Plasmodial enzymes in metabolic pathways as therapeutic targets and contemporary strategies to discover new antimalarial drugs: a review

Malaria continues to pose imminent threat to the world population, as the mortality rate associated with this disease remains high. Current treatment relies on antimalarial drugs such as Artemisinin Combination Therapy (ACT) are still effective throughout the world except in some places, where ACT-resistance has been reported, thus necessitating novel approaches to develop new anti-malarial therapy. In the light of emerging translational research, several plasmodial targets, mostly proteins or enzymes located in the parasite’s unique organelles, have been extensively explored as potential candidates for the development of novel antimalarial drugs. By targeting the metabolic pathways in mitochondrion, apicoplast or cytoplasm of Plasmodium, the possibility to discover new drugs is tremendous, as they have potentials as antimalarial therapeutic targets. This literature review summarizes pertinent information on plasmodial targets, especially enzymes involved in specific metabolic pathways, and the strategies used to discover new antimalarial drugs. © 2019, University of Malaya. All rights reserved

Ethoxyquin Inhibits the Progression of Murine Ehrlich Ascites Carcinoma through the Inhibition of Autophagy and LDH

Cancer cells exhibit an increased glycolysis rate for ATP generation (the Warburg effect) to sustain an increased proliferation rate. In tumor cells, the oxidation of pyruvate in the Krebs cycle is substituted by lactate production, catalyzed by LDH. In this study, we use ethoxyquin (EQ) as a novel inhibitor to target LDH in murine Ehrlich ascites carcinoma (EAC) and as a combination therapy to improve the therapeutic efficacy of the conventional chemotherapy drug, cisplatin (CIS). We investigated the anti-tumor effect of EQ on EAC-bearing mice and checked whether EQ can sustain the anti-tumor potential of CIS and whether it influences LDH activity. Treatment with EQ had evident anti-tumor effects on EAC as revealed by the remarkable decrease in the expression of the anti-apoptotic gene Bcl-2 and by a significant increase in the expression of apoptotic genes (BAX and caspase-3). EQ also caused a significant decrease in the autophagic activity of EAC cells, as shown by a reduction in the fluorescence intensity of the autophagosome marker. Additionally, EQ restored the altered hematological and biochemical parameters and improved the disrupted hepatic tissues of EAC-bearing mice. Co-administration of EQ and CIS showed the highest anti-tumor effect against EAC. Collectively, our findings propose EQ as a novel inhibitor of LDH in cancer cells and as a combinatory drug to increase the efficacy of cisplatin. Further studies are required to validate this therapeutic strategy in different cancer models and preclinical trials

Studies to improve in vitro transfection and infection methods of Cryptosporidium parvum and biological characterization of the putative virulence factor thrombospondin-related adhesive protein (Trap-C1)

Es geht um die Verbesserung verschiedener Infektions- und Transfektionsprotokolle zur in vitro Arbeit mit Cryptosporidium parvum in der Zellkultur. Des weiteren wurden Mausdarmorganoide ohne Hilfe eines Mikroinjektors mit C. parvum Sporozoiten infiziert. Außerdem wurde ein Protein (Trap-C1) von C. parvum welches ein putativer Virulenzfaktor ist, molekularbiologisch untersucht

Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii

Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment

Review of Experimental Compounds Demonstrating Anti-Toxoplasma Activity

Toxoplasma gondii is a ubiquitous apicomplexan parasite capable of infecting humans and other animals. Current treatment options for T. gondii infection are limited and most have drawbacks, including high toxicity and low tolerability. Additionally, no FDA-approved treatments are available for pregnant women, a high-risk population due to transplacental infection. Therefore, the development of novel treatment options is needed. To aid this effort, this review highlights experimental compounds that, at a minimum, demonstrate inhibition of in vitro growth of T. gondii. When available, host cell toxicity and in vivo data are also discussed. The purpose of this review is to facilitate additional development of anti-Toxoplasma compounds and potentially to extend our knowledge of the parasite

Functional analyses of Plasmodium Falciparum primary metabolic genes

Ph.DDOCTOR OF PHILOSOPH

Recombinant expression and initial characterisation of two Plasmodium copper binding proteins.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.Plasmodium falciparum is a protozoan parasite responsible for the most severe form of human malaria, with infection often resulting in death. Efforts to control malaria have been hindered by an increased spread of parasite resistance to previously effective antimalarial drugs, leading to an intensified search for novel antimalarial drug targets. A group of proteins suggested as potentially effective targets are the integral membrane transport proteins, since they play key roles in Plasmodium parasite growth and replication. One such membrane protein recently characterised was the P. falciparum copper efflux transporter. Treatment of cultured P. falciparum parasites with the intracellular copper chelator neocuproine inhibited parasite growth, suggesting that additional mechanisms for malaria parasite copper homoeostasis are likely to be present. Copper is an essential trace element involved in enzymatic processes requiring redox-chemistry. In higher eukaryotes copper is transported across the plasma membrane via the copper transport protein, Ctr1, and distributed intracellularly by copper metallochaperones. The mechanisms for copper acquisition and distribution in the Plasmodium parasite are, however, yet to be characterised. An in silico Basic Local Alignment Search Tool for protein (BLASTp) screen of the Plasmodium database (www.plasmodb.org) identified sequences corresponding to a putative copper transporter, and associated copper metallochaperones, in eight species of the Plasmodium parasite. Each of the Plasmodium copper transport protein sequences was found to contain features common to the well characterised copper transporters. These features included predicted copper-binding motifs in the protein's amino terminus, three membrane spanning domains and the characteristic MxxxM and GxxxG motifs located in the second and third transmembrane domains, respectively. Affinity purified anti-peptide antibodies, generated against an immunogenic peptide (CSDKQSGDDECKPILD) in the amino terminus of a putative malaria parasite copper transporter (PY00413), detected the target protein in murine malaria parasites in association with a parasite membrane. The open reading frames corresponding to the amino terminal domains of one P. berghei [PBANKA_130290 (447 bp)] and two P. falciparum [PF14_0211 (132 bp) and PF14_0369 (282 bp)] putative copper transport proteins were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with maltose binding protein (MBP). The resulting sizes for the recombinant proteins were 61kDa for MBP-PbCtrNt, 48kDa for MBP-PfCtr211Ntᵀᴰ and 55kDa for MBP-PfCtr369Ntᵀᴰ, with each protein being recognised by a corresponding anti-peptide antibody. All three recombinant proteins bound copper in vitro and in vivo, with each having a binding preference for the reduced cuprous ion. This preference has been similarly established for the characterised copper transporters. Although the results supported the expression and copper binding ability of a Plasmodium parasite copper transport protein, the directional transport of copper, by this protein, requires experimental confirmation as does its specific location. The identification of a P. falciparum copper transporter, and other copper dependent proteins, implies a parasite metabolic requirement for copper. Mammalian and yeast cells require a Cox17 metallochaperone for copper delivery to cytochrome-c oxidase. Identification of P. falciparum orthologs for Cox17 (PF10_0252) and a number of cytochrome-c oxidase subunits (PF13_0327; PF14_0288; mal_mito_1; mal_mito_2; PFI1365w; PFI1375w), suggests the existence of similar parasite mechanisms for copper delivery. Analysis of the Plasmodium Cox17-like sequences identified essential amino acids conserved in the well characterised yeast and mammalian Cox17. This included the identification of six cysteine residues essential for Cox17 function. A homology model of P. falciparum Cox17, with human Cox17 as the template [PDB ID: 2RN9 (apoCox17); 2RN8 (Cu⁺-Cox17)], suggested that Plasmodium Cox17 orthologs would adopt a similar structural conformation. The open reading frames for full-length P. yoelii [PY03823 (192 bp)] and P. falciparum [PF10_0252 (195 bp)] Cox17 were PCR amplified, ligated into pGEM®-T and then expressed as recombinant fusion proteins with either a His₆-tag or glutathione S-transferase (GST)-tag, respectively. The resulting sizes for the recombinant proteins were 11.6kDa for His₆-PyCox17 and 33.5kDa for GST-PfCox17, with each protein being recognised by a corresponding anti-peptide antibody. Both recombinant Cox17 proteins bound the cuprous ion in vitro and in vivo, similar to mammalian and yeast Cox17. This supported the likely existence of a mitochondrial copper metallochaperone pathway within the malaria parasite; however, this requires further experimental confirmation. Identification of a parasite copper transport protein, and associated metallochaperones, could provide novel targets for drug-based inhibition of parasite growth. Alternatively, the copper transporter may provide a novel mechanism for drug delivery into the Plasmodium parasite. The potential of these malaria parasite proteins being effective drug targets does, however, remain to be confirmed

Expanding the Knowledge on Oocyst Molecules of Toxoplasma gondii

The goal of this thesis is to shed more light on the oocyst stage of Toxoplasma gondii regarding several key aspects such as possible new molecules in the oocyst wall that can be harnessed for ligand-based methods for detection, molecules inside the oocyst that contribute to the increased stress resistance and molecules that can be used in serological detection methods. To this end, the project is split into three parts. The first part aims to lay the groundwork for establishment of new methods for oocyst enrichment in environmental samples by identifying new molecules binding to the oocyst wall. As a basis for further experiments, a method for robust immunofluorescence experiments using only small numbers of oocysts is developed. Using this method, molecules binding to the oocyst wall can be identified and their binding characteristics further analyzed. The generation of nanobodies targeting the oocyst wall is explored. Also, members from the Dectin-1 CLR cluster are investigated with special focus on their binding to the oocyst wall. Results from these experiments will also contribute to new insights into the molecular composition of the oocyst wall. In the second part, factors contributing to the oocysts resilience to environmental stress are investigated. Oocysts are known to stay viable in the environment under varying conditions, relatively untouched by environmental factors. This is often attributed to the rigidity of the bilayered walls that protect the oocyst and the sporocysts. However, the presence of so-called LEA proteins inside the oocyst that contribute to protection against damage induced by certain stress factors has been hypothesized. These LEA proteins are known to confer stress resistance in plants and invertebrates through their disordered structure. Through in silico, in vitro and in vivo characterization of the TgLEAs, more in depth knowledge will be acquired, collecting predictions and findings regarding their IDP characteristics, their biochemical properties as well as their effect on bacterial growth upon several stresses. The in vitro analyses will investigate the LEA protein’s potential to protect an important T. gondii pathogenesis factor from stress induced damage. The findings from these experiments will lead to a better understanding of oocyst physiology as well as opening up more possibilities to develop methods to eradicate oocysts from the environment in a targeted and more efficient manner and prevent parasite spread. Lastly, this thesis analyzes the oocyst specific TgLEAs regarding their putative role as antigens that can elicit a specific antibody response, serving as infection marker. Such a serological tool would be highly beneficial in early identification of infection clusters and efficient mounting of counter measures to prevent further spread. First, antibodies against two most promising LEA proteins will be generated by immunization of two rabbits to serve as additional controls in future experiments as well as proof of basic antigenicity of TgLEAs. Subsequently, the Luminex technology will be shortly introduced and adapted to allow feasible analyses of large numbers of chicken sera for T. gondii infections. ELISA tests will assess the TgLEAs’ antigenic potential by analysis of sera from experimentally infected chickens. Lastly, the Luminex technology will be employed to corroborate the ELISA findings and investigate on a large-scale different serum dilutions to confirm if TgLEAs are suitable antigens to differentiate different T. gondii infection routes, i.e. tachyzoite, bradyzoite or oocyst. In summary, this project will expand the knowledge on molecules (1) binding to the oocyst wall, (2) contributing to stress tolerance inside the oocyst and (3) as antigens for identification of oocyst-mediated infections

Biochemical characterization of ornithine aminotransferase and cystathionine \u3b3-lyase from Toxoplasma gondii: possible targets for drug development?

Toxoplasmosis is a widespread parasitic disease caused by Toxoplasma gondii, an obligate intracellular protozoa belonging to the phylum Apicomplexa. Toxoplasmosis is a major public health problem, infecting one-third of humans worldwide. Due to the fact that no effective vaccine is currently available and treatment is based on drugs for which resistance is emerging, there is an urgent need to discover novel drug targets that are exploitable for the design of new therapeutics against the pathogen. A recent proteomic analysis of partially sporulated oocysts of T. gondii showed that oocysts have a greater capability of de novo amino acid biosynthesis, shedding light on several stage-specific proteins whose functional profile is in accord with the oocyst need to resist various environmental stresses [1]. Herein, we focused our attention on two enzymes belonging to these putative oocyst/sporozoite-specific protein group: the ornithine aminotransferase (OAT) and the cystathionine \u3b3-lyase (CGL). OAT is involved in the polyamine metabolism and catalyzes the reversible conversion of L-ornithine into glutamate-5-semialdehyde and glutamate, while CGL catalyzes the cleavage of L-cystathionine (L-cth) to L-cysteine, \u3b1-ketobutyrate and ammonia in the reverse transsulfuration pathway. Despite the central metabolic roles of these enzymes, the functionality of none of them has so far been investigated. Herein, a biochemical characterization of OAT and CGL from T. gondii has been performed, in order to expand the very limited knowledge about the polyamine and cysteine metabolism of the parasite and to explore the possible use of these enzymes as novel drug targets against toxoplasmosis. Analysis of spectral and kinetic properties of TgOAT revealed that the enzyme is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not possess a specific ornithine aminotransferase activity, but exhibits both N-acetylornithine and \u3b3-aminobutyric acid (GABA) transaminase activity, highlighting its possible role both in arginine and GABA metabolism in vivo. The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr resulted in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The purified TgCGL is a functional enzyme which splits L-cth almost exclusively at the C\u3b3S bond to yield L-cysteine. This finding likely implies that the reverse transsulfuration pathway is operative in the parasite. The enzyme displays only marginal reactivity toward L-cysteine, which is also a mixed-type inhibitor of TgCGL activity, therefore indicating a tight regulation of cysteine intracellular levels in the parasite. Structure-guided homology modelling revealed two striking amino acid differences between human and TgCGL active sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of these two residues to the corresponding residues in human revealed their importance in modulating both substrate and reaction specificity of the parasitic enzyme. Altogether our findings could be considered as a first step toward exploring the possible use of TgOAT and TgCGL as an anti-toxoplasmosis drug targets

The role of protozoan genetic diversity in human disease : implications for the epidemiology of cryptosporidiosis and giardiasis in New Zealand : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Palmerston North, New Zealand

Cryptosporidium and Giardia are two common causes of diarrhoea in humans and livestock and responsible for multiple outbreaks of gastroenteritis every year in New Zealand and around the globe. Despite their prevalence, there are few effective therapies or vaccines, against either parasite. This is largely due to the difficulty of manipulating these parasites in vitro. The understanding of the epidemiology of this parasite in New Zealand is incomplete, due to the presence of multiple dominant subtypes of each parasite within samples from the same outbreak. In this thesis, new techniques are employed to investigate the genetic diversity of these parasites within hosts and develop an in vitro assay for comparing the infectivity of multiple subtypes of Cryptosporidium. Current methods for the purification of Giardia cysts from faecal samples do not adequately remove debris from the sample and produce low numbers of purified cysts. This hampers molecular techniques that benefit from uncontaminated samples resulting in the use of expensive methods like immunomagnetic separation. Here, a novel method for the purification of cysts from faecal samples was developed, which produced purified oocysts with negligible debris and a 10-fold increase in yield over current techniques. Epidemiological and molecular investigations of past giardiasis and cryptosporidiosis outbreaks in New Zealand have highlighted inconsistent results, where epidemiologically linked cases can have different dominant subtypes identified through Sanger sequencing. Here, amplicon-based metabarcoding was utilised to resolve Giardia and Cryptosporidium outbreak epidemiology in New Zealand. Human faecal samples from past outbreaks previously classified using Sanger sequencing were analysed using next-generation sequencing. This strategy uncovered significant within-host diversity and identified potential emerging subtypes of Cryptosporidium that could have public health significance in the future. Analysis of diversity within outbreaks provided previously unidentified genetic links between samples from the same outbreak. Previous studies show that people experience different symptoms depending on the subtype of Cryptosporidium they are infected with. Also, the dominant subtypes of the parasite in a region, like the USA and Australia, have changed multiple times within the past 20 years. This suggests there are differences in infectivity between subtypes, but further analysis of this problem has been hampered by the lack of adequate cell culture systems that allow the complete development of the parsite in vitro. To better understand the differences in infectivity between subtypes of Cryptosporidium, an analysis of the expression of Cryptosporidium genes in the COLO-680N cell line at multiple timepoints during infection was carried out using the NanoString nCounter analysis system. This was done to investigate whether differences in gene expression could account for differences in infectivity. Furthermore, utilising flow cytometry a system was developed capable of identifying and quantifying infection in infected cells with and without the use of a fluorescent antibody. A novel signal was identified in the near-infra red range that was specific to Cryptosporidium infection and showed better signalling characteristics than the fluorophore