scholarly journals The Plasmodium falciparum cytoplasmic translation apparatus: a promising therapeutic target not yet exploited by clinically approved antimalarials

2018 ◽  
Author(s):  
Christine Moore Sheridan ◽  
Valentina E. Garcia ◽  
Vida Ahyong ◽  
Joseph L. DeRisi
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Drug Target ◽  
Malaria Parasite ◽  
Experimental Methods ◽  
Mechanisms Of Action ◽  
Antimalarial Drugs ◽  
Inhibit Protein Synthesis ◽  
Indirect Measure ◽  
Wide Range

AbstractThe continued specter of resistance to existing antimalarials necessitates the pursuit of novel targets and mechanisms of action for drug development. One class of promising targets consists of the 80S ribosome and its associated components comprising the parasite translational apparatus. Development of translation-targeting therapeutics requires a greater understanding of protein synthesis and its regulation in the malaria parasite. Research in this area has been limited by the lack of appropriate experimental methods, particularly a direct measure of parasite translation. We have recently developed and optimized the PfIVT assay, an in vitro method directly measuring translation in whole-cell extracts from the malaria parasite Plasmodium falciparum.Here, we present an extensive pharmacologic assessment of the PfIVT assay using a wide range of known inhibitors, demonstrating its utility for studying activity of both ribosomal and non-ribosomal elements directly involved in translation. We further demonstrate the superiority of this assay over a historically utilized indirect measure of translation, S35-radiolabel incorporation. Additionally, we utilize the PfIVT assay to investigate a panel of clinically approved antimalarial drugs, many with unknown or unclear mechanisms of action, and show that none inhibit translation, reaffirming Plasmodium translation to be a viable alternative drug target. Within this set, we unambiguously find that mefloquine lacks translation inhibition activity, despite having been recently mischaracterized as a ribosomal inhibitor. This work exploits a direct and reproducible assay for measuring P. falciparum translation, demonstrating its value in the continued study of protein synthesis in malaria and its inhibition as a drug target.Author summaryNovel antimalarial drugs are required to combat rising resistance to current therapies. The protein synthesis machinery of the malaria parasite Plasmodium falciparum is a promising unexploited target for antimalarial development, but its study has been hindered by use of indirect experimental methods which often produce misleading and inaccurate results. We have recently developed a direct method to investigate malaria protein synthesis utilizing whole-parasite extracts. In this work, we present an extensive characterization of the assay, using a panel of pharmacologic inhibitors with known mechanisms of action. We demonstrate the specificity of the assay in various stages of protein synthesis, as well as its improved accuracy and sensitivity in comparison to an indirect measure that has been the previous standard for the field. We further demonstrate that no current clinically available antimalarial drugs inhibit protein synthesis, emphasizing its potential as a target for drugs that will overcome existing resistance. Importantly, among the antimalarials tested was mefloquine, a widely used antimalarial that has recently been mischaracterized as an inhibitor protein synthesis. Our finding that mefloquine does not inhibit protein synthesis emphasizes the importance of using direct functional measurements when determining drug targets.

scholarly journals The Antibiotic Micrococcin Is a Potent Inhibitor of Growth and Protein Synthesis in the Malaria Parasite

1998 ◽  
Vol 42 (3) ◽  
pp. 715-716 ◽  
Author(s):  
M. John Rogers ◽  
Eric Cundliffe ◽  
Thomas F. McCutchan
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Malaria Parasite ◽  
Inhibitory Concentration ◽  
Potent Inhibitor ◽  
Growth Inhibitor ◽  
Antimalarial Drugs ◽  
Human Malaria Parasite ◽  
Potent Growth ◽  
Potent Growth Inhibitor

ABSTRACT The antibiotic micrococcin is a potent growth inhibitor of the human malaria parasite Plasmodium falciparum, with a 50% inhibitory concentration of 35 nM. This is comparable to or less than the corresponding levels of commonly used antimalarial drugs. Micrococcin, like thiostrepton, putatively targets protein synthesis in the plastid-like organelle of the parasite.

scholarly journals Plasmodium falciparum Resistance to a Lead Benzoxaborole Due to Blocked Compound Activation and Altered Ubiquitination or Sumoylation

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kirthana M. V. Sindhe ◽  
Wesley Wu ◽  
Jenny Legac ◽  
Yong-Kang Zhang ◽  
Eric E. Easom ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Stress Responses ◽  
Mechanisms Of Action ◽  
Antimalarial Drugs ◽  
New Drugs ◽  
Loss Of Function ◽  
Content Type ◽  
High Level ◽  
Level Resistance

ABSTRACT New antimalarial drugs are needed. The benzoxaborole AN13762 showed excellent activity against cultured Plasmodium falciparum, against fresh Ugandan P. falciparum isolates, and in murine malaria models. To gain mechanistic insights, we selected in vitro for P. falciparum isolates resistant to AN13762. In all of 11 independent selections with 100 to 200 nM AN13762, the 50% inhibitory concentration (IC50) increased from 18–118 nM to 180–890 nM, and whole-genome sequencing of resistant parasites demonstrated mutations in prodrug activation and resistance esterase (PfPARE). The introduction of PfPARE mutations led to a similar level of resistance, and recombinant PfPARE hydrolyzed AN13762 to the benzoxaborole AN10248, which has activity similar to that of AN13762 but for which selection of resistance was not readily achieved. Parasites further selected with micromolar concentrations of AN13762 developed higher-level resistance (IC50, 1.9 to 5.0 μM), and sequencing revealed additional mutations in any of 5 genes, 4 of which were associated with ubiquitination/sumoylation enzyme cascades; the introduction of one of these mutations, in SUMO-activating enzyme subunit 2, led to a similar level of resistance. The other gene mutated in highly resistant parasites encodes the P. falciparum cleavage and specificity factor homolog PfCPSF3, previously identified as the antimalarial target of another benzoxaborole. Parasites selected for resistance to AN13762 were cross-resistant with a close analog, AN13956, but not with standard antimalarials, AN10248, or other benzoxaboroles known to have different P. falciparum targets. Thus, AN13762 appears to have a novel mechanism of antimalarial action and multiple mechanisms of resistance, including loss of function of PfPARE preventing activation to AN10248, followed by alterations in ubiquitination/sumoylation pathways or PfCPSF3. IMPORTANCE Benzoxaboroles are under study as potential new drugs to treat malaria. One benzoxaborole, AN13762, has potent activity and promising features, but its mechanisms of action and resistance are unknown. To gain insights into these mechanisms, we cultured malaria parasites with nonlethal concentrations of AN13762 and generated parasites with varied levels of resistance. Parasites with low-level resistance had mutations in PfPARE, which processes AN13762 into an active metabolite; PfPARE mutations prevented this processing. Parasites with high-level resistance had mutations in any of a number of enzymes, mostly those involved in stress responses. Parasites selected for AN13762 resistance were not resistant to other antimalarials, suggesting novel mechanisms of action and resistance for AN13762, a valuable feature for a new class of antimalarial drugs.

scholarly journals Comparison of Efficacies of Cysteine Protease Inhibitors against Five Strains of Plasmodium falciparum

2001 ◽  
Vol 45 (3) ◽  
pp. 949-951 ◽  
Author(s):  
Ajay Singh ◽  
Philip J. Rosenthal
Keyword(s):  
Plasmodium Falciparum ◽  
Protease Inhibitors ◽  
Cysteine Protease ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Antimalarial Drugs ◽  
Cross Resistance ◽  
Cysteine Protease Inhibitors ◽  
Antimalarial Agents ◽  
Parasite Strains

ABSTRACT Falcipain-2, a cysteine protease and essential hemoglobinase ofPlasmodium falciparum, is a potential antimalarial drug target. We compared the falcipain-2 sequences and sensitivities to cysteine protease inhibitors of five parasite strains that differ markedly in sensitivity to established antimalarial drugs. The sequence of falcipain-2 was highly conserved, and the sensitivities of all of the strains to falcipain-2 inhibitors were very similar. Thus, cross-resistance between cysteine protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.

scholarly journals Re-refinement of Plasmodium falciparum orotidine 5′-monophosphate decarboxylase provides a clearer picture of an important malarial drug target

2018 ◽  
Vol 74 (10) ◽  
pp. 664-668 ◽  
Author(s):  
Walter R. P. Novak ◽  
Korbin H. J. West ◽  
Lucy M. D. Kirkman ◽  
Gabriel S. Brandt
Keyword(s):  
Public Health ◽  
Plasmodium Falciparum ◽  
Drug Design ◽  
Electron Density ◽  
Antimalarial Drug ◽  
Drug Target ◽  
Antimalarial Drugs ◽  
Important Advance ◽  
Structure Based Drug Design ◽  
Monophosphate Decarboxylase

The development of antimalarial drugs remains a public health priority, and the orotidine 5′-monophosphate decarboxylase from Plasmodium falciparum (PfOMPDC) has great potential as a drug target. The crystallization of PfOMPDC with substrate bound represents an important advance for structure-based drug-design efforts [Tokuoka et al. (2008), J. Biochem. 143, 69–78]. The complex of the enzyme bound to the substrate OMP (PDB entry 2za1) would be of particular utility in this regard. However, re-refinement of this structure of the Michaelis complex shows that the bound ligand is the product rather than the substrate. Here, the re-refinement of a set of three structures, the apo enzyme and two versions of the product-bound form (PDB entries 2za1, 2za2 and 2za3), is reported. The improved geometry and fit of these structures to the observed electron density will enhance their utility in antimalarial drug design.

scholarly journals Functional Characterization of the m6A-Dependent Translational Modulator PfYTH.2 in the Human Malaria Parasite

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Ameya Sinha ◽  
Sebastian Baumgarten ◽  
Amy Distiller ◽  
Emma McHugh ◽  
Patty Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Malaria Parasite ◽  
Binding Proteins ◽  
Functional Characterization ◽  
Mrna Translation ◽  
Content Type ◽  
Mrna Transcripts

ABSTRACT Posttranscriptional regulation of gene expression is central to the development and replication of the malaria parasite, Plasmodium falciparum, within its human host. The timely coordination of RNA maturation, homeostasis, and protein synthesis relies on the recruitment of specific RNA-binding proteins to their cognate target mRNAs. One possible mediator of such mRNA-protein interactions is the N6-methylation of adenosines (m6A), a prevalent mRNA modification of parasite mRNA transcripts. Here, we used RNA protein pulldowns, RNA modification mass spectrometry, and quantitative proteomics to identify two P. falciparum YTH domain proteins (PfYTH.1 and PfYTH.2) as m6A-binding proteins during parasite blood-stage development. Interaction proteomics revealed that PfYTH.2 associates with the translation machinery, including multiple subunits of the eukaryotic initiation factor 3 (eIF3) and poly(A)-binding proteins. Furthermore, knock sideways of PfYTH.2 coupled with ribosome profiling showed that this m6A reader is essential for parasite survival and is a repressor of mRNA translation. Together, these data reveal an important missing link in the m6A-mediated mechanism controlling mRNA translation in a unicellular eukaryotic pathogen. IMPORTANCE Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis. A widespread mechanism of posttranscriptional gene regulation is the chemical modification of adenosines (m6A), through which the fate of individual mRNA transcripts can be changed. Here, we report on the protein machinery that “reads” this modification and “translates” it into a functional outcome. We provide mechanistic insight into one m6A reader protein and show that it interacts with the translational machinery and acts as a repressor of mRNA translation. This m6A-mediated phenotype has not been described in other eukaryotes as yet, and the functional characterization of the m6A interactome will ultimately open new avenues to combat the disease.

scholarly journals Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis

2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Wilson Wong ◽  
Xiao-Chen Bai ◽  
Brad E. Sleebs ◽  
Tony Triglia ◽  
Alan Brown ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Protein Synthesis ◽  
Inhibit Protein Synthesis ◽  
80S Ribosome

scholarly journals Decrypting a cryptic allosteric pocket in H. pylori glutamate racemase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Pratik Rajesh Chheda ◽  
Grant T. Cooling ◽  
Sondra F. Dean ◽  
Jonah Propp ◽  
Kathryn F. Hobbs ◽  
...  
Keyword(s):  
Life Cycle ◽  
Experimental Methods ◽  
Mechanisms Of Action ◽  
Allosteric Inhibitors ◽  
Coupled Motion ◽  
Glutamate Racemase ◽  
Wide Range ◽  
Essential Enzyme ◽  
H Pylori ◽  
Drug Leads

AbstractOne of our greatest challenges in drug design is targeting cryptic allosteric pockets in enzyme targets. Drug leads that do bind to these cryptic pockets are often discovered during HTS campaigns, and the mechanisms of action are rarely understood. Nevertheless, it is often the case that the allosteric pocket provides the best option for drug development against a given target. In the current studies we present a successful way forward in rationally exploiting the cryptic allosteric pocket of H. pylori glutamate racemase, an essential enzyme in this pathogen’s life cycle. A wide range of computational and experimental methods are employed in a workflow leading to the discovery of a series of natural product allosteric inhibitors which occupy the allosteric pocket of this essential racemase. The confluence of these studies reveals a fascinating source of the allosteric inhibition, which centers on the abolition of essential monomer-monomer coupled motion networks.

scholarly journals Involvement of δ-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum

2002 ◽  
Vol 367 (2) ◽  
pp. 321-327 ◽  
Author(s):  
S. VARADHARAJAN ◽  
S. DHANASEKARAN ◽  
Z.Q. BONDAY ◽  
P.N. RANGARAJAN ◽  
G. PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
Enzyme Activity ◽  
Drug Target ◽  
Malaria Parasite ◽  
De Novo ◽  
Parasite Growth ◽  
Pcr Analysis ◽  
Trophozoite Stage ◽  
Parasite Survival ◽  
A Cell

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for Δ-aminolaevulinate synthase (PfALAS) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The PfALAS gene is functional and vital for parasite haem synthesis and parasite survival.

scholarly journals The evolution, metabolism and functions of the apicoplast

2010 ◽  
Vol 365 (1541) ◽  
pp. 749-763 ◽  
Author(s):  
Liting Lim ◽  
Geoffrey Ian McFadden
Keyword(s):  
Plasmodium Falciparum ◽  
Drug Development ◽  
Drug Target ◽  
Malaria Parasite ◽  
Therapeutic Interventions ◽  
Parasite Cell ◽  
Metabolic Functions

The malaria parasite, Plasmodium falciparum , harbours a relict plastid known as the ‘apicoplast’. The discovery of the apicoplast ushered in an exciting new prospect for drug development against the parasite. The eubacterial ancestry of the organelle offers a wealth of opportunities for the development of therapeutic interventions. Morphological, biochemical and bioinformatic studies of the apicoplast have further reinforced its ‘plant-like’ characteristics and potential as a drug target. However, we are still not sure why the apicoplast is essential for the parasite's survival. This review explores the origins and metabolic functions of the apicoplast. In an attempt to decipher the role of the organelle within the parasite we also take a closer look at the transporters decorating the plastid to better understand the metabolic exchanges between the apicoplast and the rest of the parasite cell.

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