A dissertation submitted to the Faculty of Science University of the Witwatersrand, Johannesburg for the degree of Master of Science. Johannesburg, 1994. / Molecular mechanics calculations are used to interpret and predict metal ion discrimination by coordinating ligands. Of particular interest are chelates exhibiting characteristics that Single them out for potential medical application; Selectivity patterns for several series of ligands are investigated with the help of strain energy profiles as a function of metal-donor atom bond distance. Ligands include simple; open-chain oxygen- and nitrogen-donors ana triaza- and tetraazamacrocyeles. Results are compared with X-ray crystallographic and solution data. Factors such as chelate ring size, conformational flexibility and preferred metal coordination geometry are found to influence metal specificity. Addition of pendent donor groups to macrocyoles leads to rigid structures and selectivity predictions according to cavity size. Interpretation of specific. metal ion recognition by polyetner antibiotics is attempted. Structural and steric factors are probed as possible determinants of metal choice. both covalent and ionic bonding models are explored. The covalent approach results in predictions of metal selectivity which correlate with mown selectivity patterns. Unfortunately, inability to optimise force field parameters in the ionic bonding approach forced us to abandon this model. The main force field used is the TRIPOS (1992,1993) force field. It performs well in calculations involving a univariate scanning technique but has to be modified to obtain reasonable structure reproduction with the large antibiotics, Errors in thermodynamic data predictions are obtained, nonbonding parameters have yet to be properly parameterized and the allocation of partial atomic charges warrants closer examination . All of these factors contribute to the poor performance of the force field when ionic interactions between metal and donor atoms of the polyethers are assumed. / AC2017

With antimicrobial resistance to current drugs steadily rising, the development of new antibiotics with novel mechanisms of action has become an imperative. The majority of life-threatening infections worldwide are caused by "ESKAPE" pathogens which are encountered in more than 40% of hospital-acquired infections, and are resistant to the majority of commonly used antibiotics. Naturally occurring cyclic depsipeptides, microbial secondary metabolites that contain one or more ester bonds in addition to amide bonds, have emerged as an important source of pharmacologically active compounds or lead structures for the development of novel antibiotics. Some of those peptides are either already marketed (daptomycin) or in advanced stages of clinical development (ramoplanin). Structurally simple, yet potent, fusaricidin/LI-F and lysobactin families of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents capable of overco ming infections caused by multidrug-resistant bacteria. These natural products exhibit potent antimicrobial activity against a variety of clinically relevant fungi and Gram-positive bacteria. Therefore, access to these classes of natural products and their synthetic analogs, combined with elucidation of their mode of action represent important initial steps toward full exploitation of their antmicrobial potential. This dissertation describes a general approach toward the solid-phase synthesis of fusaricidin/LI-F and lysobactin analogs and an extensive structure-activity relationship (SAR) study. We have devised a simple and robust preparation strategy based on standard Fmoc solid-phase peptide synthesis protocols. / The SAR study revealed key structural requirements for fusaricidin/LI-F and related cyclic lipopeptides antibacterial activity, including the presence of the guanidino moietly at the end of the lipidic tail, hydrophobic amino acid residues, and peptide conformation Moreover, substitution of the ester bond with an amide bond significantly improved stability under physiologically relevant conditions and reduced toxicity. In addition, we have shown that these antibacterial peptides exert their mode of action via a novel mechanism, which invloves bacterial membrane interactions, followed by peptide internalization. Altogether, the research described in this dissertation demonstrates that new antibiotics derived from fusaricidin/LI-F natural products, have the potential to meet the challenge of antibiotic resistance in Gram-positive bacteria. / by Nina Bionda. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Autolysins are endogenous enzymes responsible for the cleavage of specific bonds in the bacterial sacculus resulting in damage to the integrity and protective properties of the cell wall. The true biological functions of these enzymes are largely unknown. However, they have been implicated in various important biological synthesis processes making their characterization important. Antibiotic susceptibility testing showed these streptococcal strains to have broad spectrum inhibitory concentrations. The major autolysins of selected streptococcal strains were detected and partially characterized by renaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (zymograms). The autolysins were isolated from the specific culture supematants using 4% SDS precipitation and were shown to have apparent molecular masses ranging from 60kDa to 20kDa. Four major autolysins named A, B, C, and D from the Streptococcus milleri 77 strain were characterized. Lytic enzymes were blotted onto polyvinylidene difluoride (PVDF) membrane and N-terminally sequenced. Sequences showed between 100% and 80% similarity to that of a muramidase, glucosaminidase and a peptidase from S. mutans, S. pyogenes and S. pneumonia respectively. Biochemical characterization confirmed autolysin A to exhibit muramidase activity with both autolysin Band C exhibiting endopeptidase activity. Autolysin D showed an 80% N-terminal sequence similarity to Millericin B, a peptidoglycan hydrolase that is known to exhibit peptidase activity. Autolysis was determined using different buffers at two optimal pHs. Assaying for autolytic activity at different growth stages showed autolysis to be moderate during the lag and early exponential phases of the growth cycle. The activities of autolysins were the highest in the late exponential phase and the stationary phase of growth. Zymogram analysis showed that the Streptococcal milleri strains had moderate autolytic expression during the early and late exponential phases of the growth cycle. Control regulatory mechanisms of autolysins were determined in the presence or absence of specific charged groups, such as teichoic acids. In each case the absence of these charged groups inhibited the rate of autolysis, suggesting that the absence of teichoic acids could play a role in the regulation of the autolysins. Two-dimensional-SDS and zymographic-electrophoresis was used to determine total protein profiles for each strain. This is the first report using twodimensional zymography. Specific proteins which were either up- or down-regulated were identified. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.