Antibacterial Target Research Articles

Characterization of the enzymatic and multifunctional properties of Acinetobacter baumannii erythrose-4-phosphate dehydrogenase (E4PDH).

Infections due to Acinetobacter baumannii (A. baumannii) are rapidly increasing worldwide and consequently therapeutic options for treatment are limited. The emergence of multi drug resistant (MDR) strains has rendered available antibiotics ineffective, necessitating the urgent discovery of new drugs and drug targets. The vitamin B6 biosynthetic pathway has been considered as a potential antibacterial drug target but it is as yet uncharacterized for A. baumannii. In the current work, we have carried out in silico and biochemical characterization of Erythrose-4-phosphate dehydrogenase (E4PDH) (EC 1.2.1.72). This enzyme catalyzes the first step in the deoxyxylulose-5-phosphate (DXP) dependent Vitamin B6 biosynthetic pathway i.e. the conversion of d-erythrose-4-phosphate (E4P) to 4-Phosphoerythronate. E4PDH also possesses an additional activity whereby it can catalyze the conversion of Glyceraldehyde-3-phosphate (G3P) to 1,3 bisphosphoglycerate (1,3BPG). Our studies have revealed that this enzyme exhibits an alternate moonlighting function as a cell surface receptor for the human iron transport proteins transferrin (Tf) and lactoferrin (Lf). The present work reports the internalization of Tf and consequent iron acquisition as an alternate strategy for iron acquisition. Given its essential role in two crucial pathways i.e. metabolism and iron acquisition, A. baumannii E4PDH may play a vital role in bacterial pathogenesis.

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives.

Isoprenoids, a diverse class of natural products, are present in all living organisms. Their two universal building blocks are synthesized via two independent pathways: the mevalonate pathway and the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway. The presence of the latter in pathogenic bacteria and its absence in humans make all its enzymes suitable targets for the development of novel antibacterial drugs. (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), the last intermediate of this pathway, is a natural ligand for the human Vγ9Vδ2 T cells and the most potent natural phosphoantigen known to date. Moreover, 5-hydroxypentane-2,3-dione, a metabolite produced by Escherichia coli 1-deoxy-ᴅ-xylulose 5-phosphate synthase (DXS), the first enzyme of the MEP pathway, structurally resembles (S)-4,5-dihydroxy-2,3-pentanedione, a signal molecule implied in bacterial cell communication. In this review, we shed light on the diversity of potential uses of the MEP pathway in antibacterial therapies, starting with an overview of the antibacterials developed for each of its enzymes. Then, we provide insight into HMBPP, its synthetic analogs, and their prodrugs. Finally, we discuss the potential contribution of the MEP pathway to quorum sensing mechanisms. The MEP pathway, providing simultaneously antibacterial drug targets and potent immunostimulants, coupled with its potential role in bacterial cell-cell communication, opens new therapeutic perspectives.

Gold(I) selenium N-heterocyclic carbene complexes as potent antibacterial agents against multidrug-resistant gram-negative bacteria via inhibiting thioredoxin reductase

Multidrug-resistant (MDR) Gram-negative bacteria have become a global threat to human life and health, and novel antibiotics are urgently needed. The thioredoxin (Trx) system can be used as an antibacterial target to combat MDR bacteria. Here, we found that two active gold(I) selenium N-heterocyclic carbene complexes H7 and H8 show more promising antibacterial effects against MDR bacteria than auranofin. Both H7 and H8 irreversibly inhibit the bacterial TrxR activity via targeting the redox-active motif, abolishing the capacity of TrxR to quench reactive oxygen species (ROS) and finally leading to oxidative stress. The increased cellular superoxide radical levels impact a variety of functions necessary for bacterial survival, such as cellular redox balance, cell membrane integrity, amino acid metabolism, and lipid peroxidation. In vivo data present much better antibacterial activity of H7 and H8 than auranofin, promoting the wound healing and prolonging the survival time of Carbapenem-resistant Acinetobacter baumannii (CRAB) induced peritonitis. Most notably in this study, we revealed the influence of gold(I) complexes on both the Trx system and the cellular metabolic states to better understand their killing mechanism and to support further antibacterial drug design.

Discovery, Validation, and Target Prediction of Antibacterial and Antidiabetic Components of Archidendron clypearia Based on a Combination of Multiple Analytical Methods.

Archidendron clypearia (A. clypearia), a Fabaceae family member, is widely used as an anti-inflammatory herbal medicine; however, its antibacterial and antidiabetic properties have not been extensively investigated. This study aimed to systematically analyze the antibacterial and antidiabetic components of A. clypearia by utilizing a combination of analytical methods. First, ten different polarity extracts were analyzed through ultra-performance liquid chromatography (UPLC), and their antibacterial and antidiabetic activities were evaluated. Then the spectrum-effect relationship between the biological activity and UPLC chromatograms was analyzed by partial least squares regression and gray relational analysis, followed by corresponding validation using isolated components. Finally, network pharmacology and molecular docking were implemented to predict the main antibacterial target components of A. clypearia and the enzyme inhibition active sites of α-amylase and α-glucosidase. P15, P16, and P20 were found to be the antibacterial and antidiabetic active components. The inhibitory effect of 7-O-galloyltricetiflavan (P15) on six bacterial species may be mediated through the lipid and atherosclerosis pathway, prostate cancer, adherens junctions, and targets such as SRC, MAPK1, and AKT1. The molecular docking results revealed that 7-O-galloyltricetiflavan and 7,4'-di-O-galloyltricetiflavan (P16/P20) can bind to α-amylase and α-glucosidase pockets with binding energies lower than -6 kcal/mol. Our study provides guidance for the development of antibacterial and antidiabetic products based on A. clypearia and can be used as a reference for the evaluation of bioactivity of other herbs.

Synthesis, molecular docking and antibacterial activity of an oxadiazole-based lipoteichoic acid inhibitor and its metabolites

Amongst drug resistant Gram-positive bacteria, Staphylococcus aureus is a pathogen of great concern as it is the leading cause of life-threatening nosocomial and community acquired infections which are often associated with implanted medical devices. The biosynthesis of lipotheicoic acid (LTA) by S. aureus has been recognized as a promising antibacterial target, owing its critical role in the growth and survival of Gram-positive bacteria. Here we report for the first time the chemical synthesis and characterisation of an oxadiazole based compound (1771), previously described as an inhibitor of LTA biosynthesis by targeting Lta synthase enzyme (LtaS). To investigate its controversial mode of action, we also performed molecular docking studies, which indicated that 1771 behaves as a competitive inhibitor against LtaS. We also synthesised and evaluated the antimicrobial activity of 1771 metabolites which we have identified from its decomposition in mouse serum, proving that the biological activity was caused by intact 1771.

Metabolite profiling, in vitro and in silico assessment of antibacterial and anticancer activities of Alternaria alternata endophytic in Jatropha heynei.

Fungal endophytes produce a range of structurally diverse metabolites with bioactive principles. In this study, an endophytic fungus Alternaria alternata was isolated from Jatropha heynei and cultured in potato dextrose liquid broth. Culture filtrate of A. alternata was extracted in ethyl acetate and metabolites were characterized by QTOF-HRLCMS. Among compounds detected, spectral compounds such as kigelinone, and levofuraltadone were reported with antibacterial property, while 2-hydroxychrasophanol, isoathyriol, glycophymoline, columbianetin and kaempferol 3-O-β-D- galactoside were reported with cytotoxic properties. Partially purified metabolites of A. alternata showed significant antibacterial activity against tested clinical bacterial strains by agar well diffusion method. High zone of inhibition was recorded against Enterococcus faecalis, Pseudomonas syringae and Klebsiella pneumoniae. In vitro anticancer activity of fungal extract by MTT assay displayed high cytotoxic effect on human lung carcinoma cancer cell line (A549) with IC50 value of 393.52µgml-1, and without any significant cytotoxic effect on human breast cancer cell line (MCF-7). Further, antibacterial and anticancer spectral compounds of A. alternata were subjected to molecular docking analysis with antibacterial target proteins such as tellurite resistance protein (2JXU), indole-3-acetaldehyde dehydrogenase (5IUU) and alkyl hydroperoxide reductase (5Y63), and anticancer target human apoptotic regulator protein (1G5M). The results of the study indicated that kigelinone, levofuraltadone, 2-hydroxychrasophanol and isoathyriolin the fungal extract have significant binding modes, with best binding energy scores with their respective antibacterial and anticancer target proteins. Alternaria alternata resident in J. heynei offers a promising source of broad-spectrum antibacterial and anticancer compounds.

Determination of Chemical Composition and Investigation of Biological Activities of Ocimum basilicum L.

This study aimed to determine the chemical composition of the essential oils (EOs) of Ocimum basilicum L., as well as to evaluate the antibacterial, antidiabetic, dermatoprotective, and anti-inflammatory properties, and the EOs and aqueous extracts of O. basilicum. The antibacterial activity was evaluated against bacterial strains, Gram-positive and Gram-negative, using the well diffusion and microdilution methods, whereas the antidiabetic activity was assessed in vitro using two enzymes involved in carbohydrate digestion, α-amylase and α-glucosidase. On the other hand, the dermatoprotective and anti-inflammatory activities were studied by testing tyrosinase and lipoxygenase inhibition activity, respectively. The results showed that the chemical composition of O. basilicum EO (OBEO) is dominated by methyl chavicol (86%) and trans-anethol (8%). OBEO exhibited significant antibacterial effects against Gram-negative and Gram-positive strains, demonstrated by considerable diameters of the inhibition zones and lower MIC and MBC values. In addition, OBEO exhibited significant inhibition of α-amylase (IC50 = 50.51 ± 0.32 μg/mL) and α-glucosidase (IC50 = 39.84 ± 1.2 μg/mL). Concerning the anti-inflammatory activity, OBEO significantly inhibited lipoxygenase activity (IC50 = 18.28 ± 0.03 μg/mL) compared to the aqueous extract (IC50 = 24.8 ± 0.01 μg/mL). Moreover, tyrosinase was considerably inhibited by OBEO (IC50 = 68.58 ± 0.03 μg/mL) compared to the aqueous extract (IC50 = 118.37 ± 0.05 μg/mL). The toxicological investigations revealed the safety of O. basilicum in acute and chronic toxicity. The finding of in silico analysis showed that methyl chavicol and trans-anethole (main compounds of OBEO) validate the pharmacokinetics of these compounds and decipher some antibacterial targets.

Computer-aided design of a cyclic di-AMP synthesizing enzyme CdaA inhibitor.

Cyclic di-AMP (c-di-AMP) is an essential secondary messenger regulating cell wall homeostasis and myriads of physiological processes in several Gram-positive and mycobacteria, including human pathogens. Hence, c-di-AMP synthesizing enzymes (DACs) have become a promising antibacterial drug target. To overcome a scarcity of small molecule inhibitors of c-di-AMP synthesizing enzyme CdaA, a computer-aided design of a new compound that should block the enzyme has been performed. This has led to the identification of a molecule comprising two thiazole rings and showing inhibitory potential based on ITC measurements. Thiazole scaffold is a good pharmacophore nucleus known due to its various pharmaceutical applications. It is contained in more than 18 FDA-approved drugs as well as in dozens of experimental drugs. Hence, the designed inhibitor can serve as a potent lead compound for further development of inhibitor against CdaA.

Exploring the Antibacterial and Antiviral Mechanisms of Baiyaozi Via Network Pharmacology

This paper aims to analyze the antibacterial and antiviral active components of Baiyaozi and predict its molecular mechanisms via network pharmacology. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature were used to screen active components of Baiyaozi (Radix Stephania cepharantha), and Pubchem and Swiss Target Prediction were used to collect the targets of the active components. The GeneCards database was used to search and screen the antibacterial and antiviral targets, intersecting with the practical components' targets to obtain the antibacterial and antiviral core targets of Baiyaozi. Then, the active ingredients and target genes were introduced into Cytoscape to construct the active component-target network regulation map of Baiyaozi. The STRING database was used for protein-protein interaction (PPI) network analysis, and the DAVID platform was used to conduct GO functional and KEGG pathway enrichment analyses of common targets. Eight active ingredients of Baiyaozi acted on 67 antibacterial targets and 130 antiviral targets. Cepharanthine, isocorydine, and berberine might be the main antibacterial and antiviral components of Baiyaozi. The GO function and KEGG pathway enrichment analyses of common targets suggested that the antibacterial and antiviral pathways of Baiyao might focus on apoptosis, autophagy, tumor necrosis factor signaling pathway, HIF-1 signaling pathway, and the PI3K-Akt pathway. Also, the comprehensive PPI, GO, and KEGG analysis indicated that HSP90AA1, SRC, and AKT1 might be the main targets. Conclusion: Baiyaozi has multi-component, multi-target, multi-pathway antibacterial and antiviral molecular mechanisms, providing a theoretical reference for further interpretation.

Enhancing allosteric inhibition of dihydrodipicolinate synthase through the design and synthesis of novel dimeric compounds.

The synthesis of the first dimeric inhibitor of E. coli dihydrodipicolinate synthase (DHDPS) is reported herein. Inspired by 2,4-thiazolidinedione based ligands previously shown to inhibit DHDPS, a series of dimeric inhibitors were designed and synthesised, incorporating various alkyl chain bridges between two 2,4-thiazolidinedione moieties. Aiming to exploit the multimeric nature of this enzyme and enhance potency, a dimeric compound with a single methylene bridge achieved the desired outcome with low micromolar inhibition of E. coli DHDPS observed. This work highlights the continued importance of investigation into DHDPS as an antibacterial target. Furthermore, we demonstrate the design of dimeric ligands can provide a promising strategy to improve potency in the search for novel bioactive compounds.

Filamentous temperature sensitive mutant Z: a putative target to combat antibacterial resistance.

In the pre-antibiotic era, common bacterial infections accounted for high mortality and morbidity. Moreover, the discovery of penicillin in 1928 marked the beginning of an antibiotic revolution, and this antibiotic era witnessed the discovery of many novel antibiotics, a golden era. However, the misuse or overuse of these antibiotics, natural resistance that existed even before the antibiotics were discovered, genetic variations in bacteria, natural selection, and acquisition of resistance from one species to another consistently increased the resistance to the existing antibacterial targets. Antibacterial resistance (ABR) is now becoming an ever-increasing concern jeopardizing global health. Henceforth, there is an urgent unmet need to discover novel compounds to combat ABR, which act through untapped pathways/mechanisms. Filamentous Temperature Sensitive mutant Z (FtsZ) is one such unique target, a tubulin homolog involved in developing a cytoskeletal framework for the cytokinetic ring. Additionally, its pivotal role in bacterial cell division and the lack of homologous structural protein in mammals makes it a potential antibacterial target for developing novel molecules. Approximately 2176 X-crystal structures of FtsZ were available, which initiated the research efforts to develop novel antibacterial agents. The literature has reported several natural, semisynthetic, peptides, and synthetic molecules as FtsZ inhibitors. This review provides valuable insights into the basic crystal structure of FtsZ, its inhibitors, and their inhibitory activities. This review also describes the available in vitro detection and quantification methods of FtsZ-drug complexes and the various approaches for determining drugs targeting FtsZ polymerization.

Targeting Essential Hypothetical Proteins of Pseudomonas aeruginosa PAO1 for Mining of Novel Therapeutics: An In Silico Approach.

As an omnipresent opportunistic bacterium, Pseudomonas aeruginosa PAO1 is responsible for acute and chronic infection in immunocompromised individuals. Currently, this bacterium is on WHO's red list where new antibiotics are urgently required for the treatment. Finding essential genes and essential hypothetical proteins (EHP) can be crucial in identifying novel druggable targets and therapeutics. This study is aimed at characterizing these EHPs and analyzing subcellular and physiochemical properties, PPI network, nonhomologous analysis against humans, virulence factor and novel drug target prediction, and finally structural analysis of the identified target employing around 42 robust bioinformatics tools/databases, the output of which was evaluated using the ROC analysis. The study discovered 18 EHPs from 336 essential genes, with domain and functional annotation revealing that 50% of these proteins belong to the enzyme category. The majority are cytoplasmic and cytoplasmic membrane proteins, with half being stable proteins subjected to PPIs network analysis. The network contains 261 nodes and 269 edges for 9 proteins of interest, with 11 hubs containing at least three nodes each. Finally, a pipeline builder predicts 7 proteins with novel drug targets, 5 nonhomologous proteins against human proteome, human antitargets, and human gut flora, and 3 virulent proteins. Among these, homology modeling of NP_249450 and NP_251676 was done, and the Ramachandran plot analysis revealed that more than 94% of the residues were in the preferred region. By analyzing functional attributes and virulence characteristics, the findings of this study may facilitate the development of innovative antibacterial drug targets and drugs of Pseudomonas aeruginosa PAO1.

Endocytosis-mediated redistribution of antibiotics targets intracellular bacteria.

The increasing emergence and dissemination of antibiotic resistance pose a severe threat to overwhelming healthcare practices worldwide. The lack of new antibacterial drugs urgently calls for alternative therapeutic strategies to combat multidrug-resistant (MDR) bacterial pathogens, especially those that survive and replicate in host cells, causing relapse and recurrence of infections. Intracellular drug delivery is a direct efficient strategy to combat invasive pathogens by increasing the accumulation of antibiotics. However, the increased accumulation of antibiotics in the infected host cells does not mean high efficacy. The difficulty of treatment lies in the efficient intracellular delivery of antibiotics to the pathogen-containing compartments. Here, we first briefly review the survival mechanisms of intracellular bacteria to facilitate the exploration of potential antibacterial targets for precise delivery. Furthermore, we provide an overview of endocytosis-mediated drug delivery systems, including the biomedical and physicochemical properties modulating the endocytosis and intracellular redistribution of antibiotics. Lastly, we summarize the targets and payloads of recently described intracellular delivery systems and their modes of action against diverse pathogenic bacteria-associated infections. This overview of endocytosis-mediated redistribution of antibiotics sheds light on the development of novel delivery platforms and alternative strategies to combat intracellular bacterial pathogens.

Efficient Antibacterial/Antifungal Activities: Synthesis, Molecular Docking, Molecular Dynamics, Pharmacokinetic, and Binding Free Energy of Galactopyranoside Derivatives.

The chemistry and biochemistry of carbohydrate esters are essential parts of biochemical and medicinal research. A group of methyl β-d-galactopyranoside (β-MGP, 1) derivatives was acylated with 3-bromobenzoyl chloride and 4-bromobenzoyl chloride in anhydrous N,N-dimethylformamide/triethylamine to obtain 6-O-substitution products, which were subsequently converted into 2,3,4-tri-O-acyl derivatives with different aliphatic and aromatic substituents. Spectroscopic and elemental data exploration of these derivatives confirmed their chemical structures. In vitro biological experiments against five bacteria and two fungi and the prediction of activity spectra for substances (PASS) revealed ascending antifungal and antibacterial activities compared with their antiviral activities. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) experiments were performed for two derivatives, 3 and 9, based on their antibacterial activities. Most of these derivatives showed >780% inhibition of fungal mycelial growth. Density functional theory (DFT) was used to calculate the chemical descriptors and thermodynamic properties, whereas molecular docking was performed against antibacterial drug targets, including PDB: 4QDI, 5A5E, 7D27, 1ZJI, 3K8E, and 2MRW, and antifungal drug targets, such as PDB: 1EA1 and 1AI9, to identify potential drug candidates for microbial pathogens. A 100 ns molecular dynamics simulation study revealed stable conformation and binding patterns in a stimulating environment by their uniform RMSD, RMSF, SASA, H-bond, and RoG profiles. In silico pharmacokinetic and quantitative structure-activity relationship (QSAR) calculations (pIC50 values 3.67~8.15) suggested that all the designed β-MGP derivatives exhibited promising results due to their improved kinetic properties with low aquatic and non-aquatic toxicities. These biological, structure-activity relationship (SAR) [lauroyl-(CH3(CH2)10CO-) group was found to have potential], and in silico computational studies revealed that the newly synthesized MGP derivatives are potential antibacterial/antifungal candidates and can serve as therapeutic targets for human and plant pathogens.

Virtual screening and molecular dynamics simulation studies to predict the binding of Sisymbrium irio L. derived phytochemicals against Staphylococcus aureus dihydrofolate reductase (DHFR)

The discovery of antibiotics initiated the era of drug innovation and implementation for human and animal health. Very soon, antibiotic resistance started evolving due to over-prescription and heavy usage of drugs leading to deleterious side effects. However, using plant extracts or medicinal plants has emerged as a new approach to dealing with the current problem. One such medicinal plant Sisymbrium irio L. is widely used in Unani therapy as an antimicrobial, analgesic, antipyretic, antioxidant, anti-inflammatory, hepatoprotective, bronchoprotective etc. The phytochemicals extracted from the aerial part of the plant have been used as a natural compound library and screened against a well-known anti-bacterial drug target Dihydrofolate reductase (DHFR) enzyme of Staphylococcus aureus. The top two phytochemicals with lower docking score along with the positive control were subjected to molecular dynamics (MD) simulation studies to examine the stabilities of the complexes over 100 ns, followed by binding free energy estimation. The Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) and Radius of Gyration (Rg) yielded established results throughout the MD run. Moreover, the derived phytochemicals exhibited lower binding free energy values than the positive control that can be tested for its in vitro efficacy, followed by further optimization to attain a potent therapeutic against S. aureus. Taken together, the present study suggests two promising phytochemicals derived from the aerial part of the plant S. irio with stable MD simulation results, strong binding affinity and no side effects.

Directions in the development of modern and promising antimicrobial agents

The use of antibiotics is becoming increasingly limited. This is mainly due to the development of resistance to pathogenic bacteria, and, over time, more and more bacteria will become resistant to antimicrobials. This problem inevitably leads to the conclusion that studies into alternative methods of combating pathogens, which are necessary to develop sufficiently reliable and effective therapies for bacterial infections, are indispensable. This review highlights some recent developments in conventional antibiotic and non-antibiotic treatment strategies. It has been shown that traditional antibacterial targets include derivatives of known antibiotic classes, new chemical classes with new targets, as well as unknown or undefined agents with unclear targets. Promising strategies for combating microbial pathogens have been identified, including new targets, namely, toxin secretion systems, biofilm formation, and adhesion mechanisms that affect quorum sensing of microbial populations. In addition, it is important to use new antimicrobial agents with other, non-antibiotic, mechanisms of action: phage and phage-derived peptides, microbiota-modulating therapies, and enhancing immune response.

Development of pharmacophore model to identify potential DNA gyrase inhibitors

There is great concern in the medical community due to rapid increase in antibiotic resistance, causing 700,000 deaths annually worldwide. Therefore, there is paramount need to develop novel and innovative antibacterial agents active against resistant bacterial strains. DNA gyrase is a crucial enzyme in bacterial replication that is absent in eukaryotes, making it effective curative target for antibacterials. To identify potential DNA gyrase inhibitors by virtual screening of NCI database using a 3-step approach. A total of 271 compounds with known IC50 values against Escherichia coli DNA GyrA were selected to develop a pharmacophore model for dual screening approach to identify new potential hits from the NCI database. In the second step, the NCI database was also screened using in-house built NN-QSAR model. Molecular docking of common 5298 compounds screened from both methods were performed against E. coli DNA GyrA (PDB id- 6RKU), and 3004 compounds are reported to exhibit lower binding energies than ciprofloxacin (−6.77 Kcal/mol). The top three compounds (NCI371878, NCI371876 and NCI142159) reported with binding energy of −13.5, −13.19 and −13.03 Kcal/mol were further subjected to MD simulation studies for 100 ns supporting the stability of the docked complexes.

Localization of Mycobacterium tuberculosis topoisomerase I C-terminal sequence motif required for inhibition by endogenous toxin MazF4.

Only about half the multi-drug resistant tuberculosis (MDR-TB) cases are successfully cured. Thus, there is an urgent need of new TB treatment against a novel target. Mycobacterium tuberculosis (Mtb) topoisomerase I (TopA) is the only type IA topoisomerase in this organism and has been validated as an essential target for TB drug discovery. Toxin-antitoxin (TA) systems participate as gene regulators within bacteria. The TA systems contribute to the long-term dormancy of Mtb within the host-cell environment. Mtb's toxin MazF4 (Rv1495) that is part of the MazEF4 TA system has been shown to have dual activities as endoribonuclease and topoisomerase I inhibitor. We have developed a complementary assay using an Escherichia coli strain with temperature-sensitive topA mutation to provide new insights into the MazF4 action. The assay showed that E. coli is not sensitive to the endoribonuclease activity of Mtb MazF4 but became vulnerable to MazF4 growth inhibition when recombinant Mtb TopA relaxation activity is required for growth. Results from the complementation by Mtb TopA mutants with C-terminal deletions showed that the lysine-rich C-terminal tail is required for interaction with MazF4. Site-directed mutagenesis is utilized to identify two lysine residues within a conserved motif in this C-terminal tail that are critical for MazF4 inhibition. We performed molecular dynamics simulations to predict the Mtb TopA-MazF4 complex. Our simulation results show that the complex is stabilized by hydrogen bonds and electrostatic interactions established by residues in the TopA C-terminal tail including the two conserved lysines. The mechanism of Mtb TopA inhibition by MazF4 could be useful for the discovery of novel inhibitors against a new antibacterial target in pathogenic mycobacteria for treatment of both TB and diseases caused by the non-tuberculosis mycobacteria (NTM).

High-performance liquid chromatography evaluation of lipophilicity and QSRR modeling of a series of dual DNA gyrase and topoisomerase IV inhibitors

AbstractBacterial DNA gyrase and topoisomerase IV control the topological state of DNA during replication and represent important antibacterial drug targets. To be successful as drug candidates, newly synthesized compounds must possess optimal lipophilicity, which enables efficient delivery to the site of action. In this study, retention behavior of twenty-three previously synthesized dual DNA gyrase and topoisomerase IV inhibitors was tested in RP-HPLC system, consisting of C8 column and acetonitrile/phosphate buffer (pH 5.5 and pH 7.4) mobile phase. logD was calculated at both pH values and the best correlation with logD was obtained for retention parameter φ0, indicating that this RP-HPLC system could be used as an alternative to the shake-flask determination of lipophilicity. Subsequent QSRR analysis revealed that intrinsic lipophilicity (logP) and molecular weight (bcutm13) have a positive, while solubility (bcutp3) has a negative influence on this retention parameter.

Genetic Engineering in Mycobacteria

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.