Characterization of an RNA active site: interactions between a Diels-Alderase ribozyme and its substrates and products

Ribozymes have recently been shown to catalyze the stereoselective formation of carbon-carbon bonds between small organic molecules. The interactions of these Diels-Alderase ribozymes with their substrates and products have now been elucidated by chemical substitution analysis by using 44 different, systematically varied analogues. RNA-diene interaction is governed by stacking interactions, while hydrogen bonding and metal ion coordination appear to be less important. The diene has to be an anthracene derivative, and substituents at defined positions are permitted, thereby shedding light on the geometry of the binding site. The dienophile must be a five-membered maleimidyl ring with an unsubstituted reactive double bond, and a hydrophobic side chain makes a major contribution to RNA binding. The ribozyme distinguishes between different enantiomers of chiral substrates and accelerates cycloadditions with both enantio- and diastereoselectivity. The stereochemistry of the reaction is controlled by RNA-diene interactions. The RNA interacts strongly and stereoselectively with the cycloaddition products, requiring several structural features to be present. Taken together, the results highlight the intricacy of ribozyme active sites which can control chemical reaction pathways based on minute differences in substrate stereochemistry and substitution pattern.     

苯并呋喃／噻吩联二苯类PTP1B抑制剂三维构效关系研究

主要采用比较分子力场分析方法（CoMFA）对苯并呋喃／噻吩联二苯类PTP1B (protein tyrosine phosphatase 1B)抑制剂进行了三维构效关系的研究，考察了 静电场、立体场和氢键场对构效关系的影响，交叉系数q^2的值达到0．58，表明 CoMFA得到的构效关系模型比较理想，同时test set中分子的预测活性也表明，模 型具有较好的预测能力，研究还表明，氢键场的加入不一定有利于模型的改善，通 过对分子场等值面图的分析，可以观察到叠合分子周围立体场和静电场对化合物活 性的影响，为改进原有化合物的结构，提高它们的活性提供了指导，还尝试采用比 较分子相似性指数分析方法（CoMFA）对这一系列化合物作了研究，结果表明虽然 CoMFA中加入了疏水场，但是对于研究的体系，CoMFA的模型质量并没有显著提高。     

氨基酸羟胺钒配合物的合成、表征及对PTP1B酶的抑制活性

合成了2种羟胺氧钒配合物--缬(亮)氨酸羟胺氧钒, 并通过元素分析、红外光谱、紫外光谱、热重分析及X射线单晶衍射对其结构进行了表征. 采用PTP1B酶筛选模型评价了它们及相关配合物的PTP1B酶抑制活性. 实验结果表明, 这2种配合物同属于三斜晶系, P1空间群, 中心钒原子与7个配位的氮氧原子形成扭曲的五角双锥构型, 进而通过氢键作用形成三维晶体结构. 这2种配合物对PTP1B酶都表现出抑制活性, 亮氨酸羟胺氧钒在浓度为20 μg/mL时对PTP1B酶的抑制率达到90.51%.     

In-situ active site formation in CO oxidation on alumina

For the development of automotive catalysts which may fit the condition of developing countries, catalytic activity of alumina for CO oxidation was studied. It was proposed that the carbon formed in-situ acted as an active site for CO oxidation. the carbon active site was also checked by methanol oxidation on alumina which showed temperature hysteresis during consecutive heating and cooling operations. Alkali-treated Alumina did not show any indication of the temperature hysteresis. The optimal temperature for maximum carbon depostion was confirmed by thermogravimetric analysis to be 450–500 C, which well explains the hysteresis. CeO₂−Al₂O₃ showed remarkably higher activities for complete oxidation. It seems that alumina has reasonably satisfactory activity in total clean-up of exhaust gas.     

Characterization by tandem mass spectrometry of stable cysteine sulfenic acid in a cysteine switch peptide of matrix metalloproteinases

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.     

Synthesis and biological evaluation of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives as PTP1B inhibitors

<正>Based on the fact that petroselinic acid showed good inhibitory activity(IC_(50) = 6.99μmol/L) against protein tyrosine phophatase 1B(PTP1B) in vitro,a series of novel N-(alkoxyphenyl)-aminocarbonylbenzoic acid derivatives were designed and synthesized.The results indicated that most of the derivatives showed more potent activities against PTP1B.Especially,compound 13 had obvious activity with an IC_(50) of 106 nmol/L in vitro.     

Antibody-catalyzed benzoin oxidation as a mechanistic probe for nucleophilic catalysis by an active site lysine

Aldolase antibody 24H6, which was obtained by reactive immunization against a 1,3-diketone hapten, is shown to catalyze additional reactions, including H/D exchange and oxidation reactions. Comparison of the H/D exchange reaction at the alpha-position of a wide range of aldehydes and ketones by 24H6 and by other aldolase antibodies, such as 38C2, pointed at the significantly larger size of the 24H6 active site. This property allowed for the catalysis of the oxidation of substituted benzoins to benzils by potassium ferricyanide. This reaction was used as a mechanistic probe to learn about the initial steps of the 24H6-catalyzed aldol condensation reaction. The Hammett correlation (rho=4.7) of log(k(cat)) versus the substituent constant, sigma, revealed that the reaction involves rapid formation of a Schiff base intermediate from the ketone and an active site lysine residue. The rate-limiting step in this oxidation reaction is the conversion of the Schiff base to an enamine intermediate. In addition, linear correlation (rho=3.13) was found between log(K(M)) and sigma, indicating that electronic rather than steric factors are dominant in the antibody-substrate binding phenomenon and confirming that the reversible formation of a Schiff base intermediate comprises part of the substrate-binding mechanism.     

1,2-萘醌类化合物抑制PTP1B的三维定量构效关系研究

蛋白酪氨酸磷酸酶1B (protein tyrosine phosphatase 1B, PTP-1B)是近年来发现的治疗II型糖尿病的新靶点, 1,2-萘醌类化合物对PTP-1B有较好的抑制活性, 具有良好的药用前景. 为了设计出本类化合物抑制效果更好的分子构型, 用比较分子力场分析(CoMFA)和比较分子相似性指数分析(CoMSIA)对该类化合物进行了三维定量构效关系(3D-QSAR)的研究, 并建立了相关的预测模型. 其中, CoMFA模型的交叉验证相关系数(q²)为0.555, 非交叉验证相关系数(r²)为0.991, 标准偏差(SEE)为0.049, F值为564.910. CoMSIA模型的q²为0.558, r²为0.991, SEE为0.050, F值为542.773. 计算结果表明, 获得的CoMFA和CoMSIA模型具有良好的预测能力, 可以应用于指导该类化合物的设计.     

PTP1B Inhibitory Secondary Metabolites from an Antarctic Fungal Strain Acremonium sp. SF-7394

Chemical investigation of the Antarctic lichen-derived fungal strain Acremonium sp. SF-7394 yielded a new amphilectane-type diterpene, acrepseudoterin (1), and a new acorane-type sesquiterpene glycoside, isocordycepoloside A (2). In addition, three known fungal metabolites, (−)-ternatin (3), [D-Leu]-ternatin (4), and pseurotin A (5), were isolated from the EtOAc extract of the fungal strain. Their structures were mainly elucidated by analyzing their NMR and MS data. The absolute configuration of 1 was proposed by electronic circular dichroism calculations, and the absolute configuration of the sugar unit in 2 was determined by a chemical method. The inhibitory effects of the isolated compounds on protein tyrosine phosphatase 1B (PTP1B) were evaluated by enzymatic assays; results indicated that acrepseudoterin (1) and [D-Leu]-ternatin (4) dose-dependently inhibited the enzyme activity with IC₅₀ values of 22.8 ± 1.1 μM and 14.8 ± 0.3 μM, respectively. Moreover, compound 1 was identified as a competitive inhibitor of PTP1B.     

Synthesis and biological evaluation of novel N-(alkyoxyphenyl)- aminocarbonylbenzoic acid derivatives as PTP1B inhibitors

Based on the fact that petroselinic acid showed good inhibitory activity (IC50=6.99 µmol/L) against protein tyrosine phophatase 1B(PTP1B) in vitro，a series of novel N-(alkoxyphenyl)-aminocarbonyl benzoic acid derivatives were designed and synthesised. The results indicated that most of the derivatives showed more potent activities against PTP1B. Especially, compound 13 had obvious activity with an IC50 of 106 nmol/L in vitro.     

A chemical model for redox regulation of protein tyrosine phosphatase 1B (PTP1B) activity

Growing evidence indicates that endogenously produced hydrogen peroxide acts as a cellular signaling molecule that (among other things) can regulate the activity of some protein phosphatases. Recent X-ray crystallographic studies revealed an unexpected chemical transformation underlying the redox regulation of protein tyrosine phosphatase 1B, in which oxidative inactivation of the enzyme yields an intrastrand protein cross-link between the catalytic cysteine residue and its neighboring amide nitrogen. This work describes a small organic molecule that serves as an effective model for the redox-sensing assembly of functional groups at the active site of PTP1B. Findings obtained using this model system suggest that the oxidative transformation of PTP1B to its "crosslinked" inactive form can proceed directly via oxidation of the active-site cysteine to a sulfenic acid (RSOH). The remarkably facile nature of this protein cross-link-forming reaction, along with the widespread cellular occurrence of protein sulfenic acids generated via oxidation of cysteine residues, suggests that the type of oxidative protein cross-link formation first seen in the context of PTP1B represents a potentially general mechanism for redox "switching" of protein function. Thus, the chemistry characterized here could have broad relevance to both redox-regulated signal transduction and the toxic effects of oxidative stress.     

A combinatorial approach to minimal peptide models of a metalloprotein active site

Screening of a "one-bead-one-compound" peptide library containing biomimetic His/Cys ligands has led to the discovery of sequences that hydrolyze ester substrates in combination with Zn2+.     

Development of an active site titration reagent for α-amylases

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining β-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent ‘glycosyl’-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities.

Amylases are among the most common classes of enzymes employed in industrial settings, being used in detergents, bread, beer, biofuel, and many other sectors. Accordingly, α-amylases account for 25% of the world''s multi-billion dollar enzyme market.^1,2 α-Amylases are endo-acting enzymes that cleave starch into malto-oligosaccharides, which are further degraded by exo-acting α-glucosidases, glucoamylases, β-amylases and α-glucan phosphorylases and lyases. They are found in CAZy GH families 13, 57, 119 and 126, with the vast majority in the large GH13 family.³ GH13 enzymes adopt a (β/α)₈ fold with three highly conserved active site carboxylic acids.^4–6 They employ a classical double-displacement mechanism⁷ in which one of the glutamic acids provides acid catalytic assistance to the leaving group departure while an aspartate attacks the anomeric centre, forming a covalent glycosyl enzyme intermediate. In a second step, water attacks the anomeric centre with base assistance from the glutamate residue (Fig. 1A and B).Open in a separate windowFig. 1Koshland mechanism of retaining β- and α-glycosidases (A & B). The same mechanism has been observed for the hydrolysis of “β”-valienols (C), and for “α”-valienols (D).Given their industrial importance, a huge amount of attention has been given to the discovery and improvement of α-amylases to attain optimal performance for particular applications. These approaches typically require high-throughput analysis of large numbers of gene products or mutants thereof.^8–10 Identification of the best candidates then ideally requires high-throughput assay coupled with a method for determining the enzyme concentration in each sample. This can be a challenging task in the absence of purification, as would be the case for truly high-throughput approaches. The “gold standard” method to quantify active enzyme concentration is active site titration.¹¹ Active site titrants react stoichiometrically with their target enzymes and release one equivalent of a quantifiable agent, which is typically either a chromophore or fluorophore. For enzymes that operate via a covalent intermediate, such as retaining glycosidases, the active site titrants are usually chromogenic or fluorogenic substrates that form this intermediate with a rate constant (k_on) that is much greater than that for its hydrolysis (k_off) – ideally with k_off approaching zero.Our lab has previously developed active site titration reagents for several retaining β-glycosidases^12,13 and neuraminidases.^14,15 By replacing the substituent on the position adjacent to the anomeric centre of the sugar (the hydroxyl at C-2 for many monosaccharides) with a fluorine atom, both the formation and the hydrolysis of the glycosyl-enzyme intermediate are slowed, largely through inductive destabilisation of the transition state. Further incorporation of a reactive fluorogenic leaving group generates a reagent that, upon covalently inactivating the glycosidase, releases a stoichiometric and quantifiable amount of fluorophore. The fluorogenic response is then measured to determine the amount of active glycosidase that is present in solution.Unfortunately, this same strategy does not work for retaining α-glycosidases. In those cases, k_off remains greater than k_on, likely due to the inherently greater reactivity of the β-glycosyl-enzyme intermediate,^16,17 and the compounds are simply substrates with low turnover numbers. By use of 2,2-dihalosugars with yet more reactive leaving groups, this problem could be solved in some cases, but their synthesis is challenging, and inactivation rates were low, or non-existent in some cases.^18,19 Alternative approaches were called for.Recently, a new class of glycosidase substrates was reported in which the sugar moiety is replaced by an equivalently hydroxylated cyclohexene.^20–23 Hydrolysis of these enol ethers likely occurs via an allylic cation of almost identical reactivity to that of the equivalent oxocarbenium ion. Glycosidases cleave these substrates via the classical Koshland mechanism⁷ (Fig. 1C and D), but considerably more slowly than their natural substrates. However, incorporation of a good leaving group will accelerate, relatively, the first step such that, in some cases, they act as mechanism-based inactivators making them candidates for development of an active site titrant for α-amylases.Since α-amylases are endo-acting enzymes that do not usually cleave monosaccharide glycosides, an ‘extended” oligosaccharide version containing a total of 2 or 3 sugar/pseudosugar moieties would be needed. Substrates longer than this would be prone to internal glycoside cleavage. Since 2-chloro-4-nitrophenyl maltotrioside (CNP-G3) functions as a substrate for most amylases, we focused on addition of a maltosyl unit to a valienol moiety containing a 6,8-difluorocoumarin (F₂MU) leaving group at its “anomeric centre”. The low pK_a of this coumarin, 4.7,¹⁴ results in a greater reactivity of the reagent and also ensures the coumarin will be deprotonated and thus fluorescent, upon release at neutral pH.Synthesis of partially protected alcohol 2 from gluconolactone 1via literature methods²⁴ was followed by attachment of F₂MU via a Mitsunobu reaction and subsequent removal of the protecting groups under acidic conditions, generating known pseudo-glycoside 3.²³ To check this concept before we synthesized the longer version, we tested compound 3 as a titrant of a simple α-glucosidase and found that it did indeed titrate the enzyme (Fig. S5^†). Since elongation of this pseudosugar via classical organic synthesis would require substantial protecting group chemistry, we elected instead to employ an enzymatic coupling strategy using the GH13 cyclodextrin transglycosidase, CGTase. This enzyme can use glycosyl fluorides, such as α-maltosyl fluoride, to effect glycosyl transfer onto suitable acceptors. However, a significant competing reaction would involve self-condensation of glycosyl fluorides ultimately forming cyclodextrins. To avoid this problem, we employed a maltosyl fluoride donor (4), in which the 4′-hydroxyl had been capped with a methyl group.^25,26 Incorporation of 4′-methoxy groups does not alter the reaction with α-amylases, as this site in the normal substrate is occupied by additional sugar residues. Thus CGTase-catalysed glycosylation between known glycosyl fluoride 4 and pseudo-glycoside 3, gave the pseudo-trisaccharide 5 in 64% isolated yield (Scheme 1).Open in a separate windowScheme 1Synthesis of titration reagent 5.With this reagent in hand, we proceeded to screen its ability to inactivate a small panel of α-amylases. As shown in Fig. 2, time-dependent inactivation was observed for all enzymes tested, with the most industrially relevant enzymes, Effusibacillus pohliae amylase (EPA) and Aspergillus oryzae amylase (AOA), being inactivated the fastest.Open in a separate windowFig. 2Time-dependent inactivation of a small panel of amylases, showing remaining % activity versus time. Red box with X: AOA (91 nM); blue square: EPA (66.7 nM); purple cross: PPA (500 nM); green triangle: HPA (125 nM). AOA = A. oryzae amylase; EPA = E. pohliae amylase; HPA = human pancreatic amylase; PPA = porcine pancreatic amylase.Kinetic parameters for inactivation were then determined by directly monitoring the release of F₂MU by UV-Vis (Table 1). To determine k_on and k_off (Scheme 2), we monitored chromophore (F₂MU) release by absorbance at 370 or 380 nm (dependent on the concentration of 5 in the measurements of each enzyme). After mixing 5 with each enzyme individually, a burst phase followed by a steady-state phase was observed. For each enzyme, this was then repeated with varying concentrations of 5. Initial rates of F₂MU release versus concentration of 5 were fit to a Michaelis–Menten equation to provide k_on. The rate constant of cyclitol release, k_off, was determined by measuring rates of the steady-state region at a saturating concentration (5× K_i). We found that several amylases: Effusibacillus pohliae amylase (EPA), Aspergillus oryzae amylase (AOA), Rhizomucor pusillus amylase (RPA) and porcine pancreatic amylase (PPA), inactivated quickly (highest k_on, lowest k_off, and greatest k_on/K_i), and are therefore ideal candidates for titration with compound 5. Human pancreatic amylase (HPA), on the other hand, while inactivating rapidly, binds the reagent relatively poorly.Open in a separate windowScheme 2Kinetic parameters for the hydrolysis of 5 by several amylases (at 25 °C for EPA, AOA, and RPA and 30 °C for human pancreatic amylase [HPA] and porcine pancreatic amylase [PPA])

Structure-based prediction of free energy changes of binding of PTP1B inhibitors

The goals were (1) to understand the driving forces in the binding of small molecule inhibitors to the active site of PTP1B and (2) to develop a molecular mechanics-based empirical free energy function for compound potency prediction. A set of compounds with known activities was docked onto the active site. The related energy components and molecular surface areas were calculated. The bridging water molecules were identified and their contributions were considered. Linear relationships were explored between the above terms and the binding free energies of compounds derived based on experimental inhibition constants. We found that minimally three terms are required to give rise to a good correlation (0.86) with predictive power in five-group cross-validation test (q2 = 0.70). The dominant terms are the electrostatic energy and non-electrostatic energy stemming from the intra- and intermolecular interactions of solutes and from those of bridging water molecules in complexes.     

Synthesis of mangiferin derivates and study their potent PTP1B inhibitory activity

Protein tyrosine phosphatase 1B (PTP1B) has received considerable attention from the drug industry as a potential treatment for diabetes mellitus.Mangiferin has been reported to possess significant antidiabetic activity.Based on the previous study,eight new mangiferin derivates were synthesized and evaluated for their PTP1B inhibitory activity.Some of them displayed good inhibitory activity on PTP1B.     

Discovery of a new phosphotyrosine mimetic for PTP1B using breakaway tethering

Protein tyrosine phosphatases play important roles in many signaling cascades involved in human disease. The identification of druglike inhibitors for these targets is a major challenge, and the discovery of suitable phosphotyrosine (pY) mimetics remains one of the key difficulties. Here we describe an extension of tethering technology, "breakaway tethering", which is ideally suited for discovering such new chemical entities. The approach involves first irreversibly modifying a protein with an extender that contains both a masked thiol and a known pY mimetic. The extender is then cleaved to release the pY mimetic, unmasking the thiol. The resulting protein is screened against a library of disulfide-containing small molecule fragments; any molecules with inherent affinity for the pY binding site will preferentially form disulfides with the extender, allowing for their identification by mass spectrometry. The ability to start from a known substrate mimimizes perturbation of protein structure and increases the opportunity to probe the active site using tethering. We applied this approach to the anti-diabetic protein PTP1B to discover a pY mimetic which belongs to a new molecular class and which binds in a novel fashion.     

Characterization and genetic manipulation of peptide synthetases in Pseudomonas aeruginosa PAO1 in order to generate novel pyoverdines

PvdD, a nonribosomal peptide synthetase (NRPS) of Pseudomonas aeruginosa PAO1, incorporates two L-threonines into the siderophore pyoverdine. A pvdD mutant did not synthesize pyoverdine and lacked a high Mr iron-regulated cytoplasmic protein (IRCP). Analysis of other IRCPs and the P. aeruginosa genome enabled the remaining pyoverdine NRPSs to be identified. The pvdD mutation could be complemented in trans, enabling design of plasmid-based systems for the generation of novel pyoverdines. Introduction of a truncated pvdD gene resulted in attenuated forms of pyoverdine, and introduction of L-threonine-incorporating NRPSs from other organisms restored pyoverdine production to mutant cells. This is the first successful rational in vivo modification of NRPS modules outside of Bacillus subtilis. The systems employed did not allow incorporation of other residues into pyoverdine, indicating that there are multiple elements contributing toward substrate specificity in NRPSs.     

Accounting of ligand–receptor interactions to explore and design novel architecture for PTP 1B inhibition: a legitimate approach

Computer‐aided drug design was performed on a diverse set of 103 biphenyl derivatives that demonstrated antidiabetic activity by restraining the protein tyrosine phosphatase 1B (PTP 1B) receptor. A four‐point pharmacophore hypothesis using the PHASE module of Schrödinger suite with one hydrogen bond acceptor (A) and three aromatic rings (R) as pharmacophoric features was generated. The hypothesis, ARRR.2, considered the best hypothesis in the present study is characterized by survival score (3.553), cross‐validated r² (Q²) (0.722), regression coefficient (0.949), Pearson R (0.867), and F value (492.6). The developed pharmacophore model was externally validated by predicting the activity of test set molecules. Docking algorithm combined with the drug–receptor binding free energetic and pharmacokinetic drug profile envisaged a novel concept, which may provide structural insights for the development of potential PTP 1B inhibitors. The study also provided a valid rapport between pharmacophore drug mapping, atom‐based three‐dimensional quantitative structure–activity relationship, molecular docking, sitemap, molecular simulations, and pharmacokinetic prediction approaches demonstrating the trends in activity. The results of these ligand–receptor relationship studies may account to design a legitimate template for the development and optimization of highly selective and potent PTP 1B inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.