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Tag: malaria

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

Donna Huber on March 20, 2019

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.


Beatriz Baragaña, Barbara Forte, Ryan Choi, Stephen Nakazawa Hewitt, Juan A. Bueren-Calabuig, João Pedro Pisco, Caroline Peet, David M. Dranow, David A. Robinson, Chimed Jansen, Neil R. Norcross, Sumiti Vinayak, Mark Anderson, Carrie F. Brooks, Caitlin A. Cooper, Sebastian Damerow, Michael Delves, Karen Dowers, James Duffy, Thomas E. Edwards, Irene Hallyburton, Benjamin G. Horst, Matthew A. Hulverson, Liam Ferguson, María Belén Jiménez-Díaz, Rajiv S. Jumani, Donald D. Lorimer, Melissa S. Love, Steven Maher, Holly Matthews, Case W. McNamara, Peter Miller, Sandra O’Neill, Kayode K. Ojo, Maria Osuna-Cabello, Erika Pinto, John Post, Jennifer Riley, Matthias Rottmann, Laura M. Sanz, Paul Scullion, Arvind Sharma, Sharon M. Shepherd, Yoko Shishikura, Frederick R. C. Simeons, Erin E. Stebbins, Laste Stojanovski, Ursula Straschil, Fabio K. Tamaki, Jevgenia Tamjar, Leah S. Torrie, Amélie Vantaux, Benoît Witkowski, Sergio Wittlin, Manickam Yogavel, Fabio Zuccotto, Iñigo Angulo-Barturen, Robert Sinden, Jake Baum, Francisco-Javier Gamo, Pascal Mäser, Dennis E. Kyle, Elizabeth A. Winzeler, Peter J. Myler, Paul G. Wyatt, David Floyd, David Matthews, Amit Sharma, Boris Striepen, Christopher D. Huston, David W. Gray, Alan H. Fairlamb, Andrei V. Pisliakov, Chris Walpole, Kevin D. Read, Wesley C. Van Voorhis, and Ian H. Gilbert. 2019. PNAS, https://doi.org/10.1073/pnas.1814685116

Targeted Inhibition of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 with a Constrained J Domain-Derived Disruptor Peptide

Donna Huber on February 19, 2019

graphical abstract

To explore the possibility of constrained peptides to target Plasmodium-infected cells, we designed a J domain mimetic derived from Plasmodium falciparum calcium-dependent protein kinase 1 ( PfCDPK1) as a strategy to disrupt J domain binding and inhibit PfCDPK1 activity. The J domain disruptor (JDD) peptide was conformationally constrained using a hydrocarbon staple and was found to selectively permeate segmented schizonts and colocalize with intracellular merozoites in late-stage parasites. In vitro analyses demonstrated that JDD could effectively inhibit the catalytic activity of recombinant PfCDPK1 in the low micromolar range. Treatment of late-stage parasites with JDD resulted in a significant decrease in parasite viability mediated by a blockage of merozoite invasion, consistent with a primary effect of PfCDPK1 inhibition. To the best of our knowledge, this marks the first use of stapled peptides designed to specifically target a Plasmodium falciparum protein and demonstrates that stapled peptides may serve as useful tools for exploring potential antimalarial agents.

Briana R. Flaherty, Tienhuei G. Ho, Sven H. Schmidt, Friedrich W. Herberg, David S. Peterson, and Eileen J. Kennedy. 2019. ACS Infectious Diseases. DOI: 10.1021/acsinfecdis.8b0034

Priority use cases for antibody-detecting assays of recent malaria exposure as tools to achieve and sustain malaria elimination

Donna Huber on February 12, 2019

Measurement of malaria specific antibody responses represents a practical and informative method for malaria control programs to assess recent exposure to infection. Technical advances in recombinant antigen production, serological screening platforms, and analytical methods have enabled the identification of several target antigens for laboratory based and point-of-contact tests. Questions remain as to how these serological assays can best be integrated into malaria surveillance activities to inform programmatic decision-making. This report synthesizes discussions from a convening at Institut Pasteur in Paris in June 2017 aimed at defining practical and informative use cases for serology applications and highlights five programmatic uses for serological assays including: documenting the absence of transmission; stratification of transmission; measuring the effect of interventions; informing a decentralized immediate response;  and testing and treating P. vivax hypnozoite carriers.

Greenhouse B, Daily J, Guinovart C, Goncalves B, Beeson J, Bell D, Chang MA, Cohen JM, Ding X, Domingo G, Eisele TP, Lammie PJ, Mayor A, Merienne N, Monteiro W, Painter J, Rodriguez I, White M, Drakeley C, Mueller I, Malaria Serology Convening. 2019. Gates Open Res.; doi: 10.12688/gatesopenres.12897.1. eCollection 2019.

Open-source discovery of chemical leads for next-generation chemoprotective antimalarials

Donna Huber on December 7, 2018

Abstract

To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.

Yevgeniya Antonova-Koch, Stephan Meister, Matthew Abraham, Madeline R. Luth, Sabine Ottilie, Amanda K. Lukens, Tomoyo Sakata-Kato, Manu Vanaerschot, Edward Owen, Juan Carlos Jado, Steven P. Maher, Jaeson Calla, David Plouffe, Yang Zhong, Kaisheng Chen, Victor Chaumeau, Amy J. Conway, Case W. McNamara, Maureen Ibanez, Kerstin Gagaring, Fernando Neria Serrano, Korina Eribez, Cullin McLean Taggard, Andrea L. Cheung, Christie Lincoln, Biniam Ambachew, Melanie Rouillier, Dionicio Siegel, François Nosten, Dennis E. Kyle, Francisco-Javier Gamo, Yingyao Zhou, Manuel Llinás, David A. Fidock, Dyann F. Wirth, Jeremy Burrows, Brice Campo, Elizabeth A. Winzeler. 2018. Science; 362(6419):eaat9446. http://science.sciencemag.org/content/362/6419/eaat9446

Cryogenically preserved RBCs support gametocytogenesis of Plasmodium falciparum in vitro and gametogenesis in mosquitoes

Donna Huber on December 6, 2018

Abstract

Background: The malaria Eradication Research Agenda (malERA) has identified human-to-mosquito transmission of Plasmodium falciparum as a major target for eradication. The cornerstone for identifying and evaluating transmission in the laboratory is standard membrane feeding assays (SMFAs) where mature gametocytes of P. falciparum generated in vitro are offered to mosquitoes as part of a blood-meal. However, propagation of “infectious” gametocytes requires 10–12 days with considerable physico-chemical demands imposed on host RBCs and thus, “fresh” RBCs that are ≤ 1-week old post-collection are generally recommended. However, in addition to the costs, physico-chemical characteristics unique to RBC donors may confound reproducibility and interpretation of SMFAs. Cryogenic storage of RBCs (“cryo-preserved RBCs”) is accepted by European and US FDAs as an alternative to refrigeration (4 °C) for preserving RBC “quality” and while cryo-preserved RBCs have been used for in vitro cultures of other Plasmodia and the asexual stages of P. falciparum, none of the studies required RBCs to support parasite development for > 4 days.

Results: Using the standard laboratory strain, P. falciparum NF54, 11 SMFAs were performed with RBCs from four separate donors to demonstrate that RBCs cryo-preserved in the gaseous phase of liquid nitrogen (− 196 °C) supported gametocytogenesis in vitro and subsequent gametogenesis in Anopheles stephensimosquitoes. Overall levels of sporogony in the mosquito, as measured by oocyst and sporozoite prevalence, as well as oocyst burden, from each of the four donors thawed after varying intervals of cryopreservation (1, 4, 8, and 12 weeks) were comparable to using ≤ 1-week old refrigerated RBCs. Lastly, the potential for cryo-preserved RBCs to serve as a suitable alternative substrate is demonstrated for a Cambodian isolate of P. falciparum across two independent SMFAs.

Conclusions: Basic guidelines are presented for integrating cryo-preserved RBCs into an existing laboratory/insectary framework for P. falciparum SMFAs with significant potential for reducing running costs while achieving greater reliability. Lastly, scenarios are discussed where cryo-preserved RBCs may be especially useful in enhancing the understanding and/or providing novel insights into the patterns and processes underlying human-to-mosquito transmission.

Ashutosh K. Pathak, Justine C. Shiau, Matthew B. Thomas and Courtney C. Murdock. 2018. Malaria Journal; 17:457. https://doi.org/10.1186/s12936-018-2612-y

Phloroglucinols from the Roots of Garcinia dauphinensis and Their Antiproliferative and Antiplasmodial Activities

Donna Huber on October 24, 2018

Graphica abstract

Abstract

Garcinia dauphinensis is a previously uninvestigated endemic plant species of Madagascar. The new phloroglucinols dauphinols A–F and 3′-methylhyperjovoinol B (1–7) and six known phloroglucinols (8–13) together with tocotrienol 14 and the three triterpenoids 15–17 were isolated from an ethanolic extract of G. dauphinensis roots using various chromatographic techniques. The structures of the isolated compounds were elucidated by NMR, MS, optical rotation, and ECD data. Theoretical ECD spectra and specific rotations for 2 were calculated and compared to experimental data in order to assign its absolute configuration. Among the compounds tested, 1showed the most promising growth inhibitory activity against A2870 ovarian cancer cells, with IC50= 4.5 ± 0.9 μM, while 2 had good antiplasmodial activity against the Dd2 drug-resistant strain of Plasmodium falciparum, with IC50 = 0.8 ± 0.1 μM.

Rolly G. Fuentes, Kirk C. Pearce, Yongle Du, Andriamalala Rakotondrafara, Ana L. Valenciano, Maria B. Cassera, Vincent E. Rasamison, T. Daniel Crawford, and David G. I. Kingston. 2018. Journal of Natural Products.
DOI: 10.1021/acs.jnatprod.8b00379

A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages

Donna Huber on October 17, 2018

Abstract

Background:Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.

Results: Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.

Conclusions: The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Carola Schafer, Nicholas Dambrauskas, Ryan W. Steel, Sara Carbonetti, Vorada Chuenchob, Erika L. Flannery, Vladimir Vigdorovich, Brian G. Oliver, Wanlapa Roobsoong, Steven P. Maher, Dennis Kyle, Jetsumon Sattabongkot, Stefan H. I. Kappe, Sebastian A. Mikolajczak and D. Noah Sather. 2018. Malaria Journal; 17:370. https://doi.org/10.1186/s12936-018-2519-7

Plasmodium falciparum cGMP-dependent protein kinase interacts with a subunit of the parasite proteasome

Donna Huber on October 15, 2018

ABSTRACT

Malaria is caused by the protozoan parasite Plasmodium, which undergoes a complex life cycle in a human host and a mosquito vector. The parasite’s cyclic GMP (cGMP)-dependent protein kinase (PKG) is essential at multiple steps of the life cycle. Phosphoproteomic studies in Plasmodium falciparum erythrocytic stages and Plasmodium berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG) and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, and the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site; a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG: invasion by sporozoites of hepatocytes. A small-molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.

K. Govindasamy, R. Khan, M. Snyder, H. J. Lou, P. Du, H. M. Kudyba, V. Muralidharan, B. E. Turk, P. Bhanot. 2018. Infection and Immunity. https://doi.org/10.1128/IAI.00523-18

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Donna Huber on September 18, 2018

ABSTRACT

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

 

Kudyba, H. M., Cobb, D. W., Florentin, A., Krakowiak, M., Muralidharan, V. 2018. J. Vis. Exp. (139), e57747, doi:10.3791/57747

 

Trainee Spotlight: Kerri Miazowicz

Donna Huber on March 8, 2018

trainee Kerri Miazgowicz

Kerri Miazowicz is a 3rd year Ph.D. trainee in Courtney Murdock‘s laboratory. She grew up in southern Michigan where she received a B.S. in microbiology from Michigan State University in 2012. After graduation, she spent more than two years as a Postbaccalaureate IRTA Fellow at the Rocky Mountain Laboratories, NIH/NIAID in Montana with the Virus Ecology Unit within the Laboratory of Virology.

Choosing the University of Georgia

Kerri chose the University of Georgia for her graduate training because she wanted to conduct interdisciplinary research related to disease ecology and vector-borne disease transmission.

“UGA hosts many experts across several scientific disciplines allowing me to link molecular biology and individual level phenotypes to population-level dynamics,” said Kerri. ” UGA is also home to the Center for Tropical and Emerging Global Diseases and the Center for Ecology of Infectious Diseases (CEID), which both provide valuable research resources and expertise.”

Research Focus

Kerri’s research focuses on environmental drivers of mosquito-borne disease transmission. Mainly, understanding how the environment affects the mosquito vector and modeling the consequences of these interactions on transmission dynamics.

“My current project revolves around temperature effects on Anopheles stephensi [the primary mosquito that transmits malaria in Asia] trait performance (longevity, biting frequency, and population growth), and mathematically exploring the implications these effects have on transmission.”

She will also investigate how Plasmodium, the parasite that causes malaria, exposure and infection modify these mosquito traits which are critical in transmission events.

“I find vector-borne diseases interesting due to the immense complexity that these systems contain,” said Kerri. “I also find it interesting to think about ‘scaling-up’ the outcome of molecular interactions and individual phenotypes to the context of population-level dynamics.”

Kerri has been able to conduct fieldwork within the Athens area to study how microclimate across an urban area can influence mosquito development along with adult mosquito traits with are important for mosquito-borne disease transmission.

“If I was able to travel for research purpose, it would be to India or Africa, where malaria is endemic, to study local mosquito populations.”

 

trainee field work

Trainee Earns Accolades

Kerri has received a number of awards recognizing her academic and research achievements.  In 2009, she was named a 2008-2009 Regional Semifinalist for the Young Epidemiology Scholars (YES) Program. In 2010, Kerri was named a National Institutes of Health Undergraduate Scholar.

In 2014, she received an OITE Travel Award to attend the NIH Graduate and Professional School Fair, which allows NIH interns and postbacs to explore where the next step in their training will be.

Since coming to UGA, she has received an American Society of Virology Travel Award (2016) and a National Science Foundation Graduate Research Fellowship that funds 3 years of research training. In 2017, she received a travel award to attend the annual meeting of the Vector Behavior Ecology Research Coordination Network (VectorBITE RCN) at Imperial College in London.

What’s Next

While Kerri has a few more years of training at UGA, she hopes to continue conducting scientific research related to disease ecology and transmission dynamics in either an academic or government setting.

 

Your financial gift to CTEGD funds the Training Innovations in Parasitologic Studies (TIPS) Fellowships which allows trainees like Kerri Miazowicz to travel to international field sites for research. Give Today!