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Enzyme assay techniques and protocols
Abstract
Enzyme assays are standardized experimental protocols, which are established in order to measure the activity or concentration of enzymes in biochemical or cell-based systems. Mostly enzymatic assays are based upon the detection of fluorescent, luminescent, or spectrophotometric endpoint signal. During recent years, florescence-based, absorbance-based, and luminescence-based high-throughput screening assays have been developed and widely used in various fields. Enzyme assays represent an important tool for screening of sample libraries in context of drug discovery. In addition, enzyme assays are valuable for diagnosis of several diseases in clinical settings due to their high sensitivity, specificity, and rapid response. In this chapter, we aim to provide an understanding of the working principle of enzymatic assays and their applications with some specific examples of enzyme assays that are used for drug discovery and diagnostic purpose.
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IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias and are associated with various regulation of molecular process. Mutations of active site in IDH1 could lead to high levels of 2-HG and the suppression of cellular differentiation, while these changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as a novel IDH1-R132H/C inhibitor, with the IC50 value of 0.41 μmol/L and 2.7 μmol/L respectively. In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to specific interaction. Further computational docking studies and structure-activity relationship (SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new insight for the pharmacological treatment of gliomas.
Elevated glycolysis remains a universal and primary character of cancer metabolism, which deeply depends on dysregulated metabolic enzymes. Lactate dehydrogenase A (LDHA) facilitates glycolytic process by converting pyruvate to lactate. Numerous researches demonstrate LDHA has an aberrantly high expression in multiple cancers, which is associated with malignant progression. In this review, we summarized LDHA function in cancer research. First, we gave an introduction of structure, location, and basic function of LDHA. Following, we discussed the transcription and activation mode of LDHA. Further, we focused on the function of LDHA in cancer bio‐characteristics. Later, we discussed the clinical practice of LDHA in cancer prevention and treatment. What we discussed gives a precise insight into LDHA especially in cancer research, which will contribute to exploring cancer pathogenesis and its handling measures. LDHA contributes to diverse bio‐characteristics of tumors via numerous mechanisms. LDHA has been used as a tumor biomarker for clinical diagnosis and treatment and is considered as a potential anticancer target.
Cytochrome P450 (CYP) is a critical drug-metabolizing enzyme superfamily. Modulation of CYP enzyme activities has the potential to cause drug–drug/herb interactions. Drug–drug/herb interactions can lead to serious adverse drug reactions (ADRs) or drug failures. Therefore, there is a need to examine the modulatory effects of new drug entities or herbal preparations on a wide range of CYP isoforms. The classic method of quantifying CYP enzyme activities is based on high-performance liquid chromatography (HPLC), which is time- and reagent-consuming. In the past two decades, high-throughput screening methods including fluorescence-based, luminescence-based, and mass-spectrometry-based assays have been developed and widely applied to estimate CYP enzyme activities. In general, these methods are faster and use lower volume of reagents than HPLC. However, each high-throughput method has its own limitations. Investigators may make a selection of these methods based on the available equipment in the laboratory, budget, and enzyme sources supplied. Furthermore, the current high-throughput systems should look into developing a reliable automation mechanism to accomplish ultra-high-throughput screening in the near future.
Nicotinamide adenine dinucleotide (NAD⁺) is an essential redox cofactor and signaling molecule that controls the activity of enzymes involved in metabolism, DNA repair, and cellular survival, such as the PARPs, CD38, and the sirtuins. Here, we describe three methods for measuring the activity of these enzymes: the etheno-NAD⁺ assay measures NAD⁺ hydrolase activity using an NAD⁺ analog to produce a fluorescent product that is measured in real time; the PNC1 assay converts a native product of NAD⁺ hydrolysis, nicotinamide, into a quantitative fluorescent readout; and liquid chromatography tandem mass spectrometry (LC-MS/MS) is used to characterize the entire NAD⁺ metabolome in a sample. These methods will enable new insights into the roles that NAD⁺ and the enzymes that utilize it play in health and disease.
Targeting metabolic enzymes is believed to provide new therapeutic opportunities for cancer therapy. Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that importantly coordinates glycolysis, pentose phosphate pathway (PPP) flux and serine biosynthesis in cancer cells and hence gains increasing interest of inhibitor discovery. Only few PGAM1 inhibitors have been reported and the molecular potency remains very limited. In an effort to discover new PGAM1 inhibitors, we carried out a biochemical assay-based screen that was focused on natural products derived small molecule compounds. (-)-Epigallocatechin-3-gallate (EGCG), the major natural catechins of green tea extract, was identified as a PGAM1 inhibitor that was tremendously more potent than known PGAM1 inhibitors. Further studies combining molecular docking and site-specific mutagenesis revealed that EGCG inhibited PGAM1 enzymatic activity in a manner independent of substrate competition. EGCG modulated the intracellular level of 2-phosphoglycerate, impaired glycolysis and PPP and inhibited proliferation of cancer cells. This study suggested EGCG as a chemical scaffold for the discovery of potent PGAM1 inhibitors and gained mechanistic insights to understand the previously appreciated anticancer properties of EGCG.
We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells.
Background:
The prognostic value of lactate dehydrogenase levels in the prognosis of colorectal cancer patients has been assessed for years, although the results remain controversial and heterogeneous. Thus, we comprehensively reviewed the evidence from studies that evaluated lactate dehydrogenase levels in colorectal cancer patients to determine their effect.
Methods:
The following databases were searched in September 2014 to identify studies that evaluated the prognostic value of lactate dehydrogenase levels in colorectal cancer: PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. We extracted hazard ratios (HRs) and the associated 95 % confidence intervals (CIs) from the identified studies, and performed random-effects model meta-analyses on the overall survival (OS) and progression-free survival (PFS). Thirty-two studies with a cumulative sample size of 8,261 patients were included in our analysis.
Results:
Our meta-analyses revealed that high levels of lactate dehydrogenase were associated with poor OS (HR, 1.75; 95 % CI, 1.52-2.02) in colorectal cancer patients. However, this effect was not obvious in the OS of non-metastatic colorectal cancer patients (HR, 1.21; 95 % CI, 0.79-1.86). The prognostic value of lactate dehydrogenase levels on PFS was also not confirmed (HR, 1.36; 95 % CI, 0.98-1.87). Subgroup analyses revealed that the prognostic significance of lactate dehydrogenase was independent of study location, patient age, number of patients, metastasis, chemotherapy with anti-angiogenesis drugs, study type, or risk of bias.
Conclusions:
Our results indicate that high lactate dehydrogenase levels are associated with poor OS among colorectal cancer patients, although these levels are not significant predictors of PFS.
The oxidative pentose phosphate pathway (PPP) contributes to tumour growth, but the precise contribution of 6-phosphogluconate dehydrogenase (6PGD), the third enzyme in this pathway, to tumorigenesis remains unclear. We found that suppression of 6PGD decreased lipogenesis and RNA biosynthesis and elevated ROS levels in cancer cells, attenuating cell proliferation and tumour growth. 6PGD-mediated production of ribulose-5-phosphate (Ru-5-P) inhibits AMPK activation by disrupting the active LKB1 complex, thereby activating acetyl-CoA carboxylase 1 and lipogenesis. Ru-5-P and NADPH are thought to be precursors in RNA biosynthesis and lipogenesis, respectively; thus, our findings provide an additional link between the oxidative PPP and lipogenesis through Ru-5-P-dependent inhibition of LKB1-AMPK signalling. Moreover, we identified and developed 6PGD inhibitors, physcion and its derivative S3, that effectively inhibited 6PGD, cancer cell proliferation and tumour growth in nude mice xenografts without obvious toxicity, suggesting that 6PGD could be an anticancer target.
Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z' value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.
This review describes the key steps and methods which are used to develop enzyme assays suitable for high-throughput screening (HTS) applications. The goals of HTS enzyme assays are defined relative to lower-throughput bench top assays and important aspects which go into constructing robust and sensitive enzyme assays are described. Methods that have been applied to common enzyme classes are reviewed and pitfalls related to assay artifacts are discussed. We also suggest a reporting format to describe the steps in HTS enzyme assays.
Colorectal adenomas are cancer precursor lesions of the large bowel. A multitude of genomic and epigenomic changes have been documented in these preinvasive lesions, but their impact on the protein effectors of biological function has not been comprehensively explored. Using shotgun quantitative MS, we exhaustively investigated the proteome of 30 colorectal adenomas and paired samples of normal mucosa. Total protein extracts were prepared from these tissues (prospectively collected during colonoscopy) and from normal (HCEC) and cancerous (SW480, SW620, Caco2, HT29, CX1) colon epithelial cell lines. Peptides were labeled with isobaric tags (iTRAQ 8-plex), separated by OFFGEL electrophoresis, and analyzed by LC-coupled tandem MS. Non-redundant protein families (4325 in tissues, 2017 in cell lines) were identified and quantified. Principal component analysis of the results clearly distinguished adenomas from normal mucosal samples, and cancer cell lines from HCEC cells. Two hundred twelve proteins displayed significant adenoma-related expression changes (q-value < 0.02, mean fold change vs. normal mucosa +/-1.4), which correlated (r=0.74) with similar changes previously identified by our group at the transcriptome level. Fifty-one (~25%) proteins displayed directionally similar expression changes in colorectal cancer cells (vs. HCEC cells) and were therefore attributed to the epithelial component of adenomas. Although benign, adenomas already exhibited cancer-associated proteomic changes: 69 (91%) of the 76 protein upregulations identified in these lesions have already been reported in cancers. One of the most striking changes involved sorbitol dehydrogenase (SORD), a key enzyme in the polyol pathway. Validation studies revealed dramatically increased SORD concentrations and activity in adenomas and cancer cell lines, along with important changes in the expression of other enzymes in the same (AKR1B1) and related (KHK) pathways. Dysregulated polyol metabolism may represent a novel facet of the metabolome remodeling associated with tumorigenesis.
Enzymes are biocatalysts and because of their remarkable properties, they are extensively used in medical diagnosis. Researches in the last two decades have concentrated more on enzymes such as creatine kinase-MB, alanine transaminase, aspartate transaminase, acid phosphatase, alkaline phosphatase etc. for clinical applications. Enzymes are the preferred markers in various disease states such as myocardial infarction, jaundice, pancreatitis, cancer, neurodegenerative disorders, etc. They provide insight into the disease process by diagnosis, prognosis and assessment of response therapy. Even though the literature on the use of enzymes in various disease conditions has accumulated, a comprehensive analysis is lacking and hence this review.
Abstract Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein silent regulator 2 (Sir2), the founding member of the sirtuin protein family, as being NAD(+)-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD(+) to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD(+) levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by poly(ADP-ribose) polymerase (PARP) enzymes or CD38/CD157, boosts NAD(+) levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD(+) metabolism, primarily in relation to sirtuin function. We highlight how NAD(+) levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.
A century after the identification of a coenzymatic activity for NAD(+), NAD(+) metabolism has come into the spotlight again due to the potential therapeutic relevance of a set of enzymes whose activity is tightly regulated by the balance between the oxidized and reduced forms of this metabolite. In fact, the actions of NAD(+) have been extended from being an oxidoreductase cofactor for single enzymatic activities to acting as substrate for a wide range of proteins. These include NAD(+)-dependent protein deacetylases, poly(ADP-ribose) polymerases, and transcription factors that affect a large array of cellular functions. Through these effects, NAD(+) provides a direct link between the cellular redox status and the control of signaling and transcriptional events. Of particular interest within the metabolic/endocrine arena are the recent results, which indicate that the regulation of these NAD(+)-dependent pathways may have a major contribution to oxidative metabolism and life span extension. In this review, we will provide an integrated view on: 1) the pathways that control NAD(+) production and cycling, as well as its cellular compartmentalization; 2) the signaling and transcriptional pathways controlled by NAD(+); and 3) novel data that show how modulation of NAD(+)-producing and -consuming pathways have a major physiological impact and hold promise for the prevention and treatment of metabolic disease.
The levels of aspartate aminotransferase (AST/GOT) and alanineaminotransferase (ALT/GPT) in serum can help people diagnose body tissues especially theheart and the liver are injured or not. This article provides a comprehensive review ofresearch activities that concentrate on AST/GOT and ALT/GPT detection techniques due totheir clinical importance. The detection techniques include colorimetric, spectrophotometric,chemiluminescence, chromatography, fluorescence and UV absorbance, radiochemical, andelectrochemical techniques. We devote the most attention on experimental principle. Insome methods a few representative devices and important conclusions are presented.
Lactate dehydrogenase-5 (LDH-5) catalyses the reversible transformation of pyruvate to lactate, having a principal position in the anaerobic cellular metabolism. Induction of LDH-5 occurs during hypoxia and LDH-5 transcription is directly regulated by the hypoxia-inducible factor 1 (HIF1). Serum LDH levels have been correlated with poor prognosis and resistance to chemotherapy and radiotherapy in various neoplastic diseases. The expression, however, of LDH in tumours has never been investigated in the past. In the present study, we established an immunohistochemical method to evaluate the LDH-5 overexpression in tumours, using two novel antibodies raised against the rat muscle LDH-5 and the human LDH-5 (Abcam, UK). The subcellular patterns of expression in cancer cells were mixed nuclear and cytoplasmic. In direct contrast to cancer cells, stromal fibroblasts were reactive for LDH-5 only in a minority of cases. Serum LDH, although positively correlated with, does not reliably reflect the intratumoral LDH-5 status. Lactate dehydrogenase-5 overexpression was directly related to HIF1alpha and 2alpha, but not with the carbonic anhydrase 9 expression. Patients with tumours bearing high LDH-5 expression had a poor prognosis. Tumours with simultaneous LDH-5 and HIF1alpha (or HIF2alpha) overexpression, indicative of a functional HIF pathway, had a particularly aggressive behaviour. It is concluded that overexpression of LDH-5 is a common event in non-small-cell lung cancer, can be easily assessed in paraffin-embedded material and provides important prognostic information, particularly when combined with other endogenous markers of hypoxia and acidity.
ABSTRACT : Microbes utilize enzymes to perform a variety of functions. Enzymes are biocatalysts working as highly efficient machines at the molecular level. In the past, enzymes have been viewed as static entities and their function has been explained on the basis of direct structural interactions between the enzyme and the substrate. A variety of experimental and computational techniques, however, continue to reveal that proteins are dynamically active machines, with various parts exhibiting internal motions at a wide range of time-scales. Increasing evidence also indicates that these internal protein motions play a role in promoting protein function such as enzyme catalysis. Moreover, the thermodynamical fluctuations of the solvent, surrounding the protein, have an impact on internal protein motions and, therefore, on enzyme function. In this review, we describe recent biochemical and theoretical investigations of internal protein dynamics linked to enzyme catalysis. In the enzyme cyclophilin A, investigations have lead to the discovery of a network of protein vibrations promoting catalysis. Cyclophilin A catalyzes peptidyl-prolyl cis/trans isomerization in a variety of peptide and protein substrates. Recent studies of cyclophilin A are discussed in detail and other enzymes (dihydrofolate reductase and liver alcohol dehydrogenase) where similar discoveries have been reported are also briefly discussed. The detailed characterization of the discovered networks indicates that protein dynamics plays a role in rate-enhancement achieved by enzymes. An integrated view of enzyme structure, dynamics and function have wide implications in understanding allosteric and co-operative effects, as well as protein engineering of more efficient enzymes and novel drug design.
1. Sarah D. Corathers, MD*
1. *Chief Resident for Internal Medicine, University of Cincinnati, Cincinnati, Ohio. Dr Corathers wrote this article while a resident in internal medicine and pediatrics at the Cincinnati Children’s Medical Center
Alkaline phosphatase is a commonly encountered laboratory value that is included in the panel of liver function tests. Although elevated concentrations generally are attributed to either liver or bone sources, the enzyme has been identified as a biomarker for a diverse range of diseases and physiologic processes. Elevated alkaline phosphatase may have implications ranging from indicating periodontal disease to predicting preterm labor.
Functionally, alkaline phosphatase removes a phosphate group from nucleotides and proteins. As the name suggests, the enzyme works optimally at basic pH levels. It is found throughout the body in a variety of isoenzymes unique to the tissue of origin. Highest concentrations are in liver and bone, but the enzyme is present in lesser amounts in placenta, kidney, intestines, and leukocytes.
Increased concentrations of alkaline phosphatase are derived from tissues that are either functionally disturbed (obstructed liver) or greatly stimulated (growing bone). Abnormally low concentrations are far less common and more likely are related to a genetic condition or nutritional deficiency. Normal levels can vary with age, sex, hormonal status, and blood type. In children, serum alkaline phosphatase concentrations are considerably higher than in adults and correlate with the rate of bone growth. In adults, values are slightly higher in men than in women, but after age 60 years, the enzyme value is equal or higher in women. Concentrations are increased during puberty and pregnancy and after menopause.
Measurement of alkaline phosphatase activity in the laboratory involves the release of 1 mmol/min of phosphate from a standard compound (p-nitrophenylphosphate). The total amount of enzyme from all …
Significant improvements in disease management have been achieved in the past few decades. Using cancer as an example, survival rates have increased dramatically in economically developed countries, according to the World Cancer Report published by WHO in 2014.1 One of the highlights is the average 5-year survival rate of children diagnosed before the age of 15 years in Britain increased from less than 30% in 1966-70 to almost 80% in 1996-2000. The improvement can be attributed to the establishment of early screening and awareness programs, identification of a large number of genetic or non-genetic risk factors or markers, and availability of diverse therapeutic strategies including surgery and treatments using radioactive reagents, small molecules, or biological drugs. Still, cancer remains one of the major causes of deaths worldwide, affecting millions of people. Survival rates in developing countries are poor, and the global and regional burden of cancer in terms of both incidence and mortality keep increasing along with the growing population and aging society. Further improvement heavily relies on early diagnosis, wide access to treatment options, and discovery of more effective biomarkers as well as anticancer drugs with high efficacy. Such demands are also applied to diagnosis and cure for all other diseases, calling for rapid advancements in high-throughput screening (HTS) technologies that can efficiently test a large number of targets and samples, monitor diverse enzymatic processes in situ, and effectively identify biologically active substances.2
Incidentally detected abnormality in liver function tests is a common situation encountered by physicians across all disciplines. Many of these patients do not have primary liver disease as most of the commonly performed markers are not specific for the liver and are affected by myriad factors unrelated to liver disease. Also, many of these tests like liver enzyme levels do not measure the function of the liver, but are markers of liver injury, which is broadly of two types: hepatocellular and cholestatic. A combination of a careful history and clinical examination along with interpretation of pattern of liver test abnormalities can often identify type and aetiology of liver disease, allowing for a targeted investigation approach. Severity of liver injury is best assessed by composite scores like the Model for End Stage Liver Disease rather than any single parameter. In this review, we discuss the interpretation of the routinely performed liver tests along with the indications and utility of quantitative tests.
High-throughput screening (HTS) is a multidisciplinary approach to investigate in parallel the pharmacological properties of a large number of small molecules, with the aim of identifying target-specific lead compounds to be progressed as bioactive probes or therapeutic drugs. The complex integration of chemistry, biology, automation, informatics, and medicinal chemistry has prompted the evolution of HTS as an autonomous discipline integrated in the drug discovery process. Thus, HTS assays are differentiated from traditional laboratory assays by the requirement of extensive assay development and by the strict quality criteria that must be fulfilled to approach a screening campaign. In this perspective, biochemistry and enzymology have provided an essential support for the design and configuration of functional HTS assays for enzymatic targets. In turn, the impressive technological improvements of HTS have made available an unprecedented array of novel methods and instrumentations that have contributed to advance the biochemical and kinetic characterization of enzymes. In this chapter, the paradigms for the configuration of enzymatic assays for HTS are reviewed, emphasizing the tight interdependence between type and quality of the HTS assay and the outcome of a screening campaign. The sequential steps of assay development for HTS are examined, providing examples of alternative approaches to configure the activity of enzyme drug targets as HTS-compatible assays.
Keywords:
assay development;
assay optimization;
drug discovery;
enzyme;
high-throughput screening
Pyruvate kinase isoenzyme M2 (PKM2) is one of the most important control point enzyme in glycolysis pathway. Hence, its inhibitors and activators are currently considered as the potential anticancer agents. The effect of 28 polyphenolic compounds on the enzyme activity was investigated in vitro. Among these compounds, neoeriocitrin, (-)-catechin gallate, fisetin, (±)-taxifolin and (-)-epicatechin have the highest inhibition effect with IC50 value within 0.65-1.33 µM range. Myricetin and quercetin 3-β-d-glucoside exhibited the highest activation effect with 0.51 and 1.34 µM AC50 values, respectively. Twelve of the compounds showed inhibition effect within 7-38 µM range of IC50 value. Sinapinic acid and p-coumaric acid showed an activation effect with 26.2 and 22.2 µM AC50 values, respectively. The results propose that the polyphenolics may be the potential PKM2 inhibitors/activators, and they may be used as lead compounds for the synthesis of new inhibitors or activators of this enzyme.
The pentose phosphate pathway (PPP), which branches from glycolysis at the first committed step of glucose metabolism, is required for the synthesis of ribonucleotides and is a major source of NADPH. NADPH is required for and consumed during fatty acid synthesis and the scavenging of reactive oxygen species (ROS). Therefore, the PPP plays a pivotal role in helping glycolytic cancer cells to meet their anabolic demands and combat oxidative stress. Recently, several neoplastic lesions were shown to have evolved to facilitate the flux of glucose into the PPP. This review summarizes the fundamental functions of the PPP, its regulation in cancer cells, and its importance in cancer cell metabolism and survival.
Aim:
Malic enzymes are oxidative decarboxylases with NAD(+) or NAD(P)(+) as cofactor that catalyze the conversion of L-malate to pyruvate and CO2. The aim of this study was to discover and characterize a potent inhibitor of human NAD(P)(+)-dependent malic enzyme 2 (ME2).
Methods:
Recombinant human ME2-His-Tag fusion protein was overexpressed in E coli and purified with Ni-NTA resin. A high-throughput screening (HTS) assay was developed to find ME2 inhibitors. Detergent Brij-35 was used to exclude false positives. The characteristics of the inhibitor were analyzed with enzyme kinetics analysis. A thermal shift assay for ME2 was carried out to verify the binding of the inhibitor with the enzyme.
Results:
An HTS system for discovering ME2 inhibitors was established with a Z' factor value of 0.775 and a signal-to-noise ratio (S/N) of 9.80. A library containing 12 683 natural products was screened. From 47 hits, NPD387 was identified as an inhibitor of ME2. The primary structure-activity relationship study on NPD387 derivatives showed that one derivative NPD389 was more potent than the parent compound NPD387 (the IC50 of NPD389 was 4.63 ± 0.36 μmol/L or 5.59 ± 0.38 μmol/L, respectively, in the absence or presence of 0.01% Brij-35 in the assay system). The enzyme kinetics analysis showed that NPD389 was a fast-binding uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.
Conclusion:
NPD389 is a potent ME2 inhibitor that binds to the enzyme in a fast-binding mode, acting as an uncompetitive inhibitor with respect to the substrate NAD(+) and a mixed-type inhibitor with respect to the substrate L-malate.
This chapter discusses cycling assay for nicotinamide adenine dinucleotides. The methods currently available for the quantitative estimation of nicotinamide adenine dinucleotides are classified into spectrophotometric (I), fluorometric (II), and enzyme cycling (III). The spectrophotometric method is dependent on the measurement of the absorption of the reduced coenzyme at 340 nm or that of the cyanide addition complex of the oxidized coenzyme at 325 nm. The fluorometric method is dependent on the fluorescence of the reduced coenzymes or that of the fluorescent derivatives made by adding methyl ethyl ketone to the oxidized coenzymes. The cyclcing assay method utilizes a cycling mixture composed of MTT, phenazine ethosulfate (PES), and ethanol and alcohol dehydrogenase for the determination of NAD⁺ (or NADH) or glucose-6-phosphate (G6P) and G6P dehydrogenase for the determination of NADP⁺ (or NADPH). NAD⁺ or NADP⁺ is reduced by the respective dehydrogenase system, and the NADH or NADPH formed reduces MTT through the mediation of PES. The rate of reduction of MTT is proportional to the concentration of coenzyme.
A non-invasive method to assess liver fibrosis by measuring liver stiffness with transient elastography (TE) has been recently introduced. The role of TE among chronic hepatitis B (CHB) patients in starting antiviral therapy in the primary care setting is still controversial because of its high cost. The AST to platelet ratio index (APRI) could be a much cheaper alternative.
This study compares the diagnostic accuracy of TE and APRI in assessing liver fibrosis in CHB patients.
A cross-sectional study in CHB patients intending to start antiviral treatment. Liver fibrosis was staged according to the METAVIR scoring system. Liver stiffness was measured by TE performed on the same day with liver biopsy, while APRI was calculated as follows: APRI=(AST/upper limit of normal)×100/platelet count (10(9)/l). Cutoff levels of liver stiffness and APRI were calculated by using the receiver operating characteristic curve to detect significant liver fibrosis, defined as fibrosis stage F2 or more.
117 patients were enrolled in the study; their mean age was 40.6±10.97 years. The median liver stiffness was 5.9 kPa (2.5-48 kPa) and the median APRI was 0.239 (0.09-2.73). The cutoff level of liver stiffness was 5.85 kPa for ≥F2 with an AUC of 0.614, 60.3% sensitivity, 63.6% specificity, 73.3% PPV, 49.1% NPV and a LR+ of 1.66. The APRI cutoff level was 0.235 for F≥2 with an AUC of 0.693, 64.4% sensitivity, 70.5% specificity, 78.3% PPV, 54.4% NPV and a LR+ of 2.18. Both methods gave comparable diagnostic accuracy.
APRI is a useful marker to screen liver fibrosis in the primary care setting when TE is not available.
Sorbitol is an intermediate in the polyol pathway, which converts from glucose to fructose by sorbitol dehydrogenase (SORD). Androgens are essential for the development of prostate cancer. We studied castration-induced gene expression changes in the human prostate using the GeneChip array, and identified SORD as being androgen-regulated in the human prostate. A putative androgen-responsive regulatory region at the SORD 5' promoter was identified using promoter deletion constructs in a luciferase reporter assay in COS-7 cells. Chromatin immunoprecipitation assay was used to assess the binding of androgen receptor to suggested androgen responsive regulatory region. Finally, the expression of SORD in the human prostate was evaluated in 29 prostate tissue samples by immunohistochemistry. The expression of SORD decreased after castration. Androgen supplementation to the LNCaP prostate cancer cell line led to a 7.5-fold increase in SORD mRNA expression. Furthermore, a chromatin immunoprecipitation assay proved that the androgen receptor can bind to this putative androgen-responsive regulatory region. Finally, the expression of SORD in the human prostate was localised to epithelial cells of both benign and malignant prostate tissue by immunohistochemistry. In prostate cancer, increased immunostaining was associated with high Gleason patterns and high serum prostate-specific antigen concentrations. These results show that SORD is a novel androgen-regulated gene in the human prostate and suggest the need for more detailed analysis of the physiological role of SORD in the prostate.
To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.
The availability of serum blood chemistries for screening both symptomatic and asymptomatic patients has resulted in a marked increase in the number of abnormal liver chemistry tests that must be interpreted by physicians. Usually the first step in the evaluation of a patient with elevated liver enzymes is to repeat the test to confirm the result. If the result is still abnormal, it seems wise to differentiate between a predominant "necrotic pattern" of liver chemistry, as indicated by an elevation of ALT- or AST-activity or a predominant "cholestatic pattern", as indicated by elevated activities of g-GT and alkaline phosphatase. In patients with elevated serum amino transferases hepatic diseases should be excluded primarily with non-invasive serologic tests. The most common causes of elevated amino transferase levels are chronic hepatitis B and C, autoimmunhepatitis, non-alcoholic steatohepatitis, hemochromatosis, Wilson-disease and (only recently recognized) celiac sprue. In the case of a dominant "cholestatic pattern", primary biliary cirrhosis, primary sclerosing cholangitis, but also drugs and granulomatose hepatitis must be excluded. If non-invasive serologic studies remain inconclusive, ultrasound, mini-laparoscopy and liver biopsy will help to establish the final diagnoses.
Accumulating evidence has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: First, it is established that NAD mediates energy metabolism and mitochondrial functions; second, NADPH is a key component in cellular antioxidation systems; and NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; third, cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; fourth, NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and fifth, NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Moreover, many recent studies have suggested novel paradigms of NAD and NADP metabolism. Future investigation into the metabolism and biological functions of NAD and NADP may expose fundamental properties of life, and suggest new strategies for treating diseases and slowing the aging process.
The new life of a centenarian: signalling functions of NAD(P)
- Berger