Identification of ligands from natural products as inhibitors of glutathione S‐transferases using enzyme immobilized mesoporous magnetic beads with high‐performance liquid chromatography plus quadrupole time‐of‐flight mass spectrometry and molecular docking

Multidrug resistance is recognized as one of the main reasons leading to the failure of chemotherapy. Studies have shown that glutathione S‐transferase inhibitors could be regarded as multidrug resistance reversal agents. Herein, a method of applying enzyme immobilization, molecular docking, and high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was employed to screen glutathione S‐transferase inhibitors from natural products. Magnetic mesoporous silica microspheres were synthesized and modified with a poly(dopamine) layer, which has a large quantity of amino, enabling further non‐covalent binding with glutathione S‐transferase. Moreover, the immobilization conditions, namely, potential of hydrogen, catalase concentration, reaction temperature and reaction time, were optimized. In total, six potential compounds were isolated and identified from Perilla frutescens (L.) Britt leaves and green tea and molecular docking was applied to identify the binding site. Rosmarinic acid, (?)‐epigallocatechin‐3‐O‐gallate and (?)‐epicatechin‐3‐gallate showed higher binding affinity than the compounds, and their half maximal inhibitory concentration values were further determined. The results suggested that this proposed method was effective and convenient for identifying glutathione S‐transferase inhibitors from natural products.     

Cobalt Phthalocyanine Molecular Electrode for the Electrochemical Investigation of the Release of Glutathione upon Copper‐Catalyzed Decomposition of S‐Nitrosoglutathione

Decomposition of S‐nitrosoglutathione (GSNO) in phosphate buffer solution at physiological pH 7.4 in the presence of cuprous ion as a catalyst and sodium borohydride as a reducing agent is analyzed by observing the transient apparition of reduced glutathione GSH through its electrooxidation. Transient formation of GSH, upon decomposition of 1 mM GSNO in presence of 0.025 mM Cu(NO₃)₂ and 1 mM NaBH₄ was detected by using an ordinary pyrolytic graphite electrode modified with an adsorbed monolayer of cobalt phthalocyanine at 0 V vs. SCE.     

Secondary‐Ion Mass Spectrometry of Genetically Encoded Targets

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added ¹⁹F‐enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The ¹⁹F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell‐culture systems, as well as small model organisms.     

Proteome‐Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a “clickable” alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide‐capped alkynyl–cysteine adducts are readily detectable by LC‐MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.     

Simultaneous analysis of catechol‐O‐methyl transferase activity,S‐adenosylhomocysteine and adenosine

Novel HPLC method utilizing UV‐detection was developed to analyse catechol‐O‐methyltransferase (COMT) products, vanillic acid and isovanillic acid, S‐adenosylhomocysteine (SAH) and adenosine formed from dihydroxybenzoic acid and S‐adenosyl‐L‐methionine (SAM) by incubation of the rat tissues. Entacapone, a COMT inhibitor, prevented the formation of SAH only partially in the striatal homogenate whereas in the kidney homogenate the increase of SAH was prevented by entacapone. In conclusion, this method was reliable, rapid and simple. COMT seemed to be partially responsible on the SAM utilizing methylations in the striatal homogenates while in the high COMT activity tissue, COMT was the main SAH producing methyltransferase. Copyright © 2009 John Wiley & Sons, Ltd.     

Computational design of S‐nitrosothiol “click” reactions

To address a long‐standing problem of finding efficient reactions for chemical labeling of protein‐based S‐nitrosothiols (RSNOs), we computationally explored hitherto unknown (3+2) cycloaddition RSNO reactions with alkynes and alkenes. Nonactivated RSNO cycloaddition reactions have high activation enthalpy (>20 kcal/mol at the CBS‐QB3 level) and compete with alternative S—N bond insertion pathway. However, the (3+2) cycloaddition reaction barriers can be dramatically lowered by coordination of a Lewis acid to the N atom of the —SNO group. To exploit this effect, we propose to use reagents with Lewis acid and a strain‐activated carbon–carbon multiple bond linked by a rigid scaffold, which can react with RSNOs with small activation enthalpies (～5 kcal/mol) and high reaction exothermicities (～40 kcal/mol). The proposed efficient RSNO cycloaddition reactions can be used for future development of practical RSNO labeling reactions. © 2013 Wiley Periodicals, Inc.     

Polyisobutylene‐based polyurethanes. VIII. Polyurethanes with ‐O‐S‐PIB‐S‐O‐ soft segments

We describe the synthesis, characterization, and select properties of a novel polyurethane (PU) prepared using a new polyisobutylene diol, HO‐CH₂CH₂‐S‐PIB‐S‐CH₂CH₂‐OH, soft segment and conventional hard segments. The diol is synthesized by terminal functionalization of ally‐telechelic PIB followed by low‐cost thiol‐ene click chemistry. Properties of ‐S‐ containing PU (PIB_S‐PU) containing 72.5% PIB were investigated and compared to similar PUs made with HO‐PIB‐OH (PIB_O‐PU). Hydrolytic resistance was studied by contact with phosphate‐buffered saline, oxidative resistance by immersing in concentrated HNO₃, and metal ion oxidation resistance by exposure to CoCl₂/H₂O₂. Hydrolytic and oxidative resistances of PIB_S‐PU and PIB_O‐PU are similar and superior to a commercial PDMS‐based PU, Elast‐Eon? E2A. According to ¹H NMR spectroscopy the ‐S‐ in PIB_S‐PUs remained unchanged upon treatment with HNO₃, however, oxidized mainly to ‐SO₂‐ by CoCl₂/H₂O_2. Static mechanical properties of PIB_S‐PU and PIB_O‐PU are similar, except creep resistance of PIB_S‐PU is surprisingly superior. The thermal stability of PIB_S‐PUs is ～15 °C higher than that of PIB_O‐PU. FTIR spectroscopy indicates H bonded S atoms (N‐H…S) between soft and hard segments, which noticeably affect properties. DSC and XRD studies suggest random low‐periodicity crystals dispersed within a soft matrix. Energy dispersive X‐ray spectroscopy–scanning electron microscopy indicates homogeneous distribution of S atoms on PIB_S‐PU surfaces. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1119–1131     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.     

Facile Chemoenzymatic Strategies for the Synthesis and Utilization of S‐Adenosyl‐L‐Methionine Analogues

A chemoenzymatic platform for the synthesis of S‐adenosyl‐L ‐methionine (SAM) analogues compatible with downstream SAM‐utilizing enzymes is reported. Forty‐four non‐native S/Se‐alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non‐native SAM analogues. As a proof of concept for the feasibility of natural product “alkylrandomization”, a small set of differentially‐alkylated indolocarbazole analogues was generated by using a coupled hMAT2–RebM system (RebM is the sugar C4′‐O‐methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.     

Glutathione Peroxidase‐Based Amperometric Biosensor for the Detection of S‐Nitrosothiols

A new biosensor is described for the detection of S‐nitrosothiols (RSNOs) based on their decomposition by immobilized glutathione peroxidase (GPx), an enzyme containing selenocysteine residue that catalytically produces nitric oxide (NO) from RSNOs. The enzyme is entrapped at the distal tip of a planar amperometric NO sensor. The new biosensor shows good sensitivity, linearity, reversibility, and response times towards various RSNO species in PBS buffer, pH 7.4 . In most cases, the response time is less than 5 min, and the response is linear up to 6 μM of the tested RSNO species. The lowest detection limit is obtained for S‐nitrosocysteine (CysNO), at approx. 0.2 μM. The biosensor's sensitivity is not affected by the addition of EDTA as a chelating agent; an advantage over other potential catalytic enzymes that contain copper ion centers, such as CuZn‐superoxide dismutase and xanthine oxidase. However, lifetime of the new sensor is limited, with sensitivity decrease of 50% after two days of use. Nonetheless, the new amperometric GPx based RSNO sensor could prove useful for detecting relative RSNO levels in biological samples, including whole blood.     

Thermoprecipitation of Glutathione S‐Transferase by Glutathione–Poly(N‐isopropylacrylamide) Prepared by RAFT Polymerization

Herein, we report an effective and rapid method to purify glutathione S‐transferase (GST) using glutathione (GSH)‐modified poly(N‐isopropylacrylamide) (pNIPAAm) and mild, thermal conditions. A chain transfer agent modified with pyridyl disulfide was employed in the reversible addition–fragmentation chain transfer (RAFT) polymerization of NIPAAm. The resulting polymer had a narrow molecular weight distribution (polydispersity index = 1.21). Conjugation of GSH to the pyridyl disulfide–pNIPAAm reached 95% within 30 min as determined by UV–Vis monitoring of the release of pyridine‐2‐thione. GST was successfully thermoprecipitated upon heating the GSH–pNIPAAm above the lower critical solution temperature (LCST). The pull down assay was repeated with bovine serum albumin (BSA) and T4 lysozyme (T4L), which demonstrated the specificity of the polymer for GST. Due to its simplicity and high efficiency, this method holds great potential for large‐scale purification of GST‐tagged proteins.

     

Synthesis of Stable Aminoacyl‐tRNA Analogues Containing Triazole as a Bioisoster of Esters

Stable analogues : An effective synthetic route involving cycloaddition between alkynes and azidonucleosides to afford a new class of stable aminoacyl‐tRNA analogues such as depicted is presented. Biological evaluation showed that theses new compounds act as potent inhibitors of FemX_Wv aminoacyl transferase, a novel drug target.

     

Synthesis and characterization of second‐generation S,S‐dialkyl‐S‐(dimethylhydroxyphenyl)sulfonium salt photoinitiators

A new, simplified method has been developed for the synthesis of S,S‐dialkyl‐S‐(dimethylhydroxyphenyl)sulfonium salt cationic photoinitiators. This novel method has successfully been used for the preparation of S,S‐dialkyl‐S‐(3,5‐dimethyl‐4‐hydroxyphenyl)sulfonium and S,S‐dialkyl‐S‐(3,5‐dimethyl‐2‐hydroxyphenyl)sulfonium salts showing a wide variation in the length and structure of the alkyl chains on the positively charged sulfur atom. These photoinitiators can also be prepared with a wide variety of different anions. The manipulation of the lengths of the alkyl chains permits the design of compatible photoinitiators for highly nonpolar monomers and oligomers such as epoxy‐functional silicones, epoxidized polybutadiene, and epoxidized vegetable oils. This article describes the synthesis and characterization of these photoinitiators. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 2556–2569, 2003     

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.     

Semiempirical simulation of a theta‐class glutathione S‐transferase‐catalyzed glutathione attack to the allelochemical DIMBOA

We evaluated by the semiempirical method PM3 possible mechanisms of a putative interaction between a cereal allelochemical, the cyclic hydroxamic acid 2,4‐dihydroxy‐7‐metoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one (DIMBOA), and the tripeptide glutathione (GSH) inside the active site of a theta‐class glutathione S‐transferase. Based on a preliminary study of transition states from DIMBOA reactions with methanethiolate as a simple model of GSH, we investigated the roles of catalytic residues of the enzyme during nucleophilic additions of GSH to the carbonyl groups of DIMBOA and of its phenol/aldehyde isomer inside the active site model. Our results suggest that a tyrosine residue, Tyr113, makes the most important contributions for the catalytic mechanism. In the modeled reaction steps, Tyr113 behaves as a double hydrogen bond donor catalyst for nucleophilic additions of GSH to substrates: It initially helps stabilize the strongly nucleophilic reduced GSH with a hydrogen bond intermediated by a water molecule; during substrate approach, small conformational changes enable the residue to make a direct hydrogen bond to the substrate group that develop negative charge after addition of reduced GSH. © 2002 Wiley Periodicals, Inc.; Int J Quantum Chem, 2002     

Detection of Protein S‐Sulfhydration by a Tag‐Switch Technique

Protein S‐sulfhydration (forming ‐S‐SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H₂S‐mediated signaling pathways. In this study we report the first selective, “tag‐switch” method which can directly label protein S‐sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H₂S alone cannot lead to S‐sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P‐SOH) or the involvement of metal centers which would facilitate the oxidation of H₂S to HS^..     

Krebs Cycle Metabolon: Structural Evidence of Substrate Channeling Revealed by Cross‐Linking and Mass Spectrometry

It has been hypothesized that the high metabolic flux in the mitochondria is due to the self‐assembly of enzyme supercomplexes (called metabolons) that channel substrates from one enzyme to another, but there has been no experimental confirmation of this structure or the channeling. A structural investigation of enzyme organization within the Krebs cycle metabolon was accomplished by in vivo cross‐linking and mass spectrometry. Eight Krebs cycle enzyme components were isolated upon chemical fixation, and interfacial residues between mitochondrial malate dehydrogenase, citrate synthase, and aconitase were identified. Using constraint protein docking, a low‐resolution structure for the three‐enzyme complex was achieved, as well as the two‐fold symmetric octamer. Surface analysis showed formation of electrostatic channeling upon protein–protein association, which is the first structural evidence of substrate channeling in the Krebs cycle metabolon.