Structure and amino acid sequence of the cyclic pentapeptide, sheptide A (1)
As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey’s method. In an in vitro assay for antimalarial potency, 1 displayed a pEC50 value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC50 value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.
Robert A Shepherd, Cody E Earp, Kristof B Cank, Huzefa A Raja, Joanna Burdette, Steven P Maher, Adriana A Marin, Anthony A Ruberto, Sarah Lee Mai, Blaise A Darveaux, Dennis E Kyle, Cedric J Pearce, Nicholas H Oberlies. J Antibiot (Tokyo). 2023 Sep 20. doi: 10.1038/s41429-023-00655-6.
The Chagas field has gone >50 years without tangible progress toward new therapies. My colleagues and I have recently reported on a benzoxaborole compound that achieves consistent parasitological cure in experimentally infected mice and in naturally infected non-human primates (NHPs). While these results do not assure success in human clinical trials, they significantly de-risk this process and form a strong justification for such trials. Highly effective drug discovery depends on a solid understanding of host and parasite biology and excellent knowledge in designing and validating chemical entities. This opinion piece seeks to provide perspectives on the process that led to the discovery of AN15368, with the hope that this will facilitate the discovery of additional clinical candidates for Chagas disease.
Acanthamoeba species, Naegleria fowleri, and Balamuthia mandrillaris are opportunistic pathogens that cause a range of brain, skin, eye, and disseminated diseases in humans and animals. These pathogenic free-living amoebae (pFLA) are commonly misdiagnosed and have sub-optimal treatment regimens which contribute to the extremely high mortality rates (>90%) when they infect the central nervous system. To address the unmet medical need for effective therapeutics, we screened kinase inhibitor chemotypes against three pFLA using phenotypic drug assays involving CellTiter-Glo 2.0. Herein, we report the activity of the compounds against the trophozoite stage of each of the three amoebae, ranging from nanomolar to low micromolar potency. The most potent compounds that were identified from this screening effort were: 2d (A. castellanii EC50: 0.92 ± 0.3 μM; and N. fowleri EC50: 0.43 ± 0.13 μM), 1c and 2b (N. fowleri EC50s: <0.63 μM, and 0.3 ± 0.21 μM), and 4b and 7b (B. mandrillaris EC50s: 1.0 ± 0.12 μM, and 1.4 ± 0.17 μM, respectively). With several of these pharmacophores already possessing blood-brain barrier (BBB) permeability properties, or are predicted to penetrate the BBB, these hits present novel starting points for optimization as future treatments for pFLA-caused diseases.
Lori Ferrins, Melissa J Buskes, Madison M Kapteyn, Hannah N Engels, Suzanne E Enos, Chenyang Lu, Dana M Klug, Baljinder Singh, Antonio Quotadamo, Kelly Bachovchin, Westley F Tear, Andrew E Spaulding, Katherine C Forbes, Seema Bag, Mitch Rivers, Catherine LeBlanc, Erin Burchfield, Jeremy R Armand, Rosario Diaz-Gonzalez, Gloria Ceballos-Perez, Raquel García-Hernández, Guiomar Pérez-Moreno, Cristina Bosch-Navarrete, Luis Miguel Ruiz-Pérez, Francisco Gamarro, Dolores González-Pacanowska, Miguel Navarro, Kojo Mensa-Wilmot, Michael P Pollastri, Dennis E Kyle, Christopher A Rice. Front Microbiol. 2023 May 10;14:1149145. doi: 10.3389/fmicb.2023.1149145. eCollection 2023.
Cripowellins from Crinum erubescens are known pesticidal and have potent antiplasmodial activity. To gain mechanistic insights to this class of natural products, studies to determine the timing of action of cripowellins within the asexual intraerythrocytic cycle of Plasmodium falciparum were performed and led to the observation that this class of natural products induced reversible cytostasis in the ring stage within the first 24 h of treatment. The transcriptional program necessary for P. falciparum to progress through the asexual intraerythrocytic life cycle is well characterized. Whole transcriptome abundance analysis showed that cripowellin B “pauses” the transcriptional program necessary to progress through the intraerythrocytic life cycle coinciding with the lack of morphological progression of drug treated parasites. In addition, cripowellin B-treated parasites re-enter transcriptional progression after treatment was removed. This study highlights the use of cripowellins as chemical probes to reveal new aspects of cell cycle progression of the asexual ring stage of P. falciparum which could be leveraged for the generation of future antimalarial therapeutics.
Joshua H Butler, Heather J Painter, Emily K Bremers, Priscilla Krai, Manuel Llinás, Maria B Cassera. Molecules. 2023 Mar 13;28(6):2600. doi: 10.3390/molecules28062600.
Seventeen new cephalotane-type diterpenoids, fortalides A-Q (1-17), along with five known analogues, were isolated from the seeds of Cephalotaxus fortunei var. alpina. Their structures were determined by extensive spectroscopic methods, as well as electronic circular dichroism (ECD) and X-ray crystallographic data analyses. Some isolates exhibited unusual structural features that were first found in cephalotane-type diterpenoids, such as the occurrence of the 7-oxabicyclo[4.1.1]octane moiety in 14 and 15 and the cis-arrangement of 3-OH and Me-19 in 9. Besides, the antiplasmodial activity of these compounds was evaluated in this study.
Zhan-Peng Ge, Bin Zhou, Flavia M Zimbres, Reagan S Haney, Qun-Fang Liu, Yan Wu, Maria B Cassera, Jin-Xin Zhao, Jian-Min Yue. Org Biomol Chem. 2022 Nov 4. doi: 10.1039/d2ob01748b
The skeletal muscle of a mouse infected with Trypanosoma cruzi is shown under a microscope. (Submitted by Fernando Sanchez)
The condition affects tens of millions across the Americas but lacks effective treatments
Researchers from the University of Georgia have discovered a potential treatment for Chagas disease, marking the first medication with promise to successfully and safely target the parasitic infection in more than 50 years.
Human clinical trials of the drug, an antiparasitic compound known as AN15368, will hopefully begin in the next few years.
Distinguished Research Professor Dr. Rick Tarleton of the Center for Tropical and Emerging Global Diseases at the Paul D. Coverdell Center for Biomedical and Health Sciences on Thursday, May 8, 2008. Dr. Tarleton is researching the effects of drug treatments on Chagas’ disease.
“I’m very optimistic,” said Rick Tarleton, corresponding author of the study and a UGA Athletic Association Distinguished Professor in the Franklin College of Arts and Sciences and member of the Center for Tropical and Emerging Global Diseases. “I think it has a really strong chance of being a real solution, not just a stand-in for something that works better than the drugs we currently have.”
The new drug works by targeting the parasite that causes the disease, Trypanosoma cruzi, also known as T. cruzi.
Nearly all people infected with the parasite experience flu-like symptoms such as fever, headaches and vomiting. However, after their immune response kicks in, their symptoms may subside.
But for 30% to 40% of patients, the infection can result in severe heart damage that can be both debilitating and life-threatening.
New drug is 100% effective in eliminating T. cruzi
Published in Nature Microbiology, the study found the new medication was 100% effective in curing mice, as well as non-human primates that were naturally infected by the parasite at a research facility in Texas. The animals also experienced no significant side effects from exposure to the drug.
Over the past several decades, previous treatment candidates went straight from experimental infections in mice to human clinical trials, where they failed to cure the infection. The new drug’s efficacy in non-human primates bodes well for how it will perform in humans.
“We’ve got something that is as close to effective as it can be in what is as close to a human as it could be, and there aren’t any side effects. That really de-risks it by a lot going into humans,” Tarleton said. “It doesn’t make it fail-safe, but it moves it much further along.”
Current medications to treat T. cruzi infection not ideal
T. cruzi is carried by blood-sucking insects known as kissing bugs. The insects can be found throughout North, Central and South America.
In addition to a nasty bite, the creatures carry the T. cruzi parasite, which is transmitted through their fecal matter. Victims can become infected when they unknowingly rub the insect’s feces into their eyes, nose or an open wound.
The infection may also be transmitted through organ transplants, from a pregnant person to their fetus or through contaminated food. However, infections from these pathways are less common.
The go-to medications used to treat Chagas aren’t terrible, Tarleton said, but they’re not ideal. They can pack some serious side effects and they’re not reliably effective, but they’re currently the only treatment option.
Patients also have to take the drugs for two months. And even the common but mild side effects like headache or nausea get old after a few weeks. As a result, about one in five people being treated for the disease stop taking their medications before they have a chance to cure the infection.
“Plus they have variable efficacy, and it’s not predictable,” Tarleton said. “I think most physicians in Latin America have to say, ‘We have a drug. It’s going to make you feel bad, and two months later after we finish it, we’re not really going to be able to tell you if it worked or not.’
“It’s really not a good inducement to take the medication.”
Chagas disease common in Latin American countries
Tens of millions of people across the Americas are infected with the parasite that causes Chagas disease. But it doesn’t get much media attention.
It’s most common in Latin American countries, particularly in low-income areas where housing isn’t ideal. Some of the countries with the highest rates of the disease include Bolivia, Venezuela, Argentina, Chile, Mexico and Brazil.
In homes with thatched roofs, mud walls or inadequate protection from the elements, kissing bugs thrive, making infection more likely.
Chagas disease poses significant risk to pets
The Centers for Disease Control and Prevention estimates around 300,000 people infected with the parasite currently live in the U.S. But because the condition isn’t a huge threat in places with good housing options, Chagas disease treatment and prevention doesn’t get much research funding.
There is growing concern about the T. cruzi infection rate among outdoor pets in the U.S., however. Working dogs and other pets that spend extended periods of time outside are contracting the parasite at an alarming rate.
“There are areas where the infection rates are 20% to 30% new infections per year,” Tarleton said. “Those tend to be severe infections where the dogs either die or develop a disease that makes them unable to work.”
Tarleton hopes to partner with veterinary pharmaceutical companies in the future to create a drug to treat the infection in pets as a means of funding diagnostics and medication purchases in Latin America.
For the present study, Tarleton partnered with colleagues at Anacor Pharmaceuticals, Texas A&M University, the University of Texas, the University of Kansas and Pfizer. Angel Padilla, Wei Wang, Dylan Orr, Brooke White, Arlene George and Huifeng Shen from UGA’s Center for Tropical and Emerging Global Diseases and the Department of Cellular Biology are co-authors on the paper.
Rick Tarleton and colleagues recently published their new study, “Discovery of an orally active benzoxaborole prodrug effective in the treatment of Chagas disease in non-human primates” in Nature Microbiology. Check out these news stories about the study.
Behind the paper: New Hope for Treatment of a Very Neglected, Neglected Tropical Disease (Nature Microbiology)
Possible new treatment identified for neglected tropical disease (Science.org)
Researchers discover potential treatment for Chagas disease (Mirage News)
Researchers discover potential treatment for Chagas disease (Science Daily)
Researchers discover potential treatment for Chagas disease (Medical Xpress)
Chagas disease potential treatment: ‘I think it has a really strong chance of being a real solution’ (Outbreak News Today)
Promising New Drug May Effectively Treat Chagas Disease (Technology Networks)
Researchers discover potential treatment for Chagas disease (Newswise)
Steve Maher, assistant research scientist in the Center for Tropical Emerging and Global Diseases, leads a team of researchers who have implemented a new screening tool to determine if a drug candidate kills hypnozoites, the cause of malaria relapses. (Photo by Donna Huber)
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.
Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.
Steven P. Maher, Amélie Vantaux, Victor Chaumeau, Adeline C. Y. Chua, Caitlin A. Cooper, Chiara Andolina, Julie Péneau, Mélanie Rouillier, Zaira Rizopoulos, Sivchheng Phal, Eakpor Piv, Chantrea Vong, Sreyvouch Phen, Chansophea Chhin, Baura Tat, Sivkeng Ouk, Bros Doeurk, Saorin Kim, Sangrawee Suriyakan, Praphan Kittiphanakun, Nana Akua Awuku, Amy J. Conway, Rays H. Y. Jiang, Bruce Russell, Pablo Bifani, Brice Campo, François Nosten, Benoît Witkowski & Dennis E. Kyle. Sci Rep 11, 19905 (2021). https://doi.org/10.1038/s41598-021-99152-9
CTEGD member Chet Joyner and CTEGD director Dennis Kyle receive a grant from the Bill & Melinda Gates Foundation for malaria drug development and testing
Two UGA researchers are working to make it easier to develop effective treatments for malaria, a disease that sickens millions worldwide and kills hundreds of thousands each year.
In tropical climates around the globe, malaria poses a grave risk to already vulnerable populations. In 2019, the World Health Organization estimated that there were 229 million clinical cases of malaria worldwide and 409,000 deaths, usually in children below the age of five.
Currently, developing and testing drugs for malaria requires scientists to work in areas where the disease is prevalent or to work with expensive, hard-to-source equipment. Chester Joyner, an Assistant Professor in the Center for Vaccines and Immunology, and Dennis Kyle, Professor of Infectious Diseases and Cellular Biology, are working to reduce those barriers to malaria drug testing and development.
Joyner and Kyle aim to establish systems that rely on equipment most researchers can obtain: a petri dish. If successful, Joyner says this new culture system will reduce costs and be distributed more easily to advance drug and vaccine research. The University of Georgia College of Veterinary Medicine received a grant for malaria drug development and testing from the Bill & Melinda Gates Foundation.
Worldwide, there are many malaria-causing parasites that result in varying degrees of illness. Joyner and Kyle’s research focuses on defeating one of the most challenging: Plasmodium vivax. Unlike many other malaria parasites, P. vivax can lie dormant in the livers of its hosts—allowing the infected to travel abroad completely unaware that they’re carrying a potentially deadly passenger.
“Most infections with P. vivax are not due to new infections,” says Joyner. “These infections come from this parasite activating and potentially causing disease and sustaining transmission.”
Malaria disproportionately affects the poorest communities in the world, creating a cycle of disease and poverty that current treatments have improved but been unable to stop. However, treating the dormant forms of P. vivax has been particularly challenging because they can cause more harm than good in at-risk populations like pregnant women and people with certain blood conditions.
“We want researchers to have access to technologies to study P. vivax and develop new approaches to control and eliminate this parasite,” Joyner explains.
