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Tag: Dennis Kyle

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro

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

UGA researchers developing new models for malaria drug development and testing

Chet Joyner and Dennis Kyle
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.

 

This article first appeared at https://give.uga.edu/uga-researchers-developing-new-models-for-malaria-drug-development-and-testing/

EdU Incorporation To Assess Cell Proliferation and Drug Susceptibility in Naegleria fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri, and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study, we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor-linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy, and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (7-deaza-2′-deoxy-7-ethynyladenosine), an adenine analog, also was incorporated by N. fowleri but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Emma V Troth, Dennis E Kyle. Antimicrob Agents Chemother. 2021 Jun 17;65(7):e0001721. doi: 10.1128/AAC.00017-21.

Dennis Kyle Featured Guest on People, Parasites & Plagues Podcast

Dr. Dennis Kyle, director of CTEGD and professor in the departments of cellular biology and infectious diseases, is the featured guest on Episode 5 of the People, Parasites & Plagues Podcast. He talks about a deadly disease caused by Naegleria fowleri, also known as the brain-eating amoeba.

The podcast is also available at AmazoniTunesGoogleSpotifyStitcherAudible, and TuneIn

People, Parasites & Plagues is a podcast aimed at delivering information about the fascinating pathogens among us from the impressive professionals who study them.

Join hosts Dr. David Peterson and Dr. Liliana Salvador, two infectious disease researchers from the University of Georgia, as they explore the past, present, and future of science.

Tune in every other week for a new and enlightening episode as they unpack the details surrounding some of Earth’s most perplexing diseases. Look for the People, Parasites & Plagues Podcast on your favorite Podcast service!

Aminoalkoxycarbonyloxymethyl Ether Prodrugs with a pH-Triggered Release Mechanism: A Case Study Improving the Solubility, Bioavailability, and Efficacy of Antimalarial 4(1 H)-Quinolones with Single Dose Cures

Preclinical and clinical development of numerous small molecules is prevented by their poor aqueous solubility, limited absorption, and oral bioavailability. Herein, we disclose a general prodrug approach that converts promising lead compounds into aminoalkoxycarbonyloxymethyl (amino AOCOM) ether-substituted analogues that display significantly improved aqueous solubility and enhanced oral bioavailability, restoring key requirements typical for drug candidate profiles. The prodrug is completely independent of biotransformations and animal-independent because it becomes an active compound via a pH-triggered intramolecular cyclization-elimination reaction. As a proof-of-concept, the utility of this novel amino AOCOM ether prodrug approach was demonstrated on an antimalarial compound series representing a variety of antimalarial 4(1H)-quinolones, which entered and failed preclinical development over the last decade. With the amino AOCOM ether prodrug moiety, the 3-aryl-4(1H)-quinolone preclinical candidate was shown to provide single-dose cures in a rodent malaria model at an oral dose of 3 mg/kg, without the use of an advanced formulation technique.

Andrii Monastyrskyi, Fabian Brockmeyer, Alexis N LaCrue, Yingzhao Zhao, Steven P Maher, Jordany R Maignan, Vivian Padin-Irizarry, Yana I Sakhno, Prakash T Parvatkar, Ami H Asakawa, Lili Huang, Debora Casandra, Sherwin Mashkouri, Dennis E Kyle, Roman Manetsch. J Med Chem. 2021 May 12. doi: 10.1021/acs.jmedchem.0c01104.

EdU incorporation to assess cell proliferation and drug susceptibility in Naegleria fowleri

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro EdA (5’ethynyl-2′-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Emma V. TrothDennis E. Kyle

Synthesis of Mono- and Bisperoxide-Bridged Artemisinin Dimers to Elucidate the Contribution of Dimerization to Antimalarial Activity

During the past decade, artemisinin as an antimalarial has been in the spotlight, in part due to the Nobel Prize in Physiology or Medicine awarded to Tu Youyou. While many studies have been completed detailing the significant increase in activity resulting from the dimerization of natural product artemisinin, activity increases unaccounted for by the peroxide bridge have yet to be researched. Here we outline the synthesis and testing for antimalarial activity of artemisinin dimers in which the peroxide bridge in one-half of the dimer is reduced, resulting in a dimer with one active and one deactivated artemisinin moiety.

Cynthia L Lichorowic, Yingzhao Zhao, Steven P Maher, Vivian Padín-Irizarry, Victoria C Mendiola, Sagan T de Castro, Jacob A Worden, Debora Casandra, Dennis E Kyle, Roman Manetsch. ACS Infect Dis. 2021 Apr 1. doi: 10.1021/acsinfecdis.1c00066

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Logan Tillery, Kayleigh Barrett, Jenna Goldstein, Jared W Lassner, Bram Osterhout, Nathan L Tran, Lily Xu, Ryan M Young, Justin Craig, Ian Chun, David M Dranow, Jan Abendroth, Silvia L Delker, Douglas R Davies, Stephen J Mayclin, Brandy Calhoun, Madison J Bolejack, Bart Staker, Sandhya Subramanian, Isabelle Phan, Donald D Lorimer, Peter J Myler, Thomas E Edwards, Dennis E Kyle, Christopher A Rice, James C Morris, James W Leahy, Roman Manetsch, Lynn K Barrett, Craig L Smith, Wesley C Van Voorhis (2021) Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba. PLoS ONE 16(3): e0241738. https://doi.org/10.1371/journal.pone.0241738

Discovery of repurposing drug candidates for the treatment of diseases caused by pathogenic free-living amoebae

Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 μM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.

Christopher A Rice, Beatrice L Colon, Emily Chen, Mitchell V Hull, Dennis E Kyle. PLoS Negl Trop Dis. 2020 Sep 24;14(9):e0008353. doi: 10.1371/journal.pntd.0008353.