Membrane proteins represent the majority of therapeutic targets for the antibody-based drugs available today. These are routinely identified via phage display screening, but traditional antigen presentation methods require membrane protein targets to be detergent-solubilised in order to preserve their native conformations post-purification. Unfortunately, detergent solubilisation can not only lead to gradual target denaturation over time, but the detergent micelles can also occlude important epitopes on the extramembranous loops and thus prevent the discovery of antibody binders. The current thesis aimed to demonstrate that, by reconstituting purified membrane proteins into spherical-supported bilayer lipid membranes (SSBLMs) deposited on nanosized substrates, a versatile platform can be constructed for performing phage display screening against membrane protein targets, while not only presenting these within a native-like lipid environment, but also eliminating detergents from the screening phase altogether. For providing proof-of-concept, 100- and 200 nm silica nanoparticles were covered with POPC SSBLMs embedding the bacterial nucleoside transporter NupC. Full substrate coverage and the correct formation of the lipid bilayer components were established via spectrofluorometry, using fluorescent labelling and small-angle X-ray scattering (SAXS) respectively, while Western blotting and high-affinity antibody binding confirmed the presence of SSBLM-embedded NupC. The platform was then used to screen designed ankyrin repeat proteins (DARPins) against a His6-tagged construct of NupC across different screening formats so as to offer a comparison to the classic 96-well plate antigen presentation method. Following that, the DARPin binders showing the highest potential affinity for NupC were purified and subjected to further binding validation assays against two other constructs – detergent-solubilised double Strep-tagged NupC and SSBLM-embedded untagged/wild-type NupC – in order to identify any binders targeting extramembranous epitopes that would be accessible in vivo as well. Ultimately, the results presented throughout indicated that SSBLMs constitute a promising means of screening antibody binders against membrane protein targets embedded in a close-to-native format.

Chapter I describes work undertaken on a Medicinal Chemistry project studying the small molecule inhibition of the protein CSF-1R. Initial computational work, using the Cresset software package, involved modeling the key steric and electrostatic properties of compounds known to bind to the active site of the target enzyme, CSF-1R. The subsequent use of a virtual (computational) screen, provided new chemotypic scaffolds, which were used as starting points for structure activity relationship (SAR) exploration. Synthetic chemistry routes were designed and optimised towards analogues of promising compound “hits” focusing on the potential to introduce diversified structural features and the diminution of synthetic steps. This included the construction of an uncommon heterocyclic core motif and efforts to understand and control the regioselectivity of its formation. Chapter II concerns a synthetic methodology project in the area of N-heterocyclic carbene (NHC) catalysis. Work on this project included the development of a novel methodology for the formation of amide bonds using an internal redox approach. This involved the activation of an α-reducible aldehyde with an NHC to form an acyl azolium and subsequent trapping of this intermediate with amine nucleophiles. The hypothesis to be studied was that previous attempts at performing similar transformations with these substrates had been hampered by incompatibility of the aldehyde and amine reagents leading to unwanted condensation side reactivity; thus the main focus of this work was to provide an in situ slow release of the amine nucleophile by masking it as a carbamate that would gradually extrude CO2 under the reaction conditions releasing the amine. The other project discussed in this chapter involves the use of hemiacetals as masked aldehydes, in particular within carbohydrates, due to their natural abundance, making them a cheap source of fixed stereocentres.

Alveolar macrophage accumulations in the lungs of rodents and dogs that have been exposed to inhaled new drug candidates is an important cause of attrition during preclinical drug development because it is very difficult to decide whether the accumulations are adverse or nonadverse. The expression of markers of alveolar macrophage endocytosis, alveolar macrophage activation and lung inflammation were investigated by means of reverse transcriptase-polymerase chain reaction, immunohistochemistry and in situ hybridisation in samples of formalin fixed paraffin embedded lungs from male rats. Messenger RNA levels of CD68, a marker of endocytosis, were increased 1.4 to 2.4-fold in lungs that contained alveolar macrophage accumulations. Cytoplasmic immunostaining related to the CD68 protein was increased 1.43 to 5.66-fold when the alveolar macrophage accumulations were numerous, large and associated with neutrophils and lymphocytes. In contrast, the immunostaining was not increased (range 0.67 to 1.21-fold) when only small and sparse alveolar macrophage accumulations that consisted entirely out of macrophages were present. Messenger RNA levels of resistin like molecule alpha and acidic mammalian chitinase – markers of wound healing (M2) activated macrophages – were found to be increased 3.9 to 8.2-fold and 1.8 to 2.4-fold respectively, in samples of lungs that contained alveolar macrophage accumulations. Messenger RNA levels of inducible nitric oxide synthase, a marker of classical (M1) activated macrophages was found to be increased 4.1-fold when neutrophils and lymphocytes were present amongst the accumulated alveolar macrophages. Messenger RNA levels of pro-inflammatory genes (such as E selectin) were found to be increased 2.2 to 4.4-fold in samples of lungs that contained alveolar macrophage accumulations but only when neutrophils and lymphocytes were visible amongst the accumulated macrophages in histological lung sections. Measuring the markers in FFPE lung samples can potentially help investigators discriminate adverse from nonadverse drug-induced alveolar macrophage accumulations with greater clarity and scientific rigour.

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No description available.

Chapter 1 introduces the Protein Arginine Methyltransferases (PRMTs) as epigenetic regulators that decorate peptidic arginine with methyl groups. Evidence for PRMT involvement in cancer pathogenesis is reviewed and their plausibility as therapeutic targets is introduced. The methylation patterns conferred by the PRMTs is described, and the existence of novel patterns is considered. Techniques for assaying PRMT activity are compared and contrasted and a discussion of current PRMT inhibitors is presented. The chapter concludes by outlining the aims of the thesis. Chapter 2 describes synthesis towards novel methylated arginine molecules that are fully protected for inclusion in peptides via solid phase peptide synthesis. Chapter 3 outlines the total synthesis of protected d-monomethylated arginine for peptide synthesis. This methylation pattern is known in yeast but has not yet been identified in humans. Chapter 4 details a new mass spectrometry-based assay that can be applied for inhibitor and substrate analyses. Synthesis of a novel histone peptide containing the d-monomethylated arginine, produced in Chapter 3, is also described and this is tested for relevance as a human epigenetic marker. Novel polymethylation patterns are also explored in a total of five histone peptides. This chapter concludes with discussion of possible methylation pattern rearrangements. Chapter 5 describes the synthesis and testing of two series of putative PRMT inhibitors based on previously identified scaffolds within this research group. Data obtained from three different assays, including that outlined in Chapter 4, are analysed and suggestions as to the direction of PRMT assay design are offered. Chapter 6 provides the experimental data to support Chapters 2-5, including all organic synthesis procedures, protein & peptide syntheses and assay methodology.

Proton dependent oligopeptide transporters (POTs) play a central role in nitrogen homeostasis by coupling the uptake of dipeptides and tripeptides to the proton electrochemical gradient across the plasma membrane. In human, members of this transporter family, PepT1 and PepT2, are critical modulators of drug pharmacokinetics as they facilitate the uptake and retention of numerous orally administered drugs including the β-lactam antibiotics. Rationally designing drugs to target these transporters is therefore an attractive approach to improving bioavailability. To this end, the binding of peptides to a bacterial homolog, PepTSt, was modelled based on recently determined crystal structures. A range of computational methods to predict the free energy of binding were evaluated and a hybrid approach, where the end-point methods were used to classify peptides into strong and poor binders and a theoretically exact method for refinement, was able to accurately predict ligand affinities. This approach was utilised to investigate the substrate preference of PepTSt and the results were validated using in vitro transport assays. To extend this study to the human peptide transporters, homology models of PepT1 and PepT2 were built using the crystal structures of PepTSo and mouse and rat extracellular domains (ECDs) as the templates. Essential residues as proposed by various mutational studies align well with the binding cavity, suggesting that the models are structurally sound. Applying the free energy methods to predict the affinities of peptides and drugs to the homology model of PepT1, however, resulted in discrepancies with experimental data, highlighting the importance of a high-resolution crystal structure in binding affinity predictions. Based on the results for PepTSt, a binding model for peptide prodrugs and β-lactam antibiotics to the human PepT1 was proposed. Overall, this thesis provides a framework for future computational studies using free energy methods to understand drug interactions with pharmaceutically relevant transporters.

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Meptazinol, dl-m (3 ethylhexahydro-1-methyl-1H-azepin-3-yl) phenol hydrochloride is a new analgesic agent, which from animal studies appears to have a low addiction liability. By contrast however, the desmethyl derivative, although still a potent analgesic was shown to have a much greater addiction potential. Metabolic studies using a novel dual labelling approach showed no evidence for the formation of normeptazinol in animals or man. The major metabolic pathway seen in all the species so far investigated involves rapid conjugation with glucuronic acid. A minor metabolite found only in the urine of monkeys and man has been tentatively identified as 6-ethyl-6-(m hydroxyphenyl)-1-methylhexahydro-azepin-(2H)-2-one This metabolite was shown to be biologically inactive. Pharmacological studies showed meptazinol to be considerably less potent after oral as compared to parenteral administration. A good correlation between the intensity of the drug's biological effects and plasma levels of the compound was demonstrated in rats, mice and monkeys. Oral administration of the drug was shown to produce much lower plasma levels of the free drug than parenteral dosage. This did not appear to be due to incomplete absorption since the drug was efficiently absorbed in all these species. However the rate of absorption was considerably slower in monkeys and man, an effect which was shown to cause more extensive first pass conjugation of the drug in these species. This slower absorption was at least partly due to the longer gastric emptying time in these species. In addition, studies in the monkey showed the drug to have a marked retarding action on stomach emptying, an effect not seen in the rat. Rectal administration of the drug has been shown to provide a means of circumventing the problem of extensive first pass metabolism and effectively increasing the drug's potency. In man plasma levels of the drug resulting from rectal dosage were five to ten times higher than when the same dose was given orally.

Drug transporters are increasingly recognised as important drivers in the pharmaco- and toxico-kinetic characteristics of therapeutic agents. Preclinical assays are used to determine the effects of transport on the pharmacokinetics and toxicology of a new compound during drug development. However in vitro cell systems and in vivo animal models often give erroneous results due to the differential expression of important proteins within them. In silico models have been employed to help bridge the gap and make more accurate predictions for drug disposition and metabolism; however such models have not incorporated transport kinetics or induction by nuclear receptors. Many current models also use a reductionist approach, whereby a multiple component pathway is described by a single mathematical term, reducing the ability of the simulation to determine the effects of single parameters. The first aim of this research was therefore to assess the differences in drug transporter expression in a variety of human and rat hepatocellular systems compared to liver. RT-PCR and protein analysis of drug transporter levels in vitro and in vivo showed huge differences, with influx transporters generally being under-expressed and efflux transporters over-expressed in vitro compared to in vivo. These differences were more pronounced at the RNA level than protein level. Transporter expression was also shown to be dynamic, changing over time in culture and in response to nuclear receptor activation. The second aim was to generate an in silico model of cellular response to stimuli, using a whole-cell approach to investigate system dynamics. Kinetics for the lifecycle rhodamiae-123 were determined in vitro and incorporated into an in silico model of human hepatocyte disposition that could accurately simulate outcomes in a variety of cell types and make useful predictions of drug disposition from in vitro results. In conclusion, the in silico model revealed that drug transporter expression and activity may affect drug disposition, with uptake processes presenting as the fragile node in the network. Such a model would be useful in the early stages of drug development to improve extrapolation between biological systems and to identify the likely consequences of different expression levels and species inhibition and induction.