Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While αβ-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

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A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03342-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 356-364 ◽

Cited By ~ 52

Author(s):

Wesley Wu ◽

Zachary Herrera ◽

Danny Ebert ◽

Katie Baska ◽

Seok H. Cho ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antimalarial Drug ◽

Drug Targets ◽

Specific Activity ◽

Drug Candidate ◽

Content Type ◽

Chemical Rescue ◽

Growth Inhibitory ◽

Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

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A Photoalkylative Fluorogenic Probe of Guttiferone A for Live Cell Imaging and Proteome Labeling in Plasmodium falciparum

Molecules ◽

10.3390/molecules25215139 ◽

2020 ◽

Vol 25 (21) ◽

pp. 5139

Author(s):

Romain Duval ◽

Kevin Cottet ◽

Magali Blaud ◽

Anaïs Merckx ◽

Sandrine Houzé ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Natural Product ◽

Drug Targets ◽

Live Cell Imaging ◽

Cell Imaging ◽

Molecular Probe ◽

Positive Functional ◽

Fluorogenic Probe ◽

Symphonia Globulifera

Guttiferone A (GA) 1, a polycyclic polyprenylated acylphloroglucinol (PPAP) isolated from the plant Symphonia globulifera (Clusiaceae), constitutes a novel hit in antimalarial drug discovery. PPAPs do not possess identified biochemical targets in malarial parasites up to now. Towards this aim, we designed and evaluated a natural product-derived photoactivatable probe AZC-GA 5, embedding a photoalkylative fluorogenic motif of the 7-azidocoumarin (AZC) type, devoted to studying the affinity proteins interacting with GA in Plasmodium falciparum. Probe 5 manifested a number of positive functional and biological features, such as (i) inhibitory activity in vitro against P. falciparum blood-stages that was superimposable to that of GA 1, dose–response photoalkylative fluorogenic properties (ii) in model conditions using bovine serum albumin (BSA) as an affinity protein surrogate, (iii) in live P. falciparum-infected erythrocytes, and (iv) in fresh P. falciparum cell lysate. Fluorogenic signals by photoactivated AZC-GA 5 in biological settings were markedly abolished in the presence of excess GA 1 as a competitor, indicating significant pharmacological specificity of the designed molecular probe relative to the native PPAP. These results open the way to identify the detected plasmodial proteins as putative drug targets for the natural product 1 by means of proteomic analysis.

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The Effect of Aqueous Extract of Cinnamon on the Metabolome ofPlasmodium falciparumUsing1HNMR Spectroscopy

Journal of Tropical Medicine ◽

10.1155/2016/3174841 ◽

2016 ◽

Vol 2016 ◽

pp. 1-5 ◽

Cited By ~ 7

Author(s):

Shirin Parvazi ◽

Sedigheh Sadeghi ◽

Mehri Azadi ◽

Maryam Mohammadi ◽

Mohammad Arjmand ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Aqueous Extract ◽

Drug Targets ◽

Inhibitory Concentration ◽

Biological Fluids ◽

Medicinal Herbs ◽

New Drugs ◽

Lysine Biosynthesis ◽

Glutathione Metabolism

Malaria is responsible for estimated 584,000 deaths in 2013. Researchers are working on new drugs and medicinal herbs due to drug resistance that is a major problem facing them; the search is on for new medicinal herbs. Cinnamon is the bark of a tree with reported antiparasitic effects. Metabonomics is the simultaneous study of all the metabolites in biological fluids, cells, and tissues detected by high throughput technology. It was decided to determine the mechanism of the effect of aqueous extract of cinnamon on the metabolome ofPlasmodium falciparum in vitrousing1HNMR spectroscopy. Prepared aqueous extract of cinnamon was added to a culture ofPlasmodium falciparum3D7 and its 50% inhibitory concentration determined, and, after collection, their metabolites were extracted and1HNMR spectroscopy by NOESY method was done. The spectra were analyzed by chemometric methods. The differentiating metabolites were identified using Human Metabolome Database and the metabolic cycles identified by Metaboanalyst. 50% inhibitory concentration of cinnamon onPlasmodium falciparumwas 1.25 mg/mL withp<0.001. The metabolites were identified as succinic acid, glutathione, L-aspartic acid, beta-alanine, and 2-methylbutyryl glycine. The main metabolic cycles detected were alanine and aspartame and glutamate pathway and pantothenate and coenzyme A biosynthesis and lysine biosynthesis and glutathione metabolism, which are all important as drug targets.

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Mapping the malaria parasite drug-able genome using in vitro evolution and chemogenomics

10.1101/139386 ◽

2017 ◽

Cited By ~ 1

Author(s):

Annie N. Cowell ◽

Eva S. Istvan ◽

Amanda K. Lukens ◽

Maria G. Gomez-Lorenzo ◽

Manu Vanaerschot ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Multidrug Resistance ◽

Resistance Genes ◽

Genome Analysis ◽

Drug Targets ◽

Malaria Parasite ◽

Resistance Mechanisms ◽

Whole Genome

AbstractChemogenetic characterization through in vitro evolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262 Plasmodium falciparum parasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of the P. falciparum resistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals how Plasmodium falciparum evolves resistance to diverse compounds and identifies new antimalarial drug targets.

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Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

The Journal of Cell Biology ◽

10.1083/jcb.201311094 ◽

2014 ◽

Vol 204 (7) ◽

pp. 1111-1121 ◽

Cited By ~ 37

Author(s):

Emmanuel Gallaud ◽

Renaud Caous ◽

Aude Pascal ◽

Franck Bazile ◽

Jean-Philippe Gagné ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Microtubule Associated Proteins ◽

Microtubule Polymerization ◽

Sister Chromatids ◽

Centrosome Separation ◽

Associated Proteins ◽

Microtubule Growth ◽

Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

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Analyzing the Essential Proteins Set of Plasmodium Falciparum PF3D7 for Novel Drug Targets Identification Against Malaria

10.21203/rs.3.rs-383876/v1 ◽

2021 ◽

Author(s):

Fawad Ali ◽

Hira Wali ◽

Saadia Jan ◽

Muneeba Aslam ◽

Imtiaz Ahmad ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Protein Interactions ◽

Drug Targets ◽

Human Life ◽

Multi Drug Resistance ◽

Essential Proteins ◽

Human Host

Abstract Background: Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. P. falciparum is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat.Methods: We retrieved the P. falciparum essential proteins from the recently published studies. Pathogen essential proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analyzed for druggability potential via in silico methods to possibly identify novel therapeutic targets.Results: The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein-protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. Conclusion: The prioritized protein targets may worthy to test in malarial drug discovery program to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria.

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Analysing the essential proteins set of Plasmodium falciparum PF3D7 for novel drug targets identification against malaria

Malaria Journal ◽

10.1186/s12936-021-03865-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Fawad Ali ◽

Hira Wali ◽

Saadia Jan ◽

Asad Zia ◽

Muneeba Aslam ◽

...

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Virtual Screening ◽

Drug Targets ◽

Human Life ◽

Multi Drug Resistance ◽

Essential Proteins ◽

Human Host

Abstract Background Plasmodium falciparum is an obligate intracellular parasite of humans that causes malaria. Falciparum malaria is a major public health threat to human life responsible for high mortality. Currently, the risk of multi-drug resistance of P. falciparum is rapidly increasing. There is a need to address new anti-malarial therapeutics strategies to combat the drug-resistance threat. Methods The P. falciparum essential proteins were retrieved from the recently published studies. These proteins were initially scanned against human host and its gut microbiome proteome sets by comparative proteomics analyses. The human host non-homologs essential proteins of P. falciparum were additionally analysed for druggability potential via in silico methods to possibly identify novel therapeutic targets. Finally, the PfAp4AH target was prioritized for pharmacophore modelling based virtual screening and molecular docking analyses to identify potent inhibitors from drug-like compounds databases. Results The analyses identified six P. falciparum essential and human host non-homolog proteins that follow the key druggability features. These druggable targets have not been catalogued so far in the Drugbank repository. These prioritized proteins seem novel and promising drug targets against P. falciparum due to their key protein–protein interactions features in pathogen-specific biological pathways and to hold appropriate drug-like molecule binding pockets. The pharmacophore features based virtual screening of Pharmit resource predicted a lead compound i.e. MolPort-045–917-542 as a promising inhibitor of PfAp4AH among prioritized targets. Conclusion The prioritized protein targets may worthy to test in malarial drug discovery programme to overcome the anti-malarial resistance issues. The in-vitro and in-vivo studies might be promising for additional validation of these prioritized lists of drug targets against malaria.

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In vitro mutagenesis defines drug targets in adolase of Plasmodium falciparum

Memórias do Instituto Oswaldo Cruz ◽

10.1590/s0074-02761992000700043 ◽

1992 ◽

Vol 87 (suppl 3) ◽

pp. 263-264

Author(s):

Ulrich Certa ◽

Christian Itin ◽

Heinz Döbeli

Keyword(s):

Plasmodium Falciparum ◽

Drug Targets ◽

In Vitro Mutagenesis

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Exploration of the Plasmodium falciparum Resistome and Druggable Genome Reveals New Mechanisms of Drug Resistance and Antimalarial Targets

Microbiology Insights ◽

10.1177/1178636118808529 ◽

2018 ◽

Vol 11 ◽

pp. 117863611880852 ◽

Cited By ~ 8

Author(s):

Annie Cowell ◽

Elizabeth Winzeler

Keyword(s):

Drug Resistance ◽

Plasmodium Falciparum ◽

Drug Targets ◽

Antimalarial Activity ◽

Genomic Analysis ◽

Microbial Pathogens ◽

Antimalarial Resistance ◽

Genetic Mechanisms ◽

Human Immune Response

Plasmodium parasites, the causative agent of malaria infections, rapidly evolve drug resistance and escape detection by the human immune response via the incredible mutability of its genome. Understanding the genetic mechanisms by which Plasmodium parasites develop antimalarial resistance is essential to understanding why most drugs fail in the clinic and designing the next generation of therapies. A systematic genomic analysis of 262 Plasmodium falciparum clones with stable in vitro resistance to 37 diverse compounds with potent antimalarial activity was undertaken with the main goal of identifying new drug targets. Despite several challenges inherent to this method of in vitro drug resistance generation followed by whole genome sequencing, the study was able to identify a likely drug target or resistance gene for every compound for which resistant parasites could be generated. Known and novel P falciparum resistance mediators were discovered along with several new promising antimalarial drug targets. Surprisingly, gene amplification events contributed to one-third of the drug resistance acquisition events. The study can serve as a model for drug discovery and resistance analyses in other similar microbial pathogens amenable to in vitro culture.

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Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200503023 ◽

2005 ◽

Vol 170 (7) ◽

pp. 1047-1055 ◽

Cited By ~ 166

Author(s):

Kazuhisa Kinoshita ◽

Tim L. Noetzel ◽

Laurence Pelletier ◽

Karl Mechtler ◽

David N. Drechsel ◽

...

Keyword(s):

Cell Cycle ◽

Xenopus Laevis ◽

Microtubule Assembly ◽

Aurora A ◽

Specific Mechanism ◽

Microtubule Polymerization ◽

Egg Extracts ◽

Microtubule Growth

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

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