Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Branched chain amino acid metabolism profiles in progressive human nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a globally widespread disease of increasing clinical significance. The pathological progression of the disease from simple steatosis to nonalcoholic steatohepatitis (NASH) has been well defined, however, the contribution of altered branched chain amino acid metabolomic profiles to the progression of NAFLD is not known. The three BCAAs: leucine, isoleucine and valine are known to mediate activation of several important hepatic metabolic signaling pathways ranging from insulin signaling to glucose regulation. The purpose of this study is to profile changes in hepatic BCAA metabolite levels with transcriptomic changes in the progression of human NAFLD to discover novel mechanisms of disease progression. Metabolomic and transcriptomic data sets representing the spectrum of human NAFLD (normal, steatosis, NASH fatty, and NASH not fatty livers) were utilized for this study. During the transition from steatosis to NASH, increases in the levels of leucine (127% of normal), isoleucine (139%), and valine (147%) were observed. Carnitine metabolites also exhibited significantly elevated profiles in NASH fatty and NASH not fatty samples and included propionyl, hexanoyl, lauryl, acetyl and butyryl carnitine. Amino acid and BCAA metabolism gene sets were significantly enriched among downregulated genes during NASH. These cumulative alterations in BCAA metabolite and amino acid metabolism gene profiles represent adaptive physiological responses to disease-induced hepatic stress in NASH patients.

Comparison of physiologic and pharmacologic parameters in Asian and mauritius cynomolgus macaques.

This comparative study was conducted to assess background physiologic and pharmacologic parameters of cynomolgus macaques (Macaca fascicularis) from Cambodia, from a mixed Asian source (Cambodia, Vietnam and Indonesia), and from Mauritius. This evaluation provides a comprehensive assessment of several of these parameters in a single study. Ten male and 10 female captive-bred, age-matched macaques from each source were evaluated. Criteria for evaluation included weight gain, assessment of drug metabolizing enzyme activity, metabolomic analysis, immunologic assessments (lymphocyte subsets, TDAR, and serum Ig isotyping), clinical pathology evaluations, physical (respiratory, neurologic, cardiovascular, and ophthalmologic) examinations, pathogen screening, organ weights, and gross and microscopic pathology analyses. The results of this evaluation indicate that, compared to macaques of Asian origin, macaques from Mauritius had the lowest incidence and/or severity of spontaneous pathologic findings in several organs and tissues (lymphoid organs, stomach, kidney, urothelium, heart, arteries and lung) and better testicular maturity at a given age with minimal variability in organ weights. Although slight differences were observed in other parameters, none were considered detrimental to the use of macaques of Asian or Mauritius origin in pharmaceutical candidate safety studies with the use of a consistent source, concomitant controls, and appropriate background knowledge and screening.

1H NMR-based lipidomics of rodent fur: species-specific lipid profiles and SCD1 inhibitor-related dermal toxicity.

A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.

Characterization of hepatocellular carcinoma related genes and metabolites in human nonalcoholic fatty liver disease.

BACKGROUND: The worldwide prevalences of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are estimated to range from 30 to 40 % and 5-17 %, respectively. Hepatocellular carcinoma (HCC) is primarily caused by hepatitis B infection, but retrospective data suggest that 4-29 % of NASH cases will progress to HCC. Currently the connection between NASH and HCC is unclear. AIMS: The purpose of this study was to identify changes in expression of HCC-related genes and metabolite profiles in NAFLD progression. METHODS: Transcriptomic and metabolomic datasets from human liver tissue representing NAFLD progression (normal, steatosis, NASH) were utilized and compared to published data for HCC. RESULTS: Genes involved in Wnt signaling were downregulated in NASH but have been reported to be upregulated in HCC. Extracellular matrix/angiogenesis genes were upregulated in NASH, similar to reports in HCC. Iron homeostasis is known to be perturbed in HCC and we observed downregulation of genes in this pathway. In the metabolomics analysis of hepatic NAFLD samples, several changes were opposite to what has been reported in plasma of HCC patients (lysine, phenylalanine, citrulline, creatine, creatinine, glycodeoxycholic acid, inosine, and alpha-ketoglutarate). In contrast, multiple acyl-lyso-phosphatidylcholine metabolites were downregulated in NASH livers, consistent with observations in HCC patient plasma. CONCLUSIONS: These data indicate an overlap in the pathogenesis of NAFLD and HCC where several classes of HCC related genes and metabolites are altered in NAFLD. Importantly, Wnt signaling and several metabolites are different, thus implicating these genes and metabolites as mediators in the transition from NASH to HCC.

Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease.

Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the 'classical' (neutral) and 'alternative' (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH.

NMR-based metabolomics of mammalian cell and tissue cultures.

NMR spectroscopy was used to evaluate growth media and the cellular metabolome in two systems of interest to biomedical research. The first of these was a Chinese hamster ovary cell line engineered to express a recombinant protein. Here, NMR spectroscopy and a quantum mechanical total line shape analysis were utilized to quantify 30 metabolites such as amino acids, Krebs cycle intermediates, activated sugars, cofactors, and others in both media and cell extracts. The impact of bioreactor scale and addition of anti-apoptotic agents to the media on the extracellular and intracellular metabolome indicated changes in metabolic pathways of energy utilization. These results shed light into culture parameters that can be manipulated to optimize growth and protein production. Second, metabolomic analysis was performed on the superfusion media in a common model used for drug metabolism and toxicology studies, in vitro liver slices. In this study, it is demonstrated that two of the 48 standard media components, choline and histidine are depleted at a faster rate than many other nutrients. Augmenting the starting media with extra choline and histidine improves the long-term liver slice viability as measured by higher tissues levels of lactate dehydrogenase (LDH), glutathione and ATP, as well as lower LDH levels in the media at time points out to 94 h after initiation of incubation. In both models, media components and cellular metabolites are measured over time and correlated with currently accepted endpoint measures.

Identification of 1- and 3-methylhistidine as biomarkers of skeletal muscle toxicity by nuclear magnetic resonance-based metabolic profiling.

Nuclear magnetic resonance (NMR)-based metabolomic profiling identified urinary 1- and 3-methylhistidine (1- and 3-MH) as potential biomarkers of skeletal muscle toxicity in Sprague-Dawley rats following 7 and 14 daily doses of 0.5 or 1mg/kg cerivastatin. These metabolites were highly correlated to sex-, dose- and time-dependent development of cerivastatin-induced myotoxicity. Subsequently, the distribution and concentration of 1- and 3-MH were quantified in 18 tissues by gas chromatography-mass spectrometry. The methylhistidine isomers were most abundant in skeletal muscle with no fiber or sex differences observed; however, 3-MH was also present in cardiac and smooth muscle. In a second study, rats receiving 14 daily doses of 1mg/kg cerivastatin (a myotoxic dose) had 6- and 2-fold elevations in 1- and 3-MH in urine and had 11- and 3-fold increases in 1- and 3-MH in serum, respectively. Selectivity of these potential biomarkers was tested by dosing rats with the cardiotoxicant isoproterenol (0.5mg/kg), and a 2-fold decrease in urinary 1- and 3-MH was observed and attributed to the anabolic effect on skeletal muscle. These findings indicate that 1- and 3-MH may be useful urine and serum biomarkers of drug-induced skeletal muscle toxicity and hypertrophy in the rat, and further investigation into their use and limitations is warranted.

Metabolomic and transcriptomic changes induced by overnight (16 h) fasting in male and female Sprague-Dawley rats.

The overnight (16-h) fast is one of the most common experimental manipulations performed in rodent studies. Despite its ubiquitous employment, a comprehensive evaluation of metabolomic and transcriptomic sequelae of fasting in conjunction with routine clinical pathology evaluation has not been undertaken. This study assessed the impact of a 16-h fast on urine and serum metabolic profiles, transcript profiles of liver, psoas muscle, and jejunum as well as on routine laboratory clinical pathology parameters. Fasting rats had an approximate 12% relative weight decrease compared to ad libitum fed animals, and urine volume was significantly increased. Fasting had no effect on hematology parameters, though several changes were evident in serum and urine clinical chemistry data. In general, metabolic changes in biofluids were modest in magnitude but broad in extent, with a majority of measured urinary metabolites and from 1/3 to 1/2 of monitored serum metabolites significantly affected. Increases in fatty acids and bile acids dominated the upregulated metabolites. Downregulated serum metabolites were dominated by diet-derived and/or gut-microflora derived metabolites. Major transcriptional changes included genes with roles in fatty acid, carbohydrate, cholesterol, and bile acid metabolism indicating decreased activity in glycolytic pathways and a shift toward increased utilization of fatty acids. Typically, several genes within these metabolic pathways, including key rate limiting genes, changed simultaneously, and those changes were frequently correlative to changes in clinical pathology parameters or metabolomic data. Importantly, up- or down-regulation of a variety of cytochrome P450s, transporters, and transferases was evident. Taken together, these data indicate profound consequences of fasting on systemic biochemistry and raise the potential for unanticipated interactions, particularly when metabolomic or transcriptomic data are primary end points.

Urinary metabolites of 2-bromoethanamine identified by stable isotope labelling: evidence for carbamoylation and glutathione conjugation.

2-Bromoethanamine (BEA) causes renal papillary necrosis (RPN) in rats after a single dose and has been widely used as a model compound for studying the lesion. Although the metabolism of BEA may be an important determinant of toxicity, the metabolic fate of the compound has not been fully elucidated. To date, the only identified BEA metabolites are aziridine, 2-oxazolidone and 5-hydroxy-2-oxazolidone. In this study, stable isotope labelling (SIL) of BEA analogs ((¹³C and ²H) were used to differentiate generated BEA metabolites from endogenous molecules which enabled the accurate liquid chromatography mass spectrometry detection of more than 180 novel metabolites. BEA metabolism was evaluated in rats after acute administration of a non-toxic dose (50 mg/kg) and a toxic dose (250 mg/kg) that caused frank RPN and polyuria. Newly identified metabolites include three carbamoylation products, two mercapturic acids and a group of amino acid conjugates. Overall, the results indicate that BEA metabolism is very complex, suggest the potential formation of reactive intermediates and establish that BEA is subject to conjugation with glutathione. The results also demonstrate the utility and sensitivity of the SIL approach for identification of metabolites from small, reactive compounds.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS-/- CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10-19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500-L and 1000-L bioreactors replicated laboratory results using 5-L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Metabolomic and quality data for early and late passages of an antibody-producing industrial CHO cell line.

In this article, we provide four data sets for an industrial Chinese Hamster Ovary (CHO) cell line producing antibodies during a 14-day bioreactor run. This cell line was selected for further evaluation because of its significant titer loss as the cells were passaged over time. Four conditions that differed in cell bank ages were run for this dataset. Specifically, cells were passaged to passage 12, 21, 25, and 37 and then used in this experiment. Once the run commenced the following datasets were gathered: 1). Glycosylation data for each reactor 2). Size Exclusion Chromatography (SEC) data for the antibodies produced which allowed for the identification of high and low molecular weight species in the samples (N-Glycan and SEC data was taken on day 14 only). 3/4). Metabolites levels measured using Nuclear Magnetic Resonance (NMR) and liquid chromatography-mass spectroscopy (LC-MS) for all reactors over the time course of days 1, 4, 6, 8, 12, and 14. We also provide a graph of the glutamine levels for cells of different ages as an example of the utility of the data. These metabolomics data provide relative amounts for 36 metabolites (NMR) and 109 metabolites (LC-MS) over the 14-day time course. These data were collected in connection with a co-submitted paper [1].

Modulation of ascorbic acid metabolism by cytochrome P450 induction revealed by metabonomics and transcriptional profiling.

In the present study, NMR-based urinary metabonomic profiles resulting from dosing with widely recognized microsomal enzyme inducers were evaluated in male rats. Wistar or Sprague-Dawley rats were dosed daily by oral gavage with phenobarbital (PB; 100 mg/kg), diallyl sulfide (DAS; 500 mg/kg), the investigational compound DMP-904 (150 mg/kg), or beta-naphthoflavone (BNF; 100 mg/kg) for 4 days, and urine was collected daily for analysis. Compounds known to increase cytochrome P450 2B enzymes, including PB, DAS and DMP-904, increased the urinary excretion of gulonic and ascorbic acid in a time-dependent manner, reaching a maximum following 3-4 days of dosing. In contrast, BNF, an agent that induces primarily Cyp1A enzymes, did not increase gulonic or ascorbic acid excretion, despite inducing Cyp1A1 more than 200-fold. Given the metabonomic results, hepatic transcriptional changes in the regulation of ascorbic acid biosynthesis were determined by RT-PCR. All Cyp2B inducers increased hepatic mRNA levels of aldo-keto reductase 1A1, an enzyme that catalyzes the formation of gulonic acid from glucuronate with concurrent decreased expression of both regucalcin (Rgn), the enzyme responsible for conversion of gulonic acid to gulono-1, 4-lactone and gulonolactone oxidase (Gulo), the rate-limiting enzyme in ascorbate biosynthesis. These effects would be expected to increase levels of gulonic acid. In addition, Cyp2B inducers also increased hepatic expression of enzymes regulating ascorbic acid reutilization including glutaredoxin reductase (Glrx2) and thioredoxin reductase (Txnrd1). In contrast, BNF did not effect hepatic expression of any enzyme regulating gulonic or ascorbic acid biosynthesis. Thus, some microsomal enzyme inducers alter transcriptional regulation of ascorbic acid biosynthesis, and these changes are detected by noninvasive metabonomic profiling. However, not all microsomal enzyme inducers appear to alter ascorbic acid metabolism. Finally, the work illustrates how metabonomic results can direct additional studies to determine the biochemical mechanisms underlying changes in urinary metabolite excretion.

Identification of N-Methyl Nicotinamide and N-Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Identification of Imidazo[1,2-b]pyridazine Derivatives as Potent, Selective, and Orally Active Tyk2 JH2 Inhibitors.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

Nuclear magnetic resonance metabonomics: methods for drug discovery and development.

Nuclear magnetic resonance (NMR)-based metabonomics is gaining popularity in drug discovery and development and in academia in a variety of settings, ranging from toxicology, preclinical, and clinical approaches to nutrition research, studies on microorganisms, and research on plants. This chapter focuses on the basic steps in a metabonomics study and emphasizes experience and lessons learned in our lab where we focused on metabonomic analyses of plant extracts, cell lines, and a variety of animal tissues and biofluids. We emphasize that a comprehensive and suitable study design is pivotal for a correct biological interpretation of the results, as well as highly controlled experimental conditions. Sample preparation and NMR protocols are detailed for a wide range of sample types. We discuss alternative data processing strategies and considerations for a general data analysis approach, paying particular attention to the statistical interpretation and validation of the results while also highlighting approaches to avoid possible pitfalls resulting from systematic and random errors. A tutorial written for the R statistical package and other small utilities are available from the authors upon request.

Metabolomic profiling distinction of human nonalcoholic fatty liver disease progression from a common rat model.

OBJECTIVE: Characteristic pathological changes define the progression of steatosis to nonalcoholic steatohepatitis (NASH) and are correlated to metabolic pathways. A common rodent model of NASH is the methionine and choline deficient (MCD) diet. The objective of this study was to perform full metabolomic analyses on liver samples to determine which pathways are altered most pronouncedly in this condition in humans, and to compare these changes to rodent models of nonalcoholic fatty liver disease (NAFLD). METHODS: A principal component analysis for all 91 metabolites measured indicated that metabolome perturbation is greater and less varied for humans than for rodents. RESULTS: Metabolome changes in human and rat NAFLD were greatest for the amino acid and bile acid metabolite families (e.g., asparagine, citrulline, gamma-aminobutyric acid, lysine); although, in many cases, the trends were reversed when compared between species (cholic acid, betaine). CONCLUSIONS: Overall, these results indicate that metabolites of specific pathways may be useful biomarkers for NASH progression, although these markers may not correspond to rodent NASH models. The MCD model may be useful when studying certain end points of NASH; however, the metabolomics results indicate important differences between humans and rodents in the biochemical pathogenesis of the disease.

Understanding and Controlling Sialylation in a CHO Fc-Fusion Process.

A Chinese hamster ovary (CHO) bioprocess, where the product is a sialylated Fc-fusion protein, was operated at pilot and manufacturing scale and significant variation of sialylation level was observed. In order to more tightly control glycosylation profiles, we sought to identify the cause of variability. Untargeted metabolomics and transcriptomics methods were applied to select samples from the large scale runs. Lower sialylation was correlated with elevated mannose levels, a shift in glucose metabolism, and increased oxidative stress response. Using a 5-L scale model operated with a reduced dissolved oxygen set point, we were able to reproduce the phenotypic profiles observed at manufacturing scale including lower sialylation, higher lactate and lower ammonia levels. Targeted transcriptomics and metabolomics confirmed that reduced oxygen levels resulted in increased mannose levels, a shift towards glycolysis, and increased oxidative stress response similar to the manufacturing scale. Finally, we propose a biological mechanism linking large scale operation and sialylation variation. Oxidative stress results from gas transfer limitations at large scale and the presence of oxygen dead-zones inducing upregulation of glycolysis and mannose biosynthesis, and downregulation of hexosamine biosynthesis and acetyl-CoA formation. The lower flux through the hexosamine pathway and reduced intracellular pools of acetyl-CoA led to reduced formation of N-acetylglucosamine and N-acetylneuraminic acid, both key building blocks of N-glycan structures. This study reports for the first time a link between oxidative stress and mammalian protein sialyation. In this study, process, analytical, metabolomic, and transcriptomic data at manufacturing, pilot, and laboratory scales were taken together to develop a systems level understanding of the process and identify oxygen limitation as the root cause of glycosylation variability.

Metabonomics classifies pathways affected by bioactive compounds. Artificial neural network classification of NMR spectra of plant extracts.

The biochemical mode-of-action (MOA) for herbicides and other bioactive compounds can be rapidly and simultaneously classified by automated pattern recognition of the metabonome that is embodied in the 1H NMR spectrum of a crude plant extract. The ca. 300 herbicides that are used in agriculture today affect less than 30 different biochemical pathways. In this report, 19 of the most interesting MOAs were automatically classified. Corn (Zea mays) plants were treated with various herbicides such as imazethapyr, glyphosate, sethoxydim, and diuron, which represent various biochemical modes-of-action such as inhibition of specific enzymes (acetohydroxy acid synthase [AHAS], protoporphyrin IX oxidase [PROTOX], 5-enolpyruvylshikimate-3-phosphate synthase [EPSPS], acetyl CoA carboxylase [ACC-ase], etc.), or protein complexes (photosystems I and II), or major biological process such as oxidative phosphorylation, auxin transport, microtubule growth, and mitosis. Crude isolates from the treated plants were subjected to 1H NMR spectroscopy, and the spectra were classified by artificial neural network analysis to discriminate the herbicide modes-of-action. We demonstrate the use and refinement of the method, and present cross-validated assignments for the metabolite NMR profiles of over 400 plant isolates. The MOA screen also recognizes when a new mode-of-action is present, which is considered extremely important for the herbicide discovery process, and can be used to study deviations in the metabolism of compounds from a chemical synthesis program. The combination of NMR metabolite profiling and neural network classification is expected to be similarly relevant to other metabonomic profiling applications, such as in drug discovery.