Development of a novel antibiotic adjuvant strategy against hypervirulent Community-Associated Methicillin-Resistant Staphylococcus aureus Público

Salam, Akram (Fall 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/s7526d76x?locale=es
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Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) represents one of the most serious infectious disease concerns worldwide, with the CDC labeling it a “serious threat” in 2019. The current arsenal of antibiotics works by targeting growth and survival, which exerts great selective pressure for the development of resistance. The development of novel anti-infectives that inhibit virulence in MRSA has been recurrently proposed as a promising therapeutic approach. In order to discover drug leads that work, we looked to nature. Previous studies on rural Italian traditional medicine confirm that select extracts of plants used in traditional medicinal preparations for the treatment of dermatological ailments exert antivirulence activity against MRSA. In a follow-up of a study examining the S. aureus quorum sensing inhibitory activity of Italian plants used in local traditional medicine, extract 224C-F2 was reported as a bioactive fraction of a Castanea sativa (European chestnut) extract. The fraction demonstrated high quorum sensing inhibitory activity against MRSA in vitro and effective attenuation of pathogenicity in a mouse model of skin infection.

Through further bioassay-guided fractionation using reverse-phase high performance liquid chromatography, a novel hydroperoxy cycloartane triterpenoid, castaneroxy A, was isolated. Its structure was confirmed by X-ray crystallography, mass spectrometry, and nuclear magnetic resonance. An isomer of castaneroxy A was also detected in an adjacent fraction, with a possible third isomer as a minor constituent. The castaneroxys are unique in that they possess not only a hydroperoxy group but also a cyclopropyl group, both of which are rare. In a series of assays assessing inhibition of markers of S. aureus virulence, castaneroxy A exerted activities in the low micromolar range. Castaneroxy A inhibited agr::P3 activation (IC50 = 31.72 µM), δ-toxin production (IC50 = 31.72 µM in NRS385), supernatant cytotoxicity to HaCaT human keratinocytes (IC50 = 7.93 µM in NRS385), and rabbit erythrocyte hemolytic activity (IC50 = 7.93 µM in LAC). Castaneroxy A also demonstrated a lack of off-target effects, exerting no effect on biofilm production and no cytotoxicity to HaCaTs at 31.72 µM and below. Finally, castaneroxy A was shown to abate MRSA-induced dermonecrosis in a mouse model of skin infection. The results establish castaneroxy A as a promising antivirulence drug lead for development against S. aureus and at the same time validate the ethnobotanical anti-infective value of C. sativa­ leaves.

Table of Contents

List of Figures and Tables. 11

List of Abbreviations. 14

CHAPTER I. 17

CHAPTER II. 33

Introduction. 34

Results. 36

Bioassay-guided isolation of castaneroxy compounds. 36

Structure elucidation of castaneroxy compounds. 41

Discussion. 53

Methods. 63

Spectra. 68

CHAPTER III. 83

Introduction. 84

Results. 86

Castanaroxy A acts on the agr system to inhibit virulence in MRSA.. 86

Assessment of other effects of castaneroxy A.. 97

Castaneroxy A abates dermonecrosis in vivo. 99

Discussion. 102

Methods. 108

CHAPTER IV.. 114

Discussion. 115

Perspective. 126

Supplementary Information S1. 128

Supplementary Information S2. 133

APPENDIX I. 142

Abstract 144

Introduction. 145

Plant secondary metabolites. 145

Ethnobotany as a drug discovery tool 151

Synergy among natural products. 152

Drug discovery from plant natural products. 154

Success in anti-virulence approaches. 156

Conclusions. 157

APPENDIX II. 159

Abstract 161

Introduction. 162

Extraction Techniques. 165

Maceration and decoction. 165

Reflux, Soxhlet extraction, and percolation. 165

Ultrasound-assisted extraction. 167

Essential oil extraction. 168

Supercritical fluid extraction. 168

Accelerated solvent extraction. 170

Microwave-Assisted Extraction. 171

Extraction efficiencies. 172

Chromatographic techniques. 176

Flash chromatography. 176

High-performance liquid chromatography (HPLC). 179

Gas chromatography (GC). 181

Supercritical fluid chromatography. 182

Prefractionation. 183

Solid phase extraction. 184

Spectroscopic Techniques. 186

Mass spectrometry. 186

Nuclear Magnetic Resonance spectroscopy. 188

Hyphenated techniques. 191

Metabolomics. 193

Final Considerations. 196

APPENDIX III. 198

Abstract 200

Introduction. 201

The Agr system as an emerging target for drug development in virulent S. aureus infection. 202

Synthetic Quorum Quenchers. 205

Biaryl hydroketones. 205

Savirin. 209

Oxacillin. 210

Peptide-conjugated locked nucleic acids. 212

Peptide quorum quenchers. 212

RNAIII inhibiting peptide (RIP) derivative. 213

Natural Product Quorum Quenchers. 214

ω-hydroxyemodin. 214

Ambuic acid. 215

Castanea sativa leaf extract 217

Schinus terebinthifolia berry extract 218

Conclusions. 219

APPENDIX IV.. 224

Emerging Trends in Antibacterial Drug Discovery From Plants. 225

Machine Learning. 226

Molecular Networking and Dereplication. 228

Metabolite Generation. 231

Endophytic Fungi 234

Structure Elucidation. 236

References 240

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