Targeting the apicoplast in malaria

Malaria continues to be one of the leading causes of human mortality in the world, and the therapies available are insufficient for eradication. Severe malaria is caused by the apicomplexan parasite Plasmodium falciparum. Apicomplexan parasites, including the Plasmodium spp., are descendants of photosynthetic algae, and therefore they possess an essential plastid organelle, named the apicoplast. Since humans and animals have no plastids, the apicoplast is an attractive target for drug development. Indeed, after its discovery, the apicoplast was found to host the target pathways of some known antimalarial drugs, which motivated efforts for further research into its biological functions and biogenesis. Initially, many apicoplast inhibitions were found to result in ‘delayed death’, whereby parasite killing is seen only at the end of one invasion-egress cycle. This slow action is not in line with the current standard for antimalarials, which seeded scepticism about the potential of compounds targeting apicoplast functions as good candidates for drug development. Intriguingly, recent evidence of apicoplast inhibitors causing rapid killing could put this organelle back in the spotlight. We provide an overview of drugs known to inhibit apicoplast pathways, alongside recent findings in apicoplast biology that may provide new avenues for drug development

Marine anti-malarial isonitriles : a synthetic and computational study

The development of Plasmodium falciparum malarial resistance to the current armoury of anti-malarial drugs requires the development of new treatments to help combat this disease. The marine environment is a well established source of potential pharmaceuticals. Of interest to us are isonitrile, isocyanate and isothiocyanate compounds isolated from marine sponges and molluscs which have exhibited nano-molar anti-plasmodial activities. Through quantitative structure-activity relation studies (QSAR), a literature precedent exists for a pseudoreceptor model from which a pharmacophore for the design of novel anti-malarial agents was proposed. The current theory suggests that these marine compounds exert their inhibitory action through interfering with the heme detoxification pathway in P. falciparum. We propose that the computational methods used to draw detailed conclusions about the mode of action of these marine compounds were inadequate. This thesis addresses this problem using contemporary computational methodologies and seeks to propose a more robust method for the rational design of new anti-malarial drug compounds that inhibit heme polymerization to hemozoin. In order to investigate the interactions of the marine compounds with their heme targets, a series of modern computational procedures were formulated, validated and then applied to theoretical systems. The validations of these algorithms, before their application to the marine compound-heme systems, were achieved through two case studies. The first was used to investigate the applicability of the statistical docking algorithm AutoDock to be used for the exploration of conformational space around the heme target. A theoretical P. falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PfDXR) enzyme model, constructed by the Biochemistry Department at Rhodes University, provided the ideal model to validate the AutoDock program. The protein model was accordingly subjected to rigorous docking simulations with over 30 different ligand molecules using the AutoDock algorithm which allowed for the docking algorithm’s limitations to be ascertained and improved upon. This investigation facilitated the successful validation of the protein model, which can now be used for the rational design of new PfDXR-inhibiting anti-plasmodial compounds, as well as enabling us to propose an improvement of the docking algorithm for application to the heme systems. The second case study was used to investigate the applicability of an ab initio molecular dynamics algorithm for simulation of bond breaking/forming events between the marine compounds and their heme target. This validation involved the exploration of intermolecular interactions in a naturally occurring nonoligomeric zipper using the Car-Parrinello Molecular Dynamics (CPMD) method. This study allowed us to propose a model for the intermolecular forces responsible for zipper self-assembly and showcased the CPMD method’s abilities to simulate and predict bond forming/breaking events. Data from the computational analyses suggested that the interactions between marine isonitriles, isocyanates and isothiocyanates occur through bond-less electrostatic attractions rather than through formal intermolecular bonds as had been previously suggested. Accordingly, a simple bicyclic tertiary isonitrile (5.14) was synthesized using Kitano et al’s relatively underutilized isonitrile synthetic method for the conversion of tertiary alcohols to their corresponding isonitriles. This compound’s potential for heme detoxification inhibition was then explored in vitro via the pyridine-hemochrome assay. The assay data suggested that the synthesized isonitrile was capable of inhibiting heme polymerization in a similar fashion to the known inhibitor chloroquine. Attempts to synthesize tricyclic analogues of 5.14 were unsuccessful and highlighted the limitation of Kitano et al’s isonitrile synthetic methodology

Data-mining of potential antitubercular activities from molecular ingredients of traditional Chinese medicines

Background. Traditional Chinese medicine encompasses a well established alternate system of medicine based on a broad range of herbal formulations and is practiced extensively in the region for the treatment of a wide variety of diseases. In recent years, several reports describe in depth studies of the molecular ingredients of traditional Chinese medicines on the biological activities including anti-bacterial activities. The availability of a well-curated dataset of molecular ingredients of traditional Chinese medicines and accurate in-silico cheminformatics models for data mining for antitubercular agents and computational filters to prioritize molecules has prompted us to search for potential hits from these datasets.Results. We used a consensus approach to predict molecules with potential antitubercular activities from a large dataset of molecular ingredients of traditional Chinese medicines available in the public domain. We further prioritized 160 molecules based on five computational filters (SMARTSfilter) so as to avoid potentially undesirable molecules. We further examined the molecules for permeability across Mycobacterial cell wall and for potential activities against non-replicating and drug tolerant Mycobacteria. Additional in-depth literature surveys for the reported antitubercular activities of the molecular ingredients and their sources were considered for drawing support to prioritization.Conclusions. Our analysis suggests that datasets of molecular ingredients of traditional Chinese medicines offer a new opportunity to mine for potential biological activities. In this report, we suggest a proof-of-concept methodology to prioritize molecules for further experimental assays using a variety of computational tools. We also additionally suggest that a subset of prioritized molecules could be used for evaluation for tuberculosis due to their additional effect against non-replicating tuberculosis as well as the additional hepato-protection offered by the source of these ingredients

Cheminformatics Models for Inhibitors of Schistosoma mansoni

Schistosomiasis is a neglected tropical disease caused by a parasite Schistosoma mansoni and affects over 200 million annually. There is an urgent need to discover novel therapeutic options to control the disease with the recent emergence of drug resistance. The multifunctional protein, thioredoxin glutathione reductase (TGR), an essential enzyme for the survival of the pathogen in the redox environment has been actively explored as a potential drug target. The recent availability of small-molecule screening datasets against this target provides a unique opportunity to learn molecular properties and apply computational models for discovery of activities in large molecular libraries. Such a prioritisation approach could have the potential to reduce the cost of failures in lead discovery. A supervised learning approach was employed to develop a cost sensitive classification model to evaluate the biological activity of the molecules. Random forest was identified to be the best classifier among all the classifiers with an accuracy of around 80 percent. Independent analysis using a maximally occurring substructure analysis revealed 10 highly enriched scaffolds in the actives dataset and their docking against was also performed. We show that a combined approach of machine learning and other cheminformatics approaches such as substructure comparison and molecular docking is efficient to prioritise molecules from large molecular datasets

Functional analyses of Plasmodium Falciparum primary metabolic genes

Ph.DDOCTOR OF PHILOSOPH

Investigation of azithromycin analogues and proteasome-like inhibitors as quick-killing antimalarials

Malaria is caused by mosquito-borne parasites of the genus Plasmodium which were responsible for ~435,000 of deaths annually, with >90% caused by the deadliest species, P. falciparum. Over the last two decades, global implementation of vector control and artemisinin combination therapies have resulted in significant reductions in the global burden of malaria. Of current concern is the spread of multi-drug resistant parasites that have severely limited the efficacy of antimalarials, including front-line artemisinins, highlighting the urgent need to identify new antimalarials for use as treatments. The aim of this thesis was to investigate novel antimalarial development avenues and identify new chemotypes that could be used in the near future as treatments. The macrolide antibiotic azithromycin is known to target the malaria parasites remnant plastid organelle (the apicoplast’s) bacterial-like ribosome and causes slow-killing ‘delayed death’, where the parasite dies in the second replication cycle (4 days). Azithromycin has also been shown to inhibit invading merozoites and kill blood stages within the first replication cycle (2 days) via an unidentified mechanism, proposed to be independent of delayed death. Thus, we hypothesised that azithromycin could be redeveloped into an antimalarial with two different mechanisms of action against parasites: delayed death and quick-killing. Over 100 azithromycin analogues that featured a high proportion of different structural profiles were obtained, leading to improved quick-killing activities over azithromycin. Quick-killing was also confirmed to be completely unrelated to delayed death, as blood stage parasites lacking the apicoplast were equally susceptible to quick-killing of azithromycin and analogues. Two different avenues were also confirmed for azithromycin’s antimalarial re-development: delayed death and quick-killing or quick-killing only, which could be modulated depending on the location of added functional groups. Azithromycin and analogues were found to be active across blood stage development, with only short treatments required to kill parasites. The metabolomics signatures of parasites treated with azithromycin and analogues suggested that quick-killing acts multi-factorially, with the parasite’s food vacuole and mitochondria being likely targets. Finally, in vitro activities of two subtypes of tri-peptide proteasome-like inhibitors, vinyl sulfone and aldehydes, were addressed against P. falciparum and the zoonotic malaria parasite P. knowlesi. All compounds exhibited low-nanomolar activities against both Plasmodium spp. and showed excellent selectivity for parasites over human cells, suggesting these inhibitors provide viable chemical scaffolds for optimisation. There was no evidence of increased protein ubiquitination upon treating parasites with these compounds, suggesting they do not target the proteasome. We also investigated whether hypoxia inducible pro-drug proteasome-like inhibitors could be used to reduce host toxicity of antimalarials. However, these pro-drugs could be not activated in in vitro culture conditions and there was limited evidence suggesting this strategy would be applicable in malaria. These studies build on previous findings on the drug-killing efficacy, mechanism of action and possible application of redeveloping azithromycin analogues as new and improved antimalarials. I also identified new proteasome inhibitor-like scaffolds as starting points for further development. This body of work provides thorough biological characterisation of a panel of compounds that could lead to new avenues for antimalarial development.Thesis (Ph.D.) -- University of Adelaide, School of Biololgical Sciences, 202

Investigations into the Metabolic Requirements for Lipoic Acid and Lipid Species During the Life Cycle of the Malarial Parasite Plasmodium Berghei

Plasmodium, like many other pathogenic organisms, relies on a balance of synthesis and scavenging of lipid species for replication. How the parasite creates this balance is particularly important to successfully intervene in transmission of the disease and to generate new chemotherapies to cure infections. This study focuses on two specific aspects in the field of lipid biology of Plasmodium parasites and their hosts. Lipoic acid is a short eight-carbon chain that serves a number of different functions with the cell. By disrupting a key enzyme in the lipoic acid synthesis pathway in the rodent species of malaria, Plasmodium berghei, we sought to investigate its role during the parasite life-cycle. Deletion of the lipoyl-octanoyl transferase enzyme, LipB in P. berghei parasites demonstrate a liver-stage specific need for this metabolic pathway. In order to explore the impact of the fatty acid and triglyceride content on the pathogenesis of Plasmodium parasites, this study tests two methods to reduce lipid content in vivo and test the propagation of P. berghei parasite in these environments. Results from this study set forth new avenues of research with implications for the development of novel antimalarials and vaccine candidates