Donna Huber, Author at Center for Tropical and Emerging Global Diseases

Design, Synthesis, and Evaluation of Novel Anti-Trypanosomal Compounds

Donna Huber on April 17, 2020

Human African trypanosomiasis (HAT) is a deadly neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. During the course of screening a collection of diverse nitrogenous heterocycles, we discovered two novel compounds that contain the tetracyclic core of the Yohimbine and Corynanthe alkaloids, were potent inhibitors of T. brucei proliferation and T. brucei methionyl-tRNA synthetase (TbMetRS) activity. Inspired by these key findings, we prepared several novel series of hydroxyalkyl δ-lactam, δ-lactam, and piperidine analogs and tested their anti-trypanosomal activity. A number of inhibitors are more potent against T. brucei than these initial hits with one hydroxyalkyl δ-lactam derivative being 25-fold more effective in our assay. Surprisingly, most of these active compounds failed to inhibit TbMetRS. This work underscores the importance of verifying, irrespective of close structural similarities, that new compounds designed from a lead with a known biological target engage the putative binding site.

Lance T. Lepovitz, Alan R. Meis, Sarah M. Thomas, Justin Wiedeman, Alexandra Pham, Kojo Mensa-Wilmot, Stephen F. Martin. Tetrahedron. 2020 Apr 17;76(16). pii: 131086. doi: 10.1016/j.tet.2020.131086

Noelia Lander receives research award

Donna Huber on April 9, 2020

Noelia Lander, a cellular biologist and postdoctoral researcher in Roberto Docampo‘s laboratory, has received the 2020 Postdoctoral Award from the UGA Research Foundation.

Lander has used her research to advance understanding of a dangerous parasite affecting millions of people worldwide. She adapted the CRISPR/Cas9 genome-editing system for the study of Trypanosoma cruzi, a human parasite that causes Chagas disease. In widely cited research, she proved the usefulness of this new gene-editing system and its range of applications in T. cruzi, which historically had been difficult to manipulate. Dozens of Chagas molecular biology labs worldwide use her CRISPR/Cas9 strategy to study the parasite’s proteins, characterize its metabolic pathways, understand its biology and search for new chemotherapeutic targets. More recently, she has used her system to study protein function and calcium signaling in T. cruzi. She has trained laboratory personnel and students in scientific research and is currently conducting the mentored phase of an NIH Pathway to Independence Award.

Created in 2011, Postdoctoral Research Awards recognize the remarkable contributions of postdoctoral research scholars to the UGA research enterprise. The UGA Research Foundation funds up to two awards a year to current scholars.

Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Donna Huber on April 6, 2020

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Roberto Docampo and several of his lab members are co-authors of this study.

Faktorová, D., Nisbet, R.E.R., Fernández Robledo, J.A. et al. Genetic tool development in marine protists: emerging model organisms for experimental cell biology. Nat Methods (2020). https://doi.org/10.1038/s41592-020-0796-x

EAT-18 is an essential auxiliary protein interacting with the non-alpha nAChR subunit EAT-2 to form a functional receptor

Donna Huber on April 3, 2020

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs.

Shivani Choudhary, Samuel K. Buxton, Sreekanth Puttachary, Saurabh Verma, Gunnar R. Mair, Ciaran J. McCoy, Barbara J. Reaves, Adrian J. Wolstenholme, Richard J. Martin, Alan P. Robertson. PLoS Pathog. 2020 Apr 3;16(4):e1008396. doi: 10.1371/journal.ppat.1008396.

Galtonosides A-E: Antiproliferative and Antiplasmodial Cholestane Glycosides from Galtonia regalis

Donna Huber on March 31, 2020

An extract of Galtonia regalis from the Natural Products Discovery Institute showed moderate antiplasmodial activity, with an IC₅₀ value less than 1.25 μg/mL. The two known cholestane glycosides 1 and 2 and the five new cholestane glycosides galtonosides A–E (3–7) were isolated after bioassay-directed fractionation. The structures of the new compounds were determined by interpretation of their NMR and mass spectra. Among these compounds, galtonoside B (4) displayed the most potent antiplasmodial activity, with an IC₅₀ value of 0.214 μM against the drug-resistant Dd2 strain of Plasmodium falciparum.

Yongle Du, Brooke A. Martin, Ana Lisa Valenciano, Jason A. Clement, Michael Goetz, Maria B. Cassera, David G. I. Kingston. J Nat Prod. 2020 Mar 31. doi: 10.1021/acs.jnatprod.9b01064.

Dennis Kyle elected as American Academy of Microbiology Fellow

Donna Huber on March 31, 2020

University of Georgia researcher Dennis Kyle has been elected as a 2020 fellow by the American Academy of Microbiology. He joins a class of 68 new fellows this year.

Kyle is a GRA Eminent Scholar in antiparasitic drug discovery, with appointments in the departments of cellular biology and infectious diseases.

“Election as a Fellow of the American Academy of Microbiology is a tremendous honor and one that was achieved by the success of all the great people I’ve work with over the years on antiparasitic drug discovery,” said Kyle, who joined UGA in 2017 as the director of the Center for Tropical and Emerging Global Diseases.

His research focuses on the discovery, development, and mechanisms of resistance to antiparasitic drugs. Currently, his laboratory is concentrating on malaria, which has become increasingly resistant to current treatments, and the brain-eating amoeba Naegleria fowleri. The Kyle laboratory has been instrumental in developing methods and tests to discover new drugs that act rapidly, effectively and can be combined with existing drugs used to treat these nearly incurable diseases.

Kyle’s work is largely funded by the National Institutes of Health, Medicines for Malaria Venture and a $9.4 million grant from the Bill & Melinda Gates Foundation. He has published more than 200 research papers, and his findings have been cited more than 14,000 times.

Kyle has received a number of awards over the course of his career, including the U.S. Army Achievement Medal in 1990, the U.S. Army Commendation Medal in 1988, and the U.S. Army Meritorious Service Award. He has been honored by the Southeastern Society of Parasitologists and is a fellow of the American Society for Tropical Medicine and Hygiene and the American Association for the Advancement of Science. In 2006, he was named Scientist of the Year by Malaria Foundation International.

Kyle joins more than 2,500 AAM fellows who are elected through a highly selective, peer-review process, based on their records of scientific achievement and original contributions that have advanced microbiology. Only 58 percent of this year’s nominees were elected to the Class of 2020, and the newly elected fellows hail from 11 different countries.

Multi-target heteroleptic palladium bisphosphonate complexes

Donna Huber on March 30, 2020

Bisphosphonates are the most commonly prescribed drugs for the treatment of osteoporosis and other bone illnesses. Some of them have also shown antiparasitic activity. In search of improving the pharmacological profile of commercial bisphosphonates, our group had previously developed first row transition metal complexes with N-containing bisphosphonates (NBPs). In this work, we extended our studies to heteroleptic palladium–NBP complexes including DNA intercalating polypyridyl co-ligands (NN) with the aim of obtaining potential multi-target species. Complexes of the formula [Pd(NBP)₂(NN)]·2NaCl·xH₂O with NBP = alendronate (ale) or pamidronate (pam) and NN = 1,10 phenanthroline (phen) or 2,2′-bipyridine (bpy) were synthesized and fully characterized. All the obtained compounds were much more active in vitro against T. cruzi (amastigote form) than the corresponding NBP ligands. In addition, complexes were nontoxic to mammalian cells up to 50–100 µM. Compounds with phen as ligand were 15 times more active than their bpy analogous. Related to the potential mechanism of action, all complexes were potent inhibitors of two parasitic enzymes of the isoprenoid biosynthetic pathway. No correlation between the anti-T. cruzi activity and the enzymatic inhibition results was observed. On the contrary, the high antiparasitic activity of phen-containing complexes could be related to their ability to interact with DNA in an intercalative-like mode. These rationally designed compounds are good candidates for further studies and good leaders for future drug developments.

Micaella Cipriani, Santiago Rostán, Ignacio León, Zhu-Hong Li, Jorge S. Gancheff, Ulrike Kemmerling, Claudio Olea Azar, Susana Etcheverry, Roberto Docampo, Dinorah Gambino & Lucía Otero. J Biol Inorg Chem. 2020 Mar 30. doi: 10.1007/s00775-020-01779-y.

CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi

Donna Huber on March 28, 2020

Chagas disease is a vector-borne tropical disease affecting millions of people worldwide, for which there is no vaccine or satisfactory treatment available. It is caused by the protozoan parasite Trypanosoma cruzi and considered endemic from North to South America. This parasite has unique metabolic and structural characteristics that make it an attractive organism for basic research. The genetic manipulation of T. cruzi has been historically challenging, as compared to other pathogenic protozoans. However, the use of the prokaryotic CRISPR/Cas9 system for genome editing has significantly improved the ability to generate genetically modified T. cruzi cell lines, becoming a powerful tool for the functional study of proteins in different stages of this parasite’s life cycle, including infective trypomastigotes and intracellular amastigotes. Using the CRISPR/Cas9 method that we adapted to T. cruzi, it has been possible to perform knockout, complementation and in situ tagging of T. cruzi genes. In our system we cotransfect T. cruzi epimastigotes with an expression vector containing the Cas9 sequence and a single guide RNA, together with a donor DNA template to promote DNA break repair by homologous recombination. As a result, we have obtained homogeneous populations of mutant epimastigotes using a single resistance marker to modify both alleles of the gene. Mitochondrial Ca²⁺ transport in trypanosomes is critical for shaping the dynamics of cytosolic Ca²⁺ increases, for the bioenergetics of the cells, and for viability and infectivity. In this chapter we describe the most effective methods to achieve genome editing in T. cruzi using as example the generation of mutant cell lines to study proteins involved in calcium homeostasis. Specifically, we describe the methods we have used for the study of three proteins involved in the calcium signaling cascade of T. cruzi: the inositol 1,4,5-trisphosphate receptor (TcIP₃R), the mitochondrial calcium uniporter (TcMCU) and the calcium-sensitive pyruvate dehydrogenase phosphatase (TcPDP), using CRISPR/Cas9 technology as an approach to establish their role in the regulation of energy metabolism.

Noelia Lander, Miguel A. Chiurillo, Roberto Docampo. Methods Mol Biol. 2020;2116:177-197. doi: 10.1007/978-1-0716-0294-2_13.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids

Donna Huber on March 28, 2020

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PP_i) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.

Guozhong Huang, Silvia N. J. Moreno, Roberto Docampo. Methods Mol Biol. 2020;2116:673-688. doi: 10.1007/978-1-0716-0294-2_40.

Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3

Donna Huber on March 24, 2020

Plasmodium vivax infects hepatocytes to form schizonts that cause blood infection, or dormant hypnozoites that can persist for months in the liver before leading to relapsing blood infections. The molecular processes that drive P. vivax schizont and hypnozoite survival remain largely unknown, but they likely involve a rich network of host-pathogen interactions, including those occurring at the host-parasite interface, the parasitophorous vacuole membrane (PVM). Using a recently developed P. vivax liver-stage model system we demonstrate that host aquaporin-3 (AQP3) localizes to the PVM of schizonts and hypnozoites within 5 days after invasion. This recruitment is also observed in P. vivax-infected reticulocytes. Chemical treatment with the AQP3 inhibitor auphen reduces P. vivax liver hypnozoite and schizont burden, and inhibits P. vivax asexual blood-stage growth. These findings reveal a role for AQP3 in P. vivax liver and blood stages and suggest that the protein may be targeted for therapeutic treatment.

Dora Posfai, Steven P. Maher, Camille Roesch, Amélie Vantaux, Kayla Sylvester, Julie Péneau, Jean Popovici, Dennis E. Kyle, Benoît Witkowski, Emily R. Derbyshire. Cell Chem Biol. 2020 Mar 24. pii: S2451-9456(20)30083-0. doi: 10.1016/j.chembiol.2020.03.009.

Author: Donna Huber