associated characteristics were imparted. When likened with positive controls and other pathogens, studies on antimicrobial activity expose that the compounds linked to Enterococcus faecalis, Staphylococcus aureus, Rhizopus stolonifer, and Penicillium notatum exhibit notable antimicrobial efficacy.

Aggregation-Prone Antimicrobial Peptides Target Gram-negative Bacterial Nucleic Acids and Protein Synthesis

Aggregation of antimicrobial peptides (AMPs) enhances their efficacy by destabilising the bacterial cell wall, membrane, and cytosolic proteins. Developing aggregation-prone AMPs offers a promising strategy to combat antibiotic resistance, though predicting such AMPs and understanding bacterial responses remain challenging. Octopus bimaculoides, a cephalopod species, lacks known AMP gene families, yet its protein fragments were used to predict AMPs via artificial intelligence tools. Four peptides (Oct-P1, Oct-P2, Oct-P3, and Oct-P4) were identified based on their aggregation propensity. Among them, Oct-P2 reduced the viability of Escherichia coli and Staphylococcus aureus by up to 90%, confirmed by confocal laser scanning microscopy and scanning electron microscopy. It further aggregated plasmid DNA in vitro, and the presence of extracellular DNA reduced their antibacterial activity. With knockout mutants, it revealed that Oct-P2 was internalised into bacterial cells, possibly through membrane transport proteins, enhancing its antibacterial effect. Aggregation-induced emission assays and molecular dynamics simulations revealed that Oct-P2 aggregates with transcription promoter DNA, inhibiting transcription and translation in vitro. This dual-target mechanism not only highlights the potential of Oct-P2 as a lead template for new antimicrobial drug development, but also opens a new window for discovering AMPs from protein fragments against the upcoming challenge of bacterial infections. Statement of significanceA popular strategy for identifying antimicrobial peptides (AMPs) in specific genomes uses the conserved regions of AMP families, but this strategy has limitations in organisms lacking classical AMP gene families, such as Octopus. Fragments from non-antimicrobial proteins serve as a rich source for the identification of new AMPs. In this study, we used artificial intelligence tools to search for potential candidate AMP sequences from non-antimicrobial proteins in Octopus bimaculoides. The successful identification of aggregation-prone AMPs was shown to decrease bacterial viability, increase permeability, and reduce biomass. One candidate, Oct-P2, kills the gram-negative bacteria E. coli by aggregating with DNA and inhibiting transcription and translation, suggesting a new intracellular mechanism of AMP activity.

Salmonella enterica serovar Typhimurium Effectors spiA and spiC Promote Replication by Modulating Iron Metabolism and Oxidative Stress

Salmonella enterica serovar Typhimurium (S. Typhimurium) poses a major threat to the health and safety of animal-derived foods worldwide. Recently, we have reported that S. Typhimurium uses iron to promote its own proliferation, leading to iron metabolism disorders. However, the mechanism by which S. Typhimurium induces iron metabolism disturbances remains unclear. In this study, we found that the S. Typhimurium effectors spiA and spiC promote the expression of iron regulatory protein 2 (IRP2), transferrin receptor 1 (TfR1) and divalent metal transporter protein 1 (DMT1) and inhibit the expression of ferroportin after transfection with the recombinant plasmids pEGFP-C1-spiA and pEGFP-C1-spiC, which in turn contributes to the accumulation of iron and oxidative stress. Furthermore, we aimed to verify the role of these two effector proteins in S. Typhimurium-induced disorders of iron metabolism. We constructed spiA or spiC mutant strains and their corresponding complementation strains. Our data showed that when spiA or spiC was knocked out, the upregulation of iron metabolism proteins (IRP2, TfR1 and DMT1), the accumulation of iron and oxidative stress caused by the wild-type strain were clearly alleviated in vitro and in vivo, which plays a key role in reducing the intracellular replication of S. Typhimurium and attenuating pathological damage to the liver and ileum of mice. Our findings highlighted that S. Typhimurium induces the disruption of iron metabolism via the virulence factors spiA and spiC, thereby facilitating S. Typhimurium proliferation and causing oxidative damage to the liver and ileum, which provides prospective insights into the search for effective antimicrobial targets for the defense against salmonellosis.

Chemical composition and antibacterial activity of the essential oils of Asphodelus microcarpus Salzm. & Viv. growing wild at different sites in Morocco: ADME-Tox, molecular docking and molecular dynamics simulations

ABSTRACT This study investigates the chemical variability and antimicrobial activity of essential oils (EOs) from Asphodelus microcarpus Salzm. & Viv. flowers, collected from five Moroccan regions. Analysis by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) revealed EO yields ranging from 0.01% to 0.12%. One hundred forty-four compounds, comprising 90.24–97.31% of the total content, were identified. A new chemotype, trans-Ferruginol (58.33%), was identified in the Meknes region. This is the first study to assess the antibacterial activity of EOs from these flowers, showing significant activity against Staphylococcus aureus, Enterococcus spp, Salmonella typhi and Escherichia coli, with MICs between 0.78 and 25 mg. mL−1. Theoretical studies, including ADME-Tox, molecular docking and dynamics, indicated that trans-Ferruginol is non-toxic and strongly interacts with antimicrobial target proteins. The findings suggest that these EOs could serve as an alternative to antibiotics, potentially mitigating bacterial resistance.

Structural, DFT, Molecular Docking and Biological Activity of New Albendazole-Norfloxacin Mixed-Ligand Complexes: Promising Metal Complexes for Combating Microbial Resistance and Inflammation.

This study presents a comprehensive characterization of the Fe(III) (C1) and Co(II) (C2) complexes that were synthesized from the Albendazole (Alb) and Norfloxacin (Nor) ligands. The complexes exhibit remarkable thermal stability, low water solubility, and a non-electrolytic nature, characteristics that enhance their suitability for diverse applications. Conductivity measurements indicate molar conductivities of 9.85 and 8.59 Ω-1cm2mol-1, confirming their status as neutral molecules. Fourier Transform Infrared (FTIR) spectroscopy reveals significant ligand-metal interactions, marked by shifts in vibrational frequencies that confirm chelation, while Ultraviolet-Visible (UV-Vis) spectroscopy supports the identification of octahedral geometries for both complexes. Magnetic moment assessments align with their electronic configurations, and stoichiometric analysis consistently shows a 1:1:1 ratio, further validated by mass spectrometry. Thermal stability studies highlight anhydrous characteristics and distinct thermal decomposition behaviors, underscoring their structural integrity. Employing Density Functional Theory (DFT) calculations using the B3LYP functional, we evaluate the electronic properties of the ligands and their metal complexes, revealing reduced energy gaps (ΔE) of 2.29eV for C1 and 2.15eV for C2, significantly lower than those of the ligands (Alb: 4.61eV, Nor: 4.17eV), indicating enhanced reactivity and potential biological activity. Additionally, molecular electrostatic potential (MEP) maps provide insights into charge distributions, suggesting critical regions for interactions with biomolecules. Notably, the results demonstrate that metal coordination significantly enhances antibacterial/anti-fungal activity surpassing both the free ligands and the standard antibiotic Ofloxacin/Fluconazole. Furthermore, the complexes show significant improvement in anti-inflammatory activity by inhibiting protein denaturation more effectively than their ligand counterparts. Molecular docking studies reveal stronger binding affinities and interactions with antimicrobial target proteins 1HNJ and 5IKT, attributed to enhanced hydrophobic interactions and hydrogen bonding. These findings position C1 and C2 as promising candidates for developing effective antimicrobial therapies, highlighting the crucial role of metal ions in enhancing biological reactivity and addressing resistant strains of pathogens.

Preparation and Biological Activity of Lignin-Silver Hybrid Nanoparticles.

Silver nanoparticles (AgNPs) are excellent antimicrobial agents and promising candidates for preventing or treating bacterial infections caused by antibiotic resistant strains. However, their increasing use in commercial products raises concerns about their environmental impact. In addition, traditional physicochemical approaches often involve harmful agents and excessive energy consumption, resulting in AgNPs with short-term colloidal stability and silver ion leaching. To address these issues, we designed stable hybrid lignin-silver nanoparticles (AgLigNPs) intended to effectively hit bacterial envelopes as a main antimicrobial target. The lignin nanoparticles (LigNPs), serving as a reducing and stabilizing agent for AgNPs, have a median size of 256 nm and a circularity of 0.985. These LigNPs were prepared using the dialysis solvent exchange method, producing spherical particles stable under alkaline conditions and featuring reducing groups oriented toward a wrinkled surface, facilitating AgNPs synthesis and attachment. Maximum accumulation of silver on the LigNP surface was observed at a mass reaction ratio mAg:mLig of 0.25, at pH 11. The AgLigNPs completely inhibited suspension growth and reduced biofilm development by 50% in three tested strains of Pseudomonas aeruginosa at a concentration of 80/9.5 (lignin/silver) mg L-1. Compared to unattached AgNPs, AgLigNPs required two to eight times lower silver concentrations to achieve complete inhibition. Additionally, our silver-containing nanosystems were effective against bacteria at safe concentrations in HEK-293 and HaCaT tissue cultures. Stability experiments revealed that the nanosystems tend to aggregate in media used for bacterial cell cultures but remain stable in media used for tissue cultures. In all tested media, the nanoparticles retained their integrity, and the presence of lignin facilitated the prevention of silver ions from leaching. Overall, our data demonstrate the suitability of AgLigNPs for further valorization in the biomedical sector.

Niche-specific metabolic phenotypes can be used to identify antimicrobial targets in pathogens.

Bacterial pathogens pose a major risk to human health, leading to tens of millions of deaths annually and significant global economic losses. While bacterial infections are typically treated with antibiotic regimens, there has been a rapid emergence of antimicrobial resistant (AMR) bacterial strains due to antibiotic overuse. Because of this, treatment of infections with traditional antimicrobials has become increasingly difficult, necessitating the development of innovative approaches for deeply understanding pathogen function. To combat issues presented by broad- spectrum antibiotics, the idea of narrow-spectrum antibiotics has been previously proposed and explored. Rather than interrupting universal bacterial cellular processes, narrow-spectrum antibiotics work by targeting specific functions or essential genes in certain species or subgroups of bacteria. Here, we generate a collection of genome-scale metabolic network reconstructions (GENREs) of pathogens through an automated computational pipeline. We used these GENREs to identify subgroups of pathogens that share unique metabolic phenotypes and determined that pathogen physiological niche plays a role in the development of unique metabolic function. For example, we identified several unique metabolic phenotypes specific to stomach pathogens. We identified essential genes unique to stomach pathogens in silico and a corresponding inhibitory compound for a uniquely essential gene. We then validated our in silico predictions with an in vitro microbial growth assay. We demonstrated that the inhibition of a uniquely essential gene, thyX, inhibited growth of stomach-specific pathogens exclusively, indicating possible physiological location-specific targeting. This pioneering computational approach could lead to the identification of unique metabolic signatures to inform future targeted, physiological location-specific, antimicrobial therapies, reducing the need for broad-spectrum antibiotics.

Novel tetrazolyl-1,2,3-triazole derivatives as potent antimicrobial targets: design, synthesis and molecular docking techniques

The main objective of this study is to produce novel triazoles–loaded tetrazoles, which are crucial in the development of prospective therapeutic agents in medicinal chemistry. Recent investigations have found a wide range of uses for these derivatives, and they are prospective lead molecules for the synthesis of substances with enormous therapeutic utility for various diseases, especially for bacterial therapy. New series of 1,2,3-triazole derivatives have been synthesized from methyl (2S,4S)-4-azido-1-(2,4-difluoro-3-methylbenzoyl)pyrrolidine-2-carboxylate (5) using a well–established click reaction that has several advantages to afford a novel heterocyclic compound based on tetrazole moieties. The structures of the new compounds were ascertained by spectral means (IR, NMR: 1H and 13C) and mass spectrum. All the synthesized compounds were assessed in vitro antimicrobial activity against Gram-+ve (S. pyogenes, S. aureus and B. subtilis), Gram-negative (E. coli and P. aeruginosa) bacterial and fungal strains A. flavus and C. albicans. The prepared compounds 7b and7f proved to have strong impact on S. aureus and S. pyogenes strains with MICs of 2.5 µg/mL and 1.5 µg/mL respectively. Among the tested compounds, hybrids 7b, 7f, 7h, and 7i exhibited exceptional antifungal susceptibilities against C. albicans with zone of inhibition 25 ± 0.2, 30 ± 0.3, 30 ± 0.1, and 28 ± 0.2 mm respectively, which is stronger than fluconazole (28 ± 0.1 mm). The capacity of ligand 7f to form a stable compound on the active site of S. aureus complex with DNA Gyrase (2XCT) was confirmed by docking studies using amino acids Ala233(A), Arg234(A), Gly283(A), Ser286(A), Lys52(A), His280(A), Gly51(A), His282(A) and Val246(A). Furthermore, the physicochemical and ADME (absorption, distribution, metabolism, and excretion) filtration molecular properties, estimation of toxicity, and bioactivity scores of these scaffolds were evaluated.

YgiV promoter mutations cause resistance to cystobactamids and reduced virulence factor expression in Escherichia coli

Antimicrobial resistance is one of the major health threats of the modern world. Thus, new structural classes of antimicrobial compounds are needed in order to overcome existing resistance. Cystobactamids represent one such new compound class that inhibit the well-established target bacterial type II topoisomerases while exhibiting superior antibacterial and resistance-breaking properties. Understanding potential mechanisms of emerging resistances is crucial in the development of novel antibiotics as they directly impact the future therapeutic application and market success. Therefore, the frequency and molecular basis of cystobactamid resistance in Escherichia coli was analyzed. High-level resistant E. coli mutants were selected and found to harbor single nucleotide polymorphisms in the promotor region of the ygiV gene, causing an upregulation of the respective protein. These stable mutations are contrary to what was observed as a resistance genotype in the structurally related albicidins, where ygiV gene amplifications were identified as causing resistance. Overexpression of YgiV in the mutants was additionally amplified upon cystobactamid exposition, showing further adaptation to this compound class under treatment. YgiV binds cystobactamids with high binding affinity, thereby preventing their interaction with the antimicrobial targets topoisomerase IV and DNA gyrase. In addition, we observed a substantial impact of YgiV on in vitro gyrase activity by leading to increased DNA cleavage and concurrent reduction in the efficacy of cystobactamids in inhibiting gyrase supercoiling activity. Furthermore, we identified co-upregulation of membrane-modifying proteins, such as EptC, and the transcriptional regulator QseB. This presumably contributes to the observed reduced motility and fimbrial protein expression in resistant mutants, resulting in a reduced expression of virulence factors and potentially pathogenicity, associated with ygiV promotor mutations.

Combatting melioidosis with chemical synthetic lethality

Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei, the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis. In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and β-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei. Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens.

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB’s low specificity for Tv20S compared to the human proteasome and demonstrate CP-17’s higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.

Novel benzothiophene‒tethered 1,3,4‒oxadiazoles as potent antimicrobial targets: Design, synthesis, biological evaluation, DFT exploration and in silico docking study

Based on previous developments of oxadiazole research programs in trying to find novel compounds with multiple biological targets, such as antibacterial and antitubercular medications. In the context, a new series of 1,3,4-oxadiazole derivatives based on benzo[b]thiophene moiety 6a‒d, 7a‒d, and 8a‒d was synthesized from 5-(3-methylbenzo[b]thiophen-2-yl)-1,3,4-oxadiazole-2-thiol. The structures of 1,3,4‒oxadiazole derivatives were elucidated via NMR (1H and 13C), IR, and HRMS spectrum data as well as physical data. Subsequently, the derivatives were evaluated for their inhibitory activity against different bacterial microorganisms, and the MICs were determined in vitro tubercular activity against the sensitive M. tuberculosis H37Rv strain. Remarkably, hybrids 6c and 7a exhibited excellent antibacterial activity against S. aureus with ZI values of 31 ± 0.99, and 30 ± 0.32 mm, and it has been noticed that 8b demonstrated the most prominent antibacterial efficiency with MICs = 3.1, 3.6, 5.6, and 5.1 μg/mL against S. pneumoniae, S. pyrogenes, E. coli, and K. pneumoniae. Among them, compound 8b, featuring a benzo[b]thiophene moiety linked to oxadiazole, exhibited a significant tubercular inhibitory effect with an MIC value of 2.2.5 μM. Additionally, the docked compound 7a showed the strongest key active key amino acids Arg44(A), Arg45(A), Ala126(A), Gly18(A), Ser49(A), Asp48(A) in the antitubercular active site of 1DG5 protein. The study of computer aided ADMET was also carried out, using SwissADME and ADMETlab2.0 to investigate the pharmacokinetic properties of the tested triazole compounds. To support the experimental data, molecular docking and density functional theory (DFT) studies help in understanding the interactions better.

Exploring Acyl Thiotriazinoindole Based Pharmacophores: Design, Synthesis, and SAR Studies with Molecular Docking and Biological Activity Profiling against Urease, α-amylase, α-glucosidase, Antimicrobial, and Antioxidant Targets.

A diminutive chemical library of acyl thiotriazinoindole (ATTI) based bioactive scaffolds was synthesized, instigated by taking the economical starting material Isatin, through a series of five steps. Isatin was first nitrated followed by the attachment of pentyl moiety via nucleophilic substitution reaction. The obtained compound was reacted with thiosemicarbazide to obtain thiosemicarbazone derivative, which was eventually cyclized using basic conditions in water as solvent. Finally, the reported series was obtained through reaction of nitrated thiotriazinoindole moiety with differently substituted phenacyl bromides. The synthesized compounds were characterized using NMR spectroscopy and elemental analysis. Finally, the synthesized motifs were scrutinized for their potential to impede urease, α-glucosidase, DPPH, and α-amylase. Compound 5h with para cyano group manifested the most pivotal biological activity among all, displaying IC50 values of 29.7 ± 0.8, 20.5 ± 0.5 and 36.8 ± 3.9µM against urease, α-glucosidase, and DPPH assay, respectively. Simultaneously, for α-amylase compound 5g possessing a p-CH3 at phenyl ring unfolded as most active, with calculated IC50 values 90.3 ± 1.1µM. The scaffolds were additionally gauged for their antifungal and antibacterial activity. Among the tested strains, 5d having bromo as substituent exhibited the most potent antibacterial activity, while it also demonstrated the highest potency against Aspergillus fumigatus. Other derivatives 5b, 5e, 5i, and 5j also exhibited dual inhibition against both antibacterial and antifungal strains. The interaction pattern of derivatives clearly displayed their SAR, and their docking scores were correlated with their IC50 values. In molecular docking studies, the importance of interactions like hydrogen bonding was further asserted. The electronic factors of various substituents engendered variety of interactions between the ligands and targets implying their importance in the structures of the synthesized heterocyclic scaffolds. To conclude, the synthesized compounds had satisfactory biological activity against various important targets. Further studies are therefore encouraged by attachment of different substitutions in the structure at various positions to enhance the activity of these compounds.

Whole genome sequencing and characterization of Corynebacterium isolated from the healthy and dry eye ocular surface

BackgroundThe purpose of this study was to characterize Corynebacterium isolated from the ocular surface of dry eye disease patients and healthy controls. We aimed to investigate the pathogenic potential of these isolates in relation to ocular surface health. To this end, we performed whole genome sequencing in combination with biochemical, enzymatic, and antibiotic susceptibility tests. In addition, we employed deferred growth inhibition assays to examine how Corynebacterium isolates may impact the growth of potentially competing microorganisms including the ocular pathogens Pseudomonas aeruginosa and Staphylococcus aureus, as well as other Corynebacterium present on the eye.ResultsThe 23 isolates were found to belong to 8 different species of Corynebacterium with genomes ranging from 2.12 mega base pairs in a novel Corynebacterium sp. to 2.65 mega base pairs in C. bovis. Whole genome sequencing revealed the presence of a range of antimicrobial targets present in all isolates. Pangenome analysis showed the presence of 516 core genes and that the pangenome is open. Phenotypic characterization showed variously urease, lipase, mucinase, protease and DNase activity in some isolates. Attention was particularly drawn to a potentially new or novel Corynebacterium species which had the smallest genome, and which produced a range of hydrolytic enzymes. Strikingly the isolate inhibited in vitro the growth of a range of possible pathogenic bacteria as well as other Corynebacterium isolates. The majority of Corynebacterium species included in this study did not seem to possess canonical pathogenic activity.ConclusionsThis study is the first reported genomic and biochemical characterization of ocular Corynebacterium. A number of potential virulence factors were identified which may have direct relevance for ocular health and contribute to the finding of our previous report on the ocular microbiome, where it was shown that DNA libraries were often dominated by members of this genus. Particularly interesting in this regard was the observation that some Corynebacterium, particularly new or novel Corynebacterium sp. can inhibit the growth of other ocular Corynebacterium as well as known pathogens of the eye.

The evolution of knowledge for treating Gram-negative bacterial infections.

Infections caused by nonprimarily pathogenic Gram-negative bacilli (GNB) have been increasingly reported from the second half of the 20th century to the present. This phenomenon has expanded during the antibiotic era and in the presence of immunodeficiency.Before the discovery of sulphonamides and penicillin G, infections caused by GNB were rare compared to Gram-positive infections. The advent of anticancer therapy, the expansion of surgical procedures, the use of corticosteroids, and the implantation of prosthetic materials, along with better control of Gram-positive infections, have promoted the current increase in GNB infections.GNB have similar antimicrobial targets to Gram-positive bacteria. However, only antibiotics that can penetrate the double membrane of GNB and remain in them for a sufficient duration have antibacterial activity against them. Sulphonamides and early penicillins had limited activity against GNB. Ampicillin and subsequent beta-lactams expanded their spectrum to treat GNB. Aminoglycosides may re-surge with less toxic drugs, as highly resistant to beta-lactams GNB rise. Polymyxins, tetracyclines, and fluoroquinolones are also used for GNB. Combinations with other agents may be needed in specific cases, such as in the central nervous system and prostate, where beta-lactams may have difficulty reaching the infection site.Alternatives to current treatments must be sought in the discovery of new drug families and therapies such as phage therapy combined with antibiotics. Narrower-spectrum immunosuppressive therapies and antibiotics, antimicrobials that minimally intervene with the human microbiota, and instant diagnostic methods are necessary to imagine a future where currently dominant bacteria in infectious pathology lose their preeminence.

A Fragment-Based Screen for Inhibitors of Escherichia coli N5-CAIR Mutase.

Although purine biosynthesis is a primary metabolic pathway, there are fundamental differences between how purines are synthesized in microbes versus humans. In humans, the purine intermediate, 4- carboxy-5-aminoimidazole ribonucleotide (CAIR) is directly synthesized from 5-aminoimidazole ribonucleotide (AIR) and carbon dioxide by the enzyme AIR carboxylase. In bacteria, yeast and fungi, CAIR is synthesized from AIR via an intermediate N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) by the enzyme N5-CAIR mutase. The difference in pathways between humans and microbes indicate that N5-CAIR mutase is a potential antimicrobial drug target. To identify inhibitors of E. coli N5-CAIR mutase, a fragment-based screening campaign was conducted using a thermal shift assay and a library of 4,500 fragments. Twenty-eight fragments were initially identified that displayed dose-dependent binding to N5-CAIR mutase with Kd values ranging from 9-309 μM. Of the 28, 14 were obtained from commercial sources for retesting; however, only 5 showed dose-dependent binding to N5-CAIR mutase. The five fragments were assessed for their ability to inhibit enzyme activity. Four out of the 5 showed inhibition with Ki values of 4.8 to 159 μM. All fragments contained nitrogen heterocycles with 3 out of the 4 containing 5-membered heterocycles like those found in the substrate of the enzyme. The identified fragments show similarities to compounds identified from studies on B. anthracis N5-CAIR synthetase and human AIR carboxylase suggesting a common pharmacophore.

6-Methoxyldihydrochelerythrine Chloride Inhibiting Intra and Extracellular Drug-Resistant Bacteria.

Vancomycin-resistant enterococcus (VRE) is a major nosocomial pathogen that exhibits enhanced infectivity due to its robust virulence and biofilm-forming capabilities. In this study, 6-methoxyldihydrochelerythrine chloride (6-MDC) inhibited the growth of exponential-phase VRE and restored VRE's sensitivity to vancomycin. 6-MDC predominantly suppressed the de novo biosynthetic pathway of pyrimidine and purine in VRE by the RNA-Seq analysis, resulting in obstructed DNA synthesis, which subsequently weakened bacterial virulence and impeded intracellular survival. Furthermore, 6-MDC inhibited biofilm formation, eradicated established biofilms, reduced virulence, and enhanced the host immune response to prevent intracellular survival and replication of VRE. Finally, 6-MDC reduced the VRE load in peritoneal fluid and cells significantly in a murine peritoneal infection model. This paper provides insight into the potential antimicrobial target of benzophenanthridine alkaloids for the first time.

Fluopsin C Promotes Biofilm Removal of XDR Acinetobacter baumannii and Presents an Additive Effect with Polymyxin B on Planktonic Cells.

Acinetobacter baumannii emerged as one of the most important pathogens for the development of new antimicrobials due to the worldwide detection of isolates resistant to all commercial antibiotics, especially in nosocomial infections. Biofilm formation enhances A. baumannii survival by impairing antimicrobial action, being an important target for new antimicrobials. Fluopsin C (FlpC) is an organocupric secondary metabolite with broad-spectrum antimicrobial activity. This study aimed to evaluate the antibiofilm activity of FlpC in established biofilms of extensively drug-resistant A. baumannii (XDRAb) and the effects of its combination with polymyxin B (PolB) on planktonic cells. XDRAb susceptibility profiles were determined by Vitek 2 Compact, disk diffusion, and broth microdilution. FlpC and PolB interaction was assessed using the microdilution checkerboard method and time-kill kinetics. Biofilms of XDRAb characterization and removal by FlpC exposure were assessed by biomass staining with crystal violet. Confocal Laser Scanning Microscopy was used to determine the temporal removal of the biofilms using DAPI, and cell viability using live/dead staining. The minimum inhibitory concentration (MIC) of FlpC on XDRAb was 3.5 µg mL-1. Combining FlpC + PolB culminated in an additive effect, increasing bacterial susceptibility to both antibiotics. FlpC-treated 24 h biofilms reached a major biomass removal of 92.40 ± 3.38% (isolate 230) using 7.0 µg mL-1 FlpC. Biomass removal occurred significantly over time through the dispersion of the extracellular matrix and decreasing cell number and viability. This is the first report of FlpC's activity on XDRAb and the compound showed a promissory response on planktonic and sessile cells, making it a candidate for the development of a new antimicrobial product.

Discovery of novel BfmR inhibitors restoring carbapenem susceptibility against carbapenem-resistant Acinetobacter baumannii by structure-based virtual screening and biological evaluation

ABSTRACT The emergence and spread of Acinetobacter baumannii pose a severe threat to public health, highlighting the urgent need for the next generation of therapeutics due to its increasing resistance to existing antibiotics. BfmR, a response regulator modulating virulence and antimicrobial resistance, shows a promising potential as a novel antimicrobial target. Developing BfmR inhibitors may propel a new therapeutic direction for intractable infection of resistant strains. In this study, we conducted a structure-based hierarchical virtual screening pipeline combining molecular docking, molecular dynamic simulation, and MM/GBSA calculation to sift the Specs chemical library and finally discover three novel potential BfmR inhibitors. The three hits can reduce the MIC of meropenem for the carbapenem-resistant Acinetobacter baumannii (CRAB) strain ZJ06. Similar to the BfmR knockout strain, Cmp-98 was demonstrated to downregulate the expression of K locus genes, indicating it as a BfmR inhibitor. Bacteria underwent harmful morphological changes after treatment with these inhibitors. Molecular dynamic simulations found that all the hits tend to dynamically bind to different positions of the phosphorylation site of BfmR. Wherein we identified a potential inhibitory-binding cleft, beside a possible activated binding cleft at the edge of the phosphorylation site. Restraining the ligand binding poses may help exert inhibitory effects. This study reports a group of new scaffold BfmR inhibitors, offering new insights for novel antibiotic therapeutics against CRAB.

Antimicrobial activity of compounds identified by artificial intelligence discovery engine targeting enzymes involved in Neisseria gonorrhoeae peptidoglycan metabolism

BackgroundNeisseria gonorrhoeae (Ng) causes the sexually transmitted disease gonorrhoea. There are no vaccines and infections are treated principally with antibiotics. However, gonococci rapidly develop resistance to every antibiotic class used and there is a need for developing new antimicrobial treatments. In this study we focused on two gonococcal enzymes as potential antimicrobial targets, namely the serine protease L,D-carboxypeptidase LdcA (NgO1274/NEIS1546) and the lytic transglycosylase LtgD (NgO0626/NEIS1212). To identify compounds that could interact with these enzymes as potential antimicrobials, we used the AtomNet virtual high-throughput screening technology. We then did a computational modelling study to examine the interactions of the most bioactive compounds with their target enzymes. The identified compounds were tested against gonococci to determine minimum inhibitory and bactericidal concentrations (MIC/MBC), specificity, and compound toxicity in vitro.ResultsAtomNet identified 74 compounds that could potentially interact with Ng-LdcA and 84 compounds that could potentially interact with Ng-LtgD. Through MIC and MBC assays, we selected the three best performing compounds for both enzymes. Compound 16 was the most active against Ng-LdcA, with a MIC50 value < 1.56 µM and MBC50/90 values between 0.195 and 0.39 µM. In general, the Ng-LdcA compounds showed higher activity than the compounds directed against Ng-LtgD, of which compound 45 had MIC50 values of 1.56–3.125 µM and MBC50/90 values between 3.125 and 6.25 µM. The compounds were specific for gonococci and did not kill other bacteria. They were also non-toxic for human conjunctival epithelial cells as judged by a resazurin assay. To support our biological data, in-depth computational modelling study detailed the interactions of the compounds with their target enzymes. Protein models were generated in silico and validated, the active binding sites and amino acids involved elucidated, and the interactions of the compounds interacting with the enzymes visualised through molecular docking and Molecular Dynamics Simulations for 50 ns and Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA).ConclusionsWe have identified bioactive compounds that appear to target the N. gonorrhoeae LdcA and LtgD enzymes. By using a reductionist approach involving biological and computational data, we propose that compound Ng-LdcA-16 and Ng-LtgD-45 are promising anti-gonococcal compounds for further development.