Membrane proteins are vital to drug discovery, being targeted by some 60% of the currently marketed therapeutic medicines, with more than half of those targeting transmembrane receptors. Identification of transmembrane receptor targets of poorly characterised ligands can provide new starting points for drug innovation, provide valuable information about off-target effects, and enhance mechanistic understanding of molecular pathways. Whereas, over the years, various methods for target identification have been developed, due to unfavourable characteristics, such as hydrophobicity, low abundance and transient ligand-interactions, identification of transmembrane proteins remains a challenge. Described herein is the design, synthesis and evaluation of four universal, trifunctional probes specifically developed to allow the covalent capture of transmembrane receptors in a process called ligand-based receptor capture (LRC). These probes contain three functional groups: (1) a ligand-coupling moiety; (2) a receptor-capturing moiety; (3) and an affinity tag. In an LRC experiment these probes would be coupled to the ligand of interest, after which the adduct would be added to cells believed to express the target receptor(s) to allow receptor-capturing. After affinity purification, captured receptors would be identified using mass spectrometry. All four probes contained an N-hydroxysuccinimide (NHS)-ester to allow ligand-coupling through free amines. For receptor capture, both a protected hydrazine moiety and the photoreactive groups benzophenone and diazirine were investigated. Protected hydrazine moieties will couple to aldehydes, present on sugar tails of glycosylated receptors after mild oxidation, whereas photoreactive groups will form covalent bonds with molecules in close proximity upon activation with UV-light. For affinity purification, probes either contained a biotin group, for purification using streptavidin, or an alkyne moiety, which would allow coupling to any reporter or affinity tag bearing an azide group using the copper-catalyzed azide-alkyne cycloaddition. The interactions between the two peptidic ligands, orexin A and substance P (SP), and their respective G-protein coupled receptors orexin 1 and neurokinin 1 (NK1), expressed in an inducible manner using the Flp-InTM T-RExTM system, were employed as test systems. Initially, these systems were used to investigate individual steps in the LRC protocol, including ligand-coupling, potential interference of the probes on the ligand-receptor interaction, and ability of the probes to covalently couple to the receptor. Only for the probe containing an NHS-ester, a diazirine moiety and a biotin group, could capture of the target receptor be demonstrated. This probe was then coupled to SP and used in a full LRC experiment to successfully identify NK1 as the only SP-binding receptor. This provides a proof of concept, demonstrating that this novel probe could be used as a general tool to help identify target receptors for a variety of ligands in the near future.

Thesis (Ph. D.)--Syracuse University, 2006. / "Publication number AAT 3241611."

Supramolecular chemistry, and in particular molecular recognition through non-covalent interactions is responsible for a vast number of vital biochemical functions. One class of these functions allows enzymes to tune the redox properties of cofactors derived from vitamin-B2 to engage in tasks specific to that protein. In our preliminary investigations, we studied a functional analog of these cofactors in the context of artificial receptors, designed to interact with the guest substance through a specific pattern of hydrogen bonding. We showed experimentally that even in systems that exhibit equivalent hydrogen bonding patterns, marked differences could be observed regarding modulation of molecular properties such as the redox potential of the guest. It was demonstrated computationally that these differences could be attributed to subtle changes in the natures of the hydrogen bonds themselves with the use of strongly polarizable hydrogen bond donors giving the greatest modulation of the physical characteristic of the guest. We then employed π-stacking units in conjunction with the same hydrogen bonding contacts to further explore the use of non-covalent interactions to tailor the physical properties of a guest substance. Experimental work showed that using two sets of non-covalent interactions in combination, marked differences could be observed in the binding of one guest oxidation state relative to another. These differences in binding manifested themselves directly as changes in the redox properties of the guest substance. The knowledge gained from this work allowed us to design and construct dual input electro- and photochemically controlled devices. In order to integrate molecular recognition with current technology, we endeavored to explore how substrate-receptor interactions would operate in the context of a macromolecular environment. Studies on the binding of an electron deficient guest molecule using a polymer appended with electron rich recognition units showed that substantial changes in polymer volume and thermal characteristics accompanied guest binding. Using specific interactions in the form of a hydrogen-bonding pattern it was demonstrated that efficient guest binding is strongly dependent on the exposure of the recognition units to the surrounding medium. Furthermore, we demonstrated that using specific interactions between recognition grafted polymers and complementary nanoparticles, extended well ordered aggregates could be produced. The efficient propagation of order was found to be promoted by compact polymers that were best able to fill the interstitial voids between the spherical particles. Finally, we studied the aggregates formed between complementary functionalized polymers. It was demonstrated that the self-assembly of complementary polymer strands lead to the formation of giant vesicles through specific interchain hydrogen bonding.

No description available.

The number of protein structures deposited in the Protein data bank is increasing almost exponentially and among these structures many of the proteins are novel with unknown function. Like Docking, Pharmacophore searching is an In-silico technique which is widely used for drug discovery. In pharmacophore searching the main focus is on the hydrogen bond interactions between the ligand and the target protein. The pharmacophore models are generated either by using the already known actives as templates or by utilizing the significant chemical features of the active site. In this thesis the pharmacophore searching has been used to find potential ligands/substrates for unknown proteins and then ligand binding is confirmed by using different biophysical techniques. In the initial phases the pharmacophore models were generated by using Cerius2 and Weblab Viewer pro programs. While in later stages more sophisticated searches were carried out by using DSV (Discovery studio visualizer, Accelrys®). Procedures were optimized for model building by using DSV, which enabled the pharmacophore searching via both the Vector and the Query atom methods. To validate the technique, it was first used on known enzymes with established function e.g. xylose reductase and shikimate kinase. The optimized pharmacophore model when search through the database successfully identified the true substrates for these enzymes among other ligands thereby demonstrating the attainment. The pharmacophore searching technique has been used to find potential ligands for proteins with unknown function on three test cases e.g. TdcF, HutD and PARI. Of the potential pharmacophore hits obtained through database search, a number of compounds were either purchased or synthesised to be tested for binding affinity. Different biophysical techniques like DSC, ITC, CD and NMR were used for this purpose. Among these techniques NMR proved to be the most sensitive technique to differentiate binders from non-binders and to further detect weak and strong bonding in terms of Kd values. For TdcF among other binders the best binder was 2-ketobutyrate with a Kd value of 200µM. In case of HutD, formyl glutamate (Kd = 92µM) and formimino glutamate (Kd = 500µM) came out to be the best binders and could be the true ligands of the protein at physiological concentration. For PARI L-glutamate appeared to be a potential ligand for the protein as confirmed through the NMR experiments. Pharmacophore modelling has been successful in identifying potential interactions provided by the protein active site which in turns specifies the required features to be present in a ligand and later on the successful binding studies further confirm its applicability. In addition, protein structures from the protein data bank (PDB) with unknown ligands (UNK) were identified and manually screened to find examples that could be used to test the applicability of pharmacophore searching methods. The diversity of structures showed that the definition of an unknown ligand is completely inconsistent with many examples where any non spherical density was labelled as unknown ligand and in most cases a single atom is labelled as an unknown ligand, which most likely can be an ion or a water molecule. It appeared that some compounds like glycerol, phosphate and citrate which co-crystallized with the protein due to their presence in the crystallization conditions were also mistakenly assigned as UNK. The pharmacophore method worked successfully in finding suitable ligand (s) for the protein.

The function of biological macromolecules is inherently linked to their complex conformational behaviour. As a consequence, the corresponding potential energy landscape encompasses multiple minima. Some of the intermediate structures between the initial and final states can be characterized by experimental techniques. Computer simulations can explore the dynamics of individual states and bring these together to rationalize the overall process. A novel method based on atomistic structure-based potentials in combination with the empirical valence bond theory (EVB-SBP) has been developed and implemented in the Amber package. The method has been successfully applied to explore various biological processes. The first application of the EVB-SBP approach involves the study of base flipping in B-DNA. The use of simple structurebased potentials are shown to reproduce structural ensembles of stable states obtained by using more accurate force field simulations. Umbrella sampling in conjunction with the energy gap reaction coordinate enables the study of alternative molecular pathways efficiently. The main application of the method is the study of the switching mechanism in a short bistable RNA. Molecular pathways, which connect the two stable states, have been elucidated, with particular interest to the characterisation of the transition state ensemble. In addition, NMR experiments have been performed to support the theoretical findings. Finally, a recent study of large-scale conformational transitions in protein kinases shows the general applicability of the method to different biomolecules.

Syntheses of 2,3-dihydrobenzofurans and 3,4-dihydrobenzopyrans normally involve several stages, therefore efficient syntheses of these compounds are desirable. A number of 3,4-dihydrobenzopyrans derivatives were prepared in one pot syntheses. The reaction is thought to proceed through cation intermediates, involving allylic cations generated by the reaction of allyl alcohols, 1,3-dienes, or diols in the presence of acids such as trifluoroacetic acid (TFA), or in a solution of glacial acetic and a metal catalyst or glacial acetic acid and sulphuric acid, followed by reaction with nucleophiles such as phenols or hydroquinones. This leads to the formation of allyl aryl ethers which rearrange via a [3,3]-sigmatropic reanangement and acid-catalysed intramolecular cyclisation, to give the conesponding 3,4-dihydrobenzopyrans. 5-Formyl-2,3-dihydrobenzofuran and 6-formylamino-3,4-dihydrobenzopyran were synthesized by several routes and the isolated products exhibited cis-trans isomerism. It was found that the introduction of an alkyl substituent at carbon-4 in the model benzopyran locks the 2,3,4-alkyl substituents into axial and equatorial orientations. This may influence the peroxyl-radical scavenging activity of these compounds by altering the extent of orbital overlap between the 2p-type lone pair on the pyran oxygen and the aromatic 1t-electron system. 2,3-Dihydrobenzofurans such as 5-hydroxy-2,3-dihydrobenzofuran have been shown to have better antioxidant activity than alpha-tocopherol due to the influence of the smaller, more strained ring, which allows better overlap of the oxygen lone pair and the 1t-electrons in the aromatic system. 5-Hydroxy-2,3-dihydrobenzofuran was efficiently synthesised in an improvement on the yields previously reported in the literature. A series of substituted 2,3-dihydrobenzofurans were synthesised by the reaction of phenols with allylic alcohols or aldehydes in the presence of trifluoroacetic acid or catalytic amounts of sulphmic acid, which also promoted the acid-catalysed intramolecular cyclisation.

The utilisation of biogas as an energy source or as a feed stock for the chemical industry would help to lower the present wasteful and environmentally unfriendly venting of greenhouse gases to the atmosphere. One obstacle to this becoming a reality include finding a catalyst that is active towards carbon dioxide reforming of methane as well as resistance to carbon deposition and sulphur poisoning. As a potential alternative to supported nickel catalysts, a nickel doped perovskite has been produced via a hydrothermal synthesis method for reforming biogas. The selected perovskite, SrZrO3, was doped with 4 mol % nickel into the structure and shown to be phase pure by XRD. This material was rigorously tested catalytically using a range of biogas conditions. 4 mol% Ni doped SrZrO3 was shown to be resistant to carbon deposition under high temperature, methane rich biogas reforming conditions with no observable trend between the amount of carbon formed and the time on stream. This catalyst showed high activity and selective towards the formation of the desired products, an equimolar mixture of hydrogen and carbon monoxide. As naturally derived biogas is not a pure mixture of methane and carbon dioxide, studies into the effect that two of the other important components of biogas, water and sulphur containing compounds, were carried out. The perovskite material was seen to be stable towards continued dry reforming of methane irrespective of the inclusion of water, and at elevated reaction temperatures was able to convert the water into the required products of hydrogen and carbon monoxide. The effect of hydrogen sulphide was studied and the perovskite material was seen to be susceptible to sulphur poisoning. However the extent and the recovery from this type of deactivation was an improvement on that seen by Ni/YSZ and 5% ceria doped Ni/YSZ.

The cyclin dependent kinase inhibitor p27 is one of the most frequently dysregulated tumour suppressor protein in human cancers. A reduction in the level of cellular p27 is frequently due to increased proteasome-dependent degradation. Recently, studies show that the macrocyclic octapeptide argyrin A induced an increase in cellular p27 levels by preventing the turnover of the protein via inhibition of proteasome function. In order to investigate this interesting biological property, this project embarked on the total synthesis of argyrin A, a naturally occurring macrocyclic peptide originally isolated from myxobacteria Archangium gephyra. Argyrin A is a non-ribosomal octapeptide containing four standard amino acids and three unusual amino acid-based subunits. The synthesis of these three unusual amino acid components was established. In particular, a novel generic synthetic route to access the optically pure N-Fmoc-4-methoxy-tryptophan and analogues thereof was developed. Key features of the synthetic route include the use of chiral Strecker amino acid synthesis and mild conditions to hydrolyse α-amino nitrile to α-amino acid. Furthermore, the total synthesis of argyrin A and analogues was accomplished by the application of modern solid-phase chemistry and macrocyclisation strategies. This platform technology will enable the robust total chemical synthesis of a focused library of argyrin analogues, which will facilitate a comprehensive SAR study. Additionally, the synthesised argyrin A and analogues thereof comprising unique tryptophan analogues were tested in a cytotoxicity assay against HCT-117 human colon cell line. The results showed that all synthetic argyrin derivatives display growth inhibitory effects at nanomolar concentrations. The best result was obtained for the argyrin A and (5-methoxy-Trp4)argyrin with GI50 value at 1.8 and 3.8 nM, respectively. In summary, it became apparent that the methoxy group at 4- or 5-position of tryptophan-5 residue is essential for the biological activity of argyrin.

The essential Smc5/6 complex is structurally related to cohesin and condensin. It is required for homologous recombination (HR), rDNA stability and telomere maintenance. In Schizosaccharomyces pombe, two hypomorphic smc6 mutants, smc6-X and smc6-74, haven been shown to be deficient in HR-dependent processing of collapsed replication forks. Collapsed replication forks can generate single-ended DNA double strand breaks (se-DSB) which require HR to restore replication. In this study the requirement for Smc5/6 at a site-specific se-DSB at the mating-type locus and in the mating-type switch process were analysed. In S.pombe mating-type switching occurs over two S phases; in the first S phase replication fork stalling at mat1 leads to an imprint, which is converted to an se-DSB during the next S phase. This initiates the copying of the donor cassette using HR. In the absences of donors the sister chromatid is used for repair. Mating-type switching analysis showed that snc6-74 had a defect in switching dependent on the genotype of the smc6-74 parent. Both smc6 mutants had reduced viability in the absence of donors, consistent with a defect in HR repair of an se-DSB. analysis in an inducible system (Holmes et al., 2005) showed that in response to a se-DSB Rad52 foci appeared with wild type kinetics but the smc6 mutants delayed entry into mitosis for approximately 2hrs, dependent on the DNA damage checkpoint kinase Chk1. In order to test whether this delay facilitated rescue by a converging replication fork a novel inducible converged fork (cf) DSB system was developed. The cf-DSB required HR and the RecQ helicase Rqh1 for repair but did not require Mus81. The converging fork rescued smc6-74 but not smc6-X showing Smc5/6 to be required for repair of both types of replication-associated DSBs.