In recent years, there has been a major movement in the pharmaceutical industry towards the development of molecules that selectivity inhibit a previously-validated specific target. This is referred to as target-based drug discovery. It was hoped that adopting this approach would usher in a new golden age of drug discovery. However, this has not been the case, with issues arising such as the target’s mechanism of action being poorly understood, with it not playing the expected role in the disease progression, or feedback resistance mechanisms causing the target to lose its role in the disease. In contrast to this, in the past 20 years it has been argued that developing drugs in a target-agnostic way and screening them against an expressed phenotype i.e. phenotypic drug discovery, has been more successful, despite fewer programs being run in the manner. The AXL kinase is a receptor tyrosine kinase (RTK) and a member of the TAM family, along with MER and TYRO3. AXL has long been associated with numerous types of cancer. Having been first discovered in 1991 in acute myeloid leukaemia (AML), it has gone on to be more associated with advanced solid tumours such as brain, breast, and lung, with the trend being that increased AXL correlates with a poorer prognosis for the patient. Upon the activation of AXL by the vitamin K ligand GAS6, a series of downstream pathways are activated that go on to encourage cell survival, proliferation, and migration. In addition to this, AXL has been shown to be involved in crosstalk with other kinase pathways, resulting in AXL expression being associated with chemoresistance and survival mechanisms. Despite the promising outlook for AXL inhibitors, to date only one selective AXL inhibitor, BGB324 (formally R428) has entered clinical trials, with selective AXL inhibitors being difficult to develop due to a lack of a crystal structure or a reliable homology model. To address the aforementioned issues that target-based approaches can suffer from, and due to AXL lacking a crystal structure, the work in this thesis utilised a pragmatic drug design method that started from ligands/existing scaffolds known to inhibit the target from the literature (publications, clinical trials and patents). A series of small libraries were prepared and then tested against a selected phenotype e.g. cell viability, in at least two cell types: one that expressed the target (e.g. AXL) and one that did not. Hits were optimised for potency against the desired phenotype. The compounds then went through target deconvolution (kinase screening) to confirm the target of the inhibitors. Employing this approach, we initially synthesised two small libraries of potential AXL inhibitors. The potency of these compounds was tested using cell-based phenotypic assays, by evaluating cell viability in both native and chemo-resistant breast cancer cells. These libraries were optimised through focused combinatorial synthesis and phenotypic screening, to yield a small collection of antiproliferative hits. These hits were then profiled against a panel of twelve select kinases. The first library, while giving some important structural information, did not inhibit the kinases screened in a meaningful manner. However, the second library gave several potent compounds, inhibiting AXL, FLT3, and RET, with one compound being selective for AXL. The leads from this series were optimised further, through SAR studies, gaining important structural information in order to improve potency and selectivity of the compounds. The flexibility of the phenotypic cell-based approach allowed the pursuit of FLT3 inhibitors, resulting in the synthesis of one of the most potent FLT3 inhibitors synthesised to date.

Leishmaniasis is a disease caused by obligate intracellular parasites of the genus Leishmania, including 20 species that are pathogenic to humans. Female sand fly is the known vector that can transmit the disease. Visceral leishmaniasis is the severe form of the disease that affects internal organs and can be fatal with inappropriate diagnosis or treatment. Leishmania donovani is the causative agent of visceral leishmaniasis. Approximately 350 million in 89 countries are at risk of infection. Around 2 million new cases are reported annually with 500,000 of these are visceral leishmaniasis. Current drug therapies are inadequate due to their toxicity, high cost, severe adverse reaction, limited availability, and the emergence of resistance. With all these limitations, the need for new drugs is urgent. Pentavalent antimonials are the first line of treatment for leishmaniasis since the 1940s. Although amphotericin B, pentamidine and paromomycin are current drugs that treat leishmaniasis, they were discovered initially as a treatment for other pathogens. Furthermore, miltefosine the only available oral drug for leishmaniasis is an anticancer drug that found to be active against Leishmania. Therefore, we used our quantitative Leishmania donovani axenic amastigote assay and the clinically relevant infected macrophage assay to identify new antileishmanial hits from unstudied or understudied natural product sources such as mangrove endophytic fungi, Antarctic deep-sea coral, and terrestrial plants. We also used the same assays to screen synthetic compounds form multiple chemical scaffolds. Our well-established assays led to the identification of new antileishmanial hits from unstudied natural products and the discovery of new classes of molecules from synthetic compounds that possess potent activity against Leishmania donovani. Finally, we conducted an in vivo hamster study on an active hit that revealed high efficacy against Leishmania donovani in this severe model leading to promising antileishmanial drug development.

Many pathogenic microorganisms are believed to stochastically switch into low metabolic states that display resistance to supra-lethal levels of antibiotics. These so-called “persister” cells have been associated with recurrent infections and the development of antibiotic resistance. Whilst a compound that eliminates Staphylococcus aureus persister cells has been described, it is not active against Gram-negative bacteria. The aim of my PhD project was to develop a high-throughput assay for compounds that eradicate persister cells in the -proteobacterium Burkholderia thailandensis. Further to this, I aimed to develop “hit” compounds from screening into lead series through investigation of structure activity relationships and, use a chemical genetics approach to elucidate potential mechanisms of action. I developed a phenotypic assay to identify compounds that eradicate persister cells. The assay was based on the reduction of the resazurin based dye PrestoBlue. Optimization of the assay gave a Z’ prime of 0.41 when screened in high throughput at the DDU. Screening of the library of 61,250 compounds identified 2,127 compounds that gave a statistically significant reduction in persister cell numbers. Follow-up assays highlighted 29 compounds with a pIC50 greater than five. Detailed investigation allowed me to down select to six “best in class” compounds, which included the licensed drug chloroxine. A time dependent killing assay showed that chloroxine reduced levels of persister cells by three orders of magnitude over 72 hours (P = 0.01). Hit expansion around chloroxine using commercially available compounds did not identify any more potent compounds, but did highlight key features of the molecule for activity. Assay protocols were provided to collaborators at DSTL who were able to iv show that chloroxine is also active against persister cells formed by the tropical pathogen and Tier 1 biological agent Burkholderia pseudomallei. Investigations into the mechanism of action of chloroxine used Next Generation Sequencing of an over expression library, identifying two putative genes involved in inhibition of persister cells by chloroxine. My findings demonstrate a phenotypic assay against persister cells in Gram-negative bacteria, which has the power to identify potent anti-persister agents to assist in chemotherapy. Structural activity relationship and mechanism of action investigations have indicated lead series and genetic starting points for future development of this research. My PhD project has concluded with sufficient data for continuation of research following a number of leads and is at an ideal stage for instigation of a medicinal chemistry program for development of chloroxine as a clinical option for treatment of persistent melioidosis.

L’étude des interactions moléculaires au niveau des membranes biologiques est un enjeu capital pour le développement et le screening de nouvelles molécules médicamenteuses. La MA est la forme de démence sénile la plus répandue dans le monde et représente le principal problème socioéconomique en matière de soins de santé. L'apparition et la progression de cette maladie neurodégénérative sont associées à l'agrégation du peptide Aβ.Une stratégie thérapeutique contre la MA consiste à développer des molécules capables d'interférer à des étapes spécifiques de l’agrégation du peptide. Pour les identifier, des méthodes expérimentales sont nécessaires pour suivre et caractériser le peptide Aβ au cours de son processus de fibrillation. Ces méthodes doivent être suffisamment simples pour rester compatibles avec une démarche de drug discovery. Dans le présent travail de thèse, nous avons proposé de combiner des méthodes expérimentales pour permettre une caractérisation multiparamétrique de modulateurs potentiels de la fibrillation du peptide Aβ1-42, en y intégrant des liposomes de composition définie, comme membranes neuronales biomimétiques. Il est en effet établi que les lipides neuronaux sont un facteur important dans la formation des fibres amyloïdes et leur toxicité. Les liposomes ont été formulés par la méthode de réhydratation de film lipidique, et leurs propriétés physico-chimiques caractérisées par RMN, DLS, potentiel ζ.La détermination expérimentale du coefficient de partage de composés d’intérêt a pu être réalisée par spectrophotométrie, y compris de façon originale, par fluorescence, en utilisant ces liposomes, dans des tests miniaturisés. Des études cinétiques de l’agrégation du peptide Aβ1-42 ont été effectuées en présence de liposomes. La fluorescence de la ThT a été mesurée pour suivre la voie de la fibrillation du peptide Aβ, utilisé dans sa forme sauvage ou celle d’un mutant oligomérique, l’oG37C. Une analyse de fuite d’un fluorophore à partir des liposomes, appuyée par des mesures en DLS, a été réalisée afin d'évaluer l'impact des interactions entre le peptide et les membranes pour prévoir tout effet de déstabilisation. Les fibres toxiques formées par Aβ étant principalement organisées en feuillets β, les données ont été corrélées à l'analyse de la structure secondaire du peptide par spectroscopie ATR-FTIR. Après avoir mis en œuvre cette approche sur différentes molécules modèles et un hit d’intérêt potentiel dans le traitement de la MA, l’ensemble de ce travail a abouti à un test multiparamétrique permettant la caractérisation de l’interactome molécules/Aβ/membranes et la discrimination de modulateurs de l'agrégation du peptide Aβ1-42. Cette approche pourra être avantageusement transposée à d'autres maladies amyloïdes. / The study of molecular interactions at the level of biological membranes is a key issue for the screening and the development of new drugs. Alzheimer's disease (AD) is the most common form of senile dementia in the world and is the leading socio-economic problem in health care. The appearance and progression of this neurodegenerative disease are associated with the aggregation of the amyloid-β peptide (Aβ). A therapeutic strategy against AD consists in the development of molecules able to interfere with specific steps of Aβ aggregation. To identify such compounds, experimental methods are required to monitor and characterize the Aβ peptide during its fibrillation process. These methods must be simple enough to remain compatible with drug discovery. In this PhD project, we have proposed to combine experimental methods to allow a multiparametric characterization of potential Aβ1-42 fibrillation modulators, by integrating liposomes of defined composition as biomimetic neuronal membranes. It is indeed established that neuronal lipids are an important factor in the formation of amyloid fibers and their toxicity. The liposomes were formulated by the lipid film rehydration method, and their physicochemical properties characterized by NMR, DLS, ζ potential. The experimental determination of the compounds partition coefficient could be carried out by spectrophotometry, including in an original way, by fluorescence, these liposomes, in miniaturized tests. Kinetic studies of Aβ1-42 peptide aggregation were performed in the presence of liposomes.The ThT fluorescence was monitored to follow the Aβ peptide fibrillation pathway, used in its wild form or with an oligomeric mutant, oG37C. A fluorophore leakage analysis from liposomes, supported by DLS measurements, was performed to evaluate the impact of peptide/membranes interactions to predict any destabilization effects. The toxic fibers formed by Aβ being mainly organized in β-sheets, the data were correlated with the analysis of the peptide secondary structure by ATR-FTIR spectroscopy. After the implementation of this approach on different model molecules and a hit of potential interest in the AD treatment, all of this work has resulted in a multiparametric test allowing the molecules/Aβ/membranes interactome characterization and the discrimination of Aβ1-42 peptide aggregation modulators. This approach may be advantageously transposed to other amyloid diseases.

The multistage life cycle of malaria-causing P. falciparum is complex, making prevention and treatment difficult. As a result of resistance to many antimalarial drugs, novel compounds with unexplored targets are constantly sought after for the purpose of treating the symptoms of malaria. Here, novel compounds were screened for antiplasmodial activity against the symptom-causing asexual intraerythrocytic malaria-causing parasites. Unfortunately, many novel compounds in the drug discovery pipeline and drugs in clinical use possess underlying pharmacological issues that makes administration challenging. These include low aqueous solubility and short half-life which negatively impact bioavailability resulting in toxicity. This, in turn, increases patient non-compliance and the emergence of drug-resistant strains. Nanoparticles (NP) have the ability to mask drugs from the external environment while increasing circulation time and often alleviate many issues at once. Furthermore, the selected drugs do not need to be modified. Drug conjugation NPs with a targeting ligand and stimuli-responsive linkers have been extensively researched in many diseases, however, none have been reported for malaria clinically. Here, the first acid-labile targeted NP (tNP) that exploits the biology of infected erythrocytes and the specialised food vacuole (FV) of P. falciparum is interrogated for ability to decrease toxicity while retaining antimalarial activity. This dissertation describes the effect of tNPs on the efficacy and toxicity of selected compounds. In vitro haemolysis and cytotoxicity assays revealed that the tNPs are biocompatible to erythrocytes and HepG2 cells. The data also shows that tNPs decrease the toxicity of drugs and the chosen novel compound against human cells. A decrease in antiplasmodial activity was observed in vitro for the tNPs when compared to the novel compound and drugs on their own. However, this was due to the biogenesis of the FV and a shortened window of release. Nonetheless, the NP backbone was not active against P. falciparum intraerythrocytic parasites whereas tNPs were, showing activity due to released drug. The targeting ligand was also not specific for antiplasmodial activity. Although a significant loss in activity is observed, the results presented here suggests that these novel acid-labile tNPs serve as an attractive starting point for targeted treatment of malaria with an improved patient tolerance. Furthermore, novel compounds with issues can be selected without having to be modified or completely discarded. Therefore, increasing the chances of finding a variety of compounds that can be used to treat malaria while keeping patients safe. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2020. / NRF / Biochemistry / MSc (Biochemistry) / Unrestricted

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions are fundamental in cell signaling and cancer progression. An increasing prevalent idea in cancer therapy is the development of small molecules to disrupt protein-protein interactions. Small molecules impart their action by binding to pockets on the protein surface of their physiological target. At protein-protein interactions, these pockets are often too large and tight to be disrupted by conventional design techniques. Residues that contribute a disproportionate amount of energy at these interfaces are known as hot spots. The successful disruption of protein-protein interactions with small molecules is attributed to the ability of small molecules to mimic and engage these hot spots. Here, the role of hot spots is explored in existing inhibitors and compared with the native protein ligand to explore how hot spot residues can be leveraged in protein-protein interactions. Few studies have explored the use of interface residues for the identification of hit compounds from structure-based virtual screening. The tight uPAR•uPA interaction offers a platform to test methods that leverage hot spots on both the protein receptor and ligand. A method is described that enriches for small molecules that both engage hot spots on the protein receptor uPAR and mimic hot spots on its protein ligand uPA. In addition, differences in chemical diversity in mimicking ligand hot spots is explored. In addition to uPAR•uPA, there are additional opportunities at unperturbed protein-protein interactions implicated in cancer. Projects such as TCGA, which systematically catalog the hallmarks of cancer across multiple platforms, provide opportunities to identify novel protein-protein interactions that are paramount to cancer progression. To that end, a census of cancer-specific binding sites in the human proteome are identified to provide opportunities for drug discovery at the system level. Finally, tumor genomic, protein-protein interaction, and protein structural data is integrated to create chemogenomic libraries for phenotypic screening to uncover novel GBM targets and generate starting points for the development of GBM therapeutic agents. / 2020-10-03

No description available.

No description available.

No description available.

Malaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel naturallike synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have identified a novel compound, 3-amino-2-piperidinequinoline (APQ), which we termed UCF401. APQ demonstrated IC[sub50] at submicromolar concentrations against Plasmodium falciparum using the SYBR Green-I fluorescence assay measuring cellular proliferation. This compound also demonstrated low cytotoxicity against the NIH3T3 and HEPG2 cells using MTS assays, showing an IC50 of 174 [micro]M and 125 [micro]M respectively, suggesting of excellent selectivity. We evaluated the compliance of APQ with Lipinski's parameters and determined the in vitro physicochemical profiles of the compound. Our results show that APQ is a Lipinski parameter compliant and has good physicochemical properties. The cellular mechanism of action of APQ was characterized through the assessment of the effects of the compound at different stages of the parasite's intraerythrocytic life cycle. This assay was done by treating a synchronized cell line with the compound at 5X the IC50 value and then imaging the cells at 12-hour intervals. We found that APQ arrests parasite development at the trophozoite stage. In addition we determined that APQ is parasitocidal after a 96 h exposure. These results demonstrate that APQ can be considered as a validated hit and/or early lead.