The rise of antibiotic resistance demands the development of new antimicrobial agents. These should exhibit a novel mechanism of action so as to overcome the resistance and be invulnerable to 'not yet acquired resistance mechanisms'. Such criteria are difficult to meet, however cationic host defence peptides (HOPs) have emerged as promising candidates. HOPs target and disrupt bacterial membranes via simple physio-chemical interactions and the fundamental nature of this target raises the possibility of antimicrobial drugs based on HOP being invulnerable to the development of widespread resistance. Despite this apparent invulnerability, localised resistance to HOP does exist and the use of HOP as drugs will create new selective pressure which bacteria may be able to respond to. Herein a new potential resistance mechanism to helical HOPs in both de novo and native systems is presented. In this work coiled-coil folding was tested as a HOP regulatory system: • Anionic anti-sequences were designed to form coiled - coil dimers with peptides from three classes of naturally occurring HOP. • Native HOP sequences were mutated to identify regions of the sequences able to resist coiled-coil formation. • Successful systems were subject to biological testing to asses viability of the complexes as a microbial HOP resistance mechanism The results provide a new insight into potential resistance mechanisms to HOP and they should serve to place constraints on the design of synthetic HOP sequences. The results may be used as inspiration in the search for novel antimicrobial drug targets.

The surfactant-like peptides are amphiphillic molecules containing polar or charged amino acids moieties as hydrophilic heads and non-polar ammo acids as hydrophobic tails, their surfactant-like amphiphilicity is attractive to a wide range of biotechnological applications including regenerative medicine and drug delivery. Short and designed peptides are easy to synthesise. They are becoming increasingly popular for self-assembly studies as model systems. It is of bith fundamental and practical significance to explore how these designed peptide molecules aggregate in solution and adsorb at the interface.

Self-assembling peptide systems provide a pathway for the formation of complex molecular assemblies from relatively simple designed molecules. Stimuli which have been used to trigger the self-assembly (SA) process in aqueous conditions include temperature, pH, ionic strength, and solvent exchange. Additionally, enzymes may be used to selectively control the self-assembly process; enzymes are uniquely chemo-, regio-, and enantioselective, and work naturally under mild conditions without disrupting biological Interactions.

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Small peptides work as neurotransmitters or hormones in the body. For example, the pentapeptide enkephalin is involved in a wide variety of physiological processes such as mediation of pain and respiratory depression. In order to understand the biological function of these molecules it is necessary to examine their molecular shape and structural preferences. The large flexibility of peptides makes them difficult to be characterized by experimental and theoretical methods. A method was developed in order to explore the conformational preferences of a polypeptide using electronic structure methods based on hierarchical selection criteria. The strategy was to vary all the torsion angles of the peptide and to create all possible conformers. The conformers were assessed according to the number of hydrogen-bonding interactions in their structure. Calculations were performed at increasingly higher levels of theory. Only a number of the most stable conformers were taken through to the next level. This hierarchical selection method was used to explore the conformational features of the dipeptide Tyr-Gly (the first two amino acids of the pentapeptide enkephalin: Tyr-Gly-Gly-Phe-Met).The conformational preferences of Tyr-Gly were also explored with a stepwise rotation method. The hierarchical selection method seemed to be superior. The most stable conformers found for Tyr-Gly are characterized by a characteristic hydrogen-bonding interaction (0-H 0) between the hydroxyl hydrogen of glycine and the carboxyl oxygen of tyrosine. The optimized structures obtained with DFT differ from the structures obtained by MP2 geometry optimizations. MP2 optimizations make the structures more folded. The method has also been used to study the conformational features of the Tyr- Gly-Gly tripeptide, Tyr-Gly-Gly-Phe tetrapeptide, Tyr-Gly-Gly-Phe-Leu pentapeptide and Gly-GIy-Gly tripeptide. The most stable conformers obtained for the Tyr-Gly-Gly tripeptide are characterized by folded structures with a characteristic hydrogen-bonding interaction between the (-OH) phenyl group of tyrosine and the carboxyl oxygen of glycine (3). The most stable conformers obtained for Tyr-Gly-Gly-Phe and Tyr-Gly-Gly-Phe-Leu are characterized by folded structures with a characteristic hydrogen-bonding interaction between the (-OH) phenyl group of tyrosine and the carboxyl oxygen of phenylalanine (4). For Tyr-Gly, Tyr-Gly-Gly, Tyr-Gly-Gly-Phe and Tyr-Gly-Gly-Phe-Leu a large variation has been observed between DFT and MP2 orders of stability of the conformers. DFT fails to describe the dispersion effects arising from interactions involving the aromatic residues. MP2 would probably describe more accurately the conformational preferences of these peptides. However, the large basis set superposition error in MP2 calculations means that also MP2 may not be suitable to characterize the conformational preferences of these peptides. The hierarchical selection method developed has been shown to be a useful method to study small peptides.

INTRODUCTION: The focus of this MD thesis has been relaxin, a member of the insulin family, which is a protein composed of two disulphide linked chains of approximately 6000 Daltons. Relaxin has been traditionally recognised as a hormone of parturition, though more recently it has been postulated that relaxin may be involved in cardiovascular regulation. We used concentrations similar to those found in the plasma in physiological (non-pregnant, pregnancy) and pathophysiological (chronic heart failure) states. Firstly, we characterised the effects of relaxin in small human resistance arteries ex vivo using wire myography obtained from gluteal biopsies taken from patients with coronary heart disease (CHD) and normal left ventricular systolic function. We also studied the same effects in larger calibre arteries (internal mammary) and veins (saphenous) using standard organ bath techniques. The effect of relaxin in veins has not previously been described. Internal mammary arteries and saphenous veins were obtained from patients undergoing coronary artery bypass surgery. Small pulmonary arteries were obtained from patients undergoing thoracotomy for bronchial carcinoma. In addition, we wished to determine if a transcardiac or transpulmonary gradient of relaxin could be measured to suggest either pulmonary or cardiac secretion or clearance of the hormone. Relaxin secretion in heart failure has previously been described. Lastly, we wished to determine whether an increased relaxin plasma concentration in patients with chronic heart failure (CHF), is of prognostic importance. METHODS AND RESULTS i)comparative potency of relaxin compared to other vasodilators: Small resistance arteries were obtained from biopsies taken from patients with CHD. Each set of vessels was preconstricted with noradrenaline. Thereafter, cumulative concentration response (relaxation) curves (CRCs) were constructed with known vasodilators 25 atrial natriuretic peptide (ANP), epoprostenol, substance P and relaxin (n=8). Relaxin was found to be a more potent vasodilator than ANP and equipotent to epoprostenol. ii) mechanism of vasorelaxation: CRCs to relaxin (as above) were constructed to identify the importance of the endothelium – following the removal of the endothelium by the established method of intraluminal rubbing with a human hair. We found that relaxin is endothelium dependent. iii) interaction of relaxin with nitric oxide and other possible mechanisms of vasodilation and importance of ACE inhibitor treatment: We identified the importance of the effect of ACE inhibitor treatment on the action of relaxin in human resistance arteries. Relaxin’s vasodilatory action was significantly reduced in those patients on ACE inhibitors (n=28) compared with those patients not on ACE inhibitors (n=30). In patients treated with an ACE inhibitor, we found that manipulation of prostanoids is important. Indomethacin, (a cyclooxygenase inhibitor) (n=8) blocked relaxin’s vasodilatory action. Manipulation of the cAMP second messenger system, with milrinone, (a cAMP phosphodiesterase inhibitor) (n=6) is also important as relaxin’s vasodilatory action was enhanced. Manipulation of cyclic GMP second messenger system is also important. ODQ, (a guanylate cyclase inhibitor) (n=10) reduced relaxin’s action while zaprinast, (a cGMP phosphodiesterase inhibitor) (n=7) enhanced relaxin’s action. Manipulation of nitric oxide with L-NAME (n=8) and L-NOARG (n=10), nitric oxide synthase (NOS) inhibitors and EDHF with apamin and charybdotoxin (potassium channel blockers) (n=7) had a curious effect causing the opposite action to that expected, by enhancing relaxin’s vasodilatory action. In patients not treated with an ACE inhibitor, we found that manipulation of nitric oxide with L-NAME (n=8) and LNOARG (n=8), is important, as both reduced relaxin’s vasodilatory action. Manipulating the cGMP second messenger system with ODQ (n=8) greatly reduced relaxin’s action. but zaprinast (n=9) did not. Manipulation of EDHF with apamin and charybdotoxin (n=8) had no effect on relaxin’s action. Manipulation of prostanoids with indomethacin (n=10) reduced relaxin’s action but manipulation of cAMP with milrinone (n=8), had no effect. 26 iv)relaxin and small human pulmonary arteries: We determined, using wire myography, that relaxin is not a vasodilator of small pulmonary resistance arteries (n=5). v)relaxin and large calibre vessels: We determined, using the organ bath technique, that relaxin is not a vasodilator of larger calibre arteries i.e. internal mammary arteries removed from patients during coronary artery bypass surgery (n=5).Relaxin is not a venodilator studying saphenous veins removed from patients during coronary artery bypass surgery (n=5). vi)transmyocardial and transpulmonary gradient of relaxin: Plasma relaxin concentrations were measured using a validated assay. Samples were taken from patients undergoing CABG surgery, from the aorta, coronary sinus, pulmonary artery and pulmonary vein. We found that in 20 patients with normal left ventricular function that there was no transpulmonary gradient but there was a transcardiac gradient suggesting net cardiac extraction of relaxin. vii)prognostic value of relaxin in patients with chronic heart failure: Relaxin was compared with N-terminal pro brain natriuretic peptide to determine whether relaxin is of prognostic importance. Plasma concentrations of the hormones were measured in 87 patients admitted with CHF. These patients were followed up for a year during which time hospitalisations due to CHF and death were recorded. While NT-proBNP was found to be a powerful and independent predictor of outcome in these patients, relaxin was not. CONCLUSION. In addition to its established role in pregnancy, relaxin has many other actions. In particular, its antihypertensive, antithrombotic and vasodilatory properties suggest that relaxin may have a central role in cardiovascular regulation.

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