烟酰胺腺嘌呤二核苷酸类CD38抑制剂的合成及生物活性评价

分别以1,3,5-三苯甲酰基-α-D-核糖、3,5-二苯甲酰基-2-脱氧-2,2-二氟戊呋喃糖-1-酮和D-木糖为原料, 经由烟酰胺核苷及烟酰胺核苷酸中间体, 合成了系列糖环经氟原子取代的烟酰胺腺嘌呤二核苷酸(NAD)类CD38抑制剂. 基于对CD38的水解抑制能力的考察, 评价了所合成氟代NAD类似物的活性. 结果表明, 糖环上氟原子取代的数目和位置对抑制剂活性的影响十分明显, 烟酰胺核苷的端基构型对活性的影响较大. 2个化合物均显示出非常好的CD38抑制活性, 其中化合物2a的抑制活性高出阳性对照物阿糖型氟代烟酰胺腺嘌呤二核苷酸2个数量级.     

Boric acid inhibits adenosine diphosphate-ribosyl cyclase non-competitively

Adenosine diphosphate-ribosyl cyclase (ADP-ribosyl cyclase) is a ubiquitous enzyme in eukaryotes that converts NAD+ to cyclic-ADP-ribose (cADPR) and nicotinamide. A quantitative assay for cADPR was developed using capillary electrophoresis to separate NAD+, cADPR, ADP-ribose, and ADP with UV detection (254 nm). Using this assay, the apparent Km and Vmax for Aplysia ADP-ribosyl cyclase were determined to be 1.24+/-0.05 mM and 131.8+/-2.0 microM/min, respectively. Boric acid inhibited ADP-ribosyl cyclase non-competitively with a Ki of 40.5+/-0.5 mM. Boric acid binding to cADPR, determined by electrospray ionization mass spectrometry, was characterized by an apparent binding constant, KA, of 655+/-99 L/mol at pH 10.3.     

Inhibitors of ADP-ribosylating bacterial toxins based on oxacarbenium ion character at their transition states

The bacterial exotoxins, cholera toxin (CT), pertussis toxin (PT), and diphtheria toxin (DT), interfere with specific host proteins to cause tissue damage for their respective infections. The common toxic mechanism for these agents is mono-ADP-ribosylation of specific amino acids in G(s)(alpha), G(i)(alpha), and eEF-2 proteins, respectively, by the catalytic A chains of the toxins (CTA, PTA, and DTA). In the absence of acceptor proteins, these toxins also act as NAD(+)-N-ribosyl hydrolases. The transition-state structures for NAD(+) hydrolysis and ADP-ribosylation reactions have oxacarbenium ion character in the ribose. We designed and synthesized analogues of NAD(+) to resemble their oxacarbenium ion transition states. Inhibitors with oxacarbenium mimics replacing the NMN-ribosyl group of NAD(+) show 200-620-fold increased affinity in the hydrolytic and N-ribosyl transferase reactions catalyzed by CTA. These analogues are also inhibitors for the hydrolysis of NAD(+) by PTA with K(i) values of 24-40 microM, but bind with similar affinity to the NAD(+) substrates. Inhibition of the NAD(+) hydrolysis and ADP-ribosyl transferase reactions of DTA gave K(i) values from 19 to 48 microM. Catalytic rate enhancements by the bacterial exotoxins are small, and thus transition-state analogues cannot capture large energies of activation. In the cases of DTA and PTA, analogues known to resemble the transition states bind with approximately the same affinity as substrates. Transition-state analogue interrogation of the bacterial toxins indicates that CTA gains catalytic efficiency from modest transition-state stabilization, but DTA and PTA catalyze ADP-ribosyl transferase reactions more from ground-state destabilization. pH dependence of inhibitor action indicated that both neutral and cationic forms of transition-state analogues bind to DTA with similar affinity. The origin of this similarity is proposed to reside in the cationic nature of NAD(+) both as substrate and at the transition state.     

Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem‐mass spectrometry

Nicotinamide adenine dinucleotide (NAD) is a major co‐factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging‐related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS‐based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.     

Covalent and noncovalent intermediates of an NAD utilizing enzyme, human CD38

Enzymatic utilization of nicotinamide adenine dinucleotide (NAD) has increasingly been shown to have fundamental roles in gene regulation, signal transduction, and protein modification. Many of the processes require the cleavage of the nicotinamide moiety from the substrate and the formation of a reactive intermediate. Using X-ray crystallography, we show that human CD38, an NAD-utilizing enzyme, is capable of catalyzing the cleavage reactions through both covalent and noncovalent intermediates, depending on the substrate used. The covalent intermediate is resistant to further attack by nucleophiles, resulting in mechanism-based enzyme inactivation. The noncovalent intermediate is stabilized mainly through H-bond interactions, but appears to remain reactive. Our structural results favor the proposal of a noncovalent intermediate during normal enzymatic utilization of NAD by human CD38 and provide structural insights into the design of covalent and noncovalent inhibitors targeting NAD-utilization pathways.     

Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide ( NAD⁺ ) has emerged as a crucial substrate for NAD⁺ -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^thAD⁺ , are described. Its preparation was accomplished by enzymatic conversion of synthetic ^th ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^thAD⁺ was reduced by alcohol dehydrogenase (ADH) to N^thADH and afforded ^thADP-ribose ( ^th ADPr ) upon hydrolysis by NAD⁺ -nucleosidase (NADase). Furthermore, N^thAD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^thADP-ribosyl)ation. Due to its high photophysical responsiveness, N^thAD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺ ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.     

烟酰型辅酶NAD(P)+和NAD(P)H再生的研究进展

大部分氧化还原酶的催化反应需要烟酰型辅酶NAD(P) 和NAD(P)H作为氧化剂或还原剂参与,由于氧化还原酶应用广泛而辅酶价格昂贵,使得辅酶再生逐渐成为研究热点.综述了近年来NAD(P) 和NAD(P)H酶法再生、电化学法及光化学法再生的研究进展,并介绍了各再生技术的应用和开发状况.     

A ligation-triggered DNAzyme cascade for amplified fluorescence detection of biological small molecules with zero-background signal

Many types of fluorescent sensing systems have been reported for biological small molecules. Particularly, several methods have been developed for the recognition of ATP or NAD(+), but they only show moderate sensitivity, and they cannot discriminate either ATP or NAD(+) from their respective analogues. We have addressed these limitations and report here a dual strategy which combines split DNAzyme-based background reduction with catalytic and molecular beacon (CAMB)-based amplified detection to develop a ligation-triggered DNAzyme cascade, resulting in ultrahigh sensitivity. First, the 8-17 DNAzyme is split into two separate oligonucleotide fragments as the building blocks for the DNA ligation reaction, thereby providing a zero-background signal to improve overall sensitivity. Next, a CAMB strategy is further employed for amplified signal detection achieved through cycling and regenerating the DNAzyme to realize the true enzymatic multiple turnover (one enzyme catalyzes the cleavage of several substrates) of catalytic beacons. This combination of zero-background signal and signal amplification significantly improves the sensitivity of the sensing systems, resulting in detection limits of 100 and 50 pM for ATP and NAD(+), respectively, much lower than those of previously reported biosensors. Moreover, by taking advantage of the highly specific biomolecule-dependence of the DNA ligation reaction, the developed DNAzyme cascades show significantly high selectivity toward the target cofactor (ATP or NAD(+)), and the target biological small molecule can be distinguished from its analogues. Therefore, as a new and universal platform for the design of DNA ligation reaction-based sensing systems, this novel ligation-triggered DNAzyme cascade method may find a broad spectrum of applications in both environmental and biomedical fields.     

A New Colorimetric Procedure for Blood Alcohol Determination

Abstract

A new colorimetric procedure for blood alcohol determination is described. Blood proteins are precipitated, and the protein-free supernatant incubated with nicotinamide-adenine dinucleotide (NAD), alcohol dehydrogenase (ADH), nitro blue tetrazolium (nitro BT) and phenazine methosulphate (PMS). Ethanol in the sample reduces the NAD, and the reduced NAD so formed reduces nitro BT to a coloured formazan, PMS serving as an intermediate electron carrier. The reaction is allowed to proceed to completion and colour formation quantitatively relates to sample alcohol concentration. An ethanol standard of known concentration is included with each batch of determinations and sample alcohol concentration calculated from this. The method permits the examination of large numbers of samples with rapidity and precision and without the need for specialized apparatus. Comparison with an ultra-violet enzymatic procedure for blood alcohol determination, gave excellent agreement.     

光催化NAD（P）H再生的研究进展

辅酶与酶催化反应紧密相关,是酶催化氧化还原反应过程中不可缺失的重要组成,其中,烟酰胺类辅酶NAD和NADP参与了大多数的酶催化氧化还原反应,是辅酶中最重要的一类。然而,辅酶的高成本限制了其实际应用。因此,烟酰胺辅酶的高效和低成本再生具有特别重要的意义。本文总结了还原型烟酰胺辅酶光催化再生方法的相关研究进展以及各种光敏剂的优缺点,提出了光催化NAD(P)H再生仍需要解决的问题。     

O-ADP-Ribosylation in the NAD/NADase system: 2-alkanols as efficient substrates

Several 2-alkanols (2-propanol, 2-butanol, 2-pentanol, etc.) were examined as substrates for ADP-ribosylation in the NAD/NADase enzymatic system. Even though these secondary alcohols have hydroxy groups that are subject to the steric influence of a methyl group, they were shown to be efficiently ADP-ribosylated. However, in the case of 3-alkanol (3-butanol), only slight ADP-ribosylation was observed. In this enzymatic reaction, 1,2-propanediol provided both 1-O- and 2-O-ADP-ribosylation products in the ratio 1:1 as determined by 1H-NMR spectrometry. On the other hand, an equimolar mixture system of 1- and 2-propanols provided major 1-O- and minor 2-O-ribosylation products in the ratio 4:1. This is the first report of O-ADP-ribosylation of terminal secondary alcohols with the NAD/NADase enzymatic system.     

Specific and Rapid Glucose Detection Using NAD‐dependent Glucose Dehydrogenase,Diaphorase, and Osmium Complex

Selective glucose measurement in serum and blood and rapid glucose measurement using nicotinamide adenine dinucleotide (NAD)‐dependent glucose dehydrogenase (NAD‐GDH) are still very challenging. Here, we report a selective and rapid glucose sensor, based on electrochemical‐enzymatic‐enzymatic (ENN) redox cycling involving bis(2,2‐bipyridyl)dichloroosmium(II) [Os(bpy)₂Cl₂], diaphorase (DI), NAD⁺, NAD‐GDH, and glucose. DI and Os(bpy)₂Cl₂ are used to obtain fast mediated oxidation of NADH that is generated as a result of glucose oxidation by NAD‐GDH. DI and NAD‐GDH are co‐immobilized via affinity binding on an avidin‐modified indium tin oxide electrode to obtain fast and stable ENN redox cycling. Two enzymes (DI and NAD‐GDH) and two electron mediators [Os(bpy)₂Cl₂ and NAD⁺] are insensitive to oxygen. The applied potential (0.0 V vs Ag/AgCl) is low enough to minimize interfering electrochemical reactions, and the redox reactions of Os(bpy)₂Cl₂ with interfering species are slow. NAD‐GDH is much less reactive to problematic monosaccharides such as xylose, fructose, galactose, and mannose than glucose. Artificial serum containing 5 % (w/v) human serum albumin shows a similar electrochemical background level in serum. All results enable us to obtain selective and reproducible glucose detection. The fast ENN redox cycling allows sensitive glucose detection with a wide range of concentrations in artificial serum with a short measuring time (5 s) without an incubation period.     

新型烟酰胺腺嘌呤二核苷酸(NAD)类似物的合成及其辅酶活性

尝试4种形成焦磷酸键的方法合成了5种结构新颖的新型烟酰胺腺嘌呤二核苷酸(NAD)类似物.初步考察了类似物的生物活性,发现苹果酸酶和醇脱氢酶以类似物3b和3d为辅酶时,活性只有以NAD为辅酶时的13%～30%;而以类似物3a,3c和3e为辅酶时,这些酶的活性均极低.     

Artificial synthetic receptors as regulators of protein activity

This article discusses most recent work and progress in the direction of a rational design of small molecule receptors that efficiently interfere with the biological function of a particular receptor or enzyme-some of which are therapeutically relevant. More specifically, the following topics are highlighted here: the inhibition of voltage-dependent potassium channels of the K(v)1.x family by designed porphyrin and calix[4]arene ligands, the structural and functional recovery of the tetramerization domain of mutated P53 protein by tailored calix[4]arene ligands and the control over LDH activity by supramolecular signaling. Finally a new way to modulate NAD(+)-dependent enzymatic activities by molecular clips and tweezers is presented.     

Enzyme monolayer- and bilayer-modified electrodes with diaphorase and dehydrogenases

Diaphorase was immobilized covalently as a monolayer on a tin(IV) oxide electrode, and the diaphorase electrode thus obtained responded to NADH amperometrically in the presence of ferricyanide or 2,6-dichloroindophenol as the electron mediator. The response was one to two orders of magnitude larger than that of a bare electrode. Further derivatization of the diaphorase electrode with a dehydrogenase (glucose, lactate or alcohol dehydrogenase), which reduces NAD to NADH by reaction with the substrate, yielded dehydrogenase/diaphorase heterobilayer-modified electrodes. These electrodes functioned as sensors for the respective substrate with NAD and ferricyanide as the mediators. Each bilayer electrode responded to the substrate only in the presence of added NAD; this provides evidence for the essential contribution of diaphorase to the sensor performance. As much as 60 to 80% of the electron mediator reduced by the enzymatic reaction was utilized in the amperometric response.     

细胞内NAD(P)H水平分析技术的研究进展

细胞内NAD(P)H水平直接控制着细胞的衰老、节律、癌变、死亡等重大生命过程,NAD(P)H水平的研究是生命过程中新的研究热点之一.本文介绍了NAD(P)H的结构、特性及检测方法,重点探讨了近年来国内外NAD(P)H水平的检测,并对其研究现状进行了综述.     

A model reaction assesses contribution of h-tunneling and coupled motions to enzyme catalysis

To assess the contribution of physical features to enzyme catalysis, the enzymatic reaction has to be compared to a relevant uncatalyzed reaction. While such comparisons have been conducted for some hydrolytic and radical reactions, it is most challenging for biological hydride transfer and redox reactions in general. Here, the same experimental tools used to study the H-tunneling and coupled motions for enzymatic hydride transfer between two carbons were used in the study of an uncatalyzed model reaction. The enzymatic oxidations of benzyl alcohol and its substituted analogues mediated by alcohol dehydrogenases were compared to the oxidations by 9-phenylxanthylium cation (PhXn(+)). The PhXn(+)serves as an NAD(+) model, while the solvent, acetonitrile, models the protein environment. Experimental comparisons included linear free energy relations with Hammett reaction constant (ρ) of zero versus -2.7; temperature-independent versus temperature-dependent primary KIEs; deflated secondary KIEs with deuteride transfer (i.e., primary-secondary coupled motion) versus no coupling between secondary KIEs and H- or D-transfer; and large versus small secondary KIEs for the enzymatic versus uncatalyzed alcohol oxidation. Some of the differences may come from differences in the order of microscopic steps between the catalyzed versus uncatalyzed reactions. However, several of these comparative experiments indicate that in contrast to the uncatalyzed reaction the transition state of the enzymatic reaction is better reorganized for H-tunneling and its H-donor is better rehybridized prior to the C-H→C transfer. These findings suggest an important role for these physical features in enzyme catalysis.     

Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence.

The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)= nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein-bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein-bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high-resolution map of cellular metabolism, that is, an image of the contribution of the protein-bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein-NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein-NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes. Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time-expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time-domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel-by-pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.     

Cofactor diversity in biological oxidations: implications and applications

Until recently, it was generally believed that enzymatic oxidation and reduction requires the participation of either a nicotinamide (NAD(P)+) or a flavin (FAD, FMN), in agreement with the existence of NAD(P)/H-dependent dehydrogenases/reductases and flavoprotein dehydrogenases/reductases/oxidases. However, during the past 20 years, the unraveling of the enzymology of the oxidation and reduction of C1-compounds by bacteria has led to the discovery of many new redox cofactors, some of them discussed here as they have a wider physiological significance than just enabling enzymatic C1-conversions to occur. A good example is the quinone cofactors, encompassing PQQ (2,7,9-tricarboxy-1H-pyrrolo[2,3-f]-quinoline-4,5-dione), TTQ (tryptophyl tryptophanquinone), TPQ (topaquinone), LTQ (lysyl topaquinone), and several others whose structures have still to be elucidated. Another example is mycothiol (1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D-myo-inosoitol), the counterpart of glutathione, once thought to be a universal coenzyme. Because these novel cofactors assist in reactions that can also be catalyzed by already known enzyme "classic cofactor" combinations, and first indications suggest that the chemistry of the reactions is not unique, one may wonder about the evolutionary background for this cofactor diversity. However, as will be illustrated by examples, from a practical point of view the diversity is beneficial, as it has increased the arsenal of enzymes suitable for application.     

Esterification of borate with NAD+ and NADH as studied by electrospray ionization mass spectrometry and 11B NMR spectroscopy

This paper describes for the first time the direct measurement of boric acid (B(OH)(3)) and borate (B(OH)(4) (-)) adduction to NAD(+) and NADH by electrospray ionization mass spectrometry (ESI-MS) and (11)B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD(+) forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD(+), and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD(+) were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD(+) and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 micro M for NAD(+) and boric acid (1 : 1) to detect NAD(+)-borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance.