Development of a novel antibiotic adjuvant strategy against hypervirulent Community-Associated Methicillin-Resistant Staphylococcus aureus Public
Salam, Akram (Fall 2020)
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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