Yes / The integrin family of cell surface extracellular matrix binding proteins are key to several physiological processes involved in tissue development, as well as cancer proliferation and dissemination. They are therefore attractive targets for drug discovery with cancer and non-cancer applications. We have developed a new integrin antagonist chemotype incorporating a functionalised cyclobutane ring as the central scaffold in an arginine–glycine–aspartic acid mimetic structure. Here, we report the synthesis of cyclobutanecarboxylic acids and cyclobutylamines with tetrahydronaphthyridine and aminopyridine arginine mimetic sidechains and masked carboxylic acid aspartic acid mimetic sidechains of varying length. Effective αvβ3 antagonists and new aspartic acid mimetics were identified in cell-based adhesion and invasion assays. A lead compound selected based on in vitro activity (IC50 < 1 μM), stability (t1/2 > 80 minutes) and synthetic tractability was well-tolerated in vivo. These results show the promise of this synthetic approach for developing αvβ3 antagonists and provide a firm foundation to progress into advanced preclinical evaluation prior to progression towards the clinic. Additionally, they highlight the use of functionalised cyclobutanes as metabolically stable core structures and a straightforward and robust method for their synthesis. This important contribution to the medicinal chemists' toolbox paves the way for increased use of cyclobutanes in drug discovery. / This work was funded by Yorkshire Cancer Research (Award reference number B002-PhD) and Prostate Cancer UK (Pilot Grant PA10-01).

This project applies structure-activity relationship (SAR), structure-based and database mining approaches to study ligand-protein interactions. To support these studies, we have developed a relational database system called EDinburgh University Ligand Selection System (EDULISS 2.0) which stores the structure-data files of +5.5 million commercially available small molecules (+4.0 million are recognised as unique) and over 1,500 various calculated molecular properties (descriptors) for each compound. A user-friendly web-based interface for EDULISS 2.0 has been established and is available at http://eduliss.bch.ed.ac.uk/. We have utilised PubChem bioassay data from an NMR based screen assay for a human FKBP12 protein (PubChem AID: 608). A prediction model using a Logistic Regression approach was constructed to relate the assay result with a series of molecular descriptors. The model reveals 38 descriptors which are found to be good predictors. These are mainly 3D-based descriptors, however, the presence of some predictive functional groups is also found to give a positive contribution to the binding interaction. The application of a neural network technique called Self Organising Maps (SOMs) succeeded in visualising the similarity of the PubChem compounds based on the 38 descriptors and clustering the 36 % of active compounds (16 out of 44) in a cluster and discriminating them from 95 % of inactive compounds. We have developed a molecular descriptor called the Atomic Characteristic Distance (ACD) to profile the distribution of specified atom types in a compound. ACD has been implemented as a pharmacophore searching tool within EDULISS 2.0. A structure-based screen succeeded in finding inhibitors for pyruvate kinase and the ligand-protein complexes have been successfully crystallised. This study also discusses the interaction of metal-binding sites in metalloproteins. We developed a database system and web-based interface to store and apply geometrical information of these metal sites. The programme is called MEtal Sites in Proteins at Edinburgh UniverSity (MESPEUS; http://eduliss.bch.ed.ac.uk/MESPEUS/). MESPEUS is an exceptionally versatile tool for the collation and abstraction of data on a wide range of structural questions. As an example we carried out a survey using this database indicating that the most common protein types which contain Mg-OATP-phosphate site are transferases and the most common pattern is linkage through the β- and γ-phosphate groups.

Recent years have seen an expansion beyond the more druggable biological targets into novel areas of biological space. However, drug discovery campaigns against these challenging targets have been afflicted with low hit rates during screening campaigns and high levels of candidate attrition during clinical trials. Subsequent studies have looked to explore the underlying factors to these challenges and have identified the lack of scaffold diversity and poor physicochemical properties in screening libraries as the leading causes. In an attempt to address this issue drug discovery strategies such as fragment-based drug discovery and lead-oriented synthesis have been developed which control and direct the compound properties within screening libraries towards relevant areas of chemical space. In addition, strategies such as diversity oriented synthesis aim to synthesise structurally complex and diverse compounds, expanding screening collections into previously under-explored areas of chemical space. This thesis reports the development of a step-efficient, modular and highly adaptable synthetic route for the synthesis of partially saturated bicyclic heteroaromatic scffolds (Figure i). The designed route takes advantage of the large chiral pool provided by amino acids, with each scaffold synthesised in just 4-6 steps from these readily available enantiopure starting materials. The mild conditions allow for excellent functional group tolerance, thus enabling the incorporation of growth vectors for chemical elaboration from the outset, a strong advantage in the drug discovery process. Overall, 29 partially saturated bicyclic heteroaromatic compounds were synthesised based around 7 different scaffolds. These demonstrated a number of possible areas for diversifation both on and around the scaffold, including variation of functional groups (Figure i, red), double (cis-diastereoisomers) and single (R2- and R3-positions) substitution patterns, variation of the 5-membered heterocycle (Figure i, green) and increased size of the saturated ring (Figure i, blue). Furthermore, careful selection of the substituents, heterocycle and size of the saturated ring would enable the synthesis of screening libraries within the constraints of fragment-like, lead-like or drug-like structures. The final library has been incorporated into the Diamond XChem high-throughput crystallography program and initial screening has identified a weakly binding hit for Activin A.

Natural products have been a fundamental source of medicinal scaffolds for decades; with sixty percent of marketed drugs. Many synthetic chemists are focused on synthesizing potent and nontoxic compounds for pharmaceutical targets, however, nature is still proving to be a source of new bioactive compounds. Produced by the host organism for defense, reproduction and communication, secondary metabolites also demonstrate promising bioactivity against human pathogens. Hence, natural product chemists continue their quest for new leads. As a continuation of these efforts, this thesis attempts to explore fungi and sponges for new chemistry, and ultimately, new drug candidates. Antarctica is largely untapped; hence herein two Antarctic sponges were chemically investigated. This resulted in isolation and characterization of two metabolites. Concurrently, chemical investigation of fungus, from Floridian mangrove species, resulted in the isolation of two structurally diverse metabolites. Further, a dereplication process was applied to MPLC fractions, which lead to the identification of known metabolites and mycotoxins. This enabled prioritization of fractions for future studies.

The process of drug discovery is very slow and expensive. There is a need for reliable in silico methods; however the performance of these methods differs. This work presents a targeted study on how the drug discovery methods used in OpenEye’s tools ROCS, EON and FRED perform on targets with small ligands. It was examined if 12 compounds (markers) somewhat similar to AMP could be detected by ROCS in a random data set comprised of 1000 compounds. It was also examined if EON could find any electrostatic similarities between the queries and the markers. The performance of FRED with respect to re-generation of bound ligand modes was examined on ten different protein/ligand complexes from the Brookhaven Protein Data Bank. It was also examined if FRED is suitable as a screening tool since several other docking methods are used in such a way. Finally it was also examined if it was possible to reduce the time requirements of ROCS when running multiconformer queries by using a combination of single conformer queries coupled with multiconformer queries. The conclusions that could be drawn from this project were that FRED is not a good screening tool, but ROCS performs well as such. It was also found that the scoring functions are the weak spots of FRED. EON is probably very sensitive to the conformers used but can in some cases strengthen the results from ROCS. A novel and simple way to reduce the time complexity with multiconformer queries to ROCS was discovered and was shown to work well.

<p>The process of drug discovery is very slow and expensive. There is a need for reliable in silico methods; however the performance of these methods differs.</p><p>This work presents a targeted study on how the drug discovery methods used in OpenEye’s tools ROCS, EON and FRED perform on targets with small ligands. It was examined if 12 compounds (markers) somewhat similar to AMP could be detected by ROCS in a random data set comprised of 1000 compounds. It was also examined if EON could find any electrostatic similarities between the queries and the markers. The performance of FRED with respect to re-generation of bound ligand modes was examined on ten different protein/ligand complexes from the Brookhaven Protein Data Bank. It was also examined if FRED is suitable as a screening tool since several other docking methods are used in such a way. Finally it was also examined if it was possible to reduce the time requirements of ROCS when running multiconformer queries by using a combination of single conformer queries coupled with multiconformer queries.</p><p>The conclusions that could be drawn from this project were that FRED is not a good screening tool, but ROCS performs well as such. It was also found that the scoring functions are the weak spots of FRED. EON is probably very sensitive to the conformers used but can in some cases strengthen the results from ROCS. A novel and simple way to reduce the time complexity with multiconformer queries to ROCS was discovered and was shown to work well.</p>

The connexins are a family of 21 proteins that represent the structural units of intercellular gap junctions and single membrane hemichannels. These channels provide a means for cells to exchange small metabolites and signaling molecules with adjacent cells and the extracellular space, respectively. Compelling evidence implicates connexins, and the more recently discovered pannexins, in the control of neural progenitor cell proliferation, survival and migration. Moreover, connexin and pannexin dysregulation following central nervous system injuries such as cerebral ischemia, spinal cord injury, and epilepsy contributes to the secondary expansion of lesions days and weeks after the initial insult. While these data suggest that connexins and pannexins represent novel therapeutic targets to both reduce the extent of neural injury and promote neural repair and regeneration, we currently lack the necessary repertoire of therapeutically useful connexin- and pannexin-specific compounds to test these hypotheses. In this thesis, I conducted targeted screening of a large, ethnobotanically-derived library to address my overarching objective of identifying compounds that selectively alter connexin and/or pannexin channel function. To accomplish this, I characterized the repertoire of connexins and pannexins expressed by neural progenitor cell-like NT2/D1 cells, quantified the intercellular flux of calcein through connexin gap junctions, and measured the uptake of lucifer yellow and propidium iodide through pannexin hemichannels. Collectively, these screens identified several promising lead compounds and ethanolic plant extracts that selectively alter connexin and pannexin channel activity.

According to the World Health Organization, malaria has been classified as one of the three most important infectious diseases in Africa. The number of malaria cases is still on the increase in various countries, such as Rwanda and Zambia, which highlights the fragility of malaria control and the need to maintain and improve control programs. An innovative strategy for developing new antimalarial agents is through targeting epigenetic regulatory mechanisms in the malarial parasite, Plasmodium falciparum. Histone posttranslational modifications (PTMs) are factors contributing to epigenetic regulation in P. falciparum parasites. The epigenetic regulatory enzyme, Lysine-specific demethylase 1 (LSD1), has the ability to remove methyl groups from mono- and dimethylated lysine residues and is a regulator of gene expression through the modulation of chromatin structure. Polyamine analogues have been described as epi-drugs that target cell cycle development by blocking epigenetic control mechanisms in mammalian cells. A library of polyamine analogues were tested in cancer cells and found to specifically inhibit LSD1. In addition, these analogues were shown to have antiplasmodial activity against a drug-sensitive parasite strain, with IC50 values ranging from 88-100 nM but were metabolically unstable in vivo. In an attempt to overcome this in vivo hurdle, the leading compound was fluorinated at four different positions and tested for improved antiplasmodial activity and selectivity towards the parasites. Furthermore, the effect of the compounds on epigenetic regulatory mechanisms, through inhibition of LSD1 activity, was investigated. The analogues showed inhibition of parasite proliferation at low nanomolar concentrations and were very selective towards the parasites with low resistance indices. The leading compound showed reversible cytotoxicity towards parasite proliferation in addition to inhibitory activity against LSD1 and therefore, epigenetic regulatory changes. The approach taken in this dissertation is novel as none of the currently available antimalarials target LSD1 and as such, adds valuable information to future perspectives for drug design. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Biochemistry / MSc / Unrestricted

Candida albicans is a fungus found in the normal biota of humans, but in immuno-compromised individuals, C. albicans forms complex biofilms on the surface of medical prosthetics, skin, oral cavities, the urinary tract, and other epithelial cell layers. Biofilms and the development of drug resistance has limited treatment options. Antimicrobial peptides (AMPs) are increasingly becoming attractive therapeutic agents for the treatment of these infections due to their multifunctional properties, multiple cellular targets, and the lower incidence of resistance development. Previous studies have shown that Os, an AMP derived from the tick defensin OsDef2, has antifungal activity against C. albicans. Preliminary antifungal mode of action studies indicated that Os induces the formation of reactive oxygen species although not a primary mode of killing. Os causes membrane permeabilization, which is inhibited by an excess of free laminarin and mannan. Furthermore, Os was shown to bind plasmid DNA but was inactive in high salt conditions. The aim of this study was to further explore the mode of action of Os in planktonic C. albicans (ATCC 90028) cells. A modified microbroth dilution assay was developed to allow rapid screening of salt sensitive AMPs such as Os. With this method the IC50 of the positive control, amphotericin B (AmpB), and Os were determined as 0.547 ± 0.056 μM and 1.163 ± 0.116 μM, respectively. The effects of AmpB and Os on cellular morphology were evaluated using scanning electron microscopy and transmission electron microscopy at their respective IC25, IC50 and IC75 values. When comparing the effects of Os with AmpB on the cell wall and membrane, Os had more severe and nonspecific effects. Os induced the formation of pits on the cell surface and pores in the cell membrane, as well as increased budding scars. Using isothermal titration calorimetry, no interaction between Os and the fungal cell wall components, mannan and laminarin, could be detected. Factors such as the lack of tryptophan and aspartate residues as well as β-sheet secondary structures may account for the lack of interaction. However, with the modified microbroth dilution assay in the presence of excess of mannan or laminarin (20 mg/mL), reduced activity from Os was observed. The formation of soluble macro-complexes between Os and the cell wall components at high concentrations may account for reduced activity. The ability of Os to cause membrane depolarization was evaluated with bis-(1,3-dibutylbarbituric acid) trimethine oxonol. The control, melittin, caused a linear increase in depolarization with a significant increase at 0.63 μM, while Os caused a sigmoidal increase in depolarization with a significant increase at 2.5 μM. Therefore, membrane depolarization occurs following membrane permeabilization which occurs at 2 μM. Finally, the localisation of 0.5 μM and 6.4 μM (IC25, IC75) 5-FAM-Os, and concurrently the effect on vacuoles loaded with CellTracker Blue-CMAC, was determined with flow cytometry and confocal laser scanning microscopy (CLSM). Findings were that Os, at a concentration below its IC50, binds to the cell membrane, then translocates and binds DNA. At a concentration above its IC50, Os accumulates in the cytoplasm and causes destruction of membranes, including that of vacuoles, leading to cell death. In conclusion, this study shows that Os is a membrane acting AMP that can be further developed for clinical application as an antifungal drug. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020 / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

The main focus of our work is to develop, apply and assess cheminformatics tools and methods. In particular, we focus on the following three areas: Integration of open source tools with application to drug discovery, usability studies to assess the efficacy of these software tools and finally, developing novel techniques for database query. Rapid globalization in the present time has sparked a need in the scientific community to interact with each other at an economic and a fast pace. This is achieved by developing and sharing open source databases using World Wide Web. A web based open source database application has been developed to incorporate freeware from varied sources. The deployment of developed database and user interface in a university lab setting is discussed. To aid in connecting the end user and the software tools, usability studies are necessary. These studies communicate the end users’ needs and desires, resulting in a user-friendly and more powerful interactive software packages. Usability studies were conducted on developed database student application and on different drawing packages to determine their effectiveness. Developing new and interactive search engines to query publicly available databases helps researchers work more efficiently. The huge volume of data available and its heterogeneous nature presents issues related to querying, integration and presentation. In aiding the retrieval process, an innovative multi faceted classification system, called ChemFacets, is developed. This system provides dynamic categorization of large result sets retrieved from multiple databases.