Enolase inhibitors as therapeutic leads for Naegleria fowleri infection.

Infections with the pathogenic free-living amoebae Naegleria fowleri can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen. Recently, human enolase 2 (ENO2) phosphonate inhibitors have been developed as lead agents to treat glioblastoma multiforme (GBM). These compounds, which cure GBM in a rodent model, are well-tolerated in mammals because enolase 1 (ENO1) is the predominant isoform used systemically. Here, we describe findings that demonstrate that these agents are potent inhibitors of N. fowleri ENO ( Nf ENO) and are lethal to amoebae. In particular, (1-hydroxy-2-oxopiperidin-3-yl) phosphonic acid (HEX) was a potent enzyme inhibitor (IC 50 value of 0.14 ± 0.04 µM) that was toxic to trophozoites (EC 50 value of 0.21 ± 0.02 µM) while the reported CC 50 was >300 µM. Molecular docking simulation revealed that HEX binds strongly to the active site of Nf ENO with a binding affinity of -8.6 kcal/mol. Metabolomic studies of parasites treated with HEX revealed a 4.5 to 78-fold accumulation of glycolytic intermediates upstream of Nf ENO. Last, nasal instillation of HEX increased longevity of amoebae-infected rodents. Two days after infection, animals were treated for 10 days with 3 mg/kg HEX, followed by one week of observation. At the conclusion of the experiment, eight of 12 HEX-treated animals remained alive (resulting in an indeterminable median survival time) while one of 12 vehicle-treated rodents remained, yielding a median survival time of 10.9 days. Brains of six of the eight survivors were positive for amoebae, suggesting the agent at the tested dose suppressed, but did not eliminate, infection. These findings suggest that HEX is a promising lead for the treatment of PAM.

Altered urinary tryptophan metabolites in alcohol-associated liver disease.

BACKGROUND: Alcohol-associated liver disease (ALD) leads to millions of deaths worldwide annually. A few potential biomarkers of ALD have been discovered through metabolomic or proteomic analysis. Tryptophan (Trp), one of nine essential amino acids, has been extensively studied and shown to play significant roles in many mammalian physiological processes. However, Trp metabolism changes in ALD are not yet fully understood. Whereas urine is an abundant and non-invasive source for disease biomarker discovery the current study investigated whether the abundance of Trp metabolites in the urine of ALD patients differs from that of healthy subjects. We also examined whether, if present in ALD, changes in urinary Trp metabolites can serve as markers for differentiating between mild/moderate and severe ALD. METHODS: We quantified the concentration of Trp and its metabolites in urine samples of healthy controls (n = 18), patients with mild or moderate alcohol-related liver injury (non-severe ALD; n = 21), and patients with severe alcohol-associated hepatitis (severe AH; n = 25) using both untargeted and targeted metabolomics. RESULTS: Eighteen Trp metabolites were identified and quantified from the untargeted metabolomics data. We developed a targeted metabolomics method to quantify the Trp and its metabolites and quantified 17 metabolites from the human urine samples. The data acquired in the untargeted and targeted platforms agreed and showed that the Trp concentration is not affected by the severity of ALD. However, the abundance of 10 Trp metabolites was correlated with the model for end-stage liver disease (MELD) score, with the abundance of nine metabolites significantly different between the healthy control and ALD patient groups. CONCLUSION: We found that Trp metabolism differs between ALD patients and healthy controls even though the concentration of Trp was not affected. Two Trp metabolites, quinolinic acid and indoxyl sulfate, correlate highly with the severity of ALD.

Stable Isotope Tracing Reveals an Altered Fate of Glucose in N-Acetyltransferase 1 Knockout Breast Cancer Cells.

Breast cancer is one of the leading causes of cancer death. Recent studies found that arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer, further suggesting NAT1 could be a potential therapeutic target for breast cancer. Previous publications have established that NAT1 knockout (KO) in breast cancer cell lines leads to growth reduction both in vitro and in vivo and metabolic changes. These reports suggest that NAT1 contributes to the energy metabolism of breast cancer cells. Proteomic analysis and non-targeted metabolomics suggested that NAT1 KO may change the fate of glucose as it relates to the TCA/KREB cycle of the mitochondria of breast cancer cells. In this current study, we used [U-13C]-glucose stable isotope resolved metabolomics to determine the effect of NAT1 KO on the metabolic profile of MDA-MB-231 breast cancer cells. We incubated breast cancer cells (MDA-MB-231 cells) and NAT1 Crispr KO cells (KO#2 and KO#5) with [U-13C]-glucose for 24 h. Tracer incubation polar metabolites from the cells were extracted and analyzed by 2DLC-MS, and metabolite differences were compared between the parental and NAT1 KO cells. Differences consistent between the two KO cells were considered changes due to the loss of NAT1. The data revealed decreases in the 13C enrichment of TCA/Krebs cycle intermediates in NAT1 KO cells compared to the MDA-MB-231 cells. Specifically, 13C-labeled citrate, isocitrate, a-ketoglutarate, fumarate, and malate were all decreased in NAT1 KO cells. We also detected increased 13C-labeled L-lactate levels in the NAT1 KO cells and decreased 13C enrichment in some nucleotides. Pathway analysis showed that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and the TCA cycle were most affected. These data provide additional evidence supporting the impacts of NAT1 knockout on cellular energy metabolism. The data suggest that NAT1 expression is important for the proper functioning of mitochondria and the flux of glucose through the TCA/Krebs cycle in breast cancer cells. The metabolism changes in the fate of glucose in NAT1 KO breast cancer cells offer more insight into the role of NAT1 in energy metabolism and the growth of breast cancer cells. These data provide additional evidence that NAT1 may be a useful therapeutic target for breast cancer.

Metabolomics analysis of urine from patients with alcohol-associated liver disease reveals dysregulated caffeine metabolism.

Excess alcohol intake causes millions of deaths annually worldwide. Asymptomatic early-stage, alcohol-associated liver disease (ALD) is easily overlooked, and ALD is usually only diagnosed in more advanced stages. We explored the possibility of using polar urine metabolites as biomarkers of ALD for early-stage diagnosis and functional assessment of disease severity by quantifying the abundance of polar metabolites in the urine samples of healthy controls (n = 18), patients with mild or moderate liver injury (n = 21), and patients with severe alcohol-associated hepatitis (n = 25). The polar metabolites in human urine were first analyzed by untargeted metabolomics, showing that 209 urine metabolites are significantly changed in patients, and 17 of these are highly correlated with patients' model for end-stage liver disease (MELD) score. Pathway enrichment analysis reveals that the caffeine metabolic pathway is the most affected in ALD. We then developed a targeted metabolomics method and measured the concentration of caffeine and its metabolites in urine using internal and external standard calibration, respectively. The described method can quantify caffeine and its 14 metabolites in 35 min. The results of targeted metabolomics analysis agree with the results of untargeted metabolomics, showing that 13 caffeine metabolites are significantly decreased in patients. In particular, the concentrations of 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil are markedly decreased with increased disease severity. We suggest that these three metabolites could serve as functional biomarkers for differentiating early-stage ALD from more advanced liver injury.NEW & NOTEWORTHY Our study using both untargeted and targeted metabolomics reveals the caffeine metabolic pathway is dysregulated in ALD. Three caffeine metabolites, 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil, can differentiate the severity of early-stage ALD.

Metabolic Analysis of Nucleosides/Bases in the Urine and Serum of Patients with Alcohol-Associated Liver Disease.

Accumulating evidence supports the important role of RNA modifications in liver disease pathogenesis. However, RNA modifications in alcohol-associated liver disease (ALD) have not yet been reported. Modified ribonucleosides/bases are products of RNA degradation; therefore, we investigated whether modified ribonucleosides/bases in human urine and serum are changed and whether these changes are associated with the severity of ALD. Human urine and serum samples from patients with ALD and appropriate controls were collected. Free nucleosides/bases were extracted from these samples and quantified using untargeted and targeted metabolomic approaches. Thirty-nine and forty free nucleosides/bases were respectively detected in human urine and serum samples. Twelve and eleven modified nucleosides are significantly changed in patients' urine and serum (q < 0.05 and fold-change > 20%). The abundance of modified nucleobase and ribonucleoside, 7,9-dimethylguanine in urine and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in serum are strongly associated with the severity of ALD. Spearman's rank correlation coefficient of these two metabolites with the Model for End-stage Liver Disease (MELD) score are 0.66 and 0.74, respectively. Notably, the abundance changes in these two metabolites are sufficiently large to distinguish severe alcohol-associate hepatitis (AH) from non-severe ALD and non-severe ALD from healthy controls.

Metabolic Profiling of Bile Acids in the Urine of Patients with Alcohol-Associated Liver Disease.

Bile acids (BAs) play important functions in the development of alcohol-associated liver disease (ALD). In the current study, urine BA concentrations in 38 patients with well-described alcohol-associated hepatitis (AH) as characterized by Model for End-Stage Liver Disease (MELD), 8 patients with alcohol-use disorder (AUD), and 19 healthy controls (HCs) were analyzed using liquid chromatography-mass spectrometry. Forty-three BAs were identified, and 22 BAs had significant changes in their abundance levels in patients with AH. The potential associations of clinical data were compared to candidate BAs in this pilot proof-of-concept study. MELD score showed positive correlations with several conjugated BAs and negative correlations with certain unconjugated BAs; taurine-conjugated chenodeoxycholic acid (CDCA) and MELD score showed the highest association. Cholic acid, CDCA, and apocholic acid had nonsignificant abundance changes in patients with nonsevere ALD compared to HCs but were significantly increased in those with severe AH. Receiver operating characteristic analysis showed that the differences in these three compounds were sufficiently large to distinguish severe AH from nonsevere ALD. Notably, the abundance levels of primary BAs were significantly increased while most of the secondary BAs were markedly decreased in AH compared to AUD. Most importantly, the amount of total BAs and the ratio of primary to secondary BAs increased while the ratio of unconjugated to conjugated BAs decreased as disease severity increased. Conclusion: Abundance changes of specific BAs are closely correlated with the severity of AH in this pilot study. Urine BAs (individually or as a group) could be potential noninvasive laboratory biomarkers for detecting early stage ALD and may have prognostic value in AH morbidity.

Analysis of sex differences in dietary copper-fructose interaction-induced alterations of gut microbial activity in relation to hepatic steatosis.

BACKGROUND: Inadequate copper intake and increased fructose consumption represent two important nutritional problems in the USA. Dietary copper-fructose interactions alter gut microbial activity and contribute to the development of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to determine whether dietary copper-fructose interactions alter gut microbial activity in a sex-differential manner and whether sex differences in gut microbial activity are associated with sex differences in hepatic steatosis. METHODS: Male and female weanling Sprague-Dawley (SD) rats were fed ad libitum with an AIN-93G purified rodent diet with defined copper content for 8 weeks. The copper content is 6 mg/kg and 1.5 mg/kg in adequate copper diet (CuA) and marginal copper diet (CuM), respectively. Animals had free access to either deionized water or deionized water containing 10% fructose (F) (w/v) as the only drink during the experiment. Body weight, calorie intake, plasma alanine aminotransferase, aspartate aminotransferase, and liver histology as well as liver triglyceride were evaluated. Fecal microbial contents were analyzed by 16S ribosomal RNA (16S rRNA) sequencing. Fecal and cecal short-chain fatty acids (SCFAs) were determined by gas chromatography-mass spectrometry (GC-MS). RESULTS: Male and female rats exhibit similar trends of changes in the body weight gain and calorie intake in response to dietary copper and fructose, with a generally higher level in male rats. Several female rats in the CuAF group developed mild steatosis, while no obvious steatosis was observed in male rats fed with CuAF or CuMF diets. Fecal 16S rRNA sequencing analysis revealed distinct alterations of the gut microbiome in male and female rats. Linear discriminant analysis (LDA) effect size (LEfSe) identified sex-specific abundant taxa in different groups. Further, total SCFAs, as well as, butyrate were decreased in a more pronounced manner in female CuMF rats than in male rats. Of note, the decreased SCFAs are concomitant with the reduced SCFA producers, but not correlated to hepatic steatosis. CONCLUSIONS: Our data demonstrated sex differences in the alterations of gut microbial abundance, activities, and hepatic steatosis in response to dietary copper-fructose interaction in rats. The correlation between sex differences in metabolic phenotypes and alterations of gut microbial activities remains elusive.

Decreased ω-6:ω-3 PUFA ratio attenuates ethanol-induced alterations in intestinal homeostasis, microbiota, and liver injury.

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies, including liver injury. ω-6 PUFAs exert pro-inflammatory activity, while ω-3 PUFAs promote anti-inflammatory activity that is mediated, in part, through specialized pro-resolving mediators [e.g., resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the ω-6:ω-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. ω-3 FA desaturase-1 (fat-1) mice, which endogenously increase ω-3 PUFA levels, were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-γ, Il-6, and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation, stem cell renewal, and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further, gut microbiome and plasma metabolomics revealed several changes in fat-1 versus WT mice that may contribute to a reduced inflammatory response. Finally, these data correlated with a significant reduction in liver injury. Our study suggests that ω-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury.

Simultaneous Quantification of Nucleosides and Nucleotides from Biological Samples.

We report a reverse phase chromatography mass spectrometry (LC-MS) method for simultaneous quantification of nucleosides and nucleotides from biological samples, where compound identification was achieved by a tier-wise approach and compound quantification was achieved via external calibration. A total of 65 authentic standards of nucleosides and nucleotides were used for the platform development. The limit of detection (LOD) of those compounds ranged from 0.05 nmol/L to 1.25 µmol/L, and their limit of quantification (LOQ) ranged from 0.10 nmol/L to 2.50 µmol/L. Using the developed method, nucleosides and nucleotides from human plasma, human urine, and rat liver were quantified. Seventy-nine nucleosides and nucleotides were identified from human urine and 28 of them were quantified with concentrations of 13.0 nmol/L-151 µmol/L. Fifty-five nucleosides and nucleotides were identified from human plasma and 22 of them were quantified with concentrations of 1.21 nmol/L-8.54 µmol/L. Fifty-one nucleosides and nucleotides were identified from rat liver and 23 were quantified with concentrations of 1.03 nmol/L-31.7 µmol/L. These results demonstrate that the developed method can be used to investigate the concentration change of nucleosides and nucleotides in biological samples for the purposes of biomarker discovery or elucidation of disease mechanisms.