Thesis (Phd (Chemistry and Polymer Science))--University of Stellenbosch, 2010. / Dissertation presented for the degree of Doctor of Philosophy (Chemistry) at Stellenbosch University. / ENGLISH ABSTRACT: The human pathogen Staphylococcus aureus is a major cause of hospital-, and more recently, community-acquired infections. The rate at which this organism is acquiring resistance to antibiotics is increasing while the development of new antibiotics is slowing down. There is therefore a desperate need for new antistaphylococcal agents, and in particular ones with novel mechanisms of action that can be used to circumvent established resistance pathways. Unlike humans, S. aureus employs the essential cofactor coenzyme A (CoA) as its major low molecular weight thiol. Together, CoA and the enzyme CoA disulfide reductase (CoADR) are responsible for maintaining the internal redox homeostasis in this organism, and disruption of this balance (or reduction of CoA levels) may therefore be potential mechanisms by which new antistaphylococcal agents may act. In this study we set out to achieve this by direct inhibition of CoADR, and by inhibition of one or more of the CoA biosynthetic enzymes. For the inhibition of CoADR CoA analogues containing Michael acceptors were designed and prepared by employing a chemo-enzymatic approach. This strategy involved the chemical synthesis of pantothenamides containing α,β-unsaturated ester, ketone and sulfone moieties as Michael acceptors, followed by their biotransformation into the corresponding CoA analogues by three CoA biosynthetic enzymes. The compounds prepared in this manner all inhibited CoADR potently. A full kinetic evaluation of the inhibition by these compounds suggested that these compounds act by alkylation of the single active site cysteine of CoADR in an irreversible fashion. In this study we also set out to determine the mechanism of action of the antistaphylococcal compound CJ-15,801, which is structurally similar to pantothenic acid, the biosynthetic precursor of CoA. Due to this similarity we proposed that the antibiotic properties of CJ-15,801 are based on the inhibition of enzymes involved in CoA biosynthesis and metabolism. Our investigations confirmed that the second enzyme of the CoA pathway, phosphopantothenoylcysteine synthetase (PPCS), acts as the main target of CJ-15,801. These studies were followed by an investigation into alternative synthetic methodologies for the preparation of CJ-15,801 and its analogues. As a result an established Pd-catalyzed coupling reaction was modified and applied in the third known total synthesis of CJ-15,801, as well as of several of its analogues. This protocol has several advantages over its predecessors, most importantly its suitability for preparing these compounds on large (up to one gram) scale. / AFRIKAANSE OPSOMMING: Die menslike patogeen Staphylococcus aureus is 'n wesenlike oorsaak van hospitaal- en meer onlangs gemeenskap-verworwe infeksies. Terwyl die tempo waarteen hierdie organisme weerstandbiedig teenoor antibiotika raak toeneem, neem die ontwikkeling van nuwe antibiotiese middels af. Dit is dus van kardinale belang dat nuwe antistafilokokale middels ontwikkel word, en meer spesifiek antibiotika met 'n nuwe meganisme van aksie wat gebruik kan word om huidige weerstandbiedende padweë te ontwyk. In teenstelling met mense, gebruik S. aureus die essensiele kofaktor koënsiem A (KoA) as sy vernaamste lae molekulere gewig tiol. Die ensiem KoA disulfied reduktase (KoADR) en KoA is saam verantwoordelik om die interne redoks homeostase in hierdie organisme te handhaaf, en ontwrigting van die balans (of vermindering van KoA vlakke) kan dus potensieel 'n meganisme van aksie wees waardeur nuwe antistafilokokale middels kan optree. In hierdie studie het ons gepoog om dit te bewerkstellig deur KoADR direk te inhibeer, asook deur inhibisie van een of meer van die KoA biosintetiese ensieme. Vir die inhibisie van KoADR is KoA-analoë wat Michael-akseptor groepe bevat ontwerp en berei deur van 'n chemo-ensiematiese benadering gebruik te maak. Met hierdie strategie is pantoteenamiede gesintetiseer wat α,β-onversadigde ester, ketoon en vinielsulfoon funksionaliteite as Michael-akseptore bevat, gevolg deur biotransformasie na die ooreenstemmende KoA-analoë met behulp van drie CoA biosintetiese ensieme. Die verbindings gesintetiseer met hierdie metode het almal KoADR potent geinhibeer. 'n Omvattende kinetiese evaluasie het voorgestel dat al hierdie verbindings funksioneer deur alkielering van die enkele aktiewe setel sisteïen van KoADR op 'n onomkeerbare wyse. In die studie het ons ook gepoog om die meganisme van aksie van die antistafilokokale verbinding CJ-15,801 te bepaal. Hierdie verbinding is struktureel soortgelyk aan pantoteensuur, die biosintetiese voorganer van KoA. As gevolg van hierdie ooreenkomste het ons voorgestel dat die antibiotiese aktiwiteit van CJ-15,801 die gevolg is van die inhibisie van een of meer van die ensieme wat verantwoordelik is vir KoA biosintese en metabolisme. Ons ondersoeke het bevestig dat die tweede ensiem in die KoA biosintetiese padweg, naamlik fosfopantotenoïelsisteïensintetase, die hoofteiken van CJ-15,801 is. Hierdie studies is gevolg deur die ondersoek van alternatiewe metodologieë vir die sintese van CJ-15,801 en analoë daarvan. 'n Gevestigde Pd-gekataliseerde koppelings reaksie was gevolglik gemodifiseer en toegepas om slegs die derde totale sintese van CJ-15,801 te bewerkstelling, asook die sintese van verskeie analoë daarvan. Hierdie protokol hou verskeie voordele in vergelyking met sy voorgangers, waarvan die mees belangrikste die bereiding van hierdie verbindings op groot (tot een gram) skaal is.

Thesis advisor: Mary F. Roberts / There is substantial interest in exploring antibiotic alternatives with a new mode of action due to the increasing rates of bacterial resistance against current antibiotics. Antimicrobial peptides (AMPs) may take up the battle against bacteria in the future because as a result of their membrane-lysis mechanism, it is more difficult for bacteria to develop resistance against AMPs. Although AMPs could preferentially bind to and disrupt negatively charged bacterial membranes through electrostatic and hydrophobic interactions, there is still a great need to further increase the potency and selective toxicity towards bacteria for clinical applications. Herein, we present two strategies to improve the selectivity: light activation and environment-responsive moiety incorporation. Along the way, we also explored the effect of structure stabilization on AMPs action. A well-characterized antimicrobial peptide magainin 2 (mag2) was used as a prototype. Chemical manipulations of mag2 sequence were achieved by incorporation of unnatural amino acids. The selectivity was then tested on liposomes as a membrane model, as well as on bacterial cells and human red blood cells (hRBCs). Different extents of selectivity enhancement were observed from the modified peptides, and within the attempts to illustrate these results, we have gained useful information revealing the membrane-lysis mechanism, which may help us to rationally design and engineer AMPs as therapeutic drugs in the future. / Thesis (MS) — Boston College, 2011. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis (M. Diploma in Technology (Chemistry))--Cape Technikon, 1994 / Some naturally occurring compounds containing the naphtho [2,3 - c] pyran ring system have been found to be useful antibiotic agents and thus the laboratory synthesis ofthese compounds and their derivatives are of importance in the medical field. Their antibiotic and even antineoplastic activities are attributed to their potential to act as alkylating agents, via a bioreductive process. This thesis deals with studies directed towards the synthesis of some benzo [c] pyrans as these compounds also possess the correct structural configuration to undergo the bioreductive process, and act as alkylating agents to cellular nucleic acids. Chapter Two describes the synthesis 00,4 dihydro - 1,3 - dimethyl- IH - benzo - [cl pyran - 5,8 - quinone (13) by employing a base induced cyclization with potassium tertiary butoxide. This compound was proven to be biologically active against both Gram positive and Gram negative organisms. (±) (1R, 3R, 4R) - 3,4 - Dihydro - 4 - hyroxy -1,3 - dimethyl- 1H - benzo [e] pyran - 5,8quinone (17) and its 4 S diastereomer (18) were synthesized with cerium (IV) ammonium nitrate as the cyclizing reagent. Antimicrobial evaluation ofcompound (17) displayed inhibitory activity against both Gram positive and Gram negative organism growth. This is discussed in Chapter Three. In Chapter Four, the synthesis ofthe benzo analogue ofthe naturally occurring naphthopyran antibiotic, hongconin, is discussed. The synthetic route used for this synthesis could well be applied to synthesise hongconin.

Bacterial infections are an increasing cause for concern as resistance spreads to the majority of our front line antibiotics. To counter antibiotic resistance, new treatment regimens and drug targets are being investigated, including directly targeting bacterial virulence (pathogen-induced harm to the host), and therapies which target resistance mechanisms. The outcome of successful treatment with these compounds is not always killing or halting growth of bacteria, therefore selection for resistance to these types of therapeutics is complex. This complexity is increased by the secretion of many virulence factors, meaning their effects are shared with neighbouring individuals. In addition virulence factors show high phenotypic plasticity due to regulation by processes like quorum sensing (QS), which further complicates treatments targeting virulence, or the regulatory processes themselves. Using the example of quorum sensing inhibitors this study shows the importance of understanding the function and ecology of targeted virulence factors, to predict the selection for resistance to anti-virulence drugs. Later chapters elaborate on this to show how quorum sensing control affects selection on secreted virulence factors. The use of anti-virulence drugs as adjuvants is discussed, with a study showing that the interaction between QS inhibition and translation inhibitors is dependent on the environment. The selection for resistance to combinations of antibiotics and adjuvants is investigated using co-amoxiclav as an example, showing that treatment with high doses of adjuvant are robust to the evolution of resistance.

Objective: To retrospectively evaluate antibiotic use in pediatric appendectomy procedures following an educational intervention in December 2001. Methodology: Demographic, clinical, and prescribing data was collected for all the patients <18 years old who have had undergone non-perforated appendectomy procedures at Princess Margaret Hospital for Children, WA. Data collection and analysis were divided into three groups. Group-I involved patients from May 2002 to April 2004 (which followed the post-intervention follow-up conducted from December 2001-April 2002 by Mallik et al.1). In May 2004, the Western Australian Therapeutic Advisory Group (WATAG) sent an advisory note which recommended a change from the use of cefotetan for surgical prophylaxis to cephazolin plus metronidazole. Group-II of the study involved patients between May 2004 (when the WATAG note was released) and June 2004; while Group-III involved patients from July 2004 to April 2005 (when the hospital issued the new guidelines and withdrawn cefotetan).Patient records were randomly selected for Group I & III and all the records were evaluated for Group III. Results: Records for 408 patients were evaluated across the three groups of the study. There no significant difference (p>0.05) between gender and age across the three groups. An appropriate prophylactic drug regimen was prescribed in 68.5%, 66.7% and 39.8% of patients in Groups I, II and III respectively, with a significant difference in appropriate drug choice between Groups I and III (p <0.05). There was no significant difference between the groups with respect to appropriate prophylactic drug dose (p>0.05). Appropriateness rates for antibiotic choices for ward treatment were high at 91.0%, 92.0% and 92.7%, with no significant differences (p>0.05). / There was a significant difference (p<0.05) between the three groups regarding the number of doses for ward treatment, with inappropriateness rates of 29.9%, 40% and 16.4%. The total appropriateness rates (drug choice plus dose in theatre and ward) across the study were 54.7%, 54.2% and 31.5%, with a significant difference (p <0.05) between Groups I and III. Conclusion: This study has identified deficiencies related to the prescribing of antibiotics for prophylaxis. There was a varied level of prescribing appropriateness in terms of antibiotic choice for prophylaxis with an increasing trend for inappropriateness towards the end of the study period. This would indicate that issuing of changed guidelines and withdrawal of the drug being replaced did not positively influence appropriate prescribing. Further interventions are required to improve compliance with hospital prescribing guidelines.

Graduation date: 1999

A method for measuring cell concentration and identity based on flow cytometry (FCM) and fluorescent marking is developed and subsequently compared with traditional plating based methods, with regards to performance, economy and ergonomy. The emphasis is on competitive growth of bacteria under antibiotic stress, but the technique could be used in any situation requiring fast, high throughput counting and identification of cellular populations. The method needs further development, but shows potential as a parallelizable and fast alternative to plating.

Thesis (M.D.S.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-107).

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: antimicrobial peptides; protegrins; synthetic genes. Includes bibliographical references (p. 56-57).