Studies on the synthesis of novel PP2A inhibitors and a GPCR detergent

Chapter 1 While targeting kinases in oncology research has been explored extensively, targeting protein phosphatases is currently in its infancy. However, a number of pharmaceutical companies are currently looking to expand their research efforts in this area. PP2A has been shown to down-regulate ERK5, a mitogen-activated protein kinase (MAPK) that has been shown to be important in driving the invasive phenotype of prostate cancer. Fostriecin and its related structural analogues PD 113,270 and 113,271 have been shown to inhibit a mitotic entry checkpoint in cell growth through the potent and selective inhibition of protein phosphatases PP1, PP2A, and PP4 (IC50 of 45 μM, 1.5 nM, and 3 nM respectively). Fostriecin is one of the most selective protein phosphatase inhibitors disclosed to date with a 104 fold selectivity for PP2A/PP4 versus PP1. Unfortunately, fostriecin and its analogues are very unstable, and this instability has effectively prevented them from being used as effective therapeutic leads. The microcystins and nodularins on the other hand, exhibit significant inhibitory activity against PP1 and PP2A (IC50 = 26 pM and 1.8 nM respectively), but their high toxicity has prevented any therapeutic application. Truncation of the ADDA chain from these polypeptides completely attenuates PP inhibitory activity. Simpler analogues incorporating the N-acylated ADDA chain and D-Ala retain moderate activity against PP1 and PP2A (IC50 = 1.0 μM and 0.17 μM respectively). The generation of a new series of fostriecin analogues to further expand its structure-activity relationship is envisaged with a view to creating new more stable PP2A inhibitors. It was hoped that by incorporating some of the more stable structural features of ADDA into fostriecin that stability and activity could be reconciled. With that in mind a series of PP2A inhibitors were synthesised and biologically evaluated. Chapter 2 GPCRs are an important area of research and are the targets of a quarter of the drugs on the market (2005). As a result, GPCRs continue to be at the forefront of research in both small and large drug companies. However one of the difficulties in studying this diverse class of membrane proteins is their tendency to denature in aqueous solution. As a result there is a pressing need to develop new detergents to solubilise, stabilise and crystallise GPCRs in their native form for further study. Cholesterol analogues have been shown to be important for stabilising membrane proteins and preventing their thermal inactivation. In addition the β2-adrenergic receptor, a GPCR membrane protein, has been crystallised in the active state with two cholesterol molecules bound between the I, II, III and IV helices of the protein. This appears to represent a distinct cholesterol binding pocket on the membrane protein that is speculated to be conserved across up to 44% of the rhodopsin class of GPCRs. CHOBIMALT is a cholesterol-based detergent that has been shown to exhibit promising GPCR-stabilising properties. When benchmarked against other cholesterol based detergents it was found to be superior to all others tested except for cholesteryl hemisuccinate.1 CHOBIMALT has an aggregation number of roughly 200 and forms 210 ± 30 kDa micelles, which are significantly larger than those of most detergents used for biological systems which is likely due to the packing constraints associated with CHOBMALT’s large polar headgroup.2 As a result, CHOBIMALT is used mostly as an additive to other commercially available detergents in order to decrease micelle size. A branched dimaltoside motif is common in recently synthesised detergents by Chae and co-workers. These detergents have shown promising detergent properties, for example the maltose neopentyl glycol (MNG) detergent synthesised by Chae. This branched dimaltoside detergent was shown to be able to solubilise and stabilise the very labile light harvesting complex I (LHI) from Rhodopsin capsulatus in its active form for 20 days with little loss of protein conformation.3 A cholesterol-based detergent was envisaged that combines the cholesterol framework of CHOBIMALT but replaces its linear tetrasaccharide with a branched dimaltoside. This detergent would then be investigated to assess its ability to solubilise, stabilise and crystallise GPCR proteins. This cholesterol-based detergent (shown below) was eventually synthesised in 9 linear steps from cholesterol.

The Scots in Ireland: culture, colonialism and memory, 1315-1826

This thesis examines three key moments in the intersecting histories of Scotland, Ireland and England, and their impact on literature. Chapter one Robert Bruce and the Last King of Ireland: Writing the Irish Invasion, 1315- 1826‘, is split into two parts. Part one, Barbour‘s (other) Bruce‘ focuses on John Barbour‘s The Bruce (1375) and its depiction of the Bruce‘s Irish campaign (1315-1318). It first examines the invasion material from the perspective of the existing Irish and Scottish relationship and their opposition to English authority. It highlights possible political and ideological motivations behind Barbour‘s negative portrait of Edward Bruce - whom Barbour presents as the catalyst for the invasion and the source of its carnage and ultimate failure - and his partisan comparison between Edward and his brother Robert I. It also probes the socio-polticial and ideological background to the Bruce and its depiction of the Irish campaign, in addition to Edward and Robert. It peers behind some of the Bruce‘s most lauded themes such as chivalry, heroism, loyalty, and patriotism, and exposes its militaristic feudal ideology, its propaganda rich rhetoric, and its illusions of freedom‘. Part one concludes with an examination of two of the Irish section‘s most marginalised figures, the Irish and a laundry woman. Part two, Cultural Memories of the Bruce Invasion of Ireland, 1375-1826‘, examines the cultural memory of the Bruce invasion in three literary works from the Medieval, Early Modern and Romantic periods. The first, and by far the most significant memorialisation of the invasion is Barbour‘s Bruce, which is positioned for the first time within the tradition of ars memoriae (art of memory) and present-day cultural memory theories. The Bruce is evaluated as a site of memory and Barbour‘s methods are compared with Icelandic literature of the same period. The recall of the invasion in late sixteenth century Anglo-Irish literature is then considered, specifically Edmund Spenser‘s A View of the State of Ireland, which is viewed in the context of contemporary Ulster politics. The final text to be considered is William Hamilton Drummond‘s Bruce’s Invasion of Ireland (1826). It is argued that Drummond‘s poem offers an alternative Irish version of the invasion; a counter-memory that responds to nineteenth-century British politics, in addition to the controversy surrounding the publication of the Ossian fragments. Chapter two, The Scots in Ulster: Policies, Proposals and Projects, 1551-1575‘, examines the struggle between Irish and Scottish Gaels and the English for dominance in north Ulster, and its impact on England‘s wider colonial ideology, strategy, literature and life writing. Part one entitled Noisy neighbours, 1551-1567‘ covers the deputyships of Sir James Croft, Sir Thomas Radcliffe, and Sir Henry Sidney, and examines English colonial writing during a crucial period when the Scots provoked an increase in militarisation in the region. Part two Devices, Advices, and Descriptions, 1567-1575‘, deals with the relationship between the Scots and Turlough O‘Neill, the influence of the 5th Earl of Argyll, and the rise of Sorley Boy MacDonnell. It proposes that a renewed Gaelic alliance hindered England‘s conquest of Ireland and generated numerous plantation proposals and projects for Ulster. Many of which exhibit a blurring‘ between the documentary and the literary; while all attest to the considerable impact of the Gaelic Scots in both motivating and frustrating various projects for that province, the most prominent of which were undertaken by Sir Thomas Smith in 1571 and Walter Devereux, 1st Earl of Essex in 1573.