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An investigation into the antifungal activities of N-thiolated beta lactams against selected Candida species

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Title:
An investigation into the antifungal activities of N-thiolated beta lactams against selected Candida species
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Language:
English
Creator:
Culbreath, Marci
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Mode of Action -- Structure Activity Relationship -- Minimum Inhibitory Concentration -- Transmission Electron Microscopy -- Fungistatic
Structure activity relationship
Minimum inhibitory concentration
Transmission electron microscopy
Fungistatic
Dissertations, Academic -- Chemistry -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Beta-lactam antibiotics have long been a reliable course of treatment for bacterial infections. However, with recent increases in resistance and rising populations of immunocompromised patients new beta-lactams have been synthesized and tested. The Turos laboratory has recently discovered novel beta-lactams that have a mode of action distinct from penicillin and other beta-lactam antibiotics as cell lysis is not observed. In the current investigations, these compounds are shown to also have antifungal properties. The rising incidence and prevalence of invasive fungal infections has become an increasing concern. The most common fungal pathogens involved in these infections are species in the genus Candida. In this study antifungal activity is observed for a wide range of N-methylthio B-lactams against C. albicans, C. tropicalis, C. keyfr, C. glabrata, C. lusitinae, C. utilis, and C. parapsilosis. The structure-activity relationship based on studies of beta-lactam derivatives leaving different substituents at various positions on the lactam ring are investigated, and the minimum inhibitory concentration values determined using standard methods. In studies towards understanding the mode of action, the products of the interaction between the drug and fungal cells in a suspension were investigated using nuclear magnetic resonance spectroscopy and transmission electron microscopy. The mode of action of these new lactams seems to be similar to that observed in bacteria, involving transfer of the methylthio group to a cellular thiol.
Thesis:
Thesis (M.A.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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by Marci Culbreath.
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Title from PDF of title page.
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Document formatted into pages; contains 68 pages.

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aleph - 001798176
oclc - 157010381
usfldc doi - E14-SFE0001633
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An Investigation into the Antifungal Activities of N-Thiolated BetaLactams Against Selected Candida Species by Marci Culbreath A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Edward Turos, Ph.D. Daniel Lim, Ph.D. Diane TeStrake, Ph.D. Date of Approval: May 12, 2006 Keywords: Mode of Action, Structure Activity Relationship, Minimum Inhibitory Concentration, Transm ission Electron Microscopy, Fungistatic Copyright 2006 Marci Culbreath

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i Table of Contents List of Tables iii List of Figures iv Abstract vi Chapter One: Introduction 1 Rising Incidence and Prevalence of Fungal Infection 1 Etiology of Invasive Fungal Infections 2 Overview of Current Therapies 4 Polyenes 4 Antimetabolites 5 Azoles 6 Echinocandins 8 Emerging Resistance 10 N-Thiolated -Lactams 12 Synthesis 14 Chapter Two: Studies on Antifungal Activity 17 Spectrum of Activity Against Candida species 17 Structure-Activity Relationships 19 Effects of C4 Substitution on Antifungal Activities on NThiolated -Lactams 20 Effects of C3 Substitution on Antifungal Activities on NThiolated -Lactams 32 Effects of N-Substitution on Antifungal Activities on NThiolated -Lactams 36 Effects of Absolute Stereochem istry on Antifungal Activities on N-Thiolated -Lactams 38 Minimum Inhibitory Concentration 42 Determined using Agar Diffusion 42 Determined using Broth Macrodilution 43 Chapter Three: Studies Leading Towards Mode of Action 45 Fungistatic versus Fungicidal 45

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ii Broth Study 47 TEM Study 49 Chapter Four: Discussion 52 References 55 Appendices 58 Appendix A: NMR Spectra of Lactams Synthesized 58 Appendix B: NMR Spectra from Broth Study 62 Appendix C: TEM Images 65

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iii List of Tables Table 1 Incubation Temperatures 18 Table 2 Effects of Monofluorination on Antifungal Activities on N-Thiolated -Lactams 22 Table 3 Effects of Monochlorination on Antifungal Activities on N-Thiolated -Lactams 24 Table 4 Effects of Monobromination on Antifungal Activities on N-Thiolated -Lactams 26 Table 5 Effects of Monoiodination on Antifungal Activities on N-Thiolated -Lactams 28 Table 6 Effects of Multiple Fluorination on Antifungal Activities on N-Thiolated -Lactams 30 Table 7 Effects of Multiple Chlorinati on on Antifungal Activities on N-Thiolated -Lactams 32 Table 8 Effects of C3 Substitution on Antifungal Activities on N-Thiolated -Lactams 35 Table 9 Effects of N Substitution on Antifungal Activities on N-Thiolated -Lactams 38 Table 10 Effects of (+) and (-) Enantiomers of Lactam 25 on Antifungal Activities of N-Thiolated -Lactams 41 Table 11 Minimum Inhibitory Concentration Values for Lactam 4 Determined by Agar Dilution 43 Table 12 Minimum Inhibitory Concentration Values for Lactam 4 Determined by Broth Macrodilution 44

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iv List of Figures Figure 1 Structure of Amphotericin B 5 Figure 2 Structure of 5-Flucytosine 6 Figure 3 Structure of Ketoconazole 7 Figure 4 Structure of Fluconazole 7 Figure 5 Structure of Caspofungin 9 Figure 6 Generalized Fungal Cell Showing Sites of Antifungal Drug Activity 10 Figure 7 Structure of Penicillan versus N-Thiolated -Lactam 12 Figure 8 Structures of Previously Reported Antifungal Lactam Containing Compounds 13 Figure 9 Synthesis of N-Thiolated Lactams 15 Figure 10 Structures of Lactams Synthesized 16 Figure 11 Effects of Monofluorinati on on Antifungal Activities on N-Thiolated -Lactams 21 Figure 12 Effects of Monochlorina tion on Antifungal Activities on N-Thiolated -Lactams 23 Figure 13 Effects of Monobromination on Antifungal Activities on N-Thiolated -Lactams 25 Figure 14 Effects of Monoiodination on Antifungal Activities on N-Thiolated -Lactams 27

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v Figure 15 Effects of Multiple Fluorination on Antifungal Activities on N-Thiolated -Lactams 29 Figure 16 Effects of Multiple Chlorination on Antifungal Activities on N-Thiolated -Lactams 31 Figure 17 Effects of C3 Substitution on Antifungal Activities on N-Thiolated -Lactams 33 Figure 18 Effects of NSubstituti on on Antifungal Activities on N-Thiolated -Lactams 37 Figure 19 Enzymatic Purification of Lactam 25 39 Figure 20 Effects of (+) and (-) Enantiomers of Lactam 25 on Antifungal Activities of N-Thiolated -Lactams 40 Figure 21 Growth Study: Effect of Lactam 4 on Growth of Candida 46 Figure 22 Proposed Mechanism of Action 51

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vi An Investigation into the Antifungal Activities of N-Thiolated Lactams Against Selected Candida Species Marci Culbreath ABSTRACT -lactam antibiotics have long been a reliable course of treatment for bacterial infections. However, with recent increases in resistance and rising populations of immunocompromised patients new -lactams have been synthesized and tested. The Turos laboratory has recently discovered novel -lactams that have a mode of action distinct from penicillin and other -lactam antibiotics as cell lysis is not observed. In the current investigations, these compounds are shown to also have antifungal properties. The rising incidence and prevalence of invasive fungal infections has become an increasing concern. The most common fungal pathogens involved in these infections are species in the genus Candida In this study antifungal activity is observed for a wide range of N-methylthio -lactams against C. albicans, C. tropicalis, C. keyfr, C. glabrata, C. lusitinae, C. utilis, and C. parapsilosis. The structure-activity relationship based on studies of lactam derivatives leaving different substituents at various positions on the lactam ring are investigated, and the minimum inhibitory concentration values determined using standard methods. In studies towards understanding the mode of action, the products of the interaction between the drug and fungal cells in a suspension were investigated using nuclear magnetic resonance spectroscopy and transmission

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vii electron microscopy. The mode of action of these new lactams seems to be similar to that observed in bacteria, involving transfer of the methylthio group to a cellular thiol.

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1 Chapter One Introduction Rising Incidence and Prevalence of Candida Infections The rising incidence and prevalence of invasive fungal infections has become an increasing concern. Candida species are the most frequently isolated human fungal pathogens.1 Usually harmless commensal organisms, they are part of the normal human microflora of the mouth and gastrointestinal tract.2 However, in a state of altered homeostasis such as occurs during treatment with broad-spectrum antibiotics or in immunocompromized patients, Candida species are potent opportunistic pathogens.3 They are capable of producing infections at almost any site, varying in intensity from acute localized in fections to serious invasive infections (candidiasis, candidemia).4 The incidence of invasive cand idiasis and candidemia has risen rapidly over the past 20 years and has become a significant problem.5 Candida is now the fourth most common cause of nosocomial bloodstream infections in the US, surpassing gram negative bacilli in frequency.6 Candida infections account for 8 % of

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2 all septicemias.7 The patient population at risk for infection by Candida has also grown to include thse undergoing solid organ and stem cell transplants, those being treated for cancer, immunosuppressive therapy, AIDS, and those from premature birth, with advanced age and recovering from major surgery.8 The tremendous impact of these infections are apparent in terms of cost, morbidity and mortality. Mo st strikingly the attributable mortality to disseminated Candida infection is almost 50%.9 Etiology of Invasive Fungal Infections The etiology of Candida infections has also changed over the past twenty years. Candida albicans has long been and continues to be the leading etiologic agent of Candida infections. However, more recently there has been a growth in the number of cases of non-albicans Candida infections.10 Non-albicans species now account for greater then fifty percent of infections.11 The increased use of azoles such as fluconazole have positively selected for such less sensitive or resistant species as Candida krusei, Candida lusitaniae, and Candida glabrata.12 Candida glabrata is now the second most frequent causative agent of candidemia in the US.13 Candida glabrata has been associated with a digestive or urinary point of entry, especially with catheters.14 Candida krusei has

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3 been found in patients with solid tumors or leukemia.14 Candida lusitaniae is associated with urinary and respiratory infections as well as those arising from intravenous catheters and use of broadspectrum antibiotics.14 Both C. parapsilosis and C. tropicalis have been associated with use of intr avenous catheters, contamination of the infusate, and colonization of health care workers.14 C. tropicalis most frequently appears in patients with cancer or leukemia, and C. parapsilosis often occurs with long term parenteral alimentation.14 Interestingly, in other parts of the world, specifically in Latin American countries, C. tropicalis and C. parapsilosis usurp C. glabrata as the second most common agent of candidemia.15 The reasons for this are not well known. Apparently, the genus Candida and the infections that result include a rather disparate group of organisms that can grow as yeasts, but are not as closely related as one might expect of species in a single genus,16 evidenced by the variation of risk factors and even geographical differences in etiology. This presents a challenge in treatmen t as even the best currently available drugs are plagued by intrinsic and acquired resistance.

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4 Overview of Current Therapies Most antifungals target ergoster ol biosynthesis. This pathway converts acetic acid to the me mbrane sterol, ergosterol, using many of the same enzymes that mammalian cells use to produce cholesterol.17 There are however several enzymatic steps that are unique to the ergosterol pathway which are the targets for antifungals. The agents current ly in use can be grouped into several classes by their mode of action. Polyenes The polyenes, including amphoteric in B (Figure 1) and nystatin, are the broadest spectrum antifungals available. They are fungicidal, causing cell death by intercalating with the ergosterol in the membrane bilayer to produce small pores.18 These pores destroy the proton gradient by leaking cations, depolarizing the cell and eventually causing cell death.19

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5 Figure 1: Structure of Amphotericin B Amphotericin B delivered intrav enously has been the drug of choice for disseminated candidiasis.20 Despite its potent antifungal properties, amphotericin B has well documented toxicity as it also binds to cholesterol.21,22 The most serious side effect is nephrotoxicity. There have also been reports of both intrinsic resistance and acquired resistance in C. lusitaniae attributable to changes in membrane sterols.23 Antimetabolites Antimetabolites such as 5-flucytosine (Figure 2) disrupt DNA and protein synthesis. 5-Flucytosine is a nucleoside analogue that causes DNA miscoding.24 Since resistance to this drug develops

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6 rapidly,25 it is used in combination wi th amphotericin B in treatment of Candida endophtalmitis.20 Figure 2: Structure of 5-flucytosine Azoles Azoles include imidizoles (clotrimazole, miconizole, and ketoconazole (Figure 3)) and tria zoles (fluconazole (Figure 4)and itraconazole). Azoles inhibit cytochrome P450 dependent enzymes, more specifically, lanosterol demethylase, which is responsible for the conversion of lanosterol into ergosterol.26 This inhibition does not completely block the pathway at this point, but rather the methylated lanosterol is acted up on by enzymes downstream in the pathway to produce a toxic intermediate which is responsible for growth inhibition.27

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7 Figure 3: Structure of Ketoconazole Figure 4: Structure of Fluconazole These drugs have substantially fewer side effects than amphotericin B and are effective against oropharangeal and vaginal candidiasis. They are also used as prophylaxis for fungal infection in preparation for bone marrow tran splantation and to prevent oral candidiasis in HIV patients.20 However, many non-albicans species such as C. krusei and C. glabrata have innate resistance, and acquired resistance has been observed through several mechanisms.1 As stated earlier it is thought that the use of azoles

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8 has contributed to the rise in inci dence of these resistant species. Echinocandins The newest classes of antifungal agents include the echinocandins and their analogues, the pneumocandins. The first of which to be commercially available is caspofungin (Figure 5) They inhibit 1,3-D-glucan synthase which is responsible for forming polymers of 1,3-D-glucan, a component of the fungal cell wall.28 These drugs are very promising because little resistance has been reported even in flucon azole-resistant strains.29 Also, since mammalian cells do not contain 1,3-D-glucan, there is little toxicity.30

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9 Figure 5: Structure of Caspofungin As discussed antifungals currently in use can be grouped by their function, figure 6 depicts a generalized fungal cell and the sites of antifungal drug action.

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10 Figure 6: Generalized Fungal Cell Showing Sites of Antifungal Drug Activity Emerging Resistance As discussed in the previous section resistance is a growing problem for even the newest of anti fungal drugs. Both primary (or intrinsic) resistance and seconda ry (or acquired) resistance have been observed in Candida species. Mechanisms of resistance have been studied in great detail in many isolates of C. albicans and to a lesser extent in C. glabrata C. tropicalis, and C. krusei.25 Acquired

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11 antifungal resistance mechanisms tend to follow three distinct strategies: increased efflux, alteration of the targeted enzyme, and alteration of metabolism. Increased efflux is the main mechanism of azole resistance and results from an overexpression of ATP-binding cassette (ABC) transporter genes CDR1 and CDR2 from C. albicans,31 CgCDR1 and PDH1 from C. glabrata,32 and the major facilitator (MF) gene MDR1.33 The major difference between these two types of transporters is that ABC transporte rs will pump out almost all azoles whereas MF transporters only accept fluconazole.25 Alteration in the target enzy me either prevents the enzyme from binding the drug or prevents allosteric inactivation of the enzyme after binding of the drug. Target enzymes can also be overexpressed, resulting in activi ty despite the presence of the drug. Resistance to capsofungin is thought to be an example of this, a result of a mutation in glucan synthase.17 Alteration of metabolism refers to a mutation that prevents the buildup of a toxic metabolite resu lting from the presence of the drug. This can also refer to tolerance pathways.17 The fourth and much less documented mechanism is the alteration in sterol concentration, in which ergosterol is replaced by 7, 22-dieneol3-ol. This results in both azole and amphotericin B

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12 resistance.17 Since even the newest drugs have documented resistance, the continued study of antifungal resi stance, the search for new cellular targets and the development of no vel antifungal drugs are of the upmost importance. N-Thiolated -Lactams Our laboratory has been investigating a new series of monocyclic N-thiolated -lactams that act selectively on drug-resistant strains of Staphylococcus bacteria.34 -Lactam antibiotics have long been a reliable course of treatment for bacterial infections. Older lactams such as penicillin act by inhibiting peptidoglycan transpeptidation which is essential for bacterial cell wall synthesis. This eventually leads to cell death.35 N S O CH3 CH3 H N COOH O R N S R O R 3 R4 Figure 7: Structure of penicillin versus N-thiolated -lactam

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13 However, no cell wall deformation or cytoplasmic leakage is observed with N-thiolated -lactams in S. aureus bacteria.34 Additionally, these compounds differ structurally from older lactams in that they lack the acid ic ring functionality required for recognition by penicillin binding proteins as shown in Figure 7, and an N-organothio substituent is st rictly required for biological activity. This indicated that these compounds display a mode of action distinct from that of older -lactams.34 Fungal cell walls do not contain peptidoglycan therefore older -lactam antibiotics are ineffective against fungal infections. Modest antifungal activity has in the past been observed in some semisynthetic -lactam containing compounds, such as those shown in Figure 8.36 Clavams have also been found to be both bacteriostatic and fungistatic by distinct modes of action.37 N S C6H5CH2NHCSCH2CONH O CO2Na CH2OCOCH3 N S R O R'NH R’=C6H5OCH2CO; R”=CHO R’=C6H5OCH(CH3)CO; R”=COSK Figure 8: Structures of Previously Reported Antifungal -lactam Containing Compounds

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14 Thus, testing was performed to ascertain the activity of Nthiolated lactams against 8 Candida species, and after finding bioactivity, preliminary studies were conducted towards determining the mode of action. Synthesis Monocyclic N-thiolated lactams can be readily synthesized from commercially available precurso rs as previously reported by our laboratory.38 The synthesis begins with a condensation of an aldehyde with p-anisidine to form an imine. A Staudinger reaction or an acid chloride and the imine produces the N-protected lactam. Cerium ammonium nitrate is then used to remove the aryl protecting group. Finally, the deprotected lactam is Nalkylthiolated with an N-alkylthio ph thalimide. This is depicted in Figure 9. All compounds in this st udy were obtained in racemic form as solely the cis-diastereomers.

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15 Figure 9: Synthesis of N-thiolated lactams Syntheses of three monocyclic N-thiolated lactams were completed in this study. Their stru ctures are depicted in Figure 10. Lactams A and B were novel analog ues. The proton NMR spectra can be found in Appendix A.

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16N O SMe MeO Cl N O S MeO N O SMe MeO Lactam 4 Lactam A Lactam B Figure 10: Structures of Lactams Synthesized Hundreds of analogues of monocyclic N-thiolated lactams have been prepared by members of our laboratory. Twenty five previously synthesized lactams numbered 1-25 were also used in this study.

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17 Chapter Two Studies on Antifungal Activity Species of Candida were chosen based on their potential for pathogenicity. The following fung i were used for the antifungal evaluation of these N-thiolated -lactams : Candida albicans (ATCC 1111), Candida glabrata (ATCC 15126), Candida tropicalis (ATCC 1111), Candida parapsilosis (ATCC 1111), Candida krusei (ATCC 14243), Candida lusitaniae (ATCC 34449), Candida kefyr (ATCC 20409), and Candida utilis (ATCC 29950). Fungi were obtained commercially. Two additional Candida isolates, C. albicans and C. tropicalis were donated by Dr. Ray Widen from the University of South Florida School of Medicine. Spectrum of activity against Candida species Previously synthesized lactams were screened against 8 different Candida species. The method of test ing used to screen for activity was disk diffusion susceptibility testing.39 Although MIC (minimum inhibitory concentration) values are often used as the ideal susceptibility measure, the insolu bility of these compounds in

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18 aqueous media made MIC determinations impractical for the preliminary study. Method Culture preparation: A culture of each organism was grown on Yeast Nutrient Agar (Difco, MI) from a freezer stock in Yeast Nutrient Broth (Difco Laboratories MI) and glycerol. Plates were incubated at the temperature indicated in Table 1 below. Table 1: Incubation Temperatures Disk diffusion testing: A suspension of each fungal strain equivalent to McFarland standard 5 (approximately 1 x 109 CFU/mL) was prepared in 5mL of sterile saline solution. Using this standard suspension, Yeast Nutrient Base Ag ar was inoculated with a uniform lawn of a single fungi with a sterile swab and allowed to dry. Disks were impregnated with 50 g of test compound in 10 L of methylene chloride. Disks were also impregnated with 50 g of clotrimazole in 10 L of methylene chloride and 10 l of methylene Species Temperature C C. albicans 37.0 C. glabrata 26.0 C. keyfr 37.0 C. krusei 26.0 C. lusitaniae 26.0 C. parapsilosis 37.0 C. tropicalis 37.0 C. utilis 26.0

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19 chloride, respectively as controls. Disks were dispensed onto plates with a maximum of three disks per plate. The plates were sealed with parafilm and incubated for 24-48 hours at the temperatures given in Table 1. After 48 hours the cleared zones of inhibition were measured in mm and recorded. Each combination was tested 3 times and an average zone was dete rmined as the average of the 3 measurements. Specific trends in structure acti vity relationships are discussed in the next section. Overall, the disk study revealed varying degrees of antifungal activity for the N-thiolated -lactams against 7 of the 8 strains tested. Most notable was the innate resistance of Candida krusei to all compounds tested. Structure-Activity Relationships Structure-activity relationships were analyzed using selections from the data obtained in the above study

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20 Effects of C4 Substitution on Antifungal Activities of N-Thiolated Lactams: The effects of C4 substitution on antifungal activity are reflected in Table 2 through 10 and figures 11 through 19 and 21. Among halogenated aryl substituents, the fluoro (lactams 1-3) is the least effective; however antifungal activity does not uniformly increase with increasing molecular weight. In fact, as found in antibacterial studies, it does not appear to be as important which halogen is on the phenyl ring, but rather how many halogens are there and where they are positioned.40 Fluoroor chlorosubstituents at the para postion on the benzene ring greatly reduce biological activity, which is not observed for the iodo-or bromosubstituents. The presence of multiple fluoroor chlorosubs tituents on the benzene ring seems to intensify activity. Also as seen in bacteria, unsaturation in the C4 side chain generally seems to decrease activity,41 with the effect of the additional double bond being more pronounced than that of a triple bond.

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21 Figure 11: Effects of Monofluorinati on on Antifungal Activities on NThiolated -Lactams 0 5 10 15 20 25 123LactamZone of Inhibition (mm) C. albicans C. tropicalis C. glabrata C. keyfr C. krusei C. lusitinae C. parapsilosis C. utilis Lactam 1 Lactam 2 Lactam 3 N SMe O MeO F N SMe O MeO F N SMe O MeO F

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22 Table 2: Effects of Monofluorination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 1 50 ug C. albicans 014139 ortho F C. tropicalis 1111 C. glabrata 0000 C. kefyr 16141515 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 15222019 C. utilis 14141514.33333 Lactam 2 50 ug C. albicans 15121413.66667 meta F C. tropicalis 1111 C. glabrata 1111 C. kefyr 13115 C. krusei 0000 C. lusitaniae 1111 C. parapsilosis 25192222 C. utilis 16105.666667 Lactam 3 50 ug C .albicans 18141515.66667 para F C. tropicalis 1111 C. glabrata 0110.666667 C. kefyr 1111 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 19241920.66667 C. utilis 1111 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm.

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23 Figure 12: Effects of Monochlorinati on on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 35 40 456LactamZone of Inhibition (mm) C. albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO Cl N SMe O MeO C l N SMe O MeO Cl Lactam 4 Lactam 5 Lactam 6

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24 Table 3: Effects of Monochlorination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 4 50 ug C. albicans 17272422.66667 Ortho Cl C. tropicalis 19232020.66667 C. glabrata 29242325.33333 C. kefyr 20151617 C. krusei 0000 C. lusitaniae 19201919.33333 C. parapsilosis 36293332.66667 C. utilis 21232121.66667 Lactam 5 50 ug C. albicans 23242122.66667 Meta Cl C. tropicalis 21192120.33333 C. glabrata 24201820.66667 C. kefyr 15111313 C. krusei 0000 C. lusitaniae 14141213.33333 C. parapsilosis 21293327.66667 C. utilis 13232119 Lactam 6 50 ug C. albicans 0000 Para Cl C. tropicalis 0110.666667 C. glabrata 0000 C. kefyr 1191010 C. krusei 0000 C. lusitaniae 12141212.66667 C. parapsilosis 0110.666667 C. utilis 0000 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm.

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25 Figure 13: Effects of Monobromination on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 35 40 45 789LactamZone of Inhibition (mm) C. albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO Br N SMe O MeO B r N SMe O MeO Br Lactam 7 Lactam 8 Lactam 9

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26 Table 4: Effects of Monobromination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 7 50 ug C. albicans 25272525.66667 ortho Br C. tropicalis 21202020.33333 C. glabrata 16161716.33333 C. kefyr 12121312.33333 C. krusei 0000 C. lusitaniae 19201819 C. parapsilosis 40404040 C. utilis 25252525 Lactam 8 50 ug C. albicans 25181720 meta Br C. tropicalis 24161518.33333 C. glabrata 16151816.33333 C. kefyr 13131413.33333 C. krusei 0000 C. lusitaniae 14151414.33333 C. parapsilosis 17171817.33333 C. utilis 14141313.66667 Lactam 9 50 ug C. albicans 26252324.66667 para Br C. tropicalis 21182019.66667 C. glabrata 34222125.66667 C. kefyr 20161517 C. krusei 0000 C. lusitaniae 16161716.33333 C. parapsilosis 23242223 C. utilis 19171617.33333 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm.

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27 Figure 14: Effects of Monoiodination on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 101112LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO I N SMe O MeO I N SMe O MeO I Lactam 10 Lactam 11 Lactam 12

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28 Table 5: Effects of Monoiodination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 10 50 ug C. albicans 20141416 ortho I C. tropicalis 25111015.33333 C. glabrata 19182219.66667 C. kefyr 15141414.33333 C. krusei 0000 C. lusitaniae 20151717.33333 C. parapsilosis 20222522.33333 C. utilis 15151414.66667 Lactam 11 50 ug C. albicans 26131518 meta I C. tropicalis 18121314.33333 C. glabrata 18192019 C. kefyr 16141515 C. krusei 0000 C. lusitaniae 20172019 C. parapsilosis 19222421.66667 C. utilis 14171515.33333 Lactam 12 50 ug C. albicans 16151716 para I C. tropicalis 12131112 C. glabrata 21222422.33333 C. kefyr 20171918.66667 C. krusei 0000 C. lusitaniae 22172120 C. parapsilosis 23242925.33333 C. utilis 19212020 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm.

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29 Figure 15: Effects of Multiple Fluorination on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 35 40 131415LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO N SMe O MeO F F F F F F N SMe O MeO F F F Lactam 13 Lactam 14 Lactam 15

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30 Table 6: Effects of Multiple Fluorination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 zone2Zone3Average Lactam 13 50 ug C. albicans 17181918 2,3,5 F C. tropicalis 23252223.33333 C. glabrata 11114.333333 C. kefyr 14161515 C. krusei 0000 C. lusitaniae 15181716.66667 C. parapsilosis 20232221.66667 C. utilis 14171615.66667 Lactam 14 50 ug C. albicans 25292125 2,4,5 F C. tropicalis 19231619.33333 C. glabrata 14141313.66667 C. kefyr 13181716 C. krusei 0000 C. lusitaniae 15141414.33333 C. parapsilosis 20232221.66667 C. utilis 13141413.66667 Lactam 15 50 ug C. albicans 21262423.66667 3,4,5 F C. tropicalis 19201819 C. glabrata 17181717.33333 C. kefyr 16181917.66667 C. krusei 0000 C. lusitaniae 20282725 C. parapsilosis 30353734 C. utilis 20333429 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm.

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31 Figure 16: Effects of Multiple Chlorina tion on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 35 40 1617LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO Cl N SMe O MeO C l Cl Cl Cl Lactam 16 Lactam 17

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32 Table 7: Effects of Multiple Chlorination on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 16 50 ug C. albicans 22232222.33333 2,4 Cl C. tropicalis 21222121.33333 C. glabrata 33293030.66667 C. kefyr 31293030 C. krusei 1010.666667 C. lusitaniae 22222322.33333 C. parapsilosis 24272826.33333 C. utilis 19181918.66667 Lactam 17 50 ug C. albicans 22232222.33333 2,3,5 Cl C. tropicalis 21222121.33333 C. glabrata 33293030.66667 C. kefyr 31293030 C. krusei 1010.666667 C. lusitaniae 22222322.33333 C. parapsilosis 24272826.33333 C. utilis 19181918.66667 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm. Effects of C3 Substitution on Antifungal Activities of N-Thiolated Lactams: The effects of methoxy, pheno xy and acetoxy groups at the C3 position of the N-methylthio -lactam were also investigated. The results are displayed in Tables 9 and 10 and Figure 17. Both the bulky phenoxy and acetoxy groups reduced the activity of the

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33 lactams with both saturated and monounsaturated groups at the C4 position. The effect of the C3 acetoxy substituent on antifungal activity was less with a saturated group at the C4 position than with an unsaturated chain. Figure 17: Effects of C3 Substitution on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 181920LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis

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34 0 5 10 15 20 25 30 35 40 212223LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N SMe O MeO N SMe O PhO N SMe O AcO Lactam 18 Lactam 19 Lactam 20 N SMe O MeO N SMe O PhO N SMe O AcO Lactam 21 Lactam 22 Lactam 23 Figure 17 (Continued)

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35 Table 8: Effects of C3 Substitution on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 18 50 ug C. albicans 29272627.33333 MeO C. tropicalis 15141414.33333 C. glabrata 15161716 C. kefyr 14141313.66667 C. krusei 0000 C. lusitaniae 14141313.66667 C. parapsilosis 13131212.66667 C. utilis 18202019.33333 Lactam 19 50 ug C. albicans 0000 A cO C. tropicalis 0000 C. glabrata 12141513.66667 C. kefyr 12111111.33333 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 0000 C. utilis 13141313.33333 Lactam 20 50 ug C. albicans 0000 PhO C. tropicalis 0000 C. glabrata 91099.333333 C. kefyr 0000 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 0000 C. utilis 0000 Lactam 21 50 ug C. albicans 34343434 MeO C. tropicalis 20151516.66667 C. glabrata 20202020 C. kefyr 22252423.66667 C. krusei 0000 C. lusitaniae 15151515 C. parapsilosis 14141313.66667 C. utilis 28272827.66667 Lactam 22 50 ug C. albicans 0000 A cO C. tropicalis 0000 C. glabrata 15151414.66667 C. kefyr 13131413.33333 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 0000 C. utilis 0000

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36 Table 8 (continued) Lactam 23 50 ug C. albicans 24252424.33333 PhO C. tropicalis 21191719 C. glabrata 19181718 C. kefyr 21191819.33333 C. krusei 0000 C. lusitaniae 14131413.66667 C. parapsilosis 14131413.66667 C. utilis 22202020.66667 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm. Effects of N-Substitution on Anti fungal Activities of N-Thiolated Lactams: The effects of methylthio versus sec-butylthio substituents were next examined. The sec-butylthio side chain resulted in a marked decrease in antifungal activity against 6 of the 8 Candida species examined as shown in Figure 18. This represents a marked difference from the lactam’s antibacterial activity against Staphylococcus aureus, in which the sec-butylthio group increased activity of the lactams toward S. aureus and other bacteria screened relative to the methylthio.

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37 Figure 18: Effects of N-Substitution on Antifungal Activities of NThiolated -Lactams 0 5 10 15 20 25 30 35 40 424LactamZone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis N O SMe MeO Cl N O S MeO Cl Lactam 4 Lactam 24

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38 Table 9: Effects of N-Substitution on Antifungal Activities of NThiolated -Lactams Compound Concentration Organism Zone1 Zone2 Zone3 Average Lactam 4 50 ug C. albicans 17272422.66667 Sme C. tropicalis 19232020.66667 C. glabrata 29242325.33333 C. kefyr 20151617 C. krusei 0000 C. lusitaniae 19201919.33333 C. parapsilosis 36293332.66667 C. utilis 21232121.66667 Lactam 24 50 ug C. albicans 16151615.66667 Sec Butyl C. tropicalis 12141212.66667 C. glabrata 0000 C. kefyr 0000 C. krusei 0000 C. lusitaniae 0000 C. parapsilosis 0000 C. utilis 0000 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm. Effect of Absolute Stereochemist ry on Antifungal Activities of NThiolated -Lactams: Chiral antibiotics are typically more active in one enantiomeric form than the other. The less ac tive enantiomer can also produce unwanted side effects. Thus, the i ssue of absolute stereochemistry of the lactams on antifungal activity was assessed using

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39 enzymatically purified enantiomers of the lactam. Lipase PS-30 was used to selectively deacylate the 3S,4R 3-acetoxy derivative of the lactam. The resulting (-) alcohol and (+) acetate compounds could then be separated and independently converted into the corresponding N-methylthio -lactam as shown in Figure 19.42 N O O C H3 A cO Lipase PS-30 acetone phosphate buffer pH 7.2N O O C H3 HO N O O C H3 AcO + N O SMe AcO +N O SMe AcO Lactam 25 (-)Lactam 25(+) Figure 19: Enzymatic Purification of Lactam 25 The (+) and (–) enantiomers were compared in a disk diffusion assay. The (-) enantiomer had sign ificantly less in activity againt C. lusitiniae and C. utilis and a more modest decrease in activity against C. parapsilosis and C. tropicalis as shown in Figure 20

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40 There was little difference in acti vity between the two enantiomers against C. albicans This is different from what was found against S. aureus where absolute stereochemistry was not found to have any effect whatsoever. This suggests that unlike in bacteria, there may be some nonbonding interactions of the lactam with the biological target in some species of Candida Figure 20: Effects of (+) and (-) Enantiomers of Lactam 25 on Antifungal Activities of N-Thiolated -Lactams 0 2 4 6 8 10 12 14 16 18 (+)(-)Lactam 25Zone of Inhibition (mm) C.albicans C. tropicalis C. glabrata C. kefyr C. krusei C. lusitaniae C. parapsilosis C. utilis

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41 Table 10: Effects of (+) and (-) Enantiomers of Lactam 25 on Antifungal Activities of N-Thiolated -Lactams Compound Concentration Organism Zone1 Zone2 zone3 Average Lactam 25 50 ug C. albicans 14151414.33333 (+) enantiomer C. tropicalis 14151514.66667 C. glabrata 0000 C. kefyr 91099.333333 C. krusei 0000 C. lusitaniae 15151414.66667 C. parapsilosis 0000 C. utilis 16151615.66667 Lactam 26 50 ug C. albicans 14131313.33333 (-) enantiomer C. tropicalis 10111311.33333 C. glabrata 0000 C. kefyr 0000 C. krusei 0000 C. lusitaniae 10111010.33333 C. parapsilosis 0000 C. utilis 16151615.66667 Zone sizes are measured in mm as the diameter around the disk where there is no visible growth. Partial inhibition (spotty zones) were defined as having a zone of 1mm. No visible zone was defined as having a zone of 0mm. The data in this study reveal some interesting trends in the structure-activity profile of these lactams. In general they support what has already been found in th e structure-activity studies of these lactams against bacteria. As in bacteria, differences in the biological activity of different lactams may be more closely related to their ability to diffuse across th e cellular membrane than to any

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42 specific binding interaction with a biological target despite there being some evidence of nonbonding interactions. Minimum Inhibitory Concentration As lactam 4 was soluble in a 1% aqueous solution of DMSO, minimum inhibitory concentrations for that compound were determined using two different methods. MICs by Agar Diffusion Lactam 4 was tested against C. albicans, C. tropicalis C. glabrata, C. lusitinae, C. parapsilosis C. utilis, and C. keyfr Yeast Nitrogen Agar was prepared and a 1mg/mL solution of lactam 4 in DMSO was prepared. Each well of a 48 well (1.5 mL) plastic plate was filled with different amounts of lactam 4, from 0-50 ug in 5 ug increments. Agar was added to br ing the total volume of each well to 1 mL, stirred, and allowed to harden overnight. Each well was then inoculated with 1uL of a standardized solution of 106 CFU/mL of fungi suspended in sterile NaCl. A separate plate prepared with wells containing 0-50 ug of lactam 4 was used for each species to prevent cross contamination. Th e results are shown in Table 11.

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43 Table 11: Minimum Inhibitory Concentration Values Determined for Lactam 4 by Agar Dilution Candida sp. MIC 24 hrs. MIC 48 hrs. C. albicans <5 ug/ml <5 ug/ml C. tropicalis 10-15 ug/ml 30-35 ug/ml C. glabrata 10-15 ug/ml 10-15 ug/ml C. kefyr 10-15 ug/ml 35-40 ug/ml C. lusitaniae 10-15 ug/ml 15-20 ug/ml C. parapsilosis <5 ug/ml <5 ug/ml C. utilis 10-15 ug/ml 15-20 ug/ml The MIC was significantly higher at 48 hours than at 24 hours. This may be an effect of the fungi overcoming the concentration of compound on the surface of th e well. This suggests that the compound may be fungistatic versus fungicidal. These MICs at both 24 and 48 hours are significantly higher than the literature value of MICs for fl uconazole, amphotericin B, and nystatin. MICs Broth Macrodilution The minimum inhibitory concentration was also determined in triplicate using the NCCLS broth macrodilution method for water insoluble antifungal agents described in document M27-A243 with incubation for24 hours. This stan dardized testing method allows for

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44 better quality control and comparison to known standards (clotrimazole and amphotericin B). Table 12: Minimum Inhibitory Concentration Values for Lactam 4 Determined by Broth Macrodilution AgentMIC (ug/mL) Clotrimazole2 Amphotericin B<0.313 Lactam 48 The MIC for Amphotericin B was within the cited range of .25 – 1.0 ug/mL against C. albicans The MIC values as determined by this method are slightly lower than that found with the agar d ilution method. Although broth and agar dilution have been found to be comparable, this could be an artifact of the two different testing methods as they were performed in different types of media (Yeast Nutient versus RPMI). However this value is still significantly high er than either drug currently in use. The comparatively high concentrations of lactam 4 necessary for activity in broth would be presum ably more difficult to achieve in vivo.

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45 Chapter Three Studies Leading Towards Mode of Action Three studies were undertaken toward finding the mode of action of these lactams; 1. An assay was performed to determine whether the drug has a fungistatic or fungicidal effect on fungi 2. A broth study was performed to investigate the products of the molecule’s interaction with fungal cells 3. A TEM study was performed to visualize any ultrastructural effects the drug might have on the cells. Fungistatic versus Fungicidal A tube with 20 mL of Yeast Nutrient Broth was inoculated with a colony of C. albicans that had been cultured for 48 hours on agar. This then was incubated at 37 oC for 18 hours. Two sets of tubes were prepared from this culture. Each tube contained 4.8 mL culture and 200 uL of either DMSO or lactam 4 dissolved in DMSO (1ug/uL). Two sets of a series of dilutions (full strength, 1:10, 1:1000) were prepared, and initial plate counts performed. These tubes were incubated at 37 oC overnight.

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46 After incubation another set of plate counts were performed to determine the effect that the drug had on the growth of the fungi. The fungal growth was slowed by th e addition of Lactam 4 as shown by the dip in the red lines in Figure 10. The cells were then washed in PBS and resuspended in nutrient broth and incubated overnight to s ee if growth would resume after removal of the compound. Figure 21: Growth Study: Effect of Lactam 4 on Growth of Candida albicans 1 10 100 1000 10000 100000 1000000 10000000 Start24 h with drug 24 h after drug removedFungal growth in CFU/mL Lactam 4 1ug/mL Lactam 4 0.01 ug/mL Lactam 4 0.001 ug/mL DMSO Control DMSO 1:10 DMSO 1:1000 The fact that growth was slowed by the drug, but resumed upon its removal, indicates that the drug has a fungistatic effect. This supports previous studies in bacter ia showing an inhibitory rather than a cidal effect. The ability of the fungi to overcome the

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47 antifungal effects of the drug is an other indicator that it would take relatively high in vivo concentrations of the drug for it to be effective in treatment. Broth Study Vials were prepared with 9 mL of yeast nutrient base broth and 1 mL of 1 mg/mL solution of lactam 4 in DMSO. Vials were inoculated with one loopful of C. albicans and vortexed. One vial was incubated for 2 hours at room temperature and another was incubated for 72 hours at room temperature. Controls were set up with 1) Yeast nutrient base broth, 1 mL of DMSO, and a loopful of C. albicans 2) Yeast nutrient base broth, 1 mg/mL solution of lactam 4 in DMSO, but no C. albicans Controls were incubated at room temperature for 72 hours. After the given incubation period each solution was extracted with 5 mL of ethyl acetate. The organic layer was removed and this was repeated twice more. The three organic layers were combined, dried with magnesium sulfate and evaporated to remove solvent. The remaining residues were examined with NMR to ascertain identity of products. This procedure was repeated with saline replacing yeast nutrient

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48 base broth with incubation times of 2 hours, 24 hours and 72 hours with controls being incubated for 72 hours. The broth study of lactam 4 revealed that a complete removal of the methylthio group from the nitr ogen occurred after 24 h in both saline as well as broth both with and without C. albicans The ring structure was left intact. No ch ange from the original lactam structure was observed in any of the controls. At 2 hours a mixture of the original lactam structure and that of the lactam with the methylthio group removed was observed. Spectra are located in Appendix B. The results of the broth study reve aled a complete conversion to the N-H analogue after 24 hours in the presence of the microbe with the ring structure left intact. This is quite different from that of older -lactams, but similar to what has been seen with these lactams towards bacteria and strongly suggests a distinct mode of action possibly involving the transf er of the methylthio group to a protein.

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49 Transmission Electron Microscopy (TEM) Study Since fluorescent labeling of one of the N-methylthio lactams in an effort to elucidate the mode of action proved difficult, a TEM study to investigate ultrastructural changes in fungl cells caused by the lactam was performed. Method: A culture of C. albicans was grown from the frozen glycerol stock on yeast nutrient agar incubated at 37 oC for 48 hours. From this culture, liquid Saraboud’s dextrose medium was inoculated and incubated at 37 oC on a reciprocal shaker until exponential growth was achieved. At the exponential phase portions of the culture were added to 2 tubes each containing the MIC of either Lactam 4 or Clotrimazole dissolved in 1% aq ueous DMSO. One tube of each antifungal compound was incubated for 1h and the other for 4 h with aeration. Tubes were also prepared as the control with only 1% aqueous DMSO added instead and incubated for 1h and 4h with aeration. After incubation the cells were pelleted and washed twice with 0.1M phosphate buffer (pH 7.2). The washed pellet from each cu lture was resuspended in 2.5% aqueous glutaraldehyde in 0.1M phosphate buffer at 4oC for 2h. The cells were pelleted and post fixed in 1.5% aqueous potassium permanganate at 4oC overnight. The cells were then washed in deionized water and agar embedded. The agar embedded cells were

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50 en-block stained with 1.5% aque ous uranyl acetate for 1.5 h at room temperature. They were th en dehydrated in a series of acetone washes, infiltrated, and embe dded in Spurr’s Plastic. Blocks were trimmed and sectioned with a Sorvall MT-2B ultamicrotome and put on copper mesh grids. Grids were stained with 2% uranyl acetate and viewed with an FEI Morgani 268D Electron Microscope. The images in Appendix C revealed little ultrastructural damage to organelles in the cells. There was no membrane degradation, no leakage of cell contents and no damage to the cell wall or mitochondria. This suggests a mode of action distinct from that of most other antifungals which target ergosterol and result in visible damage to the cell membranes. Given the observed structure-activity relationship, fungistatic effect, lack of ultratructural damage induced, and spectra of the products of the drug’s interection wi th the cell, it is postulated that the mode of action of these antif ungals is similar to that seen in bacteria37,44 consisting of a thiol transfer to a biological target (Figure 22).

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51 Figure 22: Proposed mechanism of action We postulate that these lactams, after passing through the cell membrane, interact covalently with a biological target resulting in a transfer of the sulfur side chai n. This mechamism is primarily supported by the spectra of the pr oduct isolated with the lactam intact and the sulfur side chain mi ssing. This is further supported by the structure-activity relationship in that the sulfur side chain is strictly required for activity, that groups at C3 and C4 which are highly polar exhibit little to no ac tivity and that groups which are highly lipophilic may cause trapping in the membrane or organelles and exhibit reduced activity. This interaction in fungi is more specific than in bacteria in that both absolute stereochemistry and bulky side chains at the thio group affect activity. Thus, the cellular target (Nu H) attacked is likely evolutionarily conserved such as a common metabolic enzyme or intermed iate with a slightly different structure in bacteria and fungi. Fu rther study is needed to elucidate the identity of this target and it s interaction with the N-thiolated lactam. N O S-Me R M e O N O MeOR attack on sulfur + [Nu]S-Me [Nu]-H H

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52 Chapter Four Discussion As described in the introduction, Candida infections have become a significant problem in terms of medi cal cost, morbidity and mortality. These infections are and will continue to be a growing problem as even the newest of drugs have documented resistance. Thus the continuing search for and development of novel antifungals is important. This study has investigated the antif ungal activity of a new series of nonconventional -lactams. Several analogs of these lactams were shown to have activity against the most common Candida pathogens. Unlike in other reported bactrio-and fungistatic lactams these seem to work through a similar mode of action in both S. aureus and C. albicans These lactams were found to have similar structure activity relationships in both S. aureus and C. albicans As previously reported in antibacterial structure activity studies against these monocyclic lactams, it does not appear to be as important which halogen is on the phenyl ring, but rather how many halogens are there and where they are positioned, that the sulfur side ch ain is strictly required for activity, that groups at C 3 and C 4 which are highly polar exhibit little to no activity, and that groups which are highly lipophilic exhibit reduced activity. The interaction between drug and target in fungi however

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53 seems to be more specific than in bacteria in that both absolute stereochemistry and bulky side chai ns at the thio group decrease activity in fungi, but not in bacteria. As with S. aureus the broth study revealed complete removal of the methylthio group from the nitrogen upon interaction of the drug with Candida The ring structure was left intact. Upon visualization of treated Candida cells, there was no membrane degradation, no leakage of cell contents and no damage to the cell wall or mitoch ondria. The spectrum of activity, structure-activity relationships, and preliminary studies toward determining the mode of action su pport a unique mode of action in Candida fungi similar to that observed in S. aureus We postulate that these lactams, after passing through the cell membrane, interact covalently with a biological target re sulting in a transfer of the sulfur side chain to some common target. Though the high MIC of these compounds make them a poor candidate for clinical use as antif ungals, understanding the mode of action of these as well as other novel lactams and how their mode of action affects other pathways in the cell could lead to development of new drugs or new ways to combat fungal resistance to current drugs.

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54 References 1 Perea S, Patterson, TF. Antifungal resistance in pathogenic fungi. Clinical Infectious Diseases, 2002;1;35(9):1073-80. 2 Sobel JD, Ohmit SE, Schuman P, et al. The evolution of Candida species and fluconazole susceptibility among oral and vaginal isolates recovered from human immunodeficiency virus (HIV)-seropositiv e and at-risk HIV-seronegative women. Journal of Infectious Disease, 2001; 183: 286-293. 3 Richardson MD. Changing Patterns and tr ends in systemic fungal infections. Journal of Antimicrobial Chemotherapy 2005;56;Suppl. S1; i5-i11. 4 Wenzel RP. Epidemology of No socomial Candida Infections. Infectious Diseases in Clinical Practice, 1994; 3(Suppl 2):S56-S59. 5Nucci M, and Marr KA. Emer ging Fungal Diseases. Emerging Infections 2005; 41: 521-525. 6 Gudlaugsson O, Gillespie S, Lee K, et al. Attributable mortality of nosocomial candidemia, revisited. Clinical Infectious Diseases, 2003; 37: 1172-1177. 7 Pfaller MA, Messer SA, Hollis RJ, Jones RN, Doern GV, Brandt ME, Hajjeh RA. Trends in species distribution and suscept ibility to fluconazole among blood stream isolates of Candida species in the United States. Diagnosis of Microbial Infectious Diseases, 1999; 33:121-129. 8Nucci M, and Marr KA. Emer ging Fungal Diseases. Emerging Infections 2005; 41: 521-525. 9 Wey SB, Mori M, Pfaller MA, Woolso n RF, Wenzel RP. Hospital-acquired candedemia: the attributable mortality and excess length of stay. Archives of Internal Medicine 1988; 148:2642-5. 10 Colerman DC, Rinaldi MG, Haynes KA Rex JH, Summerbell RC, Anaisse, EJ. Importance of Candida species other than Candida albicans as opportunistic pathogens. Medical Mycology, 1998; 36(Suppl 1):156-65. 11 Pappas PG, Rex JH, Lee J, et al. A prospe ctive observational study of candidemia: epidemiology, therapy, and influences on mo rtality in hospitalizd adult and pediatric patients. Clinical Infectious Diseases 2003; 37: 634-643. 12 Trick WE, Fridkin SK, Edwards JR, Hajjeh RA, Gaynes RP. Secular trend of hospital acquired candidemia among intensive care patients in the United States during 19981999. Clinical Infectious Diseases, 2002;35:627-630.

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55 13 Pfaller MA, Dickema DJ. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconaz ole susceptibility of bloodstream isolates of Candida Clinical Microbiologial Infections 2004; 10(Suppl):11-23. 14 Dupont B. Invasive fungal infections ca used by yeast as emerging pathogens. Infectious Diseases in Clinical Practice, 1994;3(Suppl 2):S78-S82. 15 Colombo AL, Nucci M, Salomao R, et al. High rate of non -albicans candidemia in Brazillan tertiary care hospitals. Diagnosis of Microbial Infectious Diseases 1999; 34: 281-286. 16 Calderone, RA, editor. Candida and candidiasis. Washington: ASM Press; 2002. 17 Akins RA. An update on antifungal ta rgets and mechanisms of resistance in Candida albicans Medical Mycology, 2005; 45:285-318. 18 Wynn RL, Jabra-Rizk MA, Meiller TF. Fungal drug resistance, biofilms and new antifungals. General Dentistry 2003; 14: 85-691. 19 Brutyan RA, McPhie P. On the one-sided action of amphotericin B on lipi bilayer membranes. Journal of Genreal Physiology 1996;107:69-78. 20 Andriole VT. Current and future antifungal therapy: new targets for antifungal agents. Journal of Antimicribial Chemotherapy 1999;44:151-161. 21 Stevens DA. Current status and future directions of antifungal therapy. Infectious Diseases in Clinical Practice 1994; 3(Suppl 2):S97-S102. 22 Lorian V, Ed. Antibiotics in Laboratory Medicine 2nd Ed. Williams and W ilkins. 1986. 223-281. 23Vanden Bossche H. Molecular mechanisms of drug resistance in fungi. Trends in Microbiology, 1994; 2:393-400. 24 Groll AH, Piscitelli SC, Walsh TJ. Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets fo r antifungal drug development. Advances in Pharmacology, 1998; 44:343-500. 25 Sanglard D, Odds FC. Resistance of Candida species to antifungal agents:molecular mechanisms and clinical consequences. The Lancet Infectious Diseases, 2002; 2: 7385. 26 Lamb D, Kelly D, Kelly S. Molecular aspects of azole antifungal action and resistance. Drug Resistance Update 1999; 2: 390-402. 27 Kelly SL, Lamb DC, Corran AJ, Baldwin BC, Ke lly DE. Mode of action and resistance to azole antifungals associated with the formation of 14 alpha-methylergosta8,24(28)dien-3-beta, 6 alpha diol. Biochemicaly and Biophysical Research Communications, 1995;207:910-915.

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56 28 Denning DW. Echinocandins: a new class of antifungal. Journal of Antimicrobial Chemotherapy 2002; 49: 889-91. 29 Cuence-Estrella M, Mellado E, Diaz-Gue rra TM, Monzon A, Rodriguez-Tudel JL. Susceptability of fluconazole resistant clinical isolates of Candida species to echunocandin LY 303366, intraconazole an d amphotericin B. Journal of Antimicrobal Chemotherapy 2000; 46:475-7. 30 Walsh TJ. Echinocandinsan advance in the primary treatment of invasive candidiasis. New England Journal of Medicine, 2002; 347:2070-2. 31 Sanglard D, Isher F, Monod M, Bille J. Cloning of Candida albicans genes conferring resistance to azole antifungal agentscharacterization of CDR2, a new multidrug ABC transporter gene. Microbiology, 1997; 143:405-16. 32 Miyazaki H, Miyazaki Y, Geber A, et al. Fl uconazole resistance associated with drug and increased transcription of a drug transporter gene, pdh1, in Candida glabrata Antimicrobial Agents and Chemotherapy 1998; 42: 1695-701. 33 Sanglard D, Kuchler K, Isher F, Pagani JL, Monod M, Bille J. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients jinvolve specific multidrug transporters. Antimicrobial Agents and Chemohterapy 1995;39: 2378-86. 34 Turos E, Long TE, Konaklieva MI, Coates C, Shim JY, Dickey S, Lim DV, and Cannons A. NThiolated -Lactams: Novel anti microbial agents for methicillinresistant Staphylococcus aureus Bioorganic and Medicinal Chemistry Letters, 2002; 12: 2229-2231. 35 Morin RB, Gorman M, Eds. Chemistry and Biology of -Lactam Antibiotics. Academic: New York, 1982 Vols. 1-3. 36 Gottstein WJ, Eachus AH, Misco PF, Cheney LC, Misiek M, Price KE. -lactam antimicrobial agents which possess antifungal activity. Journal of Medicinal Chemistry, 1971; 14 (8):770-2. 37 F. Rhl, J. Rabenhorst, and H. Zhner. Bi ological properties and mode of action of clavams. Archives of Microbiology, 1987; 147 (4): 315. 38 Turos E, Konaklieva MI, Ren R, Shi H, Gonzalez J, Dickey S, and Lim DV. N thiolated bicyclic and mo nocyclic beta-lactams. Tetrahedron 2000; 56: 5571-5578. 39 Ingroff-Espinel A. Antifung al susceptibility testing. Clinical Microbiology Newsletter 1996; 18(21): 161-7. 40 Long TE, Turos E, Konaklieva MI, Blum AL Amry A, Baker EA, Suwandi LS, McCain MD, Rahman MF, Dickey S, and Lim DV. Effe ct of aryl ring fluorination on the antibacterial properties of C4 aryl-substituted N -methylthio -lactams. Bioorganic and Medicinal Chemistry, 2003; 11: 1859-1863.

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57 41 Coates C, Long TE, Turos E, Dickey S, and Lim DV. N -Thiolated -lactam antibacterials: Defining the role of unsaturation in the C4 side chain Bioorganic and Medicinal Chemistry, 2003; 11:193-196 42 Turos, E., Coates C, Shim JY, Wang Y, Leslie JM, Long TE, Reddy GSK, Ortiz A, Culbreath M, Dickey S, Lim DV, Alonso E, and Gonzalez J. N -Methylthio -lactam antibacterials: Effects of the C3/C4 ring substituents on anti-MRSA activity. Bioorganic and Medicinal Chemistry 2005; 13:6289-6308. 43 National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts, approved standard. NCCLS document M27A. National Committee for Clin ical Laboratory Standards, Wayne, Pa; 1997. 44 Unpublished results of Kerriann Greenhalgh working in the lab of Dr. Ed Turos at the University of South Florida.

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58 Appendix A: NMR Spectra of Lactams Synthesized

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59 Lactam 4 Lactam 4 NH Intermediate

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60 Lactam A Lactam B

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61 Lactam A and B NH Intermediate Lactam A and B PMP Intermediate

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62 Appendix B: NMR Spectra from Broth Study

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63 Control Extract Broth Only: shows background from components of broth Control Extract Lactam 4 + Broth: shows intact lactam in broth after 24 hours

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64 Extract of Lactam 4 + C. albicans + Broth 24 hours: shows conversion to NH lactam Extract of Lactam 4 + C. albicans + Saline 24 hours: shows conversion to NH lactam

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65 Appendix C: TEM Images

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66 Comparison of Whole Cell Candida + Lactam 4 (4 Hour Incubation) showing no visible damage to ce ll wall, cell membranes or organelles as compared to control Candida Control

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67 Comparison of Close up of Nuclei Candida + Lactam 4 (4 Hour Incubation) showing no visible damage to nucleus as compared to control Candida Control

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68 Comparison of Close up of Mitochondria Candida + Lactam 4 (4 Hour Incubation) showing no visible damage to mitochondria compared to control Candida Control


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An investigation into the antifungal activities of N-thiolated beta lactams against selected Candida species
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2006.
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ABSTRACT: Beta-lactam antibiotics have long been a reliable course of treatment for bacterial infections. However, with recent increases in resistance and rising populations of immunocompromised patients new beta-lactams have been synthesized and tested. The Turos laboratory has recently discovered novel beta-lactams that have a mode of action distinct from penicillin and other beta-lactam antibiotics as cell lysis is not observed. In the current investigations, these compounds are shown to also have antifungal properties. The rising incidence and prevalence of invasive fungal infections has become an increasing concern. The most common fungal pathogens involved in these infections are species in the genus Candida. In this study antifungal activity is observed for a wide range of N-methylthio B-lactams against C. albicans, C. tropicalis, C. keyfr, C. glabrata, C. lusitinae, C. utilis, and C. parapsilosis. The structure-activity relationship based on studies of beta-lactam derivatives leaving different substituents at various positions on the lactam ring are investigated, and the minimum inhibitory concentration values determined using standard methods. In studies towards understanding the mode of action, the products of the interaction between the drug and fungal cells in a suspension were investigated using nuclear magnetic resonance spectroscopy and transmission electron microscopy. The mode of action of these new lactams seems to be similar to that observed in bacteria, involving transfer of the methylthio group to a cellular thiol.
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Adviser: Edward Turos, Ph.D.
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Mode of Action
Structure Activity Relationship
Minimum Inhibitory Concentration
Transmission Electron Microscopy
Fungistatic
Structure activity relationship.
Minimum inhibitory concentration.
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Fungistatic.
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