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Category: publications

Effective drug discovery in Chagas disease

Donna Huber on May 15, 2023

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.

Rick L. Tarleton. Trends Parasitol. 2023 Jun;39(6):423-431. doi: 10.1016/j.pt.2023.03.015.

Identification of novel anti-amoebic pharmacophores from kinase inhibitor chemotypes

Donna Huber on May 10, 2023

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.

Generating Genetically Modified Plasmodium berghei Sporozoites

Donna Huber on May 5, 2023

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Carson Bowers, Samarchith P Kurup. J Vis Exp. 2023 May 5;(195). doi: 10.3791/64992.

Treatment of dogs with Bravecto® (fluralaner) reduces mosquito survival and fecundity

Donna Huber on April 28, 2023

Background: Mosquitoes serve as the vector of canine heartworm (Dirofilaria immitis), which represents a significant and persistent threat to canine health. A reduction in the longevity and/or reproductive success of mosquitoes that take a blood meal from fluralaner-treated dogs may consequently reduce the local transmission of heartworm and prevent new infections. A novel secondary effect of an oral formulation of the ectoparasiticide fluralaner (Bravecto®) against a laboratory strain of the mosquito Aedes aegypti, a potential major vector of canine heartworm, was investigated in this study.

Methods: Six dogs were administered a single dose of fluralaner orally in the form of Bravecto® Chews (at the labeled fluralaner dose of 25 mg/kg body weight), while six control dogs received no treatment. Mosquitoes were fed on blood that was collected from each dog prior to treatment and weekly for 15 weeks post-treatment to assess the continued effects of fluralaner as its serum level decreased. Mosquito fitness was assessed by three parameters: rate of successful blood-feeding, survival, and egg laying.

Results: Successful blood-feeding rate was similar between control and treatment groups. In the fluralaner treatment, mosquito survival was significantly reduced within the first 24 h after blood-feeding, for the first 12 weeks post-treatment of the dogs (efficacy range = 33.2-73.3%). Survival of mosquitoes up until a potentially heartworm-infective timepoint (14 days post-blood-feeding) was significantly reduced in the fluralaner-treated group at several timepoints (1, 2, 5, 11, 12, 13, 14, and 15 weeks post-treatment; efficacy range = 49.4-91.4%), but was less consistently reduced at the other timepoints. Egg laying by mosquitoes was almost completely suppressed for the first 13 weeks following treatment of the dogs with fluralaner (treatment efficacy ≥ 99.8%).

Conclusions: Mosquitoes fed blood from fluralaner-treated dogs experienced a significant reduction in survival and fecundity. These findings support the potential for a reduction in heartworm transmission directly by lethal effects on the vector and indirectly through a reduction of the local vector population when mosquitoes are exposed to animals treated with fluralaner.

Christopher Charles Evans, Dorothy Normile, Sheryl Gamble, Frank Guerino, Michael T Dzimianski, Andrew Riddell Moorhead. Parasit Vectors. 2023 Apr 28;16(1):147. doi: 10.1186/s13071-023-05682-8.

Frequency Variation and Dose Modification of Benznidazole Administration for the Treatment of Trypanosoma cruzi Infection in Mice, Dogs, and Nonhuman Primates

Donna Huber on April 11, 2023

Trypanosoma cruzi naturally infects a broad range of mammalian species and frequently results in the pathology that has been most extensively characterized in human Chagas disease. Currently employed treatment regimens fail to achieve parasitological cure of T. cruzi infection in the majority of cases. In this study, we have extended our previous investigations of more effective, higher dose, intermittent administration protocols using the FDA-approved drug benznidazole (BNZ), in experimentally infected mice and in naturally infected dogs and nonhuman primates (NHP). Collectively, these studies demonstrate that twice-weekly administration of BNZ for more than 4 months at doses that are ~2.5-fold that of previously used daily dosing protocols, provided the best chance to obtain parasitological cure. Dosing less frequently or for shorter time periods was less dependable in all species. Prior treatment using an ineffective dosing regimen in NHPs did not prevent the attainment of parasitological cure with an intensified BNZ dosing protocol. Furthermore, parasites isolated after a failed BNZ treatment showed nearly identical susceptibility to BNZ as those obtained prior to treatment, confirming the low risk of induction of drug resistance with BNZ and the ability to adjust the treatment protocol when an initial regimen fails. These results provide guidance for the use of BNZ as an effective treatment for T. cruzi infection and encourage its wider use, minimally in high value dogs and at-risk NHP, but also potentially in humans, until better options are available.

Juan M Bustamante, Brooke E White, Gregory K Wilkerson, Carolyn L Hodo, Lisa D Auckland, Wei Wang, Stephanie McCain, Sarah A Hamer, Ashley B Saunders, Rick L Tarleton. Antimicrob Agents Chemother. 2023 Apr 11;e0013223. doi: 10.1128/aac.00132-23.

Cripowellins Pause Plasmodium falciparum Intraerythrocytic Development at the Ring Stage

Donna Huber on March 13, 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.

Analysis of the Interactome of the Toxoplasma gondii Tgj1 HSP40 Chaperone

Donna Huber on March 1, 2023

Toxoplasma gondii is an obligate intracellular apicomplexan that causes toxoplasmosis in humans and animals. Central to its dissemination and pathogenicity is the ability to rapidly divide in the tachyzoite stage and infect any type of nucleated cell. Adaptation to different cell contexts requires high plasticity in which heat shock proteins (Hsps) could play a fundamental role. Tgj1 is a type I Hsp40 of T. gondii, an ortholog of the DNAJA1 group, which is essential during the tachyzoite lytic cycle. Tgj1 consists of a J-domain, ZFD, and DNAJ_C domains with a CRQQ C-terminal motif, which is usually prone to lipidation. Tgj1 presented a mostly cytosolic subcellular localization overlapping partially with endoplasmic reticulum. Protein-protein Interaction (PPI) analysis showed that Tgj1 could be implicated in various biological pathways, mainly translation, protein folding, energy metabolism, membrane transport and protein translocation, invasion/pathogenesis, cell signaling, chromatin and transcription regulation, and cell redox homeostasis among others. The combination of Tgj1 and Hsp90 PPIs retrieved only 70 interactors linked to the Tgj1-Hsp90 axis, suggesting that Tgj1 would present specific functions in addition to those of the Hsp70/Hsp90 cycle, standing out invasion/pathogenesis, cell shape motility, and energy pathway. Within the Hsp70/Hsp90 cycle, translation-associated pathways, cell redox homeostasis, and protein folding were highly enriched in the Tgj1-Hsp90 axis. In conclusion, Tgj1 would interact with a wide range of proteins from different biological pathways, which could suggest a relevant role in them.

Jonathan Munera López, Andrés Mariano Alonso, Maria Julia Figueras, Ana María Saldarriaga Cartagena, Miryam A Hortua Triana, Luis Diambra, Laura Vanagas, Bin Deng, Silvia N J Moreno, Sergio Oscar Angel. Proteomes. 2023 Mar 1;11(1):9. doi: 10.3390/proteomes11010009.

An X-Domain Phosphoinositide Phospholipase C (PI-PLC-like) of Trypanosoma brucei Has a Surface Localization and Is Essential for Proliferation

Donna Huber on February 28, 2023

Trypanosoma brucei is the causative agent of African trypanosomiasis, a deadly disease that affects humans and cattle. There are very few drugs to treat it, and there is evidence of mounting resistance, raising the need for new drug development. Here, we report the presence of a phosphoinositide phospholipase C (TbPI-PLC-like), containing an X and a PDZ domain, that is similar to the previously characterized TbPI-PLC1. TbPI-PLC-like only possesses the X catalytic domain and does not have the EF-hand, Y, and C2 domains, having instead a PDZ domain. Recombinant TbPI-PLC-like does not hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and does not modulate TbPI-PLC1 activity in vitro. TbPI-PLC-like shows a plasma membrane and intracellular localization in permeabilized cells and a surface localization in non-permeabilized cells. Surprisingly, knockdown of TbPI-PLC-like expression by RNAi significantly affected proliferation of both procyclic and bloodstream trypomastigotes. This is in contrast with the lack of effect of downregulation of expression of TbPI-PLC1.

Núria W Negrão, Logan P Crowe, Brian S Mantilla, Rodrigo P Baptista, Sharon King-Keller, Guozhong Huang, Roberto Docampo. Pathogens. 2023 Feb 28;12(3):386. doi: 10.3390/pathogens12030386.

First bovine vaccine to prevent human schistosomiasis – a cluster randomised Phase 3 clinical trial

Donna Huber on February 21, 2023

Objective

Schistosomiasis is a neglected tropical parasitic disease caused by blood flukes of the genus Schistosoma. Schistosoma japonicum is zoonotic in China, the Philippines, and Indonesia, with bovines acting as major reservoirs of human infection. The primary objective of the trial was to examine the impact of a combination of human mass chemotherapy, snail control through mollusciciding, and SjCTPI bovine vaccination on the rate of human infection.

Methods

A 5-year phase IIIa cluster randomized control trial was conducted among 18 schistosomiasis-endemic villages comprising 18,221 residents in Northern Samar, The Philippines.

Results

Overall, bovine vaccination resulted in a statistically significant decrease in human infection (relative risk [RR] = 0.75; 95% confidence interval [CI] = 0.69 to 0.82) across all trial follow-ups. The best outcome of the trial was when bovine vaccination was combined with snail mollusciciding. This combination resulted in a 31% reduction (RR = 0.69; 95% CI = 0.61 to 0.78) in human infection.

Conclusion

This is the first trial to demonstrate the effectiveness of a bovine vaccine for schistosomiasis in reducing human schistosome infection. The trial is registered with Australian New Zealand Clinical Trials Registry (ACTRN12619001048178).

Allen G. Ross, Donald A. Harn, Delia Chy, Marianette Inobaya, Jerric R. Guevarra, Lisa Shollenberger, Yuesheng Li, Donald P. McManus, Darren J. Gray and Gail M. Williams. 2023. International Journal of Infectious Diseases. S1201-9712(23)00038-3. doi: 10.1016/j.ijid.2023.01.037. Online ahead of print.

Chemical Optimization of CBL0137 for Human African Trypanosomiasis Lead Drug Discovery

Donna Huber on January 25, 2023

The carbazole CBL0137 (1) is a lead for drug development against human African trypanosomiasis (HAT), a disease caused by Trypanosoma brucei. To advance 1 as a candidate drug, we synthesized new analogs that were evaluated for the physicochemical properties, antitrypanosome potency, selectivity against human cells, metabolism in microsomes or hepatocytes, and efflux ratios. Structure-activity/property analyses of analogs revealed eight new compounds with higher or equivalent selectivity indices (5j, 5t, 5v, 5w, 5y, 8d, 13i, and 22e). Based on the overall compound profiles, compounds 5v and 5w were selected for assessment in a mouse model of HAT; while 5v demonstrated a lead-like profile for HAT drug development, 5w showed a lack of efficacy. Lessons from these studies will inform further optimization of carbazoles for HAT and other indications.

Baljinder Singh, Amrita Sharma, Naresh Gunaganti, Mitch Rivers, Pradip K Gadekar, Brady Greene, Michael Chichioco, Carlos E Sanz-Rodriguez, Courtney Fu, Catherine LeBlanc, Erin Burchfield, Nyle Sharif, Benjamin Hoffman, Gaurav Kumar, Andrei Purmal, Kojo Mensa-Wilmot, Michael P Pollastri. J Med Chem. 2023 Jan 25. doi: 10.1021/acs.jmedchem.2c01767.