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.

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 extracellular glucosyltransferases of Streptococcus sanguis polymerise the glucosyl moiety of sucrose to form high molecular weight complex glucans. The adhesive and agglutinative properties of these glucans are important in the formation of dental plaque and, hence, in cariogenesis. The glucosyltransferases of S. sanguis 804 (NCTC) were extensively purified (182-fold) by hollow fibre ultrafiltration (Bio-Fiber 80) followed by ammonium sulphate precipitation (0–70% of saturation). The enzymes were further purified by hydroxylapatite chromatography and appeared by this technique to consist of at least three enzymes with differing specific activities. It is not known whether these enzymes are, in fact, composed of different polypeptides or are modified forms of one protein. The activity of the glucosyltransferases can be measured as the rate of release of fructose from sucrose or as the rate of synthesis of ethanol-sodium acetate-precipitable polysaccharide (glucan). Using the former method, Kapp for sucrose for (NH4) 2SO4-purified glucosyltransferases was about 6 mmol/l and, using the latter method, Kapp was about 20 mmol/l. Glucosyltransferase activity (as rate of glucan synthesis) was stimulated 2 to 4-fold by low concentrations (0.125-0.50 μmol/l) of T2000 Dextran (Pharmacia; mol. wt. 2 × 106). Glucan synthesis was inhibited slightly by nigerose and was inhibited strongly by metrizamide (85% inhibition at 170 μmol/l metrizamide). The rate of release of fructose was not affected by either xylitol or hydrogen peroxide. The rate of synthesis of precipitable glucan was strongly inhibited by high concentrations of substrate (sucrose); the rate of release of fructose was relatively unaffected. The proposed mechanism for this effect is that sucrose acts as an alternative glucosyl acceptor (as well as donor) and thus inhibits glucosyl transfers to growing glucan chains. The oral concentrations of sucrose during and after consumptions of various sweet foods and beverages were studied and were often sufficient to inhibit glucan synthesis. In such cases, the sucrose concentrations for maximum rate of glucan synthesis only occurred as sucrose was cleared from the mouth, after the food or drink was finished. Glucan synthesis by S. sanguis is important in plaque formation. Thus, these results provide an additional explanation for the clinical finding that the incidence of caries is related to the frequency of dietary intake of sucrose and not merely the total amount of sucrose consumed.

Electroanalysis has proven to be one of the most widely used technologies for point-of-care devices. Owing to the direct recording of the intrinsic properties of biochemical functions, the field has been involved in the study of biology since electrochemistry’s conception in the 1800’s. With the advent of microelectronics, humanity has welcomed self-monitoring portable devices such as the glucose sensor in its everyday routine. The sensitivity of amperometry/ voltammetry has been enhanced by the use of microelectrodes. Their arrangement into microelectrode arrays (MEAs) took a step forward into sensing biomarkers, DNA and pathogens on a multitude of sites. Integrating these devices and their operating circuits on CMOS monolithically miniaturised these systems even more, improved the noise response and achieved parallel data collection. Including microfluidics on this type of devices has led to the birth of the Lab-on-a-Chip technology. Despite the technology’s inclusion in many bioanalytical instruments there is still room for enhancing its capabilities and application possibilities. Even though research has been conducted on the selective preparation of microelectrodes with different materials in a CMOS MEA to sense several biomarkers, limited effort has been demonstrated on improving the parallel electroanalytical capabilities of these devices. Living and chemical materials have a tendency to alter their composition over time. Therefore analysing a biochemical sample using as many electroanalytical methods as possible simultaneously could offer a more complete diagnostic snapshot. This thesis describes the development of a CMOS Lab-on-a-Chip device comprised of many electrochemical cells, capable of performing simultaneous amperometric/voltammetric measurements in the same fluidic chamber. The chip is named an electrochemical cell microarray (ECM) and it contains a MEA controlled by independent integrated potentiostats. The key stages in this work were: to investigate techniques for the electrochemical cell isolation through simulations; to design and implement a CMOS ECM ASIC; to prepare the CMOS chip for use in an electrochemical environment and encapsulate it to work with liquids; to test and characterise the CMOS chip housed in an experimental system; and to make parallel measurements by applying different simultaneous electroanalytical methods. It is envisaged that results from the system could be combined with multivariate analysis to describe a molecular profile rather than only concentration levels. Simulations to determine the microelectrode structure and the potentiostat design, capable of constructing isolated electrochemical cells, were made using the Cadence CAD software package. The electrochemical environment and the microelectrode structure were modelled using a netlist of resistors and capacitors. The netlist was introduced in Cadence and it was simulated with potentiostat designs to produce 3-D potential distribution and electric field intensity maps of the chemical volume. The combination of a coaxial microelectrode structure and a fully differential potentiostat was found to result in independent electrochemical cells isolated from each other. A 4 x 4 integrated ECM controlled by on-chip fully differential potentiostats and made up by a 16 × 16 working electrode MEA (laid out with the coaxial structure) was designed in an unmodified 0.35 μm CMOS process. The working electrodes were connected to a circuit capable of multiplexing them along a voltammetric measurement, maintaining their diffusion layers during stand-by time. Two readout methods were integrated, a simple resistor for an analogue readout and a discrete time digital current-to-frequency charge-sensitive amplifier. Working electrodes were designed with a 20 μm side length while the counter and reference electrodes had an 11 μm width. The microelectrodes were designed using the aluminium top metal layer of the CMOS process. The chips were received from the foundry unmodified and passivated, thus they were post-process fabricated with photolithographic processes. The passivation layer had to be thinned over the MEA and completely removed on top of the microelectrodes. The openings were made 25 % smaller than the top metal layer electrode size to ensure a full coverage of the easily corroded Al metal. Two batches of chips were prepared, one with biocompatible Au on all the microelectrodes and one altered with Pd on the counter and Ag on the reference electrode. The chips were packaged on ceramic pin grid array packages and encapsulated using chemically resistant materials. Electroplating was verified to deposit Au with increased roughness on the microelectrodes and a cleaning step was performed prior to electrochemical experiments. An experimental setup containing a PCB, a PXIe system by National Instruments, and software programs coded for use with the ECM was prepared. The programs were prepared to conduct various voltammetric and amperometric methods as well as to analyse the results. The first batch of post-processed encapsulated chips was used for characterisation and experimental measurements. The on-chip potentiostat was verified to perform alike a commercial potentiostat, tested with microelectrode samples prepared to mimic the coaxial structure of the ECM. The on-chip potentiostat’s fully differential design achieved a high 5.2 V potential window range for a CMOS device. An experiment was also devised and a 12.3 % cell-to-cell electrochemical cross-talk was found. The system was characterised with a 150 kHz bandwidth enabling fast-scan cyclic voltammetry(CV) experiments to be performed. A relatively high 1.39 nA limit-of-detection was recorded compared to other CMOS MEAs, which is however adequate for possible applications of the ECM. Due to lack of a current polarity output the digital current readout was only eligible for amperometric measurements, thus the analogue readout was used for the rest of the measurements. The capability of the ECM system to perform independent parallel electroanalytical measurements was demonstrated with 3 different experimental techniques. The first one was a new voltammetric technique made possible by the ECM’s unique characteristics. The technique was named multiplexed cyclic voltammetry and it increased the acquisition speed of a voltammogram by a parallel potential scan on all the electrochemical cells. The second technique measured a chemical solution with 5 mM of ferrocene with constant potential amperometry, staircase cyclic voltammetry, normal pulse voltammetry, and differential pulse voltammetry simultaneously on different electrochemical cells. Lastly, a chemical solution with 2 analytes (ferrocene and decamethylferrocene) was prepared and they were sensed separately with constant potential amperometry and staircase cyclic voltammetry on different cells. The potential settings of each electrochemical cell were adjusted to detect its respective analyte.