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Author: Donna Huber

Enantiopure Benzofuran-2-carboxamides of 1-Aryltetrahydro-β-carbolines Are Potent Antimalarials In Vitro

The tetrahydro-β-carboline scaffold has proven fertile ground for the discovery of antimalarial agents (e.g., MMV008138 (1) and cipargamin (2)). Similarity searching of a publicly disclosed collection of antimalarial hits for molecules resembling 1 drew our attention to N2-acyl tetrahydro-β-carboline GNF-Pf-5009 ((±)-3b). Compound purchase, “analog by catalog”, and independent synthesis of hits indicated the benzofuran-2-yl amide portion was required for in vitro efficacy against P. falciparum. Preparation of pure enantiomers demonstrated the pharmacological superiority of (R)-3b. Synthesis and evaluation of D- and F-ring substitution variants and benzofuran isosteres indicated a clear structure-activity relationship. Ultimately (R)-3b was tested in Plasmodium berghei-infected mice; unfavorable physicochemical properties may be responsible for the lack of oral efficacy.

Hanan Almolhim, Sha Ding, Joshua H Butler, Emily K Bremers, Grant J Butschek, Carla Slebodnick, Emilio F Merino, Zaira Rizopoulos, Maxim Totrov, Maria B Cassera, Paul R Carlier. ACS Med. Chem. Lett. 2022, 13, 3, 371–376.

Malaria Box-Inspired Discovery of N-Aminoalkyl-β-carboline-3-carboxamides, a Novel Orally Active Class of Antimalarials

Virtual ligand screening of a publicly available database of antimalarial hits using a pharmacophore derived from antimalarial MMV008138 identified TCMDC-140230, a tetrahydro-β-carboline amide, as worthy of exploration. All four stereoisomers of this structure were synthesized, but none potently inhibited growth of the malaria parasite Plasmodium falciparum. Interestingly, 7e, a minor byproduct of these syntheses, proved to be potent in vitro against P. falciparum and was orally efficacious (40 mg/kg) in an in vivo mouse model of malaria.

Jopaul Mathew, Sha Ding, Kevin A Kunz, Emily E Stacy, Joshua H Butler, Reagan S Haney, Emilio F Merino, Grant J Butschek, Zaira Rizopoulos, Maxim Totrov, Maria B Cassera, Paul R Carlier. ACS Med Chem Lett. 2022 Feb 23;13(3):365-370. doi: 10.1021/acsmedchemlett.1c00663.

New insights into the role of acidocalcisomes in trypanosomatids

Acidocalcisomes are electron-dense organelles rich in polyphosphate and inorganic and organic cations that are acidified by proton pumps, and possess several channels, pumps and transporters. They are present in bacteria and eukaryotes and have been studied in greater detail in trypanosomatids. Biogenesis studies of trypanosomatid acidocalcisomes found that they share properties with lysosome-related organelles of animal cells. In addition to their described roles in autophagy, cation and phosphorus storage, osmoregulation, pH homeostasis, and pathogenesis, recent studies have defined the role of these organelles in phosphate utilization, calcium ion (Ca2+ ) signaling, and bioenergetics, and will be the main subject of this review.

Roberto Docampo, Guozhong Huang. J Eukaryot Microbiol. 2022 Feb 21;e12899. doi: 10.1111/jeu.12899.

Activity-based Crosslinking to Identify Substrates of Thioredoxin-domain Proteinsin Malaria Parasites

Malaria remains a major public health issue, infecting nearly 220 million people every year. The spread of drug-resistant strains of Plasmodium falciparum around the world threatens the progress made against this disease. Therefore, identifying druggable and essential pathways in P. falciparum parasites remains a major area of research. One poorly understood area of parasite biology is the formation of disulfide bonds, which is an essential requirement for the folding of numerous proteins. Specialized chaperones with thioredoxin (Trx) domains catalyze the redox functions necessary for breaking incorrect and forming correct disulfide bonds in proteins. Defining the substrates of these redox chaperones is difficult and immunoprecipitation based assays cannot distinguish between substrates and interacting partners. Further, the substrate or client interactions with the redox chaperones are usually transient in nature. Activity based crosslinkers that rely on the nucleophilic cysteines on Trx domains and the disulfide bond forming cysteines on clients provide an easily scalable method to trap and identify the substrates of Trx-domain containing chaperones. The cell permeable crosslinker divinyl sulfone (DVSF) is active only in the presence of nucleophilic cysteines in proteins and, therefore, traps Trx domains with their substrates, as they form mixed disulfide bonds during the course of their catalytic activity. This allows the identification of substrates that rely on Trx activity for their folding, as well as discovering small molecules that interfere with Trx domain activity. Graphic abstract: Identification of thioredoxin domain substrates via divinylsulfone crosslinking and immunoprecipitation-mass spectrometry.

David W Cobb, Grace S Woods, Vasant Muralidharan. Bio Protoc. 2022 Feb 20;12(4):e4322. doi: 10.21769/BioProtoc.4322.

Protozoan phagotrophy from predators to parasites: An overview of the enigmatic cytostome-cytopharynx complex of Trypanosoma cruzi

Eating is fundamental and from this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment. As part of the world’s aquatic ecosystems, the expansive family of heterotrophic protozoans uses self-generated currents to funnel prokaryotic prey into an ancient, yet highly enigmatic, oral apparatus known as the cytostome-cytopharynx complex prior to digestion. Despite its near ubiquitous presence in protozoans, little is known mechanistically about how this feeding organelle functions. Intriguingly, one class of these flagellated phagotrophic predators known as the kinetoplastids gave rise to a lineage of obligate parasitic protozoa, the trypanosomatids, that can infect a wide variety of organisms ranging from plants to humans. One parasitic species of humans, Trypanosoma cruzi, has retained this ancestral organelle much like its free-living relatives and continues to use it as its primary mode of endocytosis. In this review, we will highlight foundational observations made regarding the cytostome-cytopharynx complex and examine some of the most pressing questions regarding the mechanistic basis for its function. We propose that T. cruzi has the potential to serve as an excellent model system to dissect the enigmatic process of protozoal phagotrophy and thus enhance our overall understanding of fundamental eukaryotic biology.

Ronald Drew Etheridge. J Eukaryot Microbiol. 2022 Feb 17;e12896. doi: 10.1111/jeu.12896.

The good, the bad, and the ugly: From planarians to parasites

Platyhelminthes can perhaps rightly be described as a phylum of the good, the bad, and the ugly: remarkable free-living worms that colonize land, river, and sea, which are often rife with color and can display extraordinary regenerative ability; parasitic worms like schistosomes that cause devastating disease and suffering; and monstrous tapeworms that are the stuff of nightmares. In this chapter, we will explore how our research expanded beyond free-living planarians to their gruesome parasitic cousins. We start with Schistosoma mansoni, which is not a new model; however, approaching these parasites from a developmental perspective required a reinvention that may hold generalizable lessons to basic biologists interested in pivoting to disease models. We then turn to our (re)establishment of the rat tapeworm Hymenolepis diminuta, a once-favorite model that had been largely forgotten by the molecular biology revolution. Here we tell our stories in three, first-person narratives in order to convey personal views of our experiences. Welcome to the dark side.

Tania Rozario, James J Collins 3rd, Phillip A Newmark. Curr Top Dev Biol. 2022;147:345-373. doi: 10.1016/bs.ctdb.2021.12.015.

Trainee Spotlight: Justine Shiau

Justine Shiau

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.

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri

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.

IMPORTANCE Naegleria 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.

A Cassiopeia Russell, Dennis E Kyle. Microbiol Spectr. 2022 Feb 9;e0189921. doi: 10.1128/spectrum.01899-21

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E, or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes but few genes, including almost no transcripts for immune factors, were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Ellen O Martinson, Kangkang Chen, Luca Valzania, Mark R Brown, Michael R Strand. J Exp Biol. 2022 Feb 7;jeb.243460. doi: 10.1242/jeb.243460.