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Tag: Dennis Kyle

Battling Malaria

UGA is developing new drugs to fight a lethal parasite. Dennis Kyle discusses what his lab is doing to fight malaria in this brief (2:30) video.

 

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In vitro screening of the open source MMV Malaria and Pathogen Boxes to discover novel compounds with activity against Balamuthia mandrillaris

Balamuthia mandrillaris, is an under reported pathogenic free-living amoeba that causes Balamuthia amoebic encephalitis (BAE) and cutaneous skin infections. Although cutaneous infections are not typically lethal, BAE with or without cutaneous involvement usually is fatal. This is due to lack of drugs that are efficacious and that can cross the blood-brain barrier. We aimed to discover new leads for drug discovery by screening the open source MMV Malaria and MMV Pathogen boxes (800 compounds total). From an initial single point screen at 1 and 10 μM, we identified 54 hits that significantly inhibited the growth of B. mandrillaris in vitro. Hits were re-confirmed in quantitative dose response assays and 23 compounds (42.6 %) were confirmed with activity greater than miltefosine, the current standard of care.

Christopher A. RiceLuis Fernando Lares-JiménezFernando Lares-VillaDennis E. Kyle. Antimicrob Agents Chemother. 2020 Feb 18. pii: AAC.02233-19. doi: 10.1128/AAC.02233-19

An adaptable soft-mold embossing process for fabricating optically-accessible, microfeature-based culture systems and application toward liver stage antimalarial compound testing

Advanced cell culture methods for modeling organ-level structure have been demonstrated to replicate in vivo conditions more accurately than traditional in vitro cell culture. Given that the liver is particularly important to human health, several advanced culture methods have been developed to experiment with liver disease states, including infection with Plasmodium parasites, the causative agent of malaria. These models have demonstrated that intrahepatic parasites require functionally stable hepatocytes to thrive and robust characterization of the parasite populations’ response to investigational therapies is dependent on high-content and high-resolution imaging (HC/RI). We previously reported abiotic confinement extends the functional longevity of primary hepatocytes in a microfluidic platform and set out to instill confinement in a microtiter plate platform while maintaining optical accessibility for HC/RI; with an end-goal of producing an improved P. vivax liver stage culture model. We developed a novel fabrication process in which a PDMS soft mold embosses hepatocyte-confining microfeatures into polystyrene, resulting in microfeature-based hepatocyte confinement (μHEP) slides and plates. Our process was optimized to form both microfeatures and culture wells in a single embossing step, resulting in a 100 μm-thick bottom ideal for HC/RI, and was found inexpensively amendable to microfeature design changes. Microfeatures improved intrahepatic parasite infection rates and μHEP systems were used to reconfirm the activity of reference antimalarials in phenotypic dose-response assays. RNAseq of hepatocytes in μHEP systems demonstrated microfeatures sustain hepatic differentiation and function, suggesting broader utility for preclinical hepatic assays; while our tailorable embossing process could be repurposed for developing additional organ models.

Steven P. Maher, Amy J. Conway, Alison Roth, Swamy R. Adapa, Phillip Cualing, Chiara Andolina, James Hsiao, Jessica Turgeon, Victor Chaumeau, Myles Johnson, Chris Palmiotti, Naresh Singh, Samantha J. Barnes, Raahil Patel, Virginia Van Grod, Robert Carter, H.-C. Steve Sun, Jetsumon Sattabongkot, Brice Campo, François Nosten, Wajeeh M. Saadi, John H. Adams, Rays H. Y. Jiang, and Dennis E. Kyle. Lab Chip. 2020 Feb 14. doi: 10.1039/c9lc00921c

Optimal 10-Aminoartemisinins With Potent Transmission-Blocking Capabilities for New Artemisinin Combination Therapies–Activities Against Blood Stage P. falciparum Including PfKI3 C580Y Mutants and Liver Stage P. berghei Parasites

We have demonstrated previously that amino-artemisinins including artemiside and artemisone in which an amino group replaces the oxygen-bearing substituents attached to C-10 of the current clinical artemisinin derivatives dihydroartemisinin (DHA), artemether and artesunate, display potent activities in vitro against the asexual blood stages of Plasmodium falciparum (Pf). In particular, the compounds are active against late blood stage Pf gametocytes, and are strongly synergistic in combination with the redox active drug methylene blue. In order to fortify the eventual selection of optimum amino-artemisinins for development into new triple combination therapies also active against artemisinin-resistant Pf mutants, we have prepared new amino-artemisinins based on the easily accessible and inexpensive DHA-piperazine. The latter was converted into alkyl- and aryl sulfonamides, ureas and amides. These derivatives were screened together with the comparator drugs DHA and the hitherto most active amino-artemisinins artemiside and artemisone against asexual and sexual blood stages of Pf and liver stage P. berghei (Pb) sporozoites. Several of the new amino-artemisinins bearing aryl-urea and -amide groups are potently active against both asexual, and late blood stage gametocytes (IC50 0.4-1.0 nM). Although the activities are superior to those of artemiside (IC50 1.5 nM) and artemisone (IC50 42.4 nM), the latter are more active against the liver stage Pb sporozoites (IC50 artemisone 28 nM). In addition, early results indicate these compounds tend not to display reduced susceptibility against parasites bearing the Pf Kelch 13 propeller domain C580Y mutation characteristic of artemisinin-resistant Pf. Thus, the advent of the amino-artemisinins including artemiside and artemisone will enable the development of new combination therapies that by virtue of the amino-artemisinin component itself will possess intrinsic transmission-blocking capabilities and may be effective against artemisinin resistant falciparum malaria.

Ho Ning Wong, Vivian Padín-Irizarry, Mariëtte E. van der Watt, Janette Reader, Wilna Liebenberg, Lubbe Wiesner, Peter Smith, Korina Eribez, Elizabeth A. Winzeler, Dennis E. Kyle, Lyn-Marie Birkholtz, Dina Coertzen, and Richard K. Haynes. Front Chem. 2020 Jan 10;7:901. doi: 10.3389/fchem.2019.00901. eCollection 2019.

Conservation of Ancient Genetic Pathways for Intracellular Persistence Among Animal Pathogenic Bordetellae

Animal and human pathogens of the genus Bordetella are not commonly considered to be intracellular pathogens, although members of the closely related classical bordetellae are known to enter and persist within macrophages in vitro and have anecdotally been reported to be intracellular in clinical samples. B. bronchiseptica, the species closest to the ancestral lineage of the classical bordetellae, infects a wide range of mammals but is known to have an alternate life cycle, persisting, replicating and disseminating with amoeba. These observations give rise to the hypothesis that the ability for intracellular survival has an ancestral origin and is common among animal-pathogenic and environmental Bordetella species. Here we analyzed the survival of B. bronchiseptica and defined its transcriptional response to internalization by murine macrophage-like cell line RAW 264.7. Although the majority of the bacteria were killed and digested by the macrophages, a consistent fraction survived and persisted inside the phagocytes. Internalization prompted the activation of a prominent stress response characterized by upregulation of genes involved in DNA repair, oxidative stress response, pH homeostasis, chaperone functions, and activation of specific metabolic pathways. Cross species genome comparisons revealed that most of these upregulated genes are highly conserved among both the classical and non-classical Bordetella species. The diverse Bordetella species also shared the ability to survive inside RAW 264.7 cells, with the single exception being the bird pathogen B. avium, which has lost several of those genes. Knock-out mutations in genes expressed intracellularly resulted in decreased persistence inside the phagocytic cells, emphasizing the importance of these genes in this environment. These data show that the ability to persist inside macrophage-like RAW 264.7 cells is shared among nearly all Bordetella species, suggesting that resisting phagocytes may be an ancient mechanism that precedes speciation in the genus and may have facilitated the adaptation of Bordetella species from environmental bacteria to mammalian respiratory pathogens.

Israel Rivera, Bodo Linz, Kalyan K. Dewan, Longhuan Ma, Christopher A. Rice, Dennis E. Kyle and Eric T. Harvill. Front Microbiol. 2019 Dec 11;10:2839. doi: 10.3389/fmicb.2019.02839. eCollection 2019.

Protozoan persister-like cells and drug treatment failure

Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmaniaspp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as ‘persister-like cells’. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.

Michael P. Barrett, Dennis E. Kyle, L. David Sibley, Joshua B. Radke & Rick L. Tarleton. Nat Rev Microbiol. 2019 Aug 23. doi: 10.1038/s41579-019-0238-x.

Robust continuous in vitro culture of the Plasmodium cynomolgi erythrocytic stages

The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.

Chua ACY, Ong JJY, Malleret B, Suwanarusk R, Kosaisavee V, Zeeman AM, Cooper CA, Tan KSW, Zhang R, Tan BH, Abas SN, Yip A, Elliot A, Joyner CJ, Cho JS, Breyer K, Baran S, Lange A, Maher SP, Nosten F, Bodenreider C, Yeung BKS, Mazier D, Galinski MR, Dereuddre-Bosquet N, Le Grand R, Kocken CHM, Rénia L, Kyle DE, Diagana TT, Snounou G, Russell B, Bifani P. Nat Commun. 2019 Aug 12;10(1):3635. doi: 10.1038/s41467-019-11332-4.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.


Beatriz Baragaña, Barbara Forte, Ryan Choi, Stephen Nakazawa Hewitt, Juan A. Bueren-Calabuig, João Pedro Pisco, Caroline Peet, David M. Dranow, David A. Robinson, Chimed Jansen, Neil R. Norcross, Sumiti Vinayak, Mark Anderson, Carrie F. Brooks, Caitlin A. Cooper, Sebastian Damerow, Michael Delves, Karen Dowers, James Duffy, Thomas E. Edwards, Irene Hallyburton, Benjamin G. Horst, Matthew A. Hulverson, Liam Ferguson, María Belén Jiménez-Díaz, Rajiv S. Jumani, Donald D. Lorimer, Melissa S. Love, Steven Maher, Holly Matthews, Case W. McNamara, Peter Miller, Sandra O’Neill, Kayode K. Ojo, Maria Osuna-Cabello, Erika Pinto, John Post, Jennifer Riley, Matthias Rottmann, Laura M. Sanz, Paul Scullion, Arvind Sharma, Sharon M. Shepherd, Yoko Shishikura, Frederick R. C. Simeons, Erin E. Stebbins, Laste Stojanovski, Ursula Straschil, Fabio K. Tamaki, Jevgenia Tamjar, Leah S. Torrie, Amélie Vantaux, Benoît Witkowski, Sergio Wittlin, Manickam Yogavel, Fabio Zuccotto, Iñigo Angulo-Barturen, Robert Sinden, Jake Baum, Francisco-Javier Gamo, Pascal Mäser, Dennis E. Kyle, Elizabeth A. Winzeler, Peter J. Myler, Paul G. Wyatt, David Floyd, David Matthews, Amit Sharma, Boris Striepen, Christopher D. Huston, David W. Gray, Alan H. Fairlamb, Andrei V. Pisliakov, Chris Walpole, Kevin D. Read, Wesley C. Van Voorhis, and Ian H. Gilbert. 2019. PNAS, https://doi.org/10.1073/pnas.1814685116