A novel predictive model for the anti-bacterial, anti-malarial and hERG cardiac QT prolongation properties of fluoroquinolones

Abbreviations FQs Fluoroquinolones, BBB Blood brain barrier, TdP Torsades de Pointes, GI gastrointestinal tract, IC 50 Half maximal Inhibitory concentration , MIC Minimum inhibitory concentrations, ZW zwitterion, steady state uptake (R ss), F blood plasma concentrations, CBP Chronic bacterial prostatis, BSA bovine serum albumin, MRP2 Multidrug resistance-associated protein 2 or specific organic anion transporter 1 (cMOAT) or ATP-binding cassette sub-family C member 2 (ABCC2), OATP organic anion uptake transporter, OCT organic cation influx transporter, ΔG desolvation free energy of water desolvation, ΔG lipophilicity free energy of lipophilicity or hydrophobicity, ΔG desolv,CDS free energy of water desolvation of the cavitation dispersion solvent structure (CDS), ΔG lipo,CDS free energy of lipophilicity or hydrophobicity for the CDS, DM dipole moment DM, SASA solvent accessible surface area, R 2 multiple correlation coefficient, F the F test of significance, SEE standards errors for the estimates, SE(ΔG desolvation) standard errors of ΔG desolvation , SE(ΔG lipophilicity), standard errors of ΔG lipophilicity , SE(Dipole Moment) standard errors for dipole moments, SE (Molecular Volume) standard errors for molecular volumes as calculated from " t " distribution statistics, QM quantum mechanics

Rapid evolution of reduced susceptibility against a balanced dual targeting antibiotic through stepping-stone mutations

Multi-targeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets are generally considered as a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multi-targeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here we propose that such individual, stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an over 2000-fold reduction in susceptibility, while individually none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, co-selected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones towards resistance against antimicrobial compounds still under development. Our research indicates that even balanced multi-targeting antibiotics are prone to resistance evolution

Structure and Mechanism of Mycobacterial Topoisomerase I

The enzyme DNA topoisomerase I is an essential enzyme that plays an important role in eukaryotic and prokaryotic cellular processes such as DNA replication, transcription, recombination and repair. Mycobacterium tuberculosistopoisomerase I (MtTOP1) is a validated drug target for antituberculosis treatment. Mycobacterial topoisomerase I regulates the topological constraints in chromosomes and helps in maintaining the growth of mycobacteria. The N- terminal domain (NTD) of mycobacterial topoisomerase I contains conserved catalytic domains that along with the active site Tyrosine are involved in cleaving and rejoining a single strand of DNA. Magnesium is required in DNA cleavage activity of type IA topoisomerases. The C-terminal domain (CTD) of mycobacterial topoisomerase I is divided into four subdomains (D5-D8) and a positively charged tail. Each subdomain has a GxxGPY sequence motif. The DNA binding, relaxation, cleavage, religation, catenation and decatenation ability of each subdomains of CTD were studied. The present study shows that each subdomain has its own characteristics. Subdomain D8 and D7 are responsible for maintaining the relaxation activity of mycobacterial topoisomerase I. Subdomain D5 is essential to maintain the DNA cleavage, religation, catenation and decatenation activity. A new crystal structure of MtTOP1-704t (amino acids A2-T704 containing NTD+D5 domains) was obtained. Structures with ssDNA substrate bound to the active site (Y342) in the presence and absence of Mg2+ were also investigated. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition position for cleavage of a specific phosphodiester linkage to form a covalent intermediate. Meanwhile, the enzyme/DNA complex with Mg2+ bound at active site may present the post- transition state for religation in the enzyme’s multiple-state DNA relaxation activity. The critical function of a strictly conserved glutamic acid in acid-base catalysis of the DNA cleavage step was also demonstrated by site-directed mutagenesis. The present work provides new functional insights into the more stringent requirement for DNA rejoining versus cleavage by type IA topoisomerase, and further establishes the potential for select interference of DNA rejoining via specific inhibitors

Architecture and conservation of the bacterial DNA replication machinery, an underexploited drug target

New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials. DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets

Architecture and Conservation of the Bacterial DNA Replication Machinery, an Underexploited Drug Target

New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials

Molecular profile of gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana.

Doctoral Degree. University of KwaZulu-Natal, Durban.Gram-negative ESKAPE (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp) pathogens are a major healthcare concern globally due to their increasing multidrug resistance and ability to cause debilitating infections. Phenotypic and genotypic characteristics of multidrug resistant Gram-negative ESKAPE pathogens from Komfo Anokye Teaching Hospital in Ghana were investigated. Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human specimens routinely processed by the diagnostic microbiological laboratory in the hospital. Multidrug resistant (isolates resistant to at least one agent in three or more antibiotic class) isolates selected from each group of Gram-negative ESKAPE pathogens constituted the final sample. Identification and antibiotic susceptibility profiles were carried out using Vitek-2. Identity of isolates for whole genome sequencing was further confirmed by MALDI-TOF MS. Four P. aeruginosa and 10 K. pneumoniae were subjected to whole genome sequencing based on their extensively drug resistant profiles and resistance to third-generation cephalosporins respectively using Illumina MiSeq, after genomic DNA extraction using the NucliSens easyMAG®. Antibiotic resistance genes and plasmids were identified by mapping the sequence data to an online database using ResFinder and plasmidFinder respectively. MLST was also determined from the WGS data. The raw read sequences and assembled whole genome contigs have been deposited in GenBank under project number PRJNA411997. An average multidrug resistance of 89.5% was observed, ranging from 53.8% in Enterobacter spp to 100.0% in Acinetobacter spp and P. aeruginosa. Gram-negative ESKAPE bacteria constituted 48.5% (97) of the 200 isolates. P. aeruginosa (n=4) belonging to ST234 harboured blaDIM-1, blaIMP-34, blaOXA-10, blaOXA-129, blaOXA-50, blaPAO aadA1, aac4 aph(3’)-IIb, fosA, sul1, dfrB5, catB7, arr-2 conferring resistance to β-lactams, aminoglycosides, fosfomycin, sulphonamides, trimethoprim phenicols and rifampin respectively. qnrVC was detected in two of the four isolates . Both blaDIM-1 and blaIMP-34-like positive contigs showed identical DNA sequences and were linked to type 1 integron structures. BlaDIM-1 was 100% identical to the blaDIM-1 prototype gene, while blaIMP-34-like had two base pair (bp) differences T190C and C314G respectively compared to blaIMP-34, leading to one amino acid substitution in IMP-34-like indicating that, the gene may have independently evolved, perhaps due to selection pressure. Blast analysis did not reveal identical genetic structures deposited in NCBI, neither among the nucleotide collection, completed genomes nor among the completed plasmids. β-lactamases (blaCTX-M-15, blaSHV-11, blaTEM-1B) and resistance genes for aminoglycosides (aac(3)-IIa-like,aph(3')-Ia) quinolones/fluoroquinolones (oqxA-like,oqxB-like,qnrB10-like,qnrB2) and others including fosfomycin (fosA), trimethoprim (dfrA14), and sulphonamide (sul2) were found in the K. pneumoniae (n=10). Multiple and diverse mutations of the quinolone resistance-determining regions gyrA, gyrB and parC genes were detected in the K. pneumoniae (n=4), which were clonally distinct. The diversity of resistance genes expressed by Gram-negative ESKAPE pathogens conferring resistance to multiple antibiotics is problematic in a resource-constrained country like Ghana, necessitating urgent antibiotic stewardship and infection prevention and control interventions

Computational Protein-Ligand Modelling of the Enzymes DNA gyrase and IcaB

Computational modelling of proteins and their interactions with small molecule ligands is a growing field of research. Such studies provide an understanding of how protein structure relates to mechanism and function as well as informing drug discovery and design. This thesis had two main aspects: computational modelling of ciprofloxacin derivatives binding to DNA gyrase and homology modelling of the protein IcaB based on sequence alignment with a related protein, PgaB. The inhibitory activity of synthetic ciprofloxacin derivatives (with various linkage to citrate groups) was experimentally assessed by gel electrophoresis to examine the effect on DNA gyrase binding to a target DNA strand. Overall, the derivative which possessed the greatest inhibition compared to the unmodified ciprofloxacin was the c-gly-ciprofloxacin derivative, which had a 2 atom linker between the ciprofloxacin and citrate groups. This correlated with the change in interactions seen between ciprofloxacin derivatives as computationally modelled by molecular mechanics methods. The second aspect of the thesis was to generate a model of the protein IcaB to test the hypothesis that it is a deactylase of poly-N-acetyl-glucosamine (PNAG) during maturation of the poly-glycan in the extracellular matrix responsible for biofilm generation for bacteria. An initial review of deacetylase enzyme structures identified the conserved features required for activity. A homologous protein, Pga,B was then used as a template to generate a homology model of IcaB. The model maintained the orientation and positioning of the metal-binding and catalytic residues critical for proper deacetylase function. However, the PNAG binding groove, believed to be involved in the transport of the PNAG to the active site of PgaB, was not properly replicated in the IcaB model. Further modelling would require improved characterization of the binding groove of IcaB

Leishmaniasis

Leishmaniasis is a major global health challenge, affecting approximately 12 million of the poorest people in 100 countries. It is a deforming and fatal disease in the visceral form. Therapies for leishmaniasis are numerically restricted, basically consisting of the administration of miltefosine, pentavalent antimonials, amphotericin B, or pentamidine. This is an important vulnerability against therapy efficiency that must be overcome by the scientific community. This book discusses important aspects of the disease, such as treatment, epidemiology, and molecular and cell biology. The information contained herein is important for young researchers as they seek to develop safe and effective treatments for this neglected tropical disease

Structural studies of toxins and toxin-like proteins

Toxins are an ancient mechanism of interaction between cohabiting organisms: basal concentrations serve as an informal cue, enough as a warning signal; too much and the dialog is over. As such, the evolutionary race to arms led to the development of a vast trove of molecular unique biochemical mechanisms, from small molecules to protein toxins. The study of these mechanisms is not only essential for the treatment of toxin-related pathologies, but also as the potential source for novel therapeutic drugs. In this thesis, a series of studies of different toxins and toxin-like proteins are compiled. To further understand the biological function and relevance of each toxin, their detailed study and characterization were pursued. Here are described the advances made using a combination of different complementary biophysical and structural methods, chosen in each case to specifically target each molecule characteristics. In the first chapter, the general biological theme of this thesis is introduced: toxins, particularly protein toxins, their description, and classification, as well as the role of structural biology in the study of proteins in general. To set the theoretical background of the following chapters, are also described the general principles of two of the most prominent methods for the study of proteins in structural biology: nuclear magnetic resonance (NMR) spectroscopy, and X-ray diffraction. In the second chapter, the interaction between human FKBP12 chaperone protein and two similar bacterial small molecule toxins is detailed: rapamycin initially used as an anti-fungal before the discovery of its potent immunosuppressive properties as a mTOR inhibitor; and mycolactone, a bacterial toxin responsible for the disease Buruli ulcers in humans. In the third chapter, the cell-free protein expression system is introduced as a technique best suited for the expression of cytotoxic proteins and otherwise difficult targets, as explored further in the following chapters. In the fourth chapter, advancements towards the structural and conformational characterization of the membrane-inserted state of two similar pore-forming toxins are detailed: the bacterial Colicin Ia protein; and the human Bax protein, an apoptosis effector; using X-ray crystallography, solution NMR and solid-state NMR. Finally, in the fifth chapter, two FIC-domain bacterial toxins are investigated: the bacterial VbhTA toxin-antitoxin protein complex, and the structural determination with its cognate target, DNA GyraseB enzyme; and the auto-activation of the bacterial NmFIC protein; in both cases using a combination of X-ray crystallography and NMR spectroscopy, as well as other biophysical techniques