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 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

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 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

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins

Abstract: Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm

Acute Ethanol Exposure during Synaptogenesis Rapidly Alters Medium Spiny Neuron Morphology and Synaptic Protein Expression in the Dorsal Striatum

Fetal alcohol spectrum disorders are caused by the disruption of normal brain development in utero. The severity and range of symptoms is dictated by both the dosage and timing of ethanol administration, and the resulting developmental processes that are impacted. In order to investigate the effects of an acute, high-dose intoxication event on the development of medium spiny neurons (MSNs) in the striatum, mice were injected with ethanol on P6, and neuronal morphology was assessed after 24 h, or at 1 month or 5 months of age. Data indicate an immediate increase in MSN dendritic length and branching, a rapid decrease in spine number, and increased levels of the synaptic protein PSD-95 as a consequence of this neonatal exposure to ethanol, but these differences do not persist into adulthood. These results demonstrate a rapid neuronal response to ethanol exposure and characterize the dynamic nature of neuronal architecture in the MSNs. Although differences in neuronal branching and spine density induced by ethanol resolve with time, early changes in the caudate/putamen region have a potential impact on the execution of complex motor skills, as well as aspects of long-term learning and addictive behavior