The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence
Staphylococcus aureus is a Gram-positive human pathogen of significant clinical importance. The development of resistance to numerous classes of clinically important antibiotics has exacerbated the need for the development of novel therapeutic modalities for the treatment of serious, drug-resistant...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2021
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| Online Access: | https://eprints.nottingham.ac.uk/66015/ |
| _version_ | 1848800287818514432 |
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| author | Reekes, James |
| author_facet | Reekes, James |
| author_sort | Reekes, James |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Staphylococcus aureus is a Gram-positive human pathogen of significant clinical importance. The development of resistance to numerous classes of clinically important antibiotics has exacerbated the need for the development of novel therapeutic modalities for the treatment of serious, drug-resistant S. aureus infections.
One approach for developing new therapies is the discovery of anti-virulence compounds. Virulence in S. aureus is mainly regulated by the agr quorum sensing system. The agr system is a master regulator of staphylococcal virulence and utilises the thiolactone macrocyclic peptide, auto-inducing peptide (AIP) as the signal molecule. The agr system is based around two divergent promoters, agrP2 and agrP3. The agrP2 promoter drives expression of the agr operon, which consists of 4 genes, agrB, agrD, agrC and agrA. AgrB and AgrD are responsible for the synthesis of AIP. AgrD is the pre-pro-peptide of AIP and is processed in part by the membrane bound cysteine protease AgrB, a process which ultimately forms the thiolactone macrocycle within the AIP molecule. Inhibitors of AgrC and AgrA have been studied extensively, however at present, there remains limited study of inhibitors of AgrB
This thesis describes research towards the development of inhibitors of AgrB through the production of analogues of the natural product ambuic acid. This represents the first study of analogues of ambuic acid as inhibitors of staphylococcal virulence. These analogues include the synthesis of a (-)-enantiomer of a truncated ambuic acid analogue and the production of a range of ketal analogues. In addition, the attempted synthesis of a range of silyl ether analogues was also carried out, unfortunately, it was only possible to synthesise one analogue.
In addition, the full chemical synthesis of a cyclopropyl isostere of the epoxide within a truncated ambuic acid analogue was established. This isostere was produced to determine whether the epoxide is required for inhibition of the putative target AgrB in an effort to advance the knowledge of how these compounds function.
The anti-agr activity of ambuic acid has been fully characterised in vitro. Ambuic acid was found to be a potent inhibitor of staphylococcal agr activity utilising a bioluminescent S. aureus reporter. In addition, ambuic acid was found to cause a reduction in the level of expression of the staphylococcal exotoxin α-haemolysin. Furthermore, utilising a bioluminescent S. aureus reporter strain which is incapable of synthesising AIP, but capable of sensing exogenous AIP (designed for the characterisation of inhibitors of AIP sensing), ambuic acid was found to have no effect. This suggests that ambuic acid inhibits the synthesis of AIP rather than the sensing of AIP.
The testing of the synthesised analogues has been carried out in vitro using the bioluminescent S. aureus agr-activity reporter and pharmacological paramaters such as IC50 were determined. The majority of compounds were found to be potent inhibitors of agr activity, with the majority of compounds found to have IC50 values of < 10 μM. Surprisingly, the cyclopropyl isosteric analogue was found to be a potent inhibitor of agr activity, suggesting that the epoxide is not required for agr-inhibitory activity.
These data, taken together represent the first study of analogues of the natural fungal product ambuic acid as inhibitors of staphylococcal virulence. In addition, these data show that these analogues are promising inhibitors of agr-activity and the production of further analogues could be used for developing a structure-activity understanding of how this class of compounds function. |
| first_indexed | 2025-11-14T20:49:10Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-66015 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T20:49:10Z |
| publishDate | 2021 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-660152021-12-31T04:40:23Z https://eprints.nottingham.ac.uk/66015/ The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence Reekes, James Staphylococcus aureus is a Gram-positive human pathogen of significant clinical importance. The development of resistance to numerous classes of clinically important antibiotics has exacerbated the need for the development of novel therapeutic modalities for the treatment of serious, drug-resistant S. aureus infections. One approach for developing new therapies is the discovery of anti-virulence compounds. Virulence in S. aureus is mainly regulated by the agr quorum sensing system. The agr system is a master regulator of staphylococcal virulence and utilises the thiolactone macrocyclic peptide, auto-inducing peptide (AIP) as the signal molecule. The agr system is based around two divergent promoters, agrP2 and agrP3. The agrP2 promoter drives expression of the agr operon, which consists of 4 genes, agrB, agrD, agrC and agrA. AgrB and AgrD are responsible for the synthesis of AIP. AgrD is the pre-pro-peptide of AIP and is processed in part by the membrane bound cysteine protease AgrB, a process which ultimately forms the thiolactone macrocycle within the AIP molecule. Inhibitors of AgrC and AgrA have been studied extensively, however at present, there remains limited study of inhibitors of AgrB This thesis describes research towards the development of inhibitors of AgrB through the production of analogues of the natural product ambuic acid. This represents the first study of analogues of ambuic acid as inhibitors of staphylococcal virulence. These analogues include the synthesis of a (-)-enantiomer of a truncated ambuic acid analogue and the production of a range of ketal analogues. In addition, the attempted synthesis of a range of silyl ether analogues was also carried out, unfortunately, it was only possible to synthesise one analogue. In addition, the full chemical synthesis of a cyclopropyl isostere of the epoxide within a truncated ambuic acid analogue was established. This isostere was produced to determine whether the epoxide is required for inhibition of the putative target AgrB in an effort to advance the knowledge of how these compounds function. The anti-agr activity of ambuic acid has been fully characterised in vitro. Ambuic acid was found to be a potent inhibitor of staphylococcal agr activity utilising a bioluminescent S. aureus reporter. In addition, ambuic acid was found to cause a reduction in the level of expression of the staphylococcal exotoxin α-haemolysin. Furthermore, utilising a bioluminescent S. aureus reporter strain which is incapable of synthesising AIP, but capable of sensing exogenous AIP (designed for the characterisation of inhibitors of AIP sensing), ambuic acid was found to have no effect. This suggests that ambuic acid inhibits the synthesis of AIP rather than the sensing of AIP. The testing of the synthesised analogues has been carried out in vitro using the bioluminescent S. aureus agr-activity reporter and pharmacological paramaters such as IC50 were determined. The majority of compounds were found to be potent inhibitors of agr activity, with the majority of compounds found to have IC50 values of < 10 μM. Surprisingly, the cyclopropyl isosteric analogue was found to be a potent inhibitor of agr activity, suggesting that the epoxide is not required for agr-inhibitory activity. These data, taken together represent the first study of analogues of the natural fungal product ambuic acid as inhibitors of staphylococcal virulence. In addition, these data show that these analogues are promising inhibitors of agr-activity and the production of further analogues could be used for developing a structure-activity understanding of how this class of compounds function. 2021-12-31 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/66015/1/James_Reekes_PhD_Thesis.pdf Reekes, James (2021) The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence. PhD thesis, University of Nottingham. Staphylococcal virulence Synthesis Novel analogues (+)-ambuic acid |
| spellingShingle | Staphylococcal virulence Synthesis Novel analogues (+)-ambuic acid Reekes, James The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title | The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title_full | The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title_fullStr | The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title_full_unstemmed | The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title_short | The synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| title_sort | synthesis and testing of novel analogues of (+)-ambuic acid as inhibitors of staphylococcal virulence |
| topic | Staphylococcal virulence Synthesis Novel analogues (+)-ambuic acid |
| url | https://eprints.nottingham.ac.uk/66015/ |