Artemisinin combination therapies (ACTs) are critical components of malaria control worldwide. Alarmingly, ACTs have begun to fail, owing to the rise in artemisinin resistance. Thus, there is an urgent need for an expanded set of novel antimalarials to generate new combination therapies. Herein, we have identified a 1,2,4-triazole-containing carboxamide scaffold that, through scaffold hopping efforts, resulted in a nanomolar potent deuterated picolinamide (110). The lead compound of this class (110) displays moderate aqueous solubility (13.4 μM) and metabolic stability (CLintapp HLM 17.3 μL/min/mg) in vitro, as well as moderate oral bioavailability (%F 16.2) in invivo pharmacokinetic studies. Compound 110 also displayed activity against various P. falciparum isolates with different genetic backgrounds and a slow-to-moderate rate of killing (average parasite reduction ratio 2.4), making the series appealing for further development.
Alicia Wagner, Roger Trombley, Maris Podgurski, Anthony A Ruberto, Meng Cui, Caitlin A Cooper, William E Long, Gia-Bao Nguyen, Adriana A Marin, Sarah Lee Mai, Franco Lombardo, Steven P Maher, Dennis E Kyle, Roman Manetsch.Eur J Med Chem. 2025 Mar 28:291:117572. doi: 10.1016/j.ejmech.2025.117572.
Plasmodium parasites undergo development and replication within hepatocytes before infecting erythrocytes and initiating clinical malaria. Although type I interferons (IFNs) are known to hinder Plasmodium infection within the liver, the underlying mechanisms remain unclear. Here, we describe two IFN-I-driven hepatocyte antimicrobial programs controlling liver-stage malaria. First, oxidative defense by NADPH oxidases 2 and 4 triggers a pathway of lysosomal fusion with the parasitophorous vacuole (PV) to help clear Plasmodium. Second, guanylate-binding protein (GBP) 1-mediated disruption of the PV activates the caspase-1 inflammasome, inducing pyroptosis to remove infected host cells. Remarkably, both human and mouse hepatocytes enlist these cell-autonomous immune programs to eliminate Plasmodium, with their pharmacologic or genetic inhibition leading to profound malarial susceptibility in vivo. In addition to identifying IFN-I-mediated cell-autonomous immune circuits controlling Plasmodium infection in the hepatocytes, our study also extends the understanding of how non-immune cells are integral to protective immunity against malaria.
Camila Marques-da-Silva, Clyde Schmidt-Silva, Carson Bowers, Nana Appiah Essel Charles-Chess, Cristina Samuel, Justine C Shiau, Eui-Soon Park, Zhongyu Yuan, Bae-Hoon Kim, Dennis E Kyle, John T Harty, John D MacMicking, Samarchith P Kurup. Cell Host Microbe. 2025 Mar 25:S1931-3128(25)00091-5. doi: 10.1016/j.chom.2025.03.008.
Assistant Professor Chet Joyner is the featured guest on the People, Parasites, and Plagues podcast. Listen as they discuss his research with Plasmodium vivax and the curious nature of its dormant liver stage.
PfFBXO1 localization by immunofluorescence stained with anti-V5 (PfFBXO1)
Plasmodium species replicate via schizogony, which involves asynchronous nuclear divisions followed by semi-synchronous segmentation and cytokinesis. Successful segmentation requires a double-membranous structure known as the inner membrane complex (IMC). Here we demonstrate that PfFBXO1 (PF3D7_0619700) is critical for both asexual segmentation and gametocyte maturation. In Toxoplasma gondii, the FBXO1 homolog, TgFBXO1, is essential for the development of the daughter cell scaffold and a component of the daughter cell IMC. We demonstrate PfFBXO1 forming a similar IMC initiation scaffold near the apical region of developing merozoites and unilaterally positioned in gametocytes of P. falciparum. While PfFBXO1 initially localizes to the apical region of dividing parasites, it displays an IMC-like localization as segmentation progresses. Similarly, PfFBXO1 localizes to the IMC region in gametocytes. Following inducible knockout of PfFBXO1, parasites undergo abnormal segmentation and karyokinesis, generating inviable daughters. PfFBXO1-deficient gametocytes are abnormally shaped and fail to fully mature. Proteomic analysis identified PfSKP1 as one of PfBXO1’s stable interacting partners, while other major proteins included multiple IMC pellicle and membrane proteins. We hypothesize that PfFBXO1 is necessary for IMC biogenesis, chromosomal maintenance, vesicular transport, and ubiquitin-mediated translational regulation of proteins in both sexual and asexual stages of P. falciparum.
Optimization of conditions for a luciferase endpoint with in-house reagents (FLAR).
Background: Malaria, a disease caused by parasites of the genus Plasmodium, continues to impact many regions globally. The rise in resistance to artemisinin-based anti-malarial drugs highlights the need for new treatments. Ideally, new anti-malarials will kill the asymptomatic liver stages as well as the symptomatic blood stages. While blood stage screening assays are routine and efficient, liver stage screening assays are more complex and costly. To decrease the cost of liver stage screening, a previously reported luciferase detection protocol requiring only common laboratory reagents was adapted for testing against luciferase-expressing Plasmodium berghei liver stage parasites.
Methods: After optimizing cell lysis conditions, the concentration of reagents, and the density of host hepatocytes (HepG2), the protocol was validated with 28 legacy anti-malarials to show this simple protocol produces a stable signal useful for obtaining quality small molecule potency data similar to that obtained from a high content imaging endpoint. The protocol was then used to screen the Global Health Priority Box (GHPB) and confirm the potency of hits in dose-response assays. Selectivity was determined using a galactose-based, 72 h HepG2 assay to avoid missing mitochondrial-toxic compounds due to the Crabtree effect. Receiver-operator characteristic plots were used to retroactively characterize the screens’ predictive value.
Results: Optimal luciferase signal was achieved using a lower HepG2 seed density (5 × 103 cells/well of a 384-well microtitre plate) compared to many previously reported luciferase-based screens. While producing lower signal compared to a commercial alternative, this luciferase detection method was found much more stable, with a > 3 h half-life, and robust enough for producing dose-response plots with as few as 500 sporozoites/well. A screen of the GHPB resulted in 9 hits with selective activity against P. berghei liver schizonts, including MMV674132 which exhibited 30.2 nM potency. Retrospective analyses show excellent predictive value for both anti-malarial activity and cytotoxicity.
Conclusions: This method is suitable for high-throughput screening at a cost nearly 20-fold less than using commercial luciferase detection kits, thereby enabling larger liver stage anti-malarial screens and hit optimization make-test cycles. Further optimization of the hits detected using this protocol is ongoing.
Gia-Bao Nguyen, Caitlin A Cooper, Olivia McWhorter, Ritu Sharma, Anne Elliot, Anthony Ruberto, Rafael Freitas, Ashutosh K Pathak, Dennis E Kyle, Steven P Maher. Malar J. 2024 Nov 23;23(1):357. doi: 10.1186/s12936-024-05155-y.
Vasant Muralidharan, associate professor in Franklin College‘s Department of Cellular Biology and member of CTEGD, is the featured guest in this episode of People, Parasites, and Plagues. He discusses his work with the Plasmodium parasite and his personal experience with malaria.
The development of parasite resistance to both artemisinin derivatives and their partner drugs jeopardizes the effectiveness of the artemisinin combination therapy. Thus, the discovery of new antimalarial drugs, with new mechanisms of action, is urgently needed. We recently disclosed that β-carboline 1a was orally efficacious in Plasmodium berghei-infected mice and that it showed low cross-resistance between susceptible Plasmodium falciparum and four different drug-resistant strains. In this report, we describe the synthesis and in vitro antimalarial evaluation of 91 new derivatives of 1a. The asexual blood stage growth inhibition data show a clear preference for a 3,4-dihalogenated, 3,5-dihalogenated, 3,4,5-trichloro-, or 4-trifluoromethyphenyl ring at the C1-position. The most potent compound, 3,4,5-trichlorophenyl-substituted 42a, is twice as potent as 1a. Six potent analogues were assessed for their drug-like properties, and four of these were subjected to in vitro barcoded cross-resistance profiling. Compounds 1a, 1m, 42a, and 42m showed no cross-resistance to 32 resistance mutations on the Dd2 genetic background and 10 resistance mutations on the 3D7 genetic background. These data suggest that compounds in this scaffold possess a novel mechanism of antimalarial action.
Jopaul Mathew, Bo Zhou, Reagan S Haney, Kevin A Kunz, Leticia S Do Amaral, Rudraneel Roy Chowdhury, Joshua H Butler, Haibo Li, Amarraj J Chakraborty, Anika Tabassum, Emily K Bremers, Emilio F Merino, Rachael Coyle, Marcus C S Lee, Delphine Baud, Stephen Brand, Maxim Totrov, Maria Belen Cassera, Paul R Carlier. ACS Infect Dis. 2024 Oct 28. doi: 10.1021/acsinfecdis.4c00653.
Vasant Muralidharan and his research group at the University of Georgia’s Center for Tropical and Emerging Global Diseases have uncovered the role of an essential protein in Plasmodium falciparum, the parasite that causes the deadliest form of malaria. The discovery offers new insights for vaccine and drug development.
The parasite that causes malaria was discovered more than 125 years ago, but much is still unknown about this complex, single-celled organism. Researchers in the University of Georgia’s Center for Tropical and Emerging Global Diseases, however, have uncovered the role of one of the parasite’s essential proteins, offering new insights for vaccine and drug development.
Plasmodium falciparum causes the deadliest form of malaria, a disease the World Health Organization estimates killed more than 600,000 people worldwide died in 2022. A large majority of those deaths were children under the age of 5.
Historically, the parasite has been difficult to study due to its complex lifecycle, which includes three stages. One occurs in the mosquito, while the liver and blood stages take place in humans. The blood stage is when the infected person exhibits symptoms of malaria.
In the blood stage, the parasite invades red blood cells (RBCs) where they replicate and can be transmitted to the mosquito. The receptor-ligand complexes that enable RBC invasion have been well-studied and it is one of the targets of anti-malarial vaccines currently in clinical trials. But questions still remain.
“How does the parasite know it has encountered a red blood cell?” asked Vasant Muralidharan, associate professor in Franklin College’s Department of Cellular Biology and leader of the Muralidharan Research Group, where the study took place.
Interested, the team took a closer look at a protein called RON11, which is sent to a pair of unique club-shaped secretory organelles known as the rhoptry (Greek for club) that houses proteins needed to invade the RBC.
Click play to listen to an excerpt of Vasant Muralidharan discussing the cellular mechanics of malaria infection.
“When we knocked out this protein, we found that the parasite could do everything it usually does – create a putative pore in the membrane of the RBC, send proteins needed for parasite invasion through this putative opening into the RBC – but the parasite itself cannot enter the red blood cell,” Muralidharan explained. “If a parasite cannot enter the red blood cell, the life cycle is interrupted and the parasite dies.”
And then things got really interesting.
“We found that the parasites lacking RON11 were only producing half the rhoptry proteins, which are used in invasion,” Muralidharan said.
While it is known that Plasmodium parasites have two rhoptry organelles, they are so teeny-tiny they have been relatively understudied due to a lack of proper tools. However, new tools and techniques are emerging. David Anaguano, a cellular biology graduate student who led the study, traveled on a Daniel G. Colley Training in Parasitology fellowship to the Absalon Laboratory at Indiana University School of Medicine to learn a new tool known as Ultrastructure Expansion Microscopy.
Vasant Muralidharan is an associate professor in Franklin College’s Department of Cellular Biology. (Photo by Lauren Corcino)
“Electron microscopy is labor intensive, and since it uses thin slices of the parasite you are never sure if what you’re looking for really isn’t there or just not in the slice of the sample you have,” Muralidharan said. “Expansion microscopy is like using light microscopy but with a special gel to expand the cell proportionately in all directions. Thus, you don’t get the distortion you would with just an enlarged cell and you can image the entire infected cell in all dimensions. It has been a real game changer.”
As reported in the PLoS Biology paper, the Muralidharan group generated for the first time a Plasmodium cell with only one rhoptry organelle when they removed RON11 from malaria parasites.
“It’s not unusual for an organism to have a backup copy, but we can see that the parasite can create the first rhoptry just fine – without defect – but the second one that should form during the end of the replication cycle never forms,” Muralidharan said. “Why is that?”
As it appears that this second rhoptry is needed for RBC invasion, understanding the mechanisms that control its development could open up new targets for vaccine and drug treatment discovery as well as answering crucial questions like whether the two rhoptries are identical.
“This has been a long unanswered question,” Muralidharan said. “Now with this RON11 knockout parasite that doesn’t form a second rhoptry, we have the tools to answer it.”
Fig 1. RON11 is essential for intraerythrocytic growth.
Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.
David Anaguano, Opeoluwa Adewale-Fasoro, Grace W Vick, Sean Yanik, James Blauwkamp, Manuel A Fierro, Sabrina Absalon, Prakash Srinivasan, Vasant Muralidharan. PLoS Biol. 2024 Sep 18;22(9):e3002801. doi: 10.1371/journal.pbio.3002801. eCollection 2024 Sep.
A systems biology approach for antimalarial drug discovery.
We report the discovery of MED6-189, an analog of the kalihinol family of isocyanoterpene natural products that is effective against drug-sensitive and drug-resistant Plasmodium falciparum strains, blocking both asexual replication and sexual differentiation. In vivo studies using a humanized mouse model of malaria confirm strong efficacy of the compound in animals with no apparent hemolytic activity or toxicity. Complementary chemical, molecular, and genomics analyses revealed that MED6-189 targets the parasite apicoplast and acts by inhibiting lipid biogenesis and cellular trafficking. Genetic analyses revealed that a mutation in PfSec13, which encodes a component of the parasite secretory machinery, reduced susceptibility to the drug. Its high potency, excellent therapeutic profile, and distinctive mode of action make MED6-189 an excellent addition to the antimalarial drug pipeline.
Z Chahine, S Abel, T Hollin, G L Barnes, J H Chung, M E Daub, I Renard, J Y Choi, P Vydyam, A Pal, M Alba-Argomaniz, C A S Banks, J Kirkwood, A Saraf, I Camino, P Castaneda, M C Cuevas, J De Mercado-Arnanz, E Fernandez-Alvaro, A Garcia-Perez, N Ibarz, S Viera-Morilla, J Prudhomme, C J Joyner, A K Bei, L Florens, C Ben Mamoun, C D Vanderwal, K G Le Roch. Science. 2024 Sep 27;385(6716):eadm7966. doi: 10.1126/science.adm7966.
