Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Discovering properties of new DNA-binding activity of proteins

Protein-DNA interactions are an essential feature in the genetic activities of life, and the ability to predict and manipulate such interactions has applications in a wide range of fields. This Thesis presents the methods of modelling the properties of protein-DNA interactions. In particular, it investigates the methods of visualising and predicting the specificity of DNA-binding Cys2His2 zinc finger interaction. The Cys2His2 zinc finger proteins interact via their individual fingers to base pair subsites on the target DNA. Four key residue positions on the a- helix of the zinc fingers make non-covalent interactions with the DNA with sequence specificity. Mutating these key residues generates combinatorial possibilities that could potentially bind to any DNA segment of interest. Many attempts have been made to predict the binding interaction using structural and chemical information, but with only limited success. The most important contribution of the thesis is that the developed model allows for the binding properties of a given protein-DNA binding to be visualised in relation to other protein-DNA combinations without having to explicitly physically model the specific protein molecule and specific DNA sequence. To prove this, various databases were generated, including a synthetic database which includes all possible combinations of the DNA-binding Cys2His2 zinc finger interactions. NeuroScale, a topographic visualisation technique, is exploited to represent the geometric structures of the protein-DNA interactions by measuring dissimilarity between the data points. In order to verify the effect of visualisation on understanding the binding properties of the DNA-binding Cys2His2 zinc finger interaction, various prediction models are constructed by using both the high dimensional original data and the represented data in low dimensional feature space. Finally, novel data sets are studied through the selected visualisation models based on the experimental DNA-zinc finger protein database. The result of the NeuroScale projection shows that different dissimilarity representations give distinctive structural groupings, but clustering in biologically-interesting ways. This method can be used to forecast the physiochemical properties of the novel proteins which may be beneficial for therapeutic purposes involving genome targeting in general.

On-line puzzle game based assessment & training for ADHD

Attention-deficit hyperactivity disorder (ADHD) is the most prevalent and impairing neurodevelopmental disorder, with worldwide estimates of 5.29%. ADHD is clinically characterized by hyperactivity-impulsivity and inattention, with neuropsychological deficits in executive functions, attention, working memory and inhibition. These cognitive processes rely on prefrontal cortex function; cognitive training programs enhance performance of ADHD participants supporting the idea of neuronal plasticity. Here we propose the development of an on-line puzzle game based assessment and training tool in which participants must deduce the ‘winning symbol’ out of N distracters. To increase ecological validity of assessments strategically triggered Twitter/Facebook notifications will challenge the ability to ignore distracters. In the UK, significant cost for the disorder on health, social and education services, stand at £23m a year. Thus the potential impact of neuropsychological assessment and training to improve our understanding of the pathophysiology of ADHD, and hence our treatment interventions and patient outcomes, cannot be overstated.

The role of the 8-18 helix of CGRP 8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

1. The role of individual residues in the 8-18 helix of CGRP 8-37 in promoting high-affinity binding to CGRP 1 receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP-mediated increase in cyclic AMP production and the binding of [ 125I]-human αCGRP. 3. Arg 11 and Arg 18 were replaced by serines to give [Ser 11.18]CGRP 8-37. These bound with pKi values <6 to SK-N-MC cells and had apparent pA 2 values of 5.81 ± 0.04 and 5.31 ± 0.11 on SK-N-MC and L6 cells. CGRP 8-37 had a pKi of 8.22 on SK-N-MC cells and pK b values on the above cell lines of 8.95±0.04 and 8.76±0.04. 3. The arginines were replaced with glutamic acid residues. [Glu 11]CGRP 8-37 had a pK b of 7.14±0.14 on SK-N-MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu 18]CGRP 8-37 had a pK b of 7.10±0.0.08 on SK-N-MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells. 4. Leu 12, Leu 15 and Leu 16 were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA 2 values of [bpa 12]-, [bpa 15]- and [bpa 16]CGRP 8-37 were respectively 7.43±0.23, 8.34±0.11 and 5.66±0.16 (pKi values of 7.14±0.17, 7.66±0.21 and <6): on L6 cells they were 7.96±0.36, 8.28±0.21 and 6.09±0.04 (all n=3). 5. It is concluded that the Arg 11 and Arg 18 are involved in specific electrostatic interactions with other residues, either on the CGRP 1 receptors or elsewhere on CGRP 8-37. Leu 16 is in a conformationally restricted site when CGRP 8-37 binds to CGRP 1 receptors, unlike Leu 12 and Leu 15.