Conformational Studies on Some Inhibitors of Thermolysin and EC 3.4.24.11

Molecular modeling is an exciting new approach in the field of drug design. A molecule's activity as a drug is dependent upon its conformation i.e. the three dimensional arrangement of its functional groups. Molecular mechanics coupled with interactive computer graphics is an excellent technique for studying the conformations of small molecules and this thesis presents a discussion of this technique and its application to some inhibitors of the enzymes Thermolysin and EC 3.4. 24.11. An introduction to the role of molecular mechanics in computer assisted drug design is given in Chapter One. Chapter Two describes the calculation of steric energies and the potential functions used to do this, while the procedures used for conformation generation and energy minimisation are discussed in Chapter Three (Appendix A lists one of the programs used for the conformation generation). The MOL software package is an interactive modeling system for small molecules which has been developed at Glasgow and Chapter Four contains a description of this system and its options. The structure and function of many enzymes is of prime importance in drug design since drugs often act by inhibition of enzymic pathways. Since very few enzyme structures are known from X-ray crystallography the primary source of information is enzyme kinetics and Chapter Five discusses some important properties of enzymes, such as their functions as catalysts, substrate and inhibitor kinetics and evolutionary pathways. EC 3.4.24.11. (a mammalian enzyme) and Thermolysin (EC 3.4.24.4, a bacterial enzyme) have very similar substrate specificities and are inhibited at comparable rates by several inhibitors. Very little is known about the active site of EC 3.4.24.11. , however, the structure of Thermolysin is well known from X-ray crystallography and in Chapter Six the known features of active sites of both enzymes are discussed and compared. It is known that two isosteric inhibitors of Thermolysin, B-Phenylpropionyl-L-Phe and Carbobenzyoxy-L-Phe, bind in completely opposite orientations. This unexpected phenomenancan be explained by conformational studies on the two inhibitors. In both cases the enzyme does not bind the lowest energy conformer, however in each case it binds the conformer which has both low energy and gives a good fit to the active site. The results of conformational studies of inhibitors of EC 3.4.24.11. are also discussed in Chapter Six and comparison of the low energy conformers with inhibitors of Thermolysin (from X-ray data) point to differences in the positions and sizes of the S1 and S2' subsites of the two enzymes. Both enzymes are highly specific for hydrophobic groups in the S1' site, with Phe being the most favoured residue. From the X-ray studies of Thermolysin it is known that the Phe side chain is bound with torsion angles of roughly 170 and 80. It is interesting to note that these torsion angle values are very common in many of the low energy conformers and it seems reasonable to assume that the high level of specificity arises from the fact that the side chain of the Phe, Leu etc. are normally in the most favourable position for binding to the S1 hydrophobic pocket without rearrangement. Several other differences between the two enzymes lead to the conclusion that their functional similarity is more likely to be a product of evolutionary convergence on function than of divergence from a common ancestor. Chapter Seven contains a brief summary of the programming which was done during the course of this project paying particular attention to both the motivation and methods involved in programming an array processor and as previously mentioned Appendix A contains a listing of the GLOMIN program - a conformation generation program which was modified to run on an array processor during the course of this project. Finally, Chapter Eight contains a discussion of work carried out in conjunction with Professor A. Y. Meyer while he was on sabbatical leave at Glasgow University. This work shows that force fields can be modified quite simply to give more transferable non-bonding parameters by the inclusion of an electrostatic term. A paper on this work has been published in the Journal of Computational Chemistry and a copy of this paper is given in Appendix B

Late-Onset Meningeal Metastases in Liposarcoma: A Case Report and Review of the Literature

Intracranial metastases from liposarcoma are rare and almost always preceded by the development of systemic tumour spread. We report here a case of liposarcoma with spread to the cranial nervous system 23 years after treatment of the primary tumour. The literature on brain metastases from soft tissue sarcoma is also reviewed

Lifelong behavioral and neuropathological consequences of repetitive mild traumatic brain injury

Objective: Exposure to repetitive concussion, or mild traumatic brain injury (mTBI), has been linked with increased risk of long-term neurodegenerative changes, specifically chronic traumatic encephalopathy (CTE). To date, preclinical studies largely have focused on the immediate aftermath of mTBI, with no literature on the lifelong consequences of mTBI in these models. This study provides the first account of lifelong neurobehavioral and histological consequences of repetitive mTBI providing unique insight into the constellation of evolving and ongoing pathologies with late survival. Methods: Male C57BL/6J mice (aged 2–3 months) were exposed to either single or repetitive mild TBI or sham procedure. Thereafter, animals were monitored and assessed at 24 months post last injury for measures of motor coordination, learning deficits, cognitive function, and anxiety-like behavior prior to euthanasia and preparation of the brains for detailed neuropathological and protein biochemical studies. Results: At 24 months survival animals exposed to r-mTBI showed clear evidence of learning and working memory impairment with a lack of spatial memory and vestibule-motor vestibulomotor deficits compared to sham animals. Associated with these late behavioral deficits there was evidence of ongoing axonal degeneration and neuroinflammation in subcortical white matter tracts. Notably, these changes were also observed after a single mTBI, albeit to a lesser degree than repetitive mTBI. Interpretation: In this context, our current data demonstrate, for the first time, that rather than an acute, time limited event, mild TBI can precipitate a lifelong degenerative process. These data therefore suggest that successful treatment strategies should consider both the acute and chronic nature of mTBI

Nilvadipine suppresses inflammation via inhibition of P-SYK and restores spatial memory deficits in a mouse model of repetitive mild TBI

Abstract: Repeated exposure to mild TBI (mTBI) has been linked to an increased risk of Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE) and other neurodegenerative diseases. Some pathological features typically observed in AD have been found in postmortem brains of TBI and CTE, hence treatments tested for AD have a potential to be effective against r-mTBI outcomes. Neuroinflammation may present a possible answer due to its central role both in acute brain injury and in chronic degenerative-like disorders. Our previous studies have shown that drug nilvadipine, acting as an inhibitor of spleen tyrosine kinase (SYK), is effective at reducing inflammation, tau hyperphosphorylation and amyloid production in AD mouse models. To demonstrate the effect of nilvadipine in the absence of age-related variables, we introduced the same treatment to young r-mTBI mice. We further investigate therapeutic mechanisms of nilvadipine using its racemic properties. Both enantiomers, (+)-nilvadipine and (−)-nilvadipine, can lower SYK activity, whereas (+)-nilvadipine is also a potent L-type calcium channel blocker (CCB) and shown to be anti-hypertensive. All r-mTBI mice exhibited increased neuroinflammation and impaired cognitive performance and motor functions. Treatment with racemic nilvadipine mitigated the TBI-induced inflammatory response and significantly improved spatial memory, whereas (−)-enantiomer decreased microgliosis and improved spatial memory but failed to reduce the astroglial response to as much as the racemate. These results suggest the therapeutic potential of SYK inhibition that is enhanced when combined with the CCB effect, which indicate a therapeutic advantage of multi-action drugs for r-mTBI

Acute or delayed treatment with anatabine improves spatial memory and reduces pathological sequelae at late time-points after repetitive mild traumatic brain injury

Traumatic brain injury (TBI) has chronic and long-term consequences for which there are currently no approved pharmacological treatments. We have previously characterized the chronic neurobehavioral and pathological sequelae of a mouse model of repetitive mild TBI (r-mTBI) through to 2 years post-TBI. Despite the mild nature of the initial insult, secondary injury processes are initiated that involve neuroinflammatory and neurodegenerative pathways persisting and progressing for weeks and months post-injury and providing a potential window of opportunity for therapeutic intervention. In this study we examined the efficacy of a novel anti-inflammatory compound, anatabine, in modifying outcome after TBI. Our model of r-mTBI involves a series of five mild impacts (midline impact at 5 m/sec, 1 mm strike depth, 200 msec dwell time) with an interval of 48 h. Anatabine treatment was administered starting 30 min after injury and was delivered continuously through drinking water. At 6 months after TBI, anatabine treatment improved spatial memory in injured mice. Nine months after TBI, a cohort of mice was euthanized for pathological analysis that revealed reductions in astroglial (glial fibrillary acid protein, GFAP) and microglial (ionized calcium-binding adapter molecule 1, IBA1) responses in treated, injured animals. Treatments for the remaining mice were then crossed-over to assess the effects of late treatment administration and the effects of treatment termination. Nine months following crossover the remaining mice showed no effect of injury on their spatial memory, and whereas pathological analysis showed improvements in mice that had received delayed treatment, corpus callosum IBA1 increased in post-crossover placebo r-mTBI mice. These data demonstrate efficacy of both early and late initiation of treatment with anatabine in improving long term behavioral and pathology outcomes after mild TBI. Future studies will characterize the treatment window, the time course of treatment needed, and the dose needed to achieve therapeutic levels of anatabine in humans after injury

Opening doors to nature: Bringing calm and raising aspirations of vulnerable young people through nature-based intervention

This qualitative study explores the experiences of YMCA residents who participated in a nature-based intervention designed to support wellbeing run by Derbyshire Wildlife Trust and YMCA Derbyshire. The intervention ran over 9 weeks and involved taking groups of residents off site for a range of outdoor activities from allotment gardening to nature conservation in various outdoor environments.  After the intervention took place semi-structured interviews, which explored the personal journeys of 8 residents who had participated in the intervention, were conducted. An IPA analysis of the interviews identified three superordinate themes: building social relationships, developing skills and developing feelings of self-worth and managing emotions through nature. It is argued that the intervention enabled the residents to feel part of a supportive community which enabled a positive shift in identity. Furthermore, the programme helped residents manage their emotions, supporting their mental health and promoting a general sense of wellbeing. This is especially important, given that members of the intervention have a history of mental health issues and often come from a background of higher socio-economic deprivation, where opportunities for social cohesion and nature connectedness are scarce. N/

Understanding farmers' naturalistic decision making around prophylactic antibiotic use in lambs using a grounded theory and natural language processing approach

The routine use of antibiotics for prevention of disease in neonatal lambs has been highlighted as inappropriate, yet research suggests that many farmers in the UK still carry out this practice.The aim of the study was to understand farmers' naturalistic decision-making around prophylactic antibiotic use in lambs. Data from 431 posts by 133 different users of an online discussion forum were analysed quantitatively using natural language processing and qualitatively using a grounded theory approach. Results from the qualitative analysis identified five categories that influenced farmers risk perceptions around prophylactic antibiotic use in lambs: anticipated regret, negative emotions and experiential avoidance; economic considerations; farmer identity; perception of capability; and perception of social judgement. Natural language processing analysis of the posts by the study group were compared to posts on topics unrelated to antibiotic use by control groups from the same forum to understand the underlying style and tone within the text. Analytical thinking and authenticity scores were significantly lower in the study group compared to the control groups (P [less than] 0.01). Words relating to cognitive processes were significantly higher in the study group compared to the control groups (P [less than 0.01). Results of the qualitative and quantitative analysis were integrated to assess the fit of the data and enhance findings from either method alone.The key findings were the identification of reasons why farmers used antibiotics prophylactically in neonatal lambs. Farmers disassociated the use of oral antibiotics in neonatal lambs from other types of antibiotics within sheep farming. Farmers used the concept of luck and uniqueness to justify their prophylactic antibiotic use. This may explain farmers' low analytical thinking scores as this reflects a more personal style of thinking as farmers express their thoughts on antibiotic use on an individual basis. Farmers' felt that they did not have the capabilities to control neonatal disease without antibiotics during busy periods and this was exacerbated by external economic constraints. For farmers' who used antibiotics for therapeutic use, fear of social judgement was an important factor in their perceptions of risk around antibiotic use. The high frequency of negations and conjunctions suggested feelings of moral duty. As such, antibiotic use has developed into a symbol of their good farming identity which conflicted with the identity held by those who used antibiotics prophylactically. These results can be used to inform knowledge exchange around prophylactic antibiotic use in lambs to improve antibiotic stewardship in the sheep farming sector