Untargeted metabolomic profiles reveal widespread metabolic perturbations and identify candidate biomarkers in aminoaciduric canine hypoaminoacidemic hepatopathy syndrome.

OBJECTIVE: To identify metabolites and metabolic pathways affected in dogs with aminoaciduric canine hypoaminoacidemic hepatopathy syndrome (ACHES) compared to healthy control (CON) dogs of similar ages and breeds. To improve our understanding of ACHES pathophysiology and identify novel candidate biomarkers associated with ACHES. ANIMALS: A prospective case-control study. Privately owned dogs with ACHES (n = 19) and healthy (CON) dogs (n = 9) were recruited between February 18, 2015, and April 18, 2018. METHODS: A prospective case-control study. Plasma and urine were collected from ACHES and CON dogs. The Cornell University Proteomics and Metabolomics Core Facility conducted an untargeted metabolomic analysis. RESULTS: After controlling for age, sex, and weight, 111 plasma and 207 urine metabolites significantly differed between ACHES and CON dogs. Data reduction and cluster analysis revealed robust segregation between ACHES and CON dogs. Enrichment analysis of significant compounds in plasma or urine identified altered metabolic pathways, including those related to AA metabolism, cellular energetics, and lipid metabolism. Biomarker analysis identified metabolites that best-distinguished ACHES from CON dogs, including pyruvic acid isomer and glycerol-3-phosphate in the plasma and an alanine isomer and choline in the urine. CLINICAL RELEVANCE: Our findings provide an in-depth analysis of metabolic perturbations associated with ACHES. Several affected metabolic pathways (eg, lipid metabolism) offer a new understanding of ACHES pathophysiology. Novel candidate biomarkers warrant further evaluation to determine their potential to aid in ACHES diagnosis, prognosis, and treatment monitoring.

Enhanced detoxification via Cyt-P450 governs cross-tolerance to ALS-inhibiting herbicides in weed species of Centaurea.

Centaurea is a genus of winter weeds with a similar life cycle and competitive traits, which occurs in small-grains production fields in the central-southern of the Iberian Peninsula. However, most of herbicides recommended for weed management in wheat show poor control of Centaurea species. This study summarizes the biology, herbicide tolerance to acetolactate synthase (ALS) inhibitors, and recommended chemical alternatives for the control of Centaurea species. Four species (C. cyanus L., C. diluta Aiton, C. melitensis L. and C. pullata L. subsp. baetica Talavera), taxonomically characterized, were found as the main important broadleaf weeds in small-grains production fields of the Iberian Peninsula. These species showed innate tolerance to tribenuron-methyl (TM), showing LD50 values (mortality of 50% of a population) higher than the field dose of TM (20 g ai ha-1). The order of tolerance was C. diluta (LD50 = 702 g ha-1) ≫ C. pullata (LD50 = 180 g ha-1) ≫ C. cyanus (LD50 = 65 g ha-1) > C. melitensis (LD50 = 32 g ha-1). Centaurea cyanus and C. melitensis presented higher foliar retention (150-180 µL herbicide solution), absorption (14-28%) and subsequent translocation (7-12%) of TM with respect to the other two species. Centaurea spp. plants were able to metabolize 14C-TM into non-toxic forms (hydroxylated OH-metsulfuron-methyl and conjugated-metsulfuron-methyl), with cytochrome P450 (Cyt-P450) monooxygenases being responsible for herbicide detoxification. Centaurea cyanus and C. mellitensis metabolized up to 25% of TM, while C. diluta and C. pullata metabolized more than 50% of the herbicide. Centaurea species showed 80-100% survival when treated with of florasulam, imazamox and/or metsulfuron-methyl, i.e., these weeds present cross-tolerance to ALS inhibitors. In contrast, auxin mimics herbicides (2,4-D, clopyralid, dicamba, fluroxypir and MCPA) efficiently controlled the four Centaurea species. In addition, the mixture of ALS-inhibitors and auxin mimics also proved to be an interesting alternative for the control of Centaurea. These results show that plants of the genus Centaurea found in the winter cereal fields of the Iberian Peninsula have an innate tolerance to TM and cross-resistance to other ALS-inhibiting herbicides, governed by reduced absorption and translocation, but mainly by the metabolization of the herbicide via Cyt-P450.

A combined ligand and target-based virtual screening strategy to repurpose drugs as putrescine uptake inhibitors with trypanocidal activity.

Chagas disease, also known as American trypanosomiasis, is a neglected tropical disease caused by the protozoa Trypanosoma cruzi, affecting nearly 7 million people only in the Americas. Polyamines are essential compounds for parasite growth, survival, and differentiation. However, because trypanosomatids are auxotrophic for polyamines, they must be obtained from the host by specific transporters. In this investigation, an ensemble of QSAR classifiers able to identify polyamine analogs with trypanocidal activity was developed. Then, a multi-template homology model of the dimeric polyamine transporter of T. cruzi, TcPAT12, was created with Rosetta, and then refined by enhanced sampling molecular dynamics simulations. Using representative snapshots extracted from the trajectory, a docking model able to discriminate between active and inactive compounds was developed and validated. Both models were applied in a parallel virtual screening campaign to repurpose known drugs as anti-trypanosomal compounds inhibiting polyamine transport in T. cruzi. Montelukast, Quinestrol, Danazol, and Dutasteride were selected for in vitro testing, and all of them inhibited putrescine uptake in biochemical assays, confirming the predictive ability of the computational models. Furthermore, all the confirmed hits proved to inhibit epimastigote proliferation, and Quinestrol and Danazol were able to inhibit, in the low micromolar range, the viability of trypomastigotes and the intracellular growth of amastigotes.

A structure-based approach towards the identification of novel antichagasic compounds: Trypanosoma cruzi carbonic anhydrase inhibitors.

Trypanosoma cruzi carbonic anhydrase (TcCA) has recently emerged as an interesting target for the design of new compounds to treat Chagas disease. In this study we report the results of a structure-based virtual screening campaign to identify novel and selective TcCA inhibitors. The combination of properly validated computational methodologies such as comparative modelling, molecular dynamics and docking simulations allowed us to find high potency hits, with KI values in the nanomolar range. The compounds also showed trypanocidal effects against T. cruzi epimastigotes and trypomastigotes. All the candidates are selective for inhibiting TcCA over the human isoform CA II, which is encouraging in terms of possible therapeutic safety and efficacy.

Cascade Ligand- and Structure-Based Virtual Screening to Identify New Trypanocidal Compounds Inhibiting Putrescine Uptake.

Chagas disease is a neglected tropical disease endemic to Latin America, though migratory movements have recently spread it to other regions. Here, we have applied a cascade virtual screening campaign combining ligand- and structure-based methods. In order to find novel inhibitors of putrescine uptake in Trypanosoma cruzi, an ensemble of linear ligand-based classifiers obtained by has been applied as initial screening filter, followed by docking into a homology model of the putrescine permease TcPAT12. 1,000 individual linear classifiers were inferred from a balanced dataset. Subsequently, different schemes were tested to combine the individual classifiers: MIN operator, average ranking, average score, average voting, with MIN operator leading to the best performance. The homology model was based on the arginine/agmatine antiporter (AdiC) from Escherichia coli as template. It showed 64% coverage of the entire query sequence and it was selected based on the normalized Discrete Optimized Protein Energy parameter and the GA341 score. The modeled structure had 96% in the allowed area of Ramachandran's plot, and none of the residues located in non-allowed regions were involved in the active site of the transporter. Positivity Predictive Value surfaces were applied to optimize the score thresholds to be used in the ligand-based virtual screening step: for that purpose Positivity Predictive Value was charted as a function of putative yields of active in the range 0.001-0.010 and the Se/Sp ratio. With a focus on drug repositioning opportunities, DrugBank and Sweetlead databases were subjected to screening. Among 8 hits, cinnarizine, a drug frequently prescribed for motion sickness and balance disorder, was tested against T. cruzi epimastigotes and amastigotes, confirming its trypanocidal effects and its inhibitory effects on putrescine uptake. Furthermore, clofazimine, an antibiotic with already proven trypanocidal effects, also displayed inhibitory effects on putrescine uptake. Two other hits, meclizine and butoconazole, also displayed trypanocidal effects (in the case of meclizine, against both epimastigotes and amastigotes), without inhibiting putrescine uptake.