Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite’s ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesized that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalyzed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.
Alona Botnar, Grant Lawrence, Steven P Maher, Amélie Vantaux, Benoît Witkowski, Justine C Shiau, Emilio F Merino, David De Vore, Christian Yang, Cameron Murray, Maria B Cassera, James W Leahy, Dennis E Kyle. Int J Parasitol. 2022 Apr 18;S0020-7519(22)00066-2. doi: 10.1016/j.ijpara.2022.03.003.
Studies on an organic extract of a marine fungus, Periconia sp. (strain G1144), led to the isolation of three halogenated cyclopentenes along with the known and recently reported rhytidhyester D; a series of spectrometric and spectroscopic techniques were used to elucidate these structures. Interestingly, two of these compounds represent tri-halogenated cyclopentene derivatives, which have been observed only rarely from Nature. The relative and absolute configurations of the compounds were established via mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, Mosher’s esters method, optical rotation and GIAO NMR calculations, including correlation coefficient calculations and the use of both DP4+ and dJ DP4 analyses. Several of the isolated compounds were tested for activity in anti-parasitic, antimicrobial, quorum sensing inhibition, and cytotoxicity assays and were shown to be inactive.
Kristóf B Cank, Robert A Shepherd, Sonja L Knowles, Manuel Rangel-Grimaldo, Huzefa A Raja, Zoie L Bunch, Nadja B Cech, Christopher A Rice, Dennis E Kyle, Joseph O Falkinham 3rd, Joanna E Burdette, Nicholas H Oberlies. Phytochemistry . 2022 Apr 11;113200. doi: 10.1016/j.phytochem.2022.113200
The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.
Yuxiang Dong, Yogesh Sonawane, Steven P Maher, Anne-Marie Zeeman, Victor Chaumeau, Amélie Vantaux, Caitlin A Cooper, Francis C K Chiu, Eileen Ryan, Jenna McLaren, Gong Chen, Sergio Wittlin, Benoît Witkowski, François Nosten, Kamaraj Sriraghavan, Dennis E Kyle, Clemens H M Kocken, Susan A Charman, Jonathan L Vennerstrom. ACS Omega. 2022 Mar 30;7(14):12401-12411. doi: 10.1021/acsomega.2c01099.
Justine Shiau, an NIH T32 fellow in Dr. Dennis Kyle’s laboratory, is originally from Taipei, Taiwan, and moved to the states after elementary school. She received her bachelor’s degree in Biology from the Pennsylvania State University, where she became interested in disease transmission, disease ecology, and parasitology while working with Dr. Ashutosh Pathak. Upon graduation, she moved to Athens to continue her training with Dr. Pathak, who at that time was working in the transmission ecology of vector-borne diseases with Dr. Courtney Murdock. Over the next two years, she took part in research projects revolving around vector biology and mosquito-transmitted pathogens. She was accepted by the UGA Integrated Life Science graduate program in Fall 2018.
In the Kyle lab, Justine is currently working on the transmission stages of Plasmodium falciparum, a human malaria parasite that causes significant mortality worldwide, specifically on the biology of the parasite transitioning from the vector to the human and the early stages within the human, prior to disease onset. She aims to complete the parasite’s life cycle in a laboratory setting, which would be a powerful tool to help further our understanding of the host-parasite interactions. She hopes to better understand the parasite biology and the transmission dynamic that the mosquitoes could have on the downstream infection in humans, which can potentially help us better understand and combat this horrible disease.
Why did you choose UGA?
UGA has one of the finest insectary facilities that allows the transmission of Plasmodium falciparum. Additionally, the Center of Tropical and Emerging Global Diseases (CTEGD) is the hub for parasitologists. The Center provides state-of-the-art infrastructure, research equipment, and, most of all, a supportive environment to cultivate and train graduate students to meet our goals.
What is your research focus?
Plasmodium falciparum is a parasite that causes malaria, which 50% of the world’s population is at risk of getting. Many children die from malaria every year; we cannot effectively prevent diseases and transmissions without a well-rounded understanding of the parasite’s biology and the essential players (mosquitoes) to complete its life cycle. My overarching goal is to complete the parasite’s life cycle in the lab. Currently, we are focusing on the biology of the parasite and its transition from mosquito back to human and within the human: from liver-to-blood stage infections. While doing this, there are two primary objectives that I would like to meet. First, I want to better understand the important factors for the parasites to establish infection in the human liver cells. Second, I am curious whether the mosquito stage infection can also impact the parasite’s efficiency in establishing infection in the human liver.
What are your future professional plans?
After graduate school, I hope to continue my postdoctoral training. I would like to pursue interdisciplinary research, with crosstalk between disease-ecology, parasitology, and vector biology.
Any advice for a student interested in this field?
Be open-minded and respectful to people with different expertise and people with diverse backgrounds.
Support trainees like Justine by giving today to the Center for Tropical & Emerging Global Diseases.
The free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.
IMPORTANCENaegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.
Malaria is a prevalent and lethal disease. The fast emergence and spread of resistance to current therapies is a major concern and the development of a novel line of therapy that could overcome, the problem of drug resistance, is imperative. Screening of a set of compounds with drug/natural product-based sub-structural motifs led to the identification of spirocyclic chroman-4-one 1 with promising antimalarial activity against the chloroquine-resistant Dd2 and chloroquine-sensitive 3D7 strains of the parasite. Extensive structure-activity and structure-property relationship studies were conducted to identify the essential features necessary for its activity and properties.
Iredia D Iyamu, Yingzhao Zhao, Prakash T Parvatkar, Bracken F Roberts, Debora R Casandra, Lukasz Wojtas, Dennis E Kyle, Debopam Chakrabarti, Roman Manetsch. Bioorg Med Chem. 2022 Jan 14;57:116629. doi: 10.1016/j.bmc.2022.116629.
Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite’s liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype.
Plasmodium lactate dehydrogenase (pLDH) is a common target in malaria rapid diagnostic tests (RDTs). These commercial antibody capture assays target either Plasmodium falciparum-specific pLDH (PfLDH), P. vivax-specific pLDH (PvLDH), or a conserved epitope in all human malaria pLDH (PanLDH). However, there are no assays specifically targeting P. ovale, P. malariae or zoonotic parasites such as P. knowlesi and P. cynomolgi. A malaria multiplex array, carrying the specific antibody spots for PfLDH, PvLDH, and PanLDH has been previously developed. This study aimed to assess potential cross-reactivity between pLDH from various Plasmodium species and this array. We tested recombinant pLDH proteins, clinical samples for P. vivax, P. falciparum, P. ovale curtisi, and P. malariae; and in vitro cultured P. knowlesi and P. cynomolgi. P. ovale-specific pLDH (PoLDH) and P. malariae-specific pLDH (PmLDH) cross-reacted with the PfLDH and PanLDH spots. Plasmodium knowlesi-specific pLDH (PkLDH) and P. cynomolgi-specific pLDH (PcLDH) cross-reacted with the PvLDH spot, but only PkLDH was recognized by the PanLDH spot. Plasmodium ovale and P. malariae can be differentiated from P. falciparum by the concentration ratios of PanLDH/PfLDH, which had mean (range) values of 4.56 (4.07-5.16) and 4.56 (3.43-6.54), respectively, whereas P. falciparum had a lower ratio of 1.12 (0.56-2.61). Plasmodium knowlesi had a similar PanLDH/PvLDH ratio value, with P. vivax having a mean value of 2.24 (1.37-2.79). The cross-reactivity pattern of pLDH can be a useful predictor to differentiate certain Plasmodium species. Cross-reactivity of the pLDH bands in RDTs requires further investigation.
Becky Barney, Miguel Velasco, Caitlin Cooper, Andrew Rashid, Dennis Kyle, Robert Moon, Gonzalo Domingo, Ihn Kyung Jang. Am J Trop Med Hyg. 2021 Nov 15;tpmd210532. doi: 10.4269/ajtmh.21-0532
Balamuthia mandrillaris, a pathogenic free-living amoeba, causes cutaneous skin lesions as well as granulomatous amoebic encephalitis, a ‘brain-eating’ disease. As with the other known pathogenic free-living amoebas (Naegleria fowleri and Acanthamoeba species), drug discovery efforts to combat Balamuthia infections of the central nervous system are sparse; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Current treatments of encephalitis due to B. mandrillaris lack efficacy, leading to case fatality rates above 90%. Using our recently published methodology to discover potential drugs against pathogenic amoebas, we screened a collection of 85 compounds with known antiparasitic activity and identified 59 compounds that impacted the growth of Balamuthia trophozoites at concentrations below 220 µM. Since there is no fully annotated genome or proteome of B. mandrillaris, we sequenced and assembled its transcriptome from a high-throughput RNA-sequencing (RNA-Seq) experiment and located the coding sequences of the genes potentially targeted by the growth inhibitors from our compound screens. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing. These will be used in the future in combination with the identified hits in structure guided drug discovery campaigns to develop new approaches for the treatment of Balamuthia infections.
Isabelle Q Phan, Christopher A Rice, Justin Craig, Rooksana E Noorai, Jacquelyn R McDonald, Sandhya Subramanian, Logan Tillery, Lynn K Barrett, Vijay Shankar, James C Morris, Wesley C Van Voorhis, Dennis E Kyle, Peter J Myler. Sci Rep. 2021 Nov 4;11(1):21664. doi: 10.1038/s41598-021-99903-8.
Globally, efforts to control malaria caused by Plasmodium vivax are lagging behind that of other species of Plasmodium due to its unique biology. A team of researchers at the University of Georgia, the Institute Pasteur of Cambodia, and Shoklo Malaria Research Unit in Thailand detail a new screening tool and report for the first time a method capable of discovering novel experimental drug compounds for use against vivax malaria. Their study was recently published in Scientific Reports.
The parasite species P. vivax is the most widespread cause of malaria. While not as deadly as malaria caused by P. falciparum, it can cause severe disease and has a significant impact on both national economies and personal finances, in part due to this species’ propensity to cause relapses.
Relapses are caused by hypnozoites, a form of the parasite residing in the liver, which can lie dormant for a period of time before causing another symptomatic blood infection. During this period of dormancy, hypnozoites are not susceptible to standard antimalarials, meaning a patient treated for a blood infection is not fully cured.
“With this assay, we can now tell earlier on in the drug discovery process if a compound is going to work against hypnozoites,” said Steven Maher, assistant research scientist at the Center for Tropical and Emerging Global Diseases and lead researcher on the study. “In this study, we were able to identify three new drugs that kill dormant hypnozoites.”
One of the drugs identified looks promising as a possible new treatment, though Maher said it will need more testing. The other two could be useful in studying hypnozoite biology and increase understanding of such things as the mechanisms of dormancy.
The team’s report also shows how two current antimalarial drugs, chloroquine and tafenoquine, synergistically work together to kill hypnozoites. However, these drugs cannot be administered to children and pregnant women (due to their known side effects), nor to people who lack the enzyme called G6PD. Up to 20% of the population in southeast Asia are G6PD deficient.
“The current drug therapies work well to treat the symptomatic blood stage of vivax malaria,” said Steven Maher. “However, in vivax malaria we need to eliminate hypnozoites to fully cure the patient, and for that we need new therapies.”
To compound the problem, typical mouse models used in malaria drug research can’t determine if the experimental compounds work against hypnozoites because the Plasmodium species that infects mice doesn’t produce them. Additionally, because the assays used as the first step in discovering potential new drug compounds focus on the blood stage of the parasite, researchers need a different kind of assay that will allow them to test these compounds on hypnozoites, which requires a stable culture of liver cells.
“It’s a challenge because you have to get samples from where the vivax malaria is endemic,” said Maher. “Liver cells don’t stay viable in culture for long, and these assays take eight days to show results. The assay itself is difficult to run, but we have a great team of researchers in Cambodia and Thailand that has really helped to make this possible.”
The team is continuing to build better tools to overcome the challenges drug discovery in P. vivax faces as they begin to test these drugs in animal models.