repurposing the orphan drug nitisinone to control the transmission of african trypanosomiasis CORD-Papers-2022-06-02 (Version 1)

Title: Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis
Abstract: Tsetse transmit African trypanosomiasis which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore nitisinone (NTBC) an FDA-approved tyrosine catabolism inhibitor killed tsetse regardless if the drug was orally or topically applied. However oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa thus accelerating current disease elimination programmes.
Published: 2021-01-26
Journal: PLoS Biol
DOI: 10.1371/journal.pbio.3000796
Author Name: Sterkel Marcos
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Author Name: Haines Lee R
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Author Name: Casas Snchez Aitor
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Author Name: Owino Adunga Vincent
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Author Name: Vionette Amaral Raquel J
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Author Name: Quek Shannon
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Author Name: Rose Clair
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Author Name: Silva dos Santos Mariana
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Author Name: Garca Escude Natalia
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Author Name: Ismail Hanafy M
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Author Name: Paine Mark I
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Author Name: Barribeau Seth M
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Author Name: Wagstaff Simon
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Author Name: MacRae James I
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Author Name: Masiga Daniel
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Author Name: Yakob Laith
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Author Name: Oliveira Pedro L
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Author Name: Acosta Serrano lvaro
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sha: 5b9c7abf7352edb7a0236c40dbbfb13fe2fbb15a
license: cc-by
source_x: PMC
pubmed_id: 33497373
pmcid: PMC7837477
has_full_text: TRUE
Keywords Extracted from Text Content: human NTBC Line 102-104 blood PTU oral nitisinone/NTBC Line 121 TAT p450s Sarcophaga sp. Line 347 Ross-Macdonald defibrinated horse blood Line 136-143 sections Fig S6 humans horse serum HPPD Inhibitor Sensitive 1 melanin fly Line 200-208 ofProduct Line 129 Line 108 triketone Vectorbase COVID-19 R. prolixus -median-time-to-death serum masse Fig 4B HPPD flies dopa castaneum Cedo P450 tyrosine rice tissue Rhodnius prolixus Pollen liver microsomes microsome tarsal Nitisinone agriculture-based HPPD metabolomics-MS fructose-PBS abdomen dopamine O. fasciatus fructose albumin plasma human liver microsomes flesh fly Order Diptera lines 432-442 PTU + M. Sterkel bloodmeals lethal haemoglobin mice triketone herbicides human MDA D. melanogaster ... livestock Glossina PO Line microsomes horse blood Bial Rhodnius NTBC cDNA-expressed P450 HPPA solid blue bovine larvae Fig.1 haem LD50 blood blood feeders BSA Line 132-134 Rev #4 Line 243 phenoloxidase lines 355-368 amino acid fat bodies Belfiori-Carrasco Orfadin Capsules Line 267 sugar C. elegans PTU mosquitoes sub-lethal NTBC NTBC-based HAT line collateral Fly HIS1 Fig. 4 POs oral mesotrione pyrethroids Faraz human endectocide P450s human liver CYP blood feeder insect Acosta-Serrano Line 126 Line 218 human cytochrome P450 abdomens NADPH-fortified PO CYP6P3
Extracted Text Content in Record: First 5000 Characters:We would like to thank all reviewers for the constructive comments and suggestions. Reviewer #1: • This is a well-conducted and exciting study on the potential of the FDA-approved drug nitisinone to control populations of hematophagous vectors. The body of evidence provided in the paper convincingly demonstrates that nitisinone (a tyrosine degradation inhibitor) can kill tsetse flies upon blood feeding by promoting toxic accumulation of tyrosine. The potency of the drug at concentrations that are compatible with mass drug administration in the human population, the versatile application (oral or topical) and the environmental friendliness (harmless to bumble-bees) make nitisinone a very promising candidate for drug-based vector control. The paper is clearly written, and I only have minor suggestions to improve it, listed below in decreasing order of importance. Response: Many thanks for your positive comments. Response: Agree. Please see our previous response to rev #2 regarding the interpretation of our PTU experiments. We have decided to withdraw this section from the paper. Response: The word "spiked" has been replaced for "supplemented with" throughout the manuscript. • Line 102-104. State degree of mortality, and knockdown efficiency, for each treatment. Presumably the explanation for incomplete mortality is due to incomplete knockdown, but this is not stated in the text. Response: The following was added to the text (lines 99-100): "However, 100% mortality was not observed, which may be explained by incomplete gene knockdown". • Line 121: please provide specific safety information for nitisinone/NTBC. Response: The safety information and adverse effects reported for NTBC are reviewed in reference 19 • It would be interesting to discuss the likelihood that flies (or other insects) could evolve nitisinone resistance by other means than metabolic detoxification (e.g., target insensitivity). In the dose-response experiments, a small proportion (10-15%) of flies seems to survive even at high drug concentration (e.g., Fig. 1d ). This observation is consistent with the existence of some genetic variation and/or phenotypic plasticity in nitisinone tolerance. Response: We have now included two paragraphs in the Discussion section related to these two issues. First (page 15, lines 355-368), newly emerged tsetse flies can ingest bloodmeals of different sizes. We removed all flies that did not have red abdomens, however the quantity of blood ingested is difficult to estimate as a fly begins to remove water (diuresis) within 10 minutes of feeding. It is likely that the small proportion of flies that survived the high drug concentrations ingested a smaller bloodmeal, and as a result, a smaller drug dose. This issue is certainly more pronounced in younger flies. Second, regarding nitisinone tolerance (page 17, lines 408-431), we think mutations that would affect the binding of NTBC to HPPD may also alter the affinity for HPPA. Thus, due to the importance of HPPD in the physiology of hematophagous arthropods, mutations that reduce its affinity for HPPA would probably be lethal. However, we cannot rule out the existence of some genetic variation or phenotypic plasticity that could confer tolerance to hematophagous vectors towards HPPD inhibitors. Certainly, after >20 years of using common agriculture-based HPPD inhibitors like mesotrione, resistant weeds have emerged but interestingly no target-site mutations of the HPPD genes has been associated with post-emergence resistance. Moreover, no gene duplication or over-expression of HPPD, before or after herbicide treatment, was detected. In addition, a new gene called HIS1 (HPPD Inhibitor Sensitive 1) has been linked with resistance to several triketone herbicides (including mesotrione) in rice plants, but our Vectorbase search ( did not find orthologous genes in the genomes of several insects, thus suggesting the absence of this gene in invertebrates. • Since PLoS Biology has no restrictions on the number of figures, some of the data relegated to the supporting information could be included in the main body of the paper. For instance, Figs. S6, S7 and S8 could be combined and provided as an additional main figure. Response: As suggested, we have included a new figure (Fig. 4) in the main part of the manuscript, which is composed of former figures S7 and S8, and now shows the potency of HPPD inhibitors in serum and BSA. Fig S6 was excluded from the manuscript (see below). The mathematical model is now Fig. 5 . The number of figures in the supplemental material have also been changed accordingly. • It is not expected that the authors carry out additional experiments with mosquitoes, but perhaps the collateral effects of nitisinone on mosquito vector species (if they are considered plausible) could be mentioned in the discussion as an additional benefit to the proposed strategy. Response: We have included a short comment at the end of the Discussion sect
Keywords Extracted from PMC Text: isopropanol humans [28] human buff-tailed bumblebee Barchanska tube flies' Polar extracts glycerol fumarylacetoacetase intraperitoneal midgut 's ethanol body wall cuticle B-methylamino-L-Alanine patients HPPD [11 green fluorescence protein Fig 1C Glossina spp Deltamethrin testes digestive tract lipid droplets × St. Louis HPPD Inhibitor Sensitive 1 BSA tyrosine wildlife ΔCT microsomal extract gambiense HAT endectocide Trypanosoma brucei rhodesiense HPPD TAT fly thorax phosphoric acid brown melanin-like Glossina tachinoides body BioCyc [68,69 b. gambiense mesotrione acetonitrile Optima HPLC fly haemocoel pea microsomal extracts NTBC-blood tissue Fig 1E Santa Clara horse blood muscle water bloodfed Fig 2A and Umea, Sweden Fig 1B skin [ children ammonium carbonate polar NADPH goats eye G. pallidipes NBTC agarose Haem UK dsTAT- TCS Biosciences bloodmeal cells mosquitoes procyclic trypanosomes pollen Glossina palpalis Eugene dione ring [53] Cytoskeleton Inc.) Ae. Mesotrione Fig 3C 2B LC50 Inceoglu abdominal tissues Microsomes lethal ventral surface picomoles/fly HB containing Fig 1A PESTANAL carbon cytochrome b5 thorax Stock HPLA rats P450 HB Colv S14 Fig. Fig 5 pyrethroids HT-1 [57,58 fructose AAT Fig 2E LD50 Horse red blood cells 90:13 red blood cell Culex Ambrosia syrup Callistro Fig 1D 4.136 NTBC-protein gplot's non-bloodfeeders Teneral Muller DL-p-hydroxyphenyllactic acid primer-blast proventriculus organs NFW Fixed tissues fly bloodmeals Hives −80°C sheath Fold gut LC-MS/MS-identified methanol fructose-BSA defibrinated horse blood neurotoxin DNA visual- dose-matched NTBC Ivermectin CYP6P3 membranes thoracic human plasma NCBI CA LSM-880 muscles ISS110 eyes LC livestock tarsal haemoglobin B ivermectin's 45:55, v/v CYP3A4 wings parts m. morsitans GFP stock " Acyrthosiphon pisum S3A 4-hydroxyphenyl lactic acid amino acid cytosolic ticks plasma NTBC-induced serum 95:5 Solvent A ivermectin trachea oral HPPA capillary tubes chloroform peGFP-N1 Buckingham U55762.1 HPLA legs Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, MDA ovaries blood HAT defibrinated bovine blood 2-[2-nitro-4-(trifluoromethyl)benzoyl]cyclohexane-1,3-dione guinea pigs Geneva, microsomes pigs ± 4 fly Solvent A rice plants wood SiR-actin midgut epithelium humans P450s Syngenta cuticular horse serum apolar Bombus terrestris Superscript III First-strand autosampler temperature 4°C membrane Agralan sugar Collages Ketamine haemocoel NTBC 2-[4-(Methylsulfonyl)-2-nitrobenzoyl]cyclohexane-1,3-dione Fig 4B BLOSUM62 bovine serum albumin herbicide aerial bodies liver CYP abdomen Vectorbase NTBC-induced lethal Nairobi, Kenya gut epithelium Congo cattle Solvent B mesotrione- Glossina spp.-specific salivary glands deltamethrin red blood cells haematophagy [11] Fly post-NTBC 1 M NaOH amino acids fumarylacetoacetate Aldrich-Sigma 4-hydroxyphenylpyruvate dioxygenase Human Fig 3 bites abdominal Wistar rats flies corn crops bee populations GE Insect fly's humans [17] bloodfed arthropods blood 3 matrix LipidMaps [65] function(x 2e−ΔCT values ( Nguruman Rats Trypanosoma brucei gambiense Colony-reared Bombus terrestris phenylthiourea HT- I triketone herbicides NCBI ID Flies intestinal haemolymph ZIC-pHILIC low-dose dorsolateral surface Fig 1G Mosquitoes people BSA haem G. m. morsitans microsomal Unga S3C Fig humans [27 PhyML masses insect cuticle bloodmeals reactive oxygen species ketamine 15N-Valine [2] ISS100 leg sugar water β-tubulin −80 midgut tissue tissues 0.125 haemocytes Malpighian tubules rectum human blood FAH acetonitrile hindgut tyrosine Bee Fig HIS1 fat body Orfadin joint abdominal-thoracic NTBC solubilised Methanol manhattan Glossina species ATSBs homogenate Tissues PBS-
Extracted PMC Text Content in Record: First 5000 Characters:Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic disease caused predominantly by the parasite Trypanosoma brucei gambiense. These parasites are transmitted to a vertebrate host when infected tsetse flies (Glossina spp.) blood feed. HAT currently affects 3,500 people/year; most patients live in the Democratic Republic of the Congo and an estimated 70 million people remain at risk of infection in sub-Saharan Africa [1]. Tsetse also spread animal African trypanosomiasis (AAT), which causes high mortality rates in livestock and consequently severely limits animal production [2]. As no vaccine for either HAT or AAT exist, and drug treatments are often difficult to obtain, tsetse population control remains essential to limit the spread of trypanosomiasis. In the last decades, tsetse control tools such as aerial spraying of insecticides (pyrethroids), visual- and odour-baited tsetse traps, insecticide-treated livestock, live traps, insecticide-impregnated traps and targets, and sterile male releases have been employed [3–7]. Despite such efforts, because AAT and HAT persist in these endemic areas, both economic development and public health continue to be jeopardised [8]. Consequently, a novel complementary strategy to control these parasitic diseases is highly desired. Tsetse, like other blood-feeding arthropods, ingest large quantities of blood and often exceed twice their body weight in a single meal [9]. Since more than 85% of blood dry weight consists of proteins, large quantities of amino acids are released in the midgut during bloodmeal digestion [10]. Previously, we showed that blocking tyrosine catabolism after a bloodmeal is lethal in mosquitoes, ticks, and kissing bugs due to the accumulation of toxic quantities of tyrosine [11]. However, inhibiting tyrosine catabolism in non-blood-feeding insects is harmless, which further provides evidence for the essentiality of this pathway for haematophagy [11]. In the present work, we evaluated how tsetse physiology was controlled by two enzymes in the tyrosine catabolism pathway: tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD). The drug nitisinone (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3cyclohexanedione; NTBC), also known as Orfadin, is an HPPD inhibitor currently used to treat patients with the genetic disease hypertyrosinemia type I (HT-1) [12], and is under clinical evaluation for the treatment of alkaptonuria [13]. NTBC was lethal to blood-fed tsetse flies. NTBC treatment, either administered orally as an endectocide or topically to the insect cuticle, causes the accumulation of tyrosine and 4-hydroxyphenyl lactic acid (HPLA) metabolites, which leads to initial fly paralysis followed by tissue destruction within 18 hours of the bloodmeal. Our results provide evidence that NTBC could be used as an eco-friendly synergistic strategy alongside current tsetse control practices. Tyrosine catabolism is a highly conserved pathway (Fig 1A) in most eukaryote and prokaryote species with only a few exceptions such as the pea aphid, Acyrthosiphon pisum [14], and trypanosome parasites [15]. The genes encoding TAT and HPPD proteins were identified in five Glossina species [16], as well as in all hematophagous arthropod species with sequenced genomes (S1 Fig). RNA interference (RNAi), of either TAT or HPPD genes, was lethal to flies once they fed on blood. However, 100% mortality was not observed, which may be explained by incomplete knockdown (Fig 1B and S2 Fig). This lethality was further validated by feeding flies with blood supplemented with mesotrione, an HPPD inhibitor widely used as a selective herbicide on corn crops under the brand name Callistro, Syngenta (S3A Fig). The mesotrione concentration that killed 50% of the insects 24 hours after administration (LC50) was 357.7 μM (95% CI: 222.5 to 512.4) (S3A and S3C Fig). This lethal concentration of mesotrione is approximately 30× higher than the drug concentration detected in human plasma (4 μg/ml (11.78 μM)) after volunteers received an oral dose of 4 mg/kg body weight [17]. No differences in susceptibility to mesotrione (or NTBC) were observed between fly sex (S4 Fig). HT-1 is a severe human genetic disease caused by a mutation in the gene encoding for the last enzyme of the tyrosine catabolism pathway, fumarylacetoacetase (FAH). This mutation causes the accumulation of toxic metabolites in blood and tissues. The only drug available to minimise the effect of HT-1 is the orphan drug NTBC. As an HPPD inhibitor, NTBC prevents the buildup of toxic products derived from fumarylacetoacetate accumulation [18]. NTBC is remarkably safe to use with few reported side effects in <1% of patients [19,20]. When NTBC was fed to tsetse flies in an artificial bloodmeal, it was approximately 173 times more potent than mesotrione with a LC50 = 2.07 μM (95% CI: 0.709 to 4.136) (Fig 1C and 1D). This lethal concentration is approximately 12 times lower than the concentrat
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