Computational tools for the high-throughput identification of protein-targeted drugs and probes

This thesis is comprised of three projects that are driven by a common theme, which is the use of computational tools in aiding molecular probe and drug design. In the first project, the feasibility of using molecular docking and scoring to estimate binding affinity for small molecules labelled covalently with fluorophores was tested using several proof-of-concept experiments. The high-throughput nature of computational screening applications such as Hierarchical Virtual Ligand Screening (HierVLS) necessitate that, in order to screen these labelled compounds, there must be an automated way to generate the associated structures virtually from large databases of base compounds and fluorophores. A script was developed in MOE software using scientific vector language (SVL) that could identify key reactive functional groups in both reactive fluorophores and target base compounds, and create the appropriate labelled structures for screening. The final fluorescence-labelled database numbers 14,862 compounds, each tagged with the ATTO680 fluorophore. In a subsequent project, the fluorescence-tagged library was screened against carbonic anhydrase 9 (CAIX), a protein implicated as a biomarker in several cancer types. This screening was accompanied by the screening of a validation set of known CAIX ligands and appropriately chosen decoys. The best scoring protocol according to our analyses was that which used principal components analysis. Ten of the top scoring candidates are suggested for future testing as probe candidates. CAIX binding sites were compared with equivalent residues in the sequences of 24 other CA isoforms to identify sites that might confer CAIX specificity, and the top scoring ligands were ranked according to this scheme. Lastly, experimental characterization was performed on three previously identified potential ligands for a cancer-related receptor tyrosine kinase, EphB4. Two in vitro assay formats were used: a homogenous time-resolved fluorescence assay and an enzyme-coupled spectrophotometric assay. One candidate, DNP-L-Arg, was the only one of the three with some experimental evidence of affecting kinase activity. The first assays suggested that DNP-L-Arg may have an activating effect on EphB4. The plausibility of this effect discussed with respect to mechanisms found in the literature, and using predicted and experimental structures for docked ligands. The coupled assay format did not conclusively confirm this effect. The research presented underscores the ability for computational tools to be incorporated into a variety of different areas within the fields of biochemistry and drug design. Future complementary experimental work will be crucial both in evaluating and refining the suggested probe candidates and in further validating and improving the computational techniques used

Diagnostic Tests and their Application in the Management of Soil- and Water-borne Oomycete Pathogen Species

Oomycete diseases cause significant losses across a broad range of crop and aquaculture commodities worldwide. These losses can be greatly reduced by disease management practices steered by accurate and early diagnoses of pathogen presence. Determinations of disease potential can help guide optimal crop rotation regimes, varietal selections, targeted control measures, harvest timings and crop post-harvest handling. Pathogen detection prior to infection can also reduce the incidence of disease epidemics. Classical methods for the isolation of oomycete pathogens are normally deployed only after disease symptom appearance. These processes are often-time consuming, relying on culturing the putative pathogen(s) and the availability of expert taxonomic skills for accurate identification; a situation that frequently results in either delayed application, or routine ‘blanket’ over-application of control measures. Increasing concerns about pesticides in the environment and the food chain, removal or restriction of their usage combined with rising costs have focussed interest in the development and improvement of disease management systems. To be effective, these require timely, accurate and preferably quantitatve diagnoses. A wide range of rapid diagnostic tools, from point of care immunodiagnostic kits to next generation nucleotide sequencing have potential application in oomycete disease management. Here we review currently-available as well as promising new technologies in the context of commercial agricultural production systems, considering the impacts of specific biotic and abiotic and other important factors such as speed and ease of access to information and cost effectivenes

Insights into the mechanism of action of quinoline antimalarials against Plasmodium falciparum revealed by novel fluorescent analogues and chemical proteomics

For centuries, quinoline-based drugs have formed the cornerstone of antimalarial treatment. Despite recent challenges posed by resistance, interest in these molecules persists. It is thus surprising that crucial details of their mechanism of action against the most virulent malaria parasite, Plasmodium falciparum, remain unresolved. This thesis develops new tools to generate deeper insights into the modes of action of the two major classes of the quinoline antimalarials against P. falciparum. These are the quinoline methanols, represented by the diastereomeric Cinchona alkaloids quinine and quinidine, and the 4-aminoquinolines, represented by chloroquine. Mechanistic studies of these antimalarials have typically focused on the inhibition of haemozoin biocrystallisation within the acidic digestive vacuole of P. falciparum. In order to conduct a comprehensive survey of the subcellular localisation of these antimalarials across the entire infected erythrocyte, a suite of novel fluorescent derivatives was designed and synthesised. Key physicochemical properties of these antimalarials were retained in order to preserve the interactions of these drugs with their putative target, ferriprotoporphyrin IX or Fe(III)PPIX. Versatile derivatisation of the alkaloids was enabled by a regioselective radical-mediated thiolene click reduction. 7-Nitrobenz-2-oxa-1,3-diazole (NBD) was identified as a suitable reporter fluorophore and was attached to the quinoline core by nucleophilic aromatic substitution. The length of the spacer chain between the quinoline and the fluorophore was varied by preparing NBD-labelled amino acids and their corresponding succinimidyl esters. A novel NBD-labelled chloroquine derivative was prepared by using its N-dealkylated analogue as a key intermediate. A single quinine derivative with an alternative fluorophore, bimane, was also prepared

Computational simulations of enzyme dynamics and the modelling of their reaction mechanisms

Proteins and enzymes are large and complex biological molecules, characterized by unique three-dimensional structure are highly flexible and dynamic nature. Thorough understanding of protein and enzyme function requires studying of their conformational flexibility, because important physiological processes, such as ligand binding and catalysis rely on an enzyme’s dynamic nature and their ability to adopt a variety of conformational states. Computational methods are widely applied in studying enzymes and proteins structure and function providing a detailed atomistic-level of resolution data about the dynamics and catalytic processes, mechanisms in biomolecules, therefore even more nowadays a term ‘computational enzymology’ has emerged. Experimental methods often have difficulty in predicting dynamic motions of proteins. Computational simulations techniques, such as Molecular Dynamics simulations, have proven successful in simulating the conformational flexibility of proteins in studying structure-function relationships. Additionally, the binding events between two molecules, e.g. an enzyme and its substrate, can be computationally predicted with molecular docking methods. Enzymes are proteins that catalyse almost all biochemical reactions and metabolic processes in all organisms. In order to study the conformational flexibility of proteins we apply molecular dynamics simulations, and in order to simulate their reaction mechanisms we apply quantum mechanical simulations. Quantum mechanical simulations can also be used to predict the electronic structure of organic compounds, by calculating their electronic structures we perform orbital analyses and predict their optical properties. The results gained from our computational simulations can give new insights into explanation of experimental findings and data and can inspire and guide further experiments

Bio-oligomers as antibacterial agents and strategies for bacterial detection

In this thesis I examined the potential of Bio-Oligomers such as peptoids, peptides and aptamers, as therapeutic and diagnostic entities. Therapeutic Bio-Oligomers; A series of peptoid analogs have been designed and synthesised using solid phase synthesis. These peptoids have been subjected to biological evaluation to determine structure-activity relationships that define their antimicrobial activity. In total 13 peptoids were synthesised. Out of 13 different peptoids, only one peptoid called Tosyl-Octyl-Peptoid (TOP) demonstrated significant broad-spectrum bactericidal activity. TOP kills bacteria under non-dividing and dividing conditions. The Minimum Inhibitory Concentrations (MIC) values of TOP for S. epidermidis, E. coli and Klebsiella were 20 μM, whereas Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Staphylococcus aureus (MSSA) were 40 μM. The highest MIC values were observed for Pseudomonas aeruginosa (PAO1) at 80 μM. The selectivity ratio (SR) or Therapeutic index (TI) was calculated, by dividing the 10% haemolysis activity (5 mM) by the median of the MIC (50 μM) yielding a TI for TOP as 100. This TI is well above previously reported peptidomimetics TI of around 20. TOP demonstrates selective bacterial killing in co-culture systems and intracellular bacterial killing activity. Diagnostic Bio-Oligomers; In the second part of my thesis, I investigated aptamer and peptide-based molecular probes to detect MRSA. As well as screening aptamers and peptide probes against whole MRSA, I over-expressed and purified PBP2A protein. This purified protein was used as a target for aptamer and peptide probes to detect MRSA. Two different aptamer libraries were initially screened for utility. In-vitro conditions for SELEX were optimised. Biopanning with a phage derived peptides was also performed. Target sequences for both methods were identified and chemically synthesised. Evaluation of fluorescently labelled sequences with flow cytometry and confocal imaging showed no specificity for MRSA detection with either method. The Bio-Oligomers and the in-vitro selection methodology require further refinement to improve diagnostic utility

Development of novel biosensing and diagnostic platforms using nanoparticle complexes

Metal nanomaterials, such as gold nanoparticles (Au NPs), exhibit unique localised surface plasmon resonance, which can be exploited for probing biochemical and biophysical phenomena at the nanoscale and molecular level. Furthermore, the ability to control the synthesis and growth of such nanomaterials using organic and biomimetic molecules, such as nucleic acids and small molecules, facilitates deeper understanding of the interactions between biomolecules and nanomaterials. This thesis described the development of various highly sensitive and novel diagnostic platforms for detecting micro-RNA (miRNA), small molecule and protein biomarkers, by utilising the unique plasmonic properties of Au NPs, as well as modulating the morphology and size of various gold nanostructures. Au NP-conjugated nucleic acid probes, together with a poly(ethylene glycol)-functionalised microarray, enabled highly sensitive and multiplexed detection of miRNAs, conveniently under an optical microscope. Also, colorimetric detection of small molecules using the naked eye was achieved via the controlled growth of aptamer-functionalised Au NPs into various distinct nanostructures, which were dependent on aptamer–target interactions and aptamer-mediated NP growth. Lastly, the interactions between small molecules and Au seeds, and the effect on the size and aspect ratios of grown gold nanorods were investigated and elucidated. The size-modulating mechanism was further incorporated in an immunoassay for the sensitive detection of a protein biomarker, enabling its application in clinical diagnostics. The platforms developed in this thesis could serve as a basis for future development of new biosensing strategies that utilise plasmonic nanomaterials.Open Acces

Autecology of crenarchaeotal and bacterial clades in marine sediments and microbial mats

The focus of this thesis was the autecology of the Miscellaneous Crenarchaeotal Group (MCG), a phylum-level clade of Archaea occurring mostly in marine sediments. Sequences of MCG 16S rRNA genes have been retrieved from a wide range of marine and terrestrial habitats, such as deep subsurface sediments, hydrothermal sediments, mud volcanoes, estuaries, hot springs and freshwater lake sediments. MCG members seem to have no general preferences for a particular temperature or salinity. So far, not a single member of the elusive MCG has been cultured. They show a high intragroup diversity with percent identity values of 16S rRNA as low as 77%. Since MCG sequences are frequent in sulfate-methane transition zones (SMTZ) of deep sea subsurface sediments, MCG were assumed to be the dominant archaeal population which might greatly contribute to biogeochemical cycles in the deep biosphere. However, quantitative data on the abundance and activity of MCG are still largely lacking. Therefore, in this doctoral thesis, a polyphasic approach was applied for the quantification and visualization of MCG in marine habitats using different molecular methods such as slot-blot hybridization, quantitative PCR and fluorescence in situ hybridization. MCG-specific oligonucleotide probes and primers were designed and used for the quantification. It was shown that in general the relative abundance of MCG strongly increased with depth. In methane-rich surface sediments MCG abundances were below 3% of total Archaea. In contrast, MCG constituted a major part of the archaeal community with 15-100% in subsurface SMTZ. My results provide the first quantitative data on the high abundance of MCG in deep subsurface sediments and are inline with a major role of MCG in biogeochemical cycling in these habitats. In this thesis, cells of MCG were visualized for the first time by CARD-FISH. The cell shape was coccoid and the cell diameter was 0.4-0.5 µm. Signals were weak, but still clearly detectable with CARD-FISH suggesting that MCG are not only abundant, but also active in subsurface sediments. The single cell identification protocol developed in this doctoral thesis will in the future facilitate further quantitative investigations of the autecology of MCG. Further experiments performed in the course of this doctoral thesis addressed the quantification and visualization of particular bacterial populations such as Chloroflexi and Aquificae in microbial mats and marine sediments

Biosensors based on bovine odorant binding protein (bOBP)

Recombinant bovine odorant binding protein (bOBP) is a very promising platform for building protein-based biosensors. The protein possesses a broad binding specificity for hydrophobic molecules with affinities in the sub-micromolar range. Previous work has shown non-covalent binding of 1-aminoanthracene (1-AMA) in the internal cavities of bOBP that results in a large enhancement of fluorescence intensity. We have shown fluorescence titrations of recombinant bOBP with 1-AMA yielded a single type of binding site with a Kd of 0.16 ± 0.023 μM. Competitive displacement assays between 1-AMA and other ligands such as thymol were established and the results indicated their binding to bOBP. The strategy of competitive binding with 1-AMA was thus employed to quantify thymol concentration for the bOBP biosensors. Ten different solid supports for the bOBP biosensors were examined for their biocompatibility with bOBP function using 1-AMA as a probe. The result was that nitrocellulose was chosen as the best membrane for immobilization, probably due to its 3-D micro-porous matrix (sponge structure) that provides a much larger surface area for protein binding compared with 2-D surfaces. It was found that the optimum operating concentrations of bOBP and 1-AMA and the method for the immobilization was incubation of nitrocellulose with the complex of 100μM bOBP and 100μM 1-AMA solution. The amounts of the total and functional protein binding to nitrocellulose were 7 ± 0.1 and 7 ± 0.4 nmol bOBP per cm2 of membrane, respectively. A fibre-optic biosensor based on bOBP has therefore been constructed. It has been an extrinsic sensor with bOBP immobilized on a nitrocellulose membrane placed at the tip of a probe of a bifurcated fibre-optic bundle that was in turn connected to the LLS-385 LED light source and the HR2000 spectrometer. The light emitted by fluorescent 1-AMA bound bOBP was detected by 2048-element CCD array of the spectrometer. The LODs for thymol in the liquid phase were found to be 14 ± 6 μM (calculated as S/N = 3), which is less than the guideline values considered to be toxic to humans. Moreover, this fibre-optic bOBP biosensor was also capable of sensing thymol vapour, and some potential uses of this sensor will be described