ABSTRACT
Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.
Subject(s)
Antimalarials , Aspartate Carbamoyltransferase/genetics , Naphthyridines/pharmacology , Plasmodium falciparum , Protozoan Proteins/genetics , Antimalarials/pharmacology , Aspartic Acid , Plasmodium falciparum/enzymology , Plasmodium falciparum/geneticsABSTRACT
Malaria remains one of the deadliest diseases worldwide and it is caused by the protozoan parasite Plasmodium spp. Parasite visualization is an important tool for the correct detection of malarial cases but also to understand its biology. Advances in visualization techniques promote new insights into the complex life cycle and biology of Plasmodium parasites. Live cell imaging by fluorescence microscopy or flow cytometry are the foundation of the visualization technique for malaria research. In this review, we present an overview of possibilities in live cell imaging of the malaria parasite. We discuss some of the state-of-the-art techniques to visualize organelles and processes of the parasite and discuss limitation and advantages of each technique. © 2019 International Society for Advancement of Cytometry.
Subject(s)
Malaria , Parasites , Animals , Flow Cytometry , Humans , Life Cycle Stages , Plasmodium falciparumABSTRACT
The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.
ABSTRACT
Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest. In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect-particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.
Subject(s)
Antimalarials/chemistry , Drug Discovery , Malate Dehydrogenase/chemistry , Plasmodium falciparum/enzymology , Amino Acid Sequence , Antimalarials/pharmacology , Binding Sites , Conserved Sequence , Gene Expression , Humans , Malate Dehydrogenase/antagonists & inhibitors , Malate Dehydrogenase/genetics , Models, Molecular , Molecular Conformation , Mutation , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Protein Binding , Recombinant Proteins , Substrate SpecificityABSTRACT
Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.
Subject(s)
Antiparasitic Agents/chemistry , Antiparasitic Agents/pharmacology , Aspartate Carbamoyltransferase/antagonists & inhibitors , Plasmodium falciparum/enzymology , Aspartate Carbamoyltransferase/chemistry , Aspartate Carbamoyltransferase/metabolism , Catalytic Domain/drug effects , Crystallography, X-Ray , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Molecular Docking Simulation , Plasmodium falciparum/chemistry , Plasmodium falciparum/drug effectsABSTRACT
BACKGROUND: The validation of drug targets in malaria and other human diseases remains a highly difficult and laborious process. In the vast majority of cases, highly specific small molecule tools to inhibit a proteins function in vivo are simply not available. Additionally, the use of genetic tools in the analysis of malarial pathways is challenging. These issues result in difficulties in specifically modulating a hypothetical drug target's function in vivo. OBJECTIVE: The current "toolbox" of various methods and techniques to identify a protein's function in vivo remains very limited and there is a pressing need for expansion. New approaches are urgently required to support target validation in the drug discovery process. METHOD: Oligomerisation is the natural assembly of multiple copies of a single protein into one object and this self-assembly is present in more than half of all protein structures. Thus, oligomerisation plays a central role in the generation of functional biomolecules. A key feature of oligomerisation is that the oligomeric interfaces between the individual parts of the final assembly are highly specific. However, these interfaces have not yet been systematically explored or exploited to dissect biochemical pathways in vivo. RESULTS AND CONCLUSION: This mini review will describe the current state of the antimalarial toolset as well as the potentially druggable malarial pathways. A specific focus is drawn to the initial efforts to exploit oligomerisation surfaces in drug target validation. As alternative to the conventional methods, Protein Interference Assay (PIA) can be used for specific distortion of the target protein function and pathway assessment in vivo.