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Author: Donna Huber

Synthesis and in vitro evaluation of new 5-substituted 6-nitroimidazooxazoles as antikinetoplastid agents

Donna Huber on February 14, 2020

In continuation of our pharmacomodulation work on the nitroimidazooxazole series, we report the synthesis of new 5-substituted 6-nitroimidazooxazole derivatives. Our aim was to evaluate how functionalization of the 5-position of the 6-nitroimidazooxazole scaffold affects antileishmanial and antitrypanosomal in vitro activities. Twenty-one original compounds were synthesized and evaluated for their in vitro antileishmanial (L. donovani) and antitrypanosomal (T. cruzi) properties. Pallado-catalyzed cross-coupling reactions were used to introduce an aryl or ethynyl aryl substituent in 5-position from a 5-brominated-6-nitroimidazooxazole starting product. Unfortunately, the first series of compounds bearing an aryl group in 5-position presented limited in vitro activities against L. donovani and T. cruzi, with IC50 > 10 μM (vs 0.18 μM and 2.31 μM for the reference drugs amphotericin B and benznidazole respectively). Interestingly, the second series of compounds bearing an ethynyl aryl substituent in 5-position showed more promising, particularly against T. cruzi. Compounds 6a, 6b, 6c, 6g and 6h had better activity than the reference drug benznidazole (0.92 μM ≤ IC50 ≤ 2.18 μM vs IC50 = 2.31 μM), whereas the non-functionalized 2-methyl-6-nitro-2,3-dihydroimidazo [2,1-b]oxazole 2 was not active against T. cruzi (IC50 > 10 μM).

Fanny Mathias, AnitaCohen, Youssef Kabri, Núria Waddington Negrão, Maxime D.Crozet, RobertoDocampo, Nadine Azas, Patrice Vanelle. Eur J Med Chem. 2020 Feb 14;191:112146. doi: 10.1016/j.ejmech.2020.112146

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

Donna Huber on February 14, 2020

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

Survey of Schistosomiasis in Saint Lucia: Evidence for Interruption of Transmission

Donna Huber on February 10, 2020

Saint Lucia at one time had levels of schistosomiasis prevalence and morbidity as high as many countries in Africa. However, as a result of control efforts and economic development, including more widespread access to sanitation and safe water, schistosomiasis on the island has practically disappeared. To evaluate the current status of schistosomiasis in Saint Lucia, we conducted a nationally representative school-based survey of 8-11-year-old children for prevalence of Schistosoma mansoni infections using circulating antigen and specific antibody detection methods. We also conducted a questionnaire about available water sources, sanitation, and contact with fresh water. The total population of 8-11-year-old children on Saint Lucia was 8,985; of these, 1,487 (16.5%) provided urine for antigen testing, 1,455 (16.2%) provided fingerstick blood for antibody testing, and 1,536 (17.1%) answered the questionnaire. Although a few children were initially low positives by antigen or antibody detection methods, none could be confirmed positive by follow-up testing. Most children reported access to clean water and sanitary facilities in or near their homes and 48% of the children reported contact with fresh water. Together, these data suggest that schistosomiasis transmission has been interrupted on Saint Lucia. Additional surveys of adults, snails, and a repeat survey among school-age children will be necessary to verify these findings. However, in the same way that research on Saint Lucia generated the data leading to use of mass drug administration for schistosomiasis control, the island may also provide the information needed for guidelines to verify interruption of schistosomiasis transmission.

Janice Gaspard, Madelaine M. Usey, Merlene Fredericks-James, Maria J. Sanchez, Lydia Atkins, Carl H. Campbell Jr., Paul L. A. M. Corstjens, Govert J. van Dam, Daniel G. Colley and William Evan Secor. Am J Trop Med Hyg. 2020 Feb 10. doi: 10.4269/ajtmh.19-0904

Gene content evolution in the arthropods

Donna Huber on January 23, 2020

Abstract

BACKGROUND:

Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods.

RESULTS:

Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception.

CONCLUSIONS:

These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Thomas GWC, Dohmen E, Hughes DST, Murali SC, Poelchau M, Glastad K, Anstead CA, Ayoub NA, Batterham P, Bellair M, Binford GJ, Chao H, Chen YH, Childers C, Dinh H, Doddapaneni HV, Duan JJ, Dugan S, Esposito LA, Friedrich M, Garb J, Gasser RB, Goodisman MAD, Gundersen-Rindal DE, Han Y, Handler AM, Hatakeyama M, Hering L, Hunter WB, Ioannidis P, Jayaseelan JC, Kalra D, Khila A, Korhonen PK Lee CE, Lee SL, Li Y, Lindsey ARI, Mayer G, McGregor AP, McKenna DD, Misof B, Munidasa M, Munoz-Torres M, Muzny DM, Niehuis O, Osuji-Lacy N, Palli SR, Panfilio KA, Pechmann M, Perry T, Peters RS, Poynton HC, Prpic NM, Qu J, Rotenberg D, Schal C, Schoville SD, Scully ED, Skinner E, Sloan DB, Stouthamer R, Strand MR, Szucsich NU, Wijeratne A, Young ND, Zattara EE, Benoit JB, Zdobnov EM, Pfrender ME, Hackett KJ, Werren JH, Worley KC, Gibbs RA, Chipman AD, Waterhouse RM, Bornberg-Bauer E, Hahn MW, Richards S. Genome Biol. 2020 Jan 23;21(1):15. doi: 10.1186/s13059-019-1925-7.

Predaceous Toxorhynchites mosquitoes require a living gut microbiota to develop

Donna Huber on January 22, 2020

Most species of mosquitoes are detritivores that feed on decaying plant and animal materials in their aquatic environment. Studies of several detritivorous mosquito species indicate that they host relatively low diversity communities of microbes that are acquired from the environment while feeding. Our recent results also indicate that detritivorous species normally require a living gut microbiota to grow beyond the first instar. Less well known is that some mosquitoes, including those belonging to the genus Toxorhynchites, are predators that feed on other species of mosquitoes and nektonic prey. In this study, we asked whether predaceous Toxorhynchites amboinensis larvae still require living microbes in their gut in order to develop. Using the detritivorous mosquito Aedes aegypti as prey, we found that T. amboinensis larvae harbour bacterial communities that are highly similar to that of their prey. Functional assays showed that T. amboinensis first instars provided axenic (i.e. bacteria-free) prey failed to develop, while two bacterial species present in gnotobiotic (i.e. colonized by one or more known bacterial species) prey successfully colonized the T. amboinensis gut and rescued development. Axenic T. amboinensis larvae also displayed defects in growth consistent with previously identified roles for microbe-mediated gut hypoxia in nutrient acquisition and assimilation in A. aegypti. Collectively, these results support a conserved role for gut microbes in regulating the development of mosquitoes with different feeding strategies.

Kerri L. Coon, Luca Valzania, Mark R. Brown and Michael R. Strand. Proc Biol Sci. 2020 Jan 29;287(1919):20192705. doi: 10.1098/rspb.2019.2705

Identification and Localization of the First Known Proteins of the Trypanosoma cruzi Cytostome Cytopharynx Endocytic Complex

Donna Huber on January 17, 2020

The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 7 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease continues to receive insufficient attention and resources in order to be effectively combatted. Unlike the other parasitic trypanosomatids that infect humans (Trypanosoma brucei and Leishmania spp.), T. cruzi retains an ancestral mode of phagotrophic feeding via an endocytic organelle known as the cytostome-cytopharynx complex (SPC). How this tubular invagination of the plasma membrane functions to bring in nutrients is poorly understood at a mechanistic level, partially due to a lack of knowledge of the protein machinery specifically targeted to this structure. Using a combination of CRISPR/Cas9 mediated endogenous tagging, fluorescently labeled overexpression constructs and endocytic assays, we have identified the first known SPC targeted protein (CP1). The CP1 labeled structure co-localizes with endocytosed protein and undergoes disassembly in infectious forms and reconstitution in replicative forms. Additionally, through the use of immunoprecipitation and mass spectrometry techniques, we have identified two additional CP1-associated proteins (CP2 and CP3) that also target to this endocytic organelle. Our localization studies using fluorescently tagged proteins and surface lectin staining have also allowed us, for the first time, to specifically define the location of the intriguing pre-oral ridge (POR) surface prominence at the SPC entrance through the use of super-resolution light microscopy. This work is a first glimpse into the proteome of the SPC and provides the tools for further characterization of this enigmatic endocytic organelle. A better understanding of how this deadly pathogen acquires nutrients from its host will potentially direct us toward new therapeutic targets to combat infection.

Nathan Michael Chasen, Isabelle Coppens and Ronald Drew Etheridge. Front Cell Infect Microbiol. 2020 Jan 17;9:445. doi: 10.3389/fcimb.2019.00445. eCollection 2019.

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

Donna Huber on January 10, 2020

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.

Environmental Predictors of Schistosomiasis Persistent Hotspots following Mass Treatment with Praziquantel

Donna Huber on December 30, 2019

Schistosomiasis control programs rely heavily on mass drug administration (MDA) campaigns with praziquantel for preventative chemotherapy. Areas where the prevalence and/or intensity of schistosomiasis infection remains high even after several rounds of treatment, termed “persistent hotspots” (PHSs), have been identified in trials of MDA effectiveness conducted by the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) in Kenya, Mozambique, Tanzania, and Côte d’Ivoire. In this analysis, we apply a previously developed set of criteria to classify the PHS status of 531 study villages from five SCORE trials. We then fit logistic regression models to data from SCORE and publically available georeferenced datasets to evaluate the influence of local environmental and population features, pre-intervention infection burden, and treatment scheduling on PHS status in each trial. The frequency of PHS in individual trials ranged from 35.3% to 71.6% in study villages. Significant relationships between PHS status and MDA frequency, distance to freshwater, rainfall, baseline schistosomiasis burden, elevation, land cover type, and village remoteness were each observed in at least one trial, although the strength and direction of these relationships was not always consistent among study sites. These findings suggest that PHSs are driven in part by environmental conditions that modify the risk and frequency of reinfection.

Joseph W. Walker, Nupur Kittur, Sue Binder, Jennifer D. Castleman, John M. Drake, Carl H. Campbell Jr., Charles H. King and Daniel G. Colley. Am J Trop Med Hyg. 2019 Dec 30. doi: 10.4269/ajtmh.19-0658.

Neurochemical and neuroinflammatory perturbations in two Gulf War Illness models: Modulation by the immunotherapeutic LNFPIII

Donna Huber on December 19, 2019

Gulf War Illness (GWI) manifests a multitude of symptoms, including neurological and immunological, and approximately a third of the 1990–1991 Gulf War (GW) veterans suffer from it. This study sought to characterize the acute neurochemical (monoamine) and neuroinflammatory profiles of two established GWI animal models and examine the potential modulatory effects of the novel immunotherapeutic Lacto-N-fucopentaose III (LNFPIII). In Model 1, male C57BL/6 J mice were treated for 10 days with pyridostigmine bromide (PB) and permethrin (PM). In Model 2, a separate cohort of mice were treated for 14 days with PB and N,N-Diethyl-methylbenzamide (DEET), plus corticosterone (CORT) via drinking water on days 8–14 and diisopropylfluorophosphate (DFP) on day 15. LNFPIII was administered concurrently with GWI chemicals treatments. Brain and spleen monoamines and hippocampal inflammatory marker expression were examined by, respectively, HPLC-ECD and qPCR, 6 h post treatment cessation. Serotonergic (5-HT) and dopaminergic (DA) dyshomeostasis caused by GWI chemicals was apparent in multiple brain regions, primarily in the nucleus accumbens (5-HT) and hippocampus (5-HT, DA) for both models. Splenic levels of 5-HT (both models) and norepinephrine (Model 2) were also disrupted by GWI chemicals. LNFPIII treatment prevented many of the GWI chemicals induced monoamine alterations. Hippocampal inflammatory cytokines were increased in both models, but the magnitude and spread of inflammation was greater in Model 2; LNFPIII was anti-inflammatory, more so in the apparently milder Model 1. Overall, in both models, GWI chemicals led to monoamine disbalance and neuroinflammation. LNFPIII co-treatment prevented many of these disruptions in both models, which is indicative of its promise as a potential GWI therapeutic.

J.M. Carpenter, H.E. Gordon, H.D. Ludwig, J.J. Wagner, D.A. Harn, T. Norberg, N.M. Filipov. Neurotoxicology. 2019 Dec 19;77:40-50. doi: 10.1016/j.neuro.2019.12.012