Integration Analysis of Pharmacokinetics and Metabolomics to Predict Metabolic Phenotype and Drug Exposure of Remdesivir

Remdesivir has displayed pharmacological activity against SARS-CoV-2. However, no pharmacometabolomics (PM) or correlation analysis with pharmacokinetics (PK) was revealed. Rats were intravenously administered remdesivir, and a series of blood samples were collected before and after treatment. Comprehensive metabolomics profile and PK were investigated and quantitated simultaneously using our previous reliable HPLC-MS/MS method. Both longitudinal and transversal metabolic analyses were conducted, and the correlation between PM and PK parameters was evaluated using Pearson’s correlation analysis and the PLS model. Multivariate statistical analysis was employed for discovering candidate biomarkers which predicted drug exposure or toxicity of remdesivir. The prominent metabolic profile variation was observed between pre- and posttreatment, and significant changes were found in 65 metabolites. A total of 15 metabolites—12 carnitines, one N-acetyl-D-glucosamine, one allantoin, and one corticosterone—were significantly correlated with the concentration of Nuc (active metabolite of remdesivir). Adenosine, spermine, guanosine, sn-glycero-3-phosphocholine, and l-homoserine may be considered potential biomarkers for predicting drug exposure or toxicity. This study is the first attempt to apply PM and PK to study remdesivir response/toxicity, and the identified candidate biomarkers might be used to predict the AUC and Cmax, indicating capability of discriminating good or poor responders. Currently, this study originally offers considerable evidence to metabolite reprogramming of remdesivir and sheds light on precision therapy development in fighting COVID-19.

Integrative Profiling of Amyotrophic Lateral Sclerosis Lymphoblasts Identifies Unique Metabolic and Mitochondrial Disease Fingerprints

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease with a rapid progression and no effective treatment. Metabolic and mitochondrial alterations in peripheral tissues of ALS patients may present diagnostic and therapeutic interest. We aimed to identify mitochondrial fingerprints in lymphoblast from ALS patients harboring SOD1 mutations (mutSOD1) or with unidentified mutations (undSOD1), compared with age/sex matched controls. Three groups of lymphoblasts, from mutSOD1 or undSOD1 ALS patients and age/sex-matched controls, were obtained from Coriell Biobank and divided into 3 age/sex-matched cohorts. Mitochondria-associated metabolic pathways were analyzed using Seahorse MitoStress and ATP Rate assays, complemented with metabolic phenotype microarrays, metabolite levels, gene expression, and protein expression and activity. Pooled (all cohorts) and paired (intra-cohort) analyses were performed by using bioinformatic tools, and the features with higher information gain values were selected and used for principal component analysis and Naïve Bayes classification. Pooled analysis revealed that undSOD1 patients had statistically higher glycolytic ATP production rate and lower Tfam protein content compared to controls, which were also the experimental features highlighted by multidimensional analysis. Metabolic phenotypic profiles in lymphoblasts from ALS patients with mutSOD1 and undSOD1 revealed unique age-dependent different substrate oxidation profiles. For most parameters, different patterns of variation were found between cohorts, which may be due to age or sex. In the present work, we investigated several metabolic and mitochondrial hallmarks in lymphoblasts from each donor and, although a high heterogeneity of results was found, we identified specific metabolic and mitochondrial fingerprints that may have a diagnostic and therapeutic interest.

Discovery of Small Molecule NSC290956 as a Therapeutic Agent for KRas Mutant Non-Small-Cell Lung Cancer

HRas-GTP has a transient intermediate state with a “non-signaling open conformation” in GTP hydrolysis and nucleotide exchange. Due to the same hydrolysis process and the structural homology, it can be speculated that the active KRas adopts the same characteristics with the “open conformation.” This implies that agents locking this “open conformation” may theoretically block KRas-dependent signaling. Applying our specificity-affinity drug screening approach, NSC290956 was chosen by high affinity and specificity interaction with the “open conformation” structure HRasG60A-GppNp. In mutant KRas-driven non-small-cell lung cancer (NSCLC) model system, NSC290956 effectively suppresses the KRas-GTP state and gives pharmacological KRas inhibition with concomitant blockages of both the MAPK-ERK and AKT-mTOR pathways. The dual inhibitory effects lead to the metabolic phenotype switching from glycolysis to mitochondrial metabolism, which promotes the cancer cell death. In the xenograft model, NSC290956 significantly reduces H358 tumor growth in nude mice by mechanisms similar to those observed in the cells. Our work indicates that NSC290956 can be a promising agent for the mutant KRas-driven NSCLC therapy.

Integrating in vitro metabolomics with a 96-well high-throughput screening platform

Introduction High-throughput screening (HTS) is emerging as an approach to support decision-making in chemical safety assessments. In parallel, in vitro metabolomics is a promising approach that can help accelerate the transition from animal models to high-throughput cell-based models in toxicity testing. Objective In this study we establish and evaluate a high-throughput metabolomics workflow that is compatible with a 96-well HTS platform employing 50,000 hepatocytes of HepaRG per well. Methods Low biomass cell samples were extracted for metabolomics analyses using a newly established semi-automated protocol, and the intracellular metabolites were analysed using a high-resolution spectral-stitching nanoelectrospray direct infusion mass spectrometry (nESI-DIMS) method that was modified for low sample biomass. Results The method was assessed with respect to sensitivity and repeatability of the entire workflow from cell culturing and sampling to measurement of the metabolic phenotype, demonstrating sufficient sensitivity (> 3000 features in hepatocyte extracts) and intra- and inter-plate repeatability for polar nESI-DIMS assays (median relative standard deviation < 30%). The assays were employed for a proof-of-principle toxicological study with a model toxicant, cadmium chloride, revealing changes in the metabolome across five sampling times in the 48-h exposure period. To allow the option for lipidomics analyses, the solvent system was extended by establishing separate extraction methods for polar metabolites and lipids. Conclusions Experimental, analytical and informatics workflows reported here met pre-defined criteria in terms of sensitivity, repeatability and ability to detect metabolome changes induced by a toxicant and are ready for application in metabolomics-driven toxicity testing to complement HTS assays.

The neural cell adhesion molecule NrCAM regulates development of hypothalamic tanycytes

Hypothalamic tanycytes are neural stem and progenitor cells, but little is known of how they are regulated. Here we provide evidence that the cell adhesion molecule, NrCAM, regulates tanycytes in the adult niche. NrCAM is strongly expressed in adult mouse tanycytes. Immunohistochemical and in situ hybridization analysis revealed that NrCAM loss of function leads to both a reduced number of tanycytes and reduced expression of tanycyte-specific cell markers, along with a small reduction in tyrosine hydroxylase-positive arcuate neurons. Similar analyses of NrCAM mutants at E16 identify few changes in gene expression or cell composition, indicating that NrCAM regulates tanycytes, rather than early embryonic hypothalamic development. Neurosphere and organotypic assays support the idea that NrCAM governs cellular homeostasis. Single-cell RNA sequencing (scRNA-Seq) shows that tanycyte-specific genes, including a number that are implicated in thyroid hormone metabolism, show reduced expression in the mutant mouse. However, the mild tanycyte depletion and loss of markers observed in NrCAM-deficient mice were associated with only a subtle metabolic phenotype.

Urine-Based Metabolomics and Machine Learning Reveals Metabolites Associated with Renal Cell Carcinoma Stage

Urine metabolomics profiling has potential for non-invasive RCC staging, in addition to providing metabolic insights into disease progression. In this study, we utilized liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and machine learning (ML) for the discovery of urine metabolites associated with RCC progression. Two machine learning questions were posed in the study: Binary classification into early RCC (stage I and II) and advanced RCC stages (stage III and IV), and RCC tumor size estimation through regression analysis. A total of 82 RCC patients with known tumor size and metabolomic measurements were used for the regression task, and 70 RCC patients with complete tumor-nodes-metastasis (TNM) staging information were used for the classification tasks under ten-fold cross-validation conditions. A voting ensemble regression model consisting of elastic net, ridge, and support vector regressor predicted RCC tumor size with a R2 value of 0.58. A voting classifier model consisting of random forest, support vector machines, logistic regression, and adaptive boosting yielded an AUC of 0.96 and an accuracy of 87%. Some identified metabolites associated with renal cell carcinoma progression included 4-guanidinobutanoic acid, 7-aminomethyl-7-carbaguanine, 3-hydroxyanthranilic acid, lysyl-glycine, glycine, citrate, and pyruvate. Overall, we identified a urine metabolic phenotype associated with renal cell carcinoma stage, exploring the promise of a urine-based metabolomic assay for staging this disease.

Combined intermittent and sustained hypoxia is a novel and deleterious cardio-metabolic phenotype

Study objectives Chronic obstructive pulmonary disease and obstructive sleep apnea overlap syndrome is associated with excess mortality, and outcomes are related to the degree of hypoxemia. People at high altitude are susceptible to periodic breathing, and hypoxia at altitude is associated with cardio-metabolic dysfunction. Hypoxemia in these scenarios may be described as superimposed sustained plus intermittent hypoxia, or overlap hypoxia (OH), the effects of which have not been investigated. We aimed to characterize the cardio-metabolic consequences of OH in mice. Methods C57BL/6J mice were subjected to either sustained hypoxia (SH, FiO2=0.10), intermittent hypoxia (IH, FiO2=0.21 for 12 hours, and FiO2 oscillating between 0.21 and 0.06, 60 times/hour, for 12 hours), OH (FiO2=0.13 for 12 hours, and FiO2 oscillating between 0.13 and 0.06, 60 times/hour, for 12 hours), or room air (RA), n=8/group. Blood pressure and intraperitoneal glucose tolerance test were measured serially, and right ventricular systolic pressure (RVSP) was assessed. Results Systolic blood pressure transiently increased in IH and OH relative to SH and RA. RVSP did not increase in IH, but increased in SH and OH by 52% (p&lt;0.001) and 20% (p=0.001). Glucose disposal worsened in IH and improved in SH, with no change in OH. Serum LDL and VLDL increased in OH and SH, but not in IH. Hepatic oxidative stress increased in all hypoxic groups, with the highest increase in OH. Conclusions Overlap hypoxia may represent a unique and deleterious cardio-metabolic stimulus, causing systemic and pulmonary hypertension, and without protective metabolic effects characteristic of sustained hypoxia.

Investigations of Intercellular Mitochondrial Transfer in Neural Cells by Applied Single Molecule Genotyping

<p>Over the past decade and a half, evidence for transfer of whole mitochondria between mammalian cells has emerged in the literature. The notion that mitochondria are restricted to the cell of origin has been overturned by this curious phenomenon, yet the physiological relevance of these transfer events remains unclear. This thesis investigates intercellular mitochondrial transfer in co-cultures of neural cells in vitro, to understand whether neural cells placed under stress demonstrate an enhanced rate of intercellular mitochondrial transfer. This would implicate the phenomenon as a cellular response to stress. Reliable techniques for quantitative study of intercellular mitochondrial transfer are limited so far in this field. To address this, a novel quantitative approach was developed to detect intercellular mitochondrial transfer, based on single molecule genotyping by target-primed rolling circle amplification. This enabled imaging of individual mitochondrial DNA molecules in situ, to detect those molecules which had moved between cells. Through this strategy, intercellular mitochondrial transfer was detected in new in vitro co-culture models. Primary murine pericytes derived from brain microvessels, were found to readily transfer mitochondria to a murine astrocyte cell line in vitro. Cisplatin, a DNA damaging agent; and chloramphenicol, a mitochondrial ribosome inhibitor, used to induce acute cellular injuries in the murine astrocyte cell line. These injuries were characterised and found to induce apoptosis, cause changes in growth characteristics, mitochondrial gene expression, and alter the metabolic phenotype of the cells. A derivative of the astrocyte cell line which completely lacks mitochondrial respiration, was found to model a chronic metabolic injury. As pericytes are prevalent throughout the brain, the pericyte/astrocyte co-culture model was selected to evaluate how the rate of intercellular mitochondrial transfer was altered, when the astrocytes were injured prior to co-culture. Through in situ single molecule genotyping and high throughput confocal microscopy, quantitative data was produced on how the rate of intercellular mitochondrial transfer was altered by injury in these models. The rate of intercellular mitochondrial transfer remained unaltered by chloramphenicol, however both cisplatin and the chronic metabolic injury model demonstrated reduced numbers of pericyte mitochondrial DNAs transferred into the injured astrocytes. These studies demonstrate successful application of a novel approach to study intercellular mitochondrial transfer and enable quantitative studies of this phenomenon.</p>