Antibacterial pyrazoles: tackling resistant bacteria.

Bacterial resistance to antibiotics threatens our progress in healthcare, modern medicine, food production and ultimately life expectancy. Antibiotic resistance is a global concern, which spreads rapidly across borders and continents due to rapid travel of people, animals and goods. Derivatives of metabolically stable pyrazole nucleus are known for their wide range of pharmacological properties, including antibacterial activities. This review highlights recent reports of pyrazole derivatives targeting different bacterial strains focusing on the drug-resistant variants. Pyrazole derivatives target different metabolic pathways of both Gram-positive and Gram-negative bacteria.

Rhizosphere enzyme activities and microorganisms drive the transformation of organic and inorganic carbon in saline-alkali soil region.

Western Jilin Province is one of the world's three major saline-alkali land distribution areas, and is also an important area of global climate change and carbon cycle research. Rhizosphere soil microorganisms and enzymes are the most active components in soil, which are closely related to soil carbon cycle and can reflect soil organic carbon (SOC) dynamics sensitively. Soil inorganic carbon (SIC) is the main existing form of soil carbon pool in arid saline-alkali land, and its quantity distribution affects the pattern of soil carbon accumulation and storage. Previous studies mostly focus on SOC, and pay little attention to SIC. Illumina Miseq high-throughput sequencing technology was used to reveal the changes of community structure in three maize fields (M1, M2 and M3) and three rice fields (R1, R2 and R3), which were affected by different levels of salinization during soil development. It is a new research topic of soil carbon cycle in saline-alkali soil region to investigate the effects of soil microorganisms and soil enzymes on the transformation of SOC and SIC in the rhizosphere. The results showed that the root-soil-microorganism interaction was changed by saline-alkali stress. The activities of catalase, invertase, amylase and ß-glucosidase decreased with increasing salinity. At the phylum level, most bacterial abundance decreases with increasing salinity. However, the relative abundance of Proteobacteria and Firmicutes in maize field and Firmicutes, Proteobacteria and Nitrospirae in rice field increased sharply under saline-alkali stress. The results of redundancy analysis showed that the differences of rhizosphere soil between the three maize and three rice fields were mainly affected by ESP, pH and soil salt content. In saline-alkali soil region, ß-glucosidase activity and amylase were significantly positively correlated with SOC content in maize fields, while catalase and ß-glucosidase were significantly positively correlated with SOC content in rice fields. Actinobacteria, Bacteroidetes and Verrucomicrobia had significant positive effects on SOC content of maize and rice fields. Proteobacteria, Gemmatimonadetes and Nitrospirae were positively correlated with SIC content. These enzymes and microorganisms are beneficial to soil carbon sequestration in saline-alkali soils.

Inhibitors of bacterial RNA polymerase transcription complex.

A series of hybrid compounds that incorporated anthranilic acid with activated 1H-indoles through a glyoxylamide linker were designed to target bacterial RNA polymerase holoenzyme formation using computational docking. Synthesis, in vitro transcription inhibition assays, and biological testing of the hybrids identified a range of potent anti-transcription inhibitors with activity against a range of pathogenic bacteria with MICs as low as 3.1 µM. A structure activity relationship study identified the key structural components necessary for inhibition of both bacterial growth and transcription. Correlation of in vitro transcription inhibition activity with in vivo mechanism of action was established using fluorescence microscopy and resistance passaging using Gram-positive bacteria showed no resistance development over 30 days. Furthermore, no toxicity was observed from the compounds in a wax moth larvae model, establishing a platform for the development of a series of new antibacterial drugs with an established mode of action.

Cholic acid: a novel steroidal uncompetitive inhibitor against ß-lactamase produced by multidrug-resistant isolates.

ß-lactam antibiotics are the most frequently prescribed class of drugs worldwide, due to its efficacy and good safety profile. However, the emergence of ß-lactamase producing bacterial strains eliminated the use of ß-lactam antibiotics as a chemotherapeutic choice. To restore their usability, a non-antibiotic adjuvant in conjunction with ß-lactam antibiotics is now being utilised. Cholic acid potentially acts as an adjuvant since it can blunt the pro-inflammatory activity in human. Our main objective is to scrutinise the inhibition of ß-lactamase-producing bacteria by adjuvant cholic acid, synergism of the test drugs and the primary mechanism of enzymatic reaction. Antibacterial effect of the cholic acid-ampicillin (CA-AMP) on 7 ß-lactamase positive isolates were evaluated accordingly to disc diffusion assay, antibiotic susceptibility test, as well as checkerboard analysis. Then, all activities were compared with ampicillin alone, penicillin alone, cholic acid alone and cholic acid-penicillin combination. The CA-AMP displayed notable antibiotic activity on all test bacteria and depicted synergistic influence by representing low fractional inhibitory concentration index (FIC ≤ 0.5). According to kinetic analyses, CA-AMP behaved as an uncompetitive inhibitor against beta lactamase, with reducing values of Michaelis constant (Km) and maximal velocity (Vmax) recorded. The inhibitor constant (Ki) of CA-AMP was equal to 4.98 ± 0.3 µM, which slightly lower than ampicillin (5.00 ± 0.1 µM).

Modular and Single-Cell Sensors of Bacterial Ser/Thr Kinase Activity.

At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation. We demonstrate the utility of these sensors in measuring the activity of PrkC, a conserved bacterial Ser/Thr kinase, in different growth conditions from single cells to colonies. We also show that PrkC activity increases in response to a cell-wall active antibiotic that blocks the late steps in peptidoglycan synthesis (cefotaxime), but not the early steps (fosfomycin). These sensors have a modular design that should generalize to other bacterial signaling systems in the future.

Antibacterial Activities and Molecular Docking of Novel Sulfone Biscompound Containing Bioactive 1,2,3-Triazole Moiety.

In this study, a new synthetic 1,2,3-triazole-containing disulfone compound was derived from dapsone. Its chemical structure was confirmed using microchemical and analytical data, and it was tested for its in vitro antibacterial potential. Six different pathogenic bacteria were selected. MICs values and ATP levels were determined. Further, toxicity performance was measured using MicroTox Analyzer. In addition, a molecular docking study was performed against two vital enzymes: DNA gyrase and Dihydropteroate synthase. The results of antibacterial abilities showed that the studied synthetic compound had a strong bactericidal effect against all tested bacterial strains, as Gram-negative species were more susceptible to the compound than Gram-positive species. Toxicity results showed that the compound is biocompatible and safe without toxic impact. The molecular docking of the compound showed interactions within the pocket of two enzymes, which are able to stabilize the compound and reveal its antimicrobial activity. Hence, from these results, this study recommends that the established compound could be an outstanding candidate for fighting a broad spectrum of pathogenic bacterial strains, and it might therefore be used for biomedical and pharmaceutical applications.

Gold(I) and Gold(III) N-Heterocyclic Carbene Complexes as Antibacterial Agents and Inhibitors of Bacterial Thioredoxin Reductase.

A series of (NHC)Au(I)Cl monocarbene complexes and their gold(III) analogues (NHC)Au(III)Cl3 were prepared and investigated as antibacterial agents and inhibitors of bacterial TrxR. The complexes showed stronger antibacterial effects against the Gram-positive MRSA and E. faecium strains than against several Gram-negative bacteria. All complexes were efficient inhibitors of bacterial thioredoxin reductase, indicating that inhibition of this enzyme might be involved in their mechanism of action. The efficacy of gold(I) and gold(III) analogues was comparable in most of the assays. The cytotoxicity of the gold NHC compounds against cancer and human cells was overall weaker than the activity against the Gram-positive bacteria, suggesting that their optimization as antibacterials warrants further investigation.

Biodistributions of l,d-Transpeptidases in Gut Microbiota Revealed by In Vivo Labeling with Peptidoglycan Analogs.

By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to ß-lactams in some pathogens in vitro. However, the prevalence of Ldt and Ldt-mediated responses to different ß-lactams in vivo have never been explored. Here, we apply an in vivo metabolic labeling strategy to study their biodistributions and Ldt-induced bacterial responses to ß-lactams in the mouse gut microbiota. A tetrapeptide-based fluorescent probe that functions as a substrate for Ldts in Gram-positive bacteria efficiently labels ∼18% of total gut bacteria after gavage, suggesting Ldts' high prevalence in gut microbiota. The cellular distributions of 3-3 cross-links on three gut bacterial species were then identified with the aid of fluorescence in situ hybridization to identify the bacterial taxa. After oral administration of two ß-lactams, ampicillin and meropenem, only the latter efficiently inhibits the tetrapeptide labeling, suggesting that Ldts may be able to cause resistance to some ß-lactams in the mammalian gut.

In Vitro and In Silico Screening and Characterization of Antimicrobial Napin Bioactive Protein in Brassica juncea and Moringa oleifera.

The study aimed to investigate the antibacterial activity of Mustard (Brassica juncea) and Moringa (Moringa oleifera) leaf extracts and coagulant protein for their potential application in water treatment. Bacterial cell aggregation and growth kinetics studies were employed for thirteen bacterial strains with different concentrations of leaf extracts and coagulant protein. Moringa oleifera leaf extract (MOS) and coagulant protein showed cell aggregation against ten bacterial strains, whereas leaf extract alone showed growth inhibition of five bacterial strains for up to 6 h and five bacterial strains for up to 3 h. Brassica juncea leaf extract (BJS) showed growth inhibition for up to 6 h, and three bacterial strains showed inhibition for up to 3 h. The highest inhibition concentration with 2.5 mg/mL was 19 mm, and furthermore, the minimum inhibitory concentration (MIC) (0.5 mg/mL) and MBC (1.5 mg/mL) were determined to have a higher antibacterial effect for <3 KDa peptides. Based on LCMS analysis, napin was identified in both MOS and BJS; furthermore, the mode of action of napin peptide was determined on lipoprotein X complex (LpxC) and four-chained structured binding protein of bacterial type II topoisomerase (4PLB). The docking analysis has exhibited moderate to potent inhibition with a range of dock score -912.9 Kcal/mol. Thus, it possesses antibacterial-coagulant potential bioactive peptides present in the Moringa oleifera purified protein (MOP) and Brassica juncea purified protein (BJP) that could act as an effective antimicrobial agent to replace currently available antibiotics. The result implies that MOP and Brassica juncea purified coagulant (BJP) proteins may perform a wide degree of antibacterial functions against different pathogens.

Building blocks and blueprints for bacterial autolysins.

Bacteria utilize a wide variety of endogenous cell wall hydrolases, or autolysins, to remodel their cell walls during processes including cell division, biofilm formation, and programmed death. We here systematically investigate the composition of these enzymes in order to gain insights into their associated biological processes, potential ways to disrupt them via chemotherapeutics, and strategies by which they might be leveraged as recombinant antibacterial biotherapies. To do so, we developed LEDGOs (lytic enzyme domains grouped by organism), a pipeline to create and analyze databases of autolytic enzyme sequences, constituent domain annotations, and architectural patterns of multi-domain enzymes that integrate peptidoglycan binding and degrading functions. We applied LEDGOs to eight pathogenic bacteria, gram negatives Acinetobacter baumannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aeruginosa; and gram positives Clostridioides difficile, Enterococcus faecium, Staphylococcus aureus, and Streptococcus pneumoniae. Our analysis of the autolytic enzyme repertoires of these pathogens reveals commonalities and differences in their key domain building blocks and architectures, including correlations and preferred orders among domains in multi-domain enzymes, repetitions of homologous binding domains with potentially complementarity recognition modalities, and sequence similarity patterns indicative of potential divergence of functional specificity among related domains. We have further identified a variety of unannotated sequence regions within the lytic enzymes that may themselves contain new domains with important functions.

Glycoconjugate pathway connections revealed by sequence similarity network analysis of the monotopic phosphoglycosyl transferases.

The monotopic phosphoglycosyl transferase (monoPGT) superfamily comprises over 38,000 nonredundant sequences represented in bacterial and archaeal domains of life. Members of the superfamily catalyze the first membrane-committed step in en bloc oligosaccharide biosynthetic pathways, transferring a phosphosugar from a soluble nucleoside diphosphosugar to a membrane-resident polyprenol phosphate. The singularity of the monoPGT fold and its employment in the pivotal first membrane-committed step allows confident assignment of both protein and corresponding pathway. The diversity of the family is revealed by the generation and analysis of a sequence similarity network for the superfamily, with fusion of monoPGTs with other pathway members being the most frequent and extensive elaboration. Three common fusions were identified: sugar-modifying enzymes, glycosyl transferases, and regulatory domains. Additionally, unexpected fusions of the monoPGT with members of the polytopic PGT superfamily were discovered, implying a possible evolutionary link through the shared polyprenol phosphate substrate. Notably, a phylogenetic reconstruction of the monoPGT superfamily shows a radial burst of functionalization, with a minority of members comprising only the minimal PGT catalytic domain. The commonality and identity of the fusion partners in the monoPGT superfamily is consistent with advantageous colocalization of pathway members at membrane interfaces.

Undecaprenol kinase: Function, mechanism and substrate specificity of a potential antibiotic target.

The bifunctional undecaprenol kinase/phosphatase (UdpK) is a small, prokaryotic, integral membrane kinase, homologous with Escherichia coli diacylglycerol kinase and expressed by the dgkA gene. In Gram-positive bacteria, UdpK is involved in the homeostasis of the bacterial undecaprenoid pool, where it converts undecaprenol to undecaprenyl phosphate (C55P) and also catalyses the reverse process. C55P is the universal lipid carrier and critical to numerous glycopolymer and glycoprotein biosynthetic pathways in bacteria. DgkA gene expression has been linked to facilitating bacterial growth and survival in response to environmental stressors, as well being implicated as a resistance mechanism to the topical antibiotic bacitracin, by providing an additional route to C55P. Therefore, identification of UdpK inhibitors could lead to novel antibiotic treatments. A combination of homology modelling and mutagenesis experiments on UdpK have been used to identify residues that may be involved in kinase/phosphatase activity. In this review, we will summarise recent work on the mechanism and substrate specificity of UdpK.

Tailored Cofactor Traps for the in Situ Detection of Hemithioacetal-Forming Pyridoxal Kinases.

Pyridoxal kinases (PLK) are crucial enzymes for the biosynthesis of pyridoxal phosphate, an important cofactor in a plethora of enzymatic reactions. The evolution of these enzymes resulted in different catalytic designs. In addition to the active site, the importance of a cysteine, embedded within a distant flexible lid region, was recently demonstrated. This cysteine forms a hemithioacetal with the pyridoxal aldehyde and is essential for catalysis. Despite the prevalence of these enzymes in various organisms, no tools were yet available to study the relevance of this lid residue. Here, we introduce pyridoxal probes, each equipped with an electrophilic trapping group in place of the aldehyde to target PLK reactive lid cysteines as a mimic of hemithioacetal formation. The addition of alkyne handles placed at two different positions within the pyridoxal structure facilitates enrichment of PLKs from living cells. Interestingly, depending on the position, the probes displayed a preference for either Gram-positive or Gram-negative PLK enrichment. By applying the cofactor traps, we were able to validate not only previously investigated Staphylococcus aureus and Enterococcus faecalis PLKs but also Escherichia coli and Pseudomonas aeruginosa PLKs, unravelling a crucial role of the lid cysteine for catalysis. Overall, our tailored probes facilitated a reliable readout of lid cysteine containing PLKs, qualifying them as chemical tools for mining further diverse proteomes for this important enzyme class.

Development of Peptides that Inhibit Aminoglycoside-Modifying Enzymes and ß-Lactamases for Control of Resistant Bacteria.

Aminoglycosides and ß-lactams are the most commonly used antimicrobial agents in clinical practice. This occurs because they are capable of acting in the treatment of acute bacterial infections. However, the effectiveness of antibiotics has been constantly threatened due to bacterial pathogens producing resistance enzymes. Among them, the aminoglycoside-modifying enzymes (AMEs) and ß-lactamase enzymes are the most frequently reported resistance mechanisms. AMEs can inactivate aminoglycosides by adding specific chemical molecules in the compound, whereas ß-lactamases hydrolyze the ß-lactams ring, preventing drug-target interaction. Thus, these enzymes provide a scenario of multidrug-resistance and a significant threat to public health at a global level. In response to this challenge, in recent decades, several studies have focused on the development of inhibitors that can restore aminoglycosides and ß-lactams activity. In this context, peptides appear as a promising approach in the field of inhibitors for future antibacterial therapies, as multiresistant bacteria may be susceptible to these molecules. Therefore, this review focused on the most recent findings related to peptide-based inhibitors that act on AMEs and ß-lactamases, and how these molecules could be used for future treatment strategies.

Merging Natural Products: Muraymycin-Sansanmycin Hybrid Structures as Novel Scaffolds for Potential Antibacterial Agents.

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so-called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine-derived core motifs. Some sub-classes also show specific selectivities towards different Gram-positive and Gram-negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5'-defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin-sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa.

From plant to probe: semi-synthesis of labelled undecaprenol analogues allows rapid access to probes for antibiotic targets.

Undecaprenol-containing glycolipids (UCGs) are essential precursors of bacterial glycopolymers and glycoproteins. We report a novel semi-synthetic strategy to prepare labelled UCGs directly from undecaprenol. This one-size-fits-all approach offers a concise and efficient method for obtaining labelled-UCGs, which will allow new mechanistic studies and inhibitor screens to be performed on novel antibiotic targets.

Antimicrobial evaluation of thiadiazino and thiazolo quinoxaline hybrids as potential DNA gyrase inhibitors; design, synthesis, characterization and morphological studies.

A series of thiadiazino[5,6-b]quinoxaline and thiazolo[4,5-b]quinoxaline derivatives was designed and synthetized from the reaction of 2,3-dichloro-6-(morpholinosulfonyl)quinoxaline (2) with thiosemicarbazide or thiocarbohydrazide and thiourea derivatives to give nineteen quinoxaline derivatives 3-16. All the synthesized compounds were evaluated for in vitro antimicrobial potential against various bacteria and fungi strains that showed considerable antimicrobial activity against tested microorganisms. The most potent compounds 2, 7, 9, 10, 12 and 13c were exhibited bactericidal activity, in addition to fungistatic activity by dead live assay. Moreover, these compounds showed a significant result against all multi-drug resistance (MDRB) used especially compound 13c that displayed the best results with MICs of MDRB (1.95, 3.9, 2.6, 3.9 µg/mL) for stains used in this study, compared with Norfloxacin (1.25, 0.78, 1.57, 3.13 µg/mL). Also, cytotoxicity on normal cell (Vero cells ATCC CCL-81) by MTT assay was performed with lower toxicity results. Additionally, morphological studies, immunostimulatory potency and DNA gyrase inhibition assay of most active compounds was done. A molecular docking study has also been carried out to support the effective binding of the most promising compounds at the active site of the target enzyme S. aureus DNA gyrase (2XCT).

Poly(cis-1,4-isoprene)-cleavage enzymes from natural rubber-utilizing bacteria.

Natural rubber and synthetic poly(cis-1,4-isoprene) are used industrially in the world. Microbial utilization for the isoprene rubbers has been reported in gram-positive and gram-negative bacteria. Poly(cis-1,4-isoprene)-cleavage enzymes that are secreted by rubber-utilizing bacteria cleave the poly(cis-1,4-isoprene) chain to generate low-molecular-weight oligo(cis-1,4-isoprene) derivatives containing aldehyde and ketone groups. The resulting products are converted to the compounds including carboxyl groups, which could then be further catabolized through ß-oxidation pathway. One of poly(cis-1,4-isoprene)-cleavage enzymes is latex-clearing protein (Lcp) that was found in gram-positive rubber degraders including Streptomyces, Gordonia, Rhodococcus, and Nocardia species. The other one is rubber oxygenase A and B (RoxA/RoxB) which have been identified from gram-negative rubber degraders such as Steroidobacter cummioxidans and Rhizobacter gummiphilus. Recently, the transcriptional regulation mechanisms for Lcp-coding genes in gram-positive bacteria have been characterized. Here, the current knowledge of genes and enzymes for the isoprene rubber catabolism were summarized.

Worldwide surveillance of Iclaprim activity: In Vitro susceptibility of gram-positive pathogens collected from patients with skin and skin structure infections from 2013 to 2017.

Iclaprim is a novel diaminopyrimidine, which inhibits bacterial dihydrofolate reductase, and it is active against Gram-positive pathogens including emerging drug-resistant pathogens. In vitro activity of iclaprim and comparators against 1365 Gram-positive clinical isolates from patients with skin and skin structure infections (SSSI) from the United States, Asia Pacific, Latin America, Europe, Africa or Middle East collected between 2013 and 2017 were tested. Susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) interpretations were based on CLSI criteria. MIC90 for all S.aureus, methicillin-susceptible S. aureus, methicillin-resistant S. aureus, Streptococcus pyogenes, S. agalactiae, S. anginosus, S. constellatus, S. dysgalactiae and S. intermedius were 0.12, 0.12, 0.5, 0.03, 0.5, ≤0.004, ≤0.004, 0.12, and 0.008 µg/ml, respectively. The MIC for iclaprim was 8 to 32-fold lower than trimethoprim, the only FDA approved dihydrofolate reductase inhibitor, against all Gram-positive isolates including resistant phenotypes. Iclaprim demonstrated lower MICs than trimethoprim against a collection (2013-2017) of Gram-positive clinical isolates from patients with SSSI from the United States, Asia Pacific, Latin America, and Europe.

Second-generation 4,5,6,7-tetrahydrobenzo[d]thiazoles as novel DNA gyrase inhibitors.

Aim: DNA gyrase and topoisomerase IV are essential bacterial enzymes, and in the fight against bacterial resistance, they are important targets for the development of novel antibacterial drugs. Results: Building from our first generation of 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase inhibitors, we designed and prepared an optimized series of analogs that show improved inhibition of DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli, with IC50 values in the nanomolar range. Importantly, these inhibitors also show improved antibacterial activity against Gram-positive strains. Conclusion: The most promising inhibitor, 29, is active against Enterococcus faecalis, Enterococcus faecium and S. aureus wild-type and resistant strains, with minimum inhibitory concentrations between 4 and 8 µg/ml, which represents good starting point for development of novel antibacterials.