The accumulation of triglycerides is associated with several disease states, consequently, modulation of lipid droplet biology by augmenting targets that affect triglycerides represents an attractive area of research. Unfortunately, there are limited options for high (er) throughput lipase activity readouts, especially those which do not involve radiolabeled tracers. The use of liquid chromatography-mass spectrometry analyses could facilitate screening efforts aimed at developing small molecule drug candidates. As a detection method, LC-MS is generally sensitive, highly specific and affords scalability to enhance throughput. Herein, we developed and validated an assay that enables studies of Patatin-like phospholipase domain-containing protein 3 (PNPLA3) activity. Since the literature implicates PNPLA3 mutations in the pathophysiology of fatty liver, we optimized the assay to enable studies of wild type and I148 M mutant forms of the enzyme. Initial studies utilized the detection of oleic acid but to improve sensitivity (and therein drive down protein requirements, especially for the I148 M mutant), the final version of the assay measured the production of diolein in 384-well plates. Good reproducibility (Z' ∼ 0.78) and sufficient signal:background ∼ 60:1 were observed. In summary, the hydrolysis of triolein, with subsequent quantitation of diolein, offers a robust lipase activity assay.

The invention of a therapeutic begins by characterizing features that differentiate healthy versus diseased states; this often presents as changes in the concentration of an analyte. Examples include elevated blood glucose in diabetes, high cholesterol in heart disease, and protein aggregation in neurodegeneration. Analyte concentrations reflect the (im)balance of synthetic and degradation rates; as such, aberrant biochemical kinetics drive the changes in endpoint concentration that define disease biology. Therapeutics aim to reset the concentration of a disease marker via modulation of biochemical kinetics. This is easy to understand for drugs directly targeting an enzyme in a pathway but, although less obvious, this can also be at the core of protein: protein interactions. For instance, stimulation of the insulin receptor changes the flux of several biochemical substrates (across multiple tissues); similarly, modulation of proprotein convertase subtilisin/kexin type 9-low density lipoprotein (PCSK9-LDL) receptor interactions alters cholesterol trafficking. These classic examples underscore the importance of studying biochemical kinetics at a clinical level. Here, we discuss how kinetic studies link disease biology with mechanism of action elucidation and screening. This has an immediate impact on (i) enabling in vitro-in vivo correlations in early discovery, (ii) enhancing exposure-response models aiding in human dose prediction, and (iii) providing support for biomarker plans, including clinical diagnostics. Mechanism of action studies can also influence modality selection; e.g., knowledge regarding target kinetics is needed when making decisions surrounding the development of a reversible inhibitor vs. an irreversible covalent modifier, or an intervention that affects target levels such as those which enhance protein degradation or reduce protein synthesis.

Microdroplet chemistry has recently gained much attention, as reactions can be greatly accelerated in microdroplets. This study reports its first application in pharmaceutical bioanalysis settings to measure glucose homeostasis with unprecedented speed and sensitivity. Quantifying metabolic flux is critical for understanding drug action, but traditional isotope kinetic assays face challenges including the need of lengthy sample preparation and using radioactive tracers. To tackle these challenges, we used microdroplet reaction to facilitate on-the-fly derivatization in microseconds during mass spectrometry (MS) analysis, which increased sample throughput and sensitivity and avoided the use of radioactive tracers. This technique allowed us to elucidate a mechanism through which Semaglutide affects the levels of endogenous 12C-glucose and exogenous [U-13C6]glucose in mouse plasma and to quantify Pioglitazone-mediated changes of glucose uptake in mouse hearts. Such microdroplet-based bioanalysis would have an immediate high impact in characterizing disease phenotypes and guiding a mechanistic understanding of new drug discovery.

Protein kinetics can be quantified by coupling stable isotope tracer methods with mass spectrometry readouts; however, interconnected decision points in the experimental design affect the complexity of the workflow and impact data interpretations. For example, choosing between a single bolus (pulse-chase) or a continuous exposure protocol influences subsequent decisions regarding when to measure and how to model the temporal labeling of a target protein. Herein, we examine the merits of in vivo tracer protocols, and we direct attention toward stable isotope tracer experiments that rely on administering a single bolus since these are generally more practical to use as compared with continuous administration protocols. We demonstrate how the interplay between precursor and product kinetics impacts downstream analytics and calculations by contrasting fast versus slow turnover precursors (e.g., 13C-leucine vs. 2H-water, respectively). Although the data collected here underscore certain advantages of using longer-lived precursors (e.g., 2H- or 18O-water), the results also highlight the influence of tracer recycling on measures of protein turnover. We discuss the impact of tracer recycling and consider how the sampling interval is critical for interpreting studies. Finally, we demonstrate that tracer recycling does not limit the ability to perform back-to-back studies of protein kinetics. It is possible to run experiments in which subjects are used as their own controls even though the precursor and product remain labeled following an initial tracer dosing.NEW & NOTEWORTHY We demonstrate a simple and robust protocol for measuring protein synthesis, the work considers problems encountered in experimental design. The logic can enable biologists with limited resources and/or can facilitate scenarios where higher throughput experiments are needed.

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.

Introduction Our untargeted metabolic data unveiled that Acyl-CoAs undergo dephosphorylation, however little is known about these novel metabolites and their physiology/pathology relevance. Objectives To understand the relationship between acyl-CoAs dephosphorylation and energy status as implied in our previous work, we seek to investigate how ischemia (energy depletion) triggers metabolic changes, specifically acyl-CoAs dephosphorylation in this work. Methods Rat hearts were isolated and perfused in Langendorff mode for 15 min followed by 0, 5, 15, and 30 minutes of global ischemia. The heart tissues were harvested for metabolic analysis. Results As expected, ATP and phosphocreatine were significantly decreased during ischemia. Most short- and medium-chain acyl-CoAs progressively increased with ischemic time from 0 to 15 min, whereas a 30-minute ischemia did not lead to further change. Unlike other acyl-CoAs, propionyl-CoA accumulated progressively in the hearts that underwent ischemia from 0 to 30 min. Progressive dephosphorylation occurred to all assayed acyl-CoAs and free CoA regardless their level changes during the ischemia. Conclusion The present work further confirms that dephosphorylation of acyl-CoAs is an energy-dependent process and how this dephosphorylation is mediated warrants further investigations. It is plausible that dephosphorylation of acyl-CoAs and limited anaplerosis are involved in ischemic injuries to heart. Further investigations are warranted to examine the mechanisms of acyl-CoA dephosphorylation and how the dephosphorylation is possibly involved in ischemic injuries.

A novel method to test human intestinal lipid handling using stable isotope labeling is presented and, for the first time, plasma appearance and lipid turnover were quantified in 12 healthy men following meals with varying amounts of fat. The method can be applied to studies in precision nutrition characterizing individual response to support basic science research or drug development. This report discusses key questions for consideration in precision nutrition that were highlighted by the data.

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

Natural product congeners serve a useful role in the understanding of natural product biosynthesis and structure-activity relationships. A minor congener with superior activity, selectivity, and modifiable functional groups could serve as a more effective lead structure and replace even the original lead molecule that was used for medicinal chemistry modifications. Currently, no effective method exists to discover targeted congeners rapidly, specifically, and selectively from producing sources. Herein, a new method based on liquid-chromatography tandem-mass spectrometry combination is evaluated for targeted discovery of congeners of platensimycin and platencin from the extracts of Streptomyces platensis. By utilizing a precursor-ion searching protocol, tandem mass spectrometry not only confirmed the presence of known congeners but also provided unambiguous detection of many previously unknown congeners of platensimycin and platencin. This high-throughput and quantitative method can be rapidly and broadly applied for dereplication and congener discovery from a variety of producing sources, even when the targeted compounds are obscured by the presence of unrelated natural products.

Screening campaigns, especially those aimed at modulating enzyme activity, often rely on measuring substrate→product conversions. Unfortunately, the presence of endogenous substrates and/or products can limit one's ability to measure conversions. As well, coupled detection systems, often used to facilitate optical readouts, are subject to interference. Stable isotope labeled substrates can overcome background contamination and yield a direct readout of enzyme activity. Not only can isotope kinetic assays enable early screening, but they can also be used to follow hit progression in translational (pre)clinical studies. Herein, we consider a case study surrounding lipid biology to exemplify how metabolic flux analyses can connect stages of drug development, caveats are highlighted to ensure reliable data interpretations. For example, when measuring enzyme activity in early biochemical screening it may be enough to quantify the formation of a labeled product. In contrast, cell-based and in vivo studies must account for variable exposure to a labeled substrate (or precursor) which occurs via tracer dilution and/or isotopic exchange. Strategies are discussed to correct for these complications. We believe that measures of metabolic flux can help connect structure-activity relationships with pharmacodynamic mechanisms of action and determine whether mechanistically differentiated biophysical interactions lead to physiologically relevant outcomes. Adoption of this logic may allow research programs to (i) build a critical bridge between primary screening and (pre)clinical development, (ii) elucidate biology in parallel with screening and (iii) suggest a strategy aimed at in vivo biomarker development.

Lipid oxidation and biosynthesis are crucial for cell survival, especially for rapidly proliferating cancer cells in a heterogeneous metabolic environment. The storage of high-energy lipid reservoirs competitively advantages the cancer cell over non-neoplastic tissue. Disrupting lipid biosynthetic processes, through modulation of fatty acid (FA) esterification or de novo lipogenesis (DNL), is of interest in drug discovery. Mimicking the in vivo environment in vitro is also vital for testing the efficacy of potential drug compounds. We present here a stable isotope tracer-based approach for examining the impact of exogenous FA and oxygen tension on the pathways that affect lipid biosynthesis, including the rates of metabolic flux. By applying tandem mass spectrometry (MS/MS) analyses to studies using parallel tracers, we characterized the impact of FA bioavailability on the positional enrichment within specific lipids. Our observations suggest that adding bioavailable FA as a carbon source preferentially biases the cellular metabolism away from DNL and toward esterification of free fatty acid pools. Additionally, we have found that this FA addition, under hypoxic conditions, led to a biased increase in the total triglyceride pool (nearly 5-fold, as compared to phospholipids), regardless of the isotope tracer utilized. We discuss the implications of this metabolic flexibility on studies that aim to characterize apparent drug efficacy.

Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-β, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-β signaling and fibrosis.

The ability to target specific proteins for degradation may open a new door toward developing therapeutics. Although effort in chemistry is essential for advancing this modality, i.e., one needs to generate proteolysis targeting chimeras (bifunctional molecules, also referred to as PROTACS) or "molecular glues" to accelerate protein degradation, we suspect that investigations could also benefit by directing attention toward physiological regulation surrounding protein homeostasis, including the methods that can be used to examine changes in protein kinetics. This perspective will first consider some metabolic scenarios that might be of importance when one aims to change protein abundance by increasing protein degradation. Specifically, could protein turnover impact the apparent outcome? We will then outline how to study protein dynamics by coupling stable isotope tracer methods with mass spectrometry-based detection; since the experimental conditions could have a dramatic effect on protein turnover, special attention is directed toward the application of methods for quantifying protein kinetics using in vitro and in vivo models. Our goal is to present key concepts that should enable mechanistically informed studies which test targeted protein degradation strategies.

Cellular availability of acetyl-CoA, a central intermediate of metabolism, regulates histone acetylation. The impact of a high-fat diet (HFD) on the turnover rates of acetyl-CoA and acetylated histones is unknown. We developed a method for simultaneous measurement of acetyl-CoA and acetylated histones kinetics using a single 2H2O tracer, and used it to examine effect of HFD-induced perturbations on hepatic histone acetylation in LDLR-/- mice, a mouse model of non-alcoholic fatty liver disease (NAFLD). Mice were given 2H2O in the drinking water and the kinetics of hepatic acetyl-CoA, histones, and acetylated histones were quantified based on their 2H-labeling. Consumption of a high fat Western-diet (WD) for twelve weeks led to decreased acetylation of hepatic histones (p< 0.05), as compared to a control diet. These changes were associated with 1.5-3-fold increased turnover rates of histones without any change in acetyl-CoA flux. Acetylation significantly reduced the stability of histones and the turnover rates of acetylated peptides were correlated with the number of acetyl groups in neighboring lysine sites. We conclude that 2H2O-method can be used to study metabolically controlled histone acetylation and acetylated histone turnover in vivo.

BACKGROUND: The development of treatments for cognitive deficits associated with central nervous system disorders is currently a significant medical need. Despite the great need for such therapeutics, a significant challenge in the drug development process is the paucity of robust biomarkers to assess target modulation and guide clinical decisions. We developed a novel, translatable biomarker of neuronal glutamate metabolism, the C-13-glutamate+glutamine (Glx) H3:H4 labeling ratio, in nonhuman primates using localized H-1-magnetic resonance spectroscopy combined with C-13-glucose infusions. METHODS: We began with numerical simulations in an established model of brain glutamate metabolism, showing that the C-13-Glx H3:H4 ratio should be a sensitive biomarker of neuronal tricarboxylic acid cycle activity, a key measure of overall neuronal metabolism. We showed that this biomarker can be measured reliably using a standard H-1-magnetic resonance spectroscopy method (point-resolved spectroscopy sequence/echo time = 20 ms), obviating the need for specialized hardware and pulse sequences typically used with C-13-magnetic resonance spectroscopy, thus improving overall clinical translatability. Finally, we used this biomarker in 8 male rhesus macaques before and after administration of the compound BNC375, a positive allosteric modulator of the alpha 7 nicotinic acetylcholine receptor that enhances glutamate signaling ex vivo and elicits procognitive effects in preclinical species. RESULTS: The C-13-Glx H3:H4 ratios in the monkeys showed that BNC375 increases neuronal metabolism in nonhuman primates in vivo, detectable on an individual basis. CONCLUSIONS: This study demonstrates that the ratio of C-13-Glx H3:H4 labeling is a biomarker that may provide an objective readout of compounds affecting glutamatergic neurotransmission and could improve decision making for the development of therapeutic agents.

"Omics"-based analyses are widely used in numerous areas of research, advances in instrumentation (both hardware and software) allow investigators to collect a wealth of data and therein characterize metabolic systems. Although analyses generally examine differences in absolute or relative (fold-) changes in concentrations, the ability to extract mechanistic insight would benefit from the use of isotopic tracers. Herein, we discuss important concepts that should be considered when stable isotope tracers are used to capture biochemical flux. Special attention is placed on in vivo systems, however, many of the general ideas have immediate impact on studies in cellular models or isolated-perfused tissues. While it is somewhat trivial to administer labeled precursor molecules and measure the enrichment of downstream products, the ability to make correct interpretations can be challenging. We will outline several critical factors that may influence choices when developing and/or applying a stable isotope tracer method. For example, is there a "best" tracer for a given study? How do I administer a tracer? When do I collect my sample(s)? While these questions may seem straightforward, we will present scenarios that can have dramatic effects on conclusions surrounding apparent rates of metabolic activity.

Spatial characterization of triglyceride metabolism is an area of significant interest which can be enabled by mass spectrometry imaging via recent advances in neutral lipid laser desorption analytical approaches. Here, we extend recent advancements in gold-assisted neutral lipid imaging and demonstrate the potential to map lipid flux in rodents. We address here critical issues surrounding the analytical configuration and interpretation of the data for a group of select triglycerides. Specifically, we examined how the signal intensity and spatial resolution would impact the apparent isotope ratio in a given analyte (which is an important consideration when performing MS based kinetics studies of this kind) with attention given to molecular ions and not fragments. We evaluated the analytics by contrasting lipid flux in well characterized mouse models, including fed vs fed states and different dietary perturbations. In total, the experimental paradigm described here should enable studies of hepatic lipogenesis; presumably, this logic can be enhanced via the inclusion of ion mobility and/or fragmentation. Although this study was carried out in robust models of liver lipogenesis, we expect that the model system could be expanded to a variety of tissues where zonated (or heterogeneous) lipid synthesis may occur, including solid tumor metabolism.

Stable isotope tracers can be used to quantify the activity of metabolic pathways. Specifically, 2H-water is quite versatile, and its incorporation into various products can enable measurements of carbohydrate, lipid, protein and nucleic acid kinetics. However, since there are limits on how much 2H-water can be administered and since some metabolic processes may be slow, it is possible that one may be challenged with measuring small changes in isotopic enrichment. We demonstrate an advantage of the isotope fractionation that occurs during gas chromatography, namely, setting tightly bounded integration regions yields a powerful approach for determining isotope ratios. We determined how the degree of isotope fractionation, chromatographic peak width and mass spectrometer dwell time can increase the apparent isotope labeling. Relatively simple changes in the logic surrounding data acquisition and processing can enhance gas chromatography-mass spectrometry measures of low levels of 2H-labeling, this is especially useful when asymmetrical peaks are recorded at low signal:background. Although we have largely focused attention on alanine (which is of interest in studies of protein synthesis), it should be possible to extend the concepts to other analytes and/or hardware configurations.

The regulation of nutrient homeostasis, i.e., the ability to transition between fasted and fed states, is fundamental in maintaining health. Since food is typically consumed over limited (anabolic) periods, dietary components must be processed and stored to counterbalance the catabolic stress that occurs between meals. Herein, we contrast tissue- and pathway-specific metabolic activity in fasted and fed states. We demonstrate that knowledge of biochemical kinetics that is obtained from opposite ends of the energetic spectrum can allow mechanism-based differentiation of healthy and disease phenotypes. Rat models of type 1 and type 2 diabetes serve as case studies for probing spatial and temporal patterns of metabolic activity via [2H]water labeling. Experimental designs that capture integrative whole body metabolism, including meal-induced substrate partitioning, can support an array of research surrounding metabolic disease; the relative simplicity of the approach that is discussed here should enable routine applications in preclinical models.

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.