Harnessing the Oxidation Susceptibility of Deubiquitinases for Inhibition with Small Molecules

Deubiquitinases (DUBs) counteract ubiquitination by removing or trimming ubiquitin chains to alter the signal. Their diverse role in biological processes and involvement in diseases have recently attracted great interest with regard to their mechanism and inhibition. It has been shown that some DUBs are regulated by reactive oxygen species (ROS) in which the catalytic Cys residue undergoes reversible oxidation, hence modulating DUBs activity under oxidative stress. Reported herein for the first time, the observation that small molecules, which are capable of generating ROS efficiently, inhibit DUBs by selective and nonreversible oxidation of the catalytic Cys residue. Interestingly, the small molecule beta‐lapachone, which is currently in clinical trials for cancer, is among the potent inhibitors, thus suggesting possible new cellular targets for its therapeutic effects. Our study describes a novel mechanism of DUBs inhibition and opens new opportunities in exploiting them for cancer therapy.     

Elucidating the Dual Mode of Action of Dipeptidyl Enoates in the Inhibition of Rhodesain Cysteine Proteases

A computational study of the two possible inhibition mechanisms of rhodesain cysteine protease by the dipeptidyl enoate Cbz-Phe-Leu-CH=CH−CO₂C₂H₅ has been carried out by means of molecular dynamics simulations with hybrid QM/MM potentials. The low free energy barriers confirm that the Cys25 residue can attack both C_β and C1 atoms of the inhibitor, confirming a dual mode of action in the inhibition of the rhodesain by enoates. According to the results, the inhibition process through the Cys25 attack on the C_β atom of the inhibitor is an exergonic and irreversible process, while the inhibition process when Cys25 attacks on the C1 atom of the inhibitor is and exergonic but reversible process. The interactions between the inhibitor and rhodesain suggest that P2 is the most important fragment to consider in the design of new efficient inhibitors of rhodesain. These results may be useful for the design of new inhibitors of rhodesain and other related cysteine proteases based on dipeptidyl enoates scaffolds.     

Docking-based virtual screening studies aiming at the covalent inhibition of SARS-CoV-2 MPro by targeting the cysteine 145

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 which has infected millions of people worldwide. The main protease of SARS-CoV-2 (M^Pro) has been recognized as a key target for the development of antiviral compounds. Taking advantage of the X-ray crystal complex with reversible covalent inhibitors interacting with the catalytic cysteine 145 (Cys145), we explored flexible docking studies to select alternative compounds able to target this residue as covalent inhibitors. First, docking studies of three known electrophilic compounds led to results consistent with co-crystallized data validating the method for SARS-CoV-2 M^Pro covalent inhibition. Then, libraries of soft electrophiles (overall 41 757 compounds) were submitted to docking-based virtual screening resulting in the identification of 17 molecules having their electrophilic group close to the Cys145 residue. We also investigated flexible docking studies of a focused approved covalent drugs library including 32 compounds with various electrophilic functional groups. Among them, the calculations resulted in the identification of four compounds, namely dimethylfumarate, fosfomycin, ibrutinib and saxagliptin, able first, to bind to the active site of the protein and second, to form a covalent bond with the catalytic cysteine.     

Reactions of an organoruthenium anticancer complex with 2-mercaptobenzanilide--a model for the active-site cysteine of protein tyrosine phosphatase 1B

The organometallic anticancer complex [(η(6)-p-cymene)Ru(en)Cl]PF(6) (1, en = ethylenediamine) readily reacts with thiols and forms stable sulfenate/sulfinate adducts which may be important for its biological activity. Protein tyrosine phosphatase 1B (PTP1B), a therapeutic target, contains a catalytic cysteinyl thiol and is involved in the regulation of insulin signaling and the balance of protein tyrosine kinase activity. On oxidation, the catalytic Cys215 can form an unusual sulfenyl-amide intermediate which can subsequently be reduced by glutathione. Here we study reactions of 1 with 2-mercaptobenzanilide, 2, a recognized model for the active site of PTP1B. We have characterized crystallographically compound 2 and its oxidized sulfenyl-amide derivative 2-phenyl-1,2-benzisothiazol-3(2H)-one (4), which shows a close structural similarity to the sulfenyl-amide in oxidized PTP1B. At pH 7.4 and 5.3, 1 reacted with 2, affording a mono-ruthenium thiolato complex [(η(6)-cym)Ru(en)(S-RS)](+) (7(+), R = (C(6)H(4))CONH(C(6)H(5))) and a triply-S-bridged thiolato complex [((η(6)-cym)Ru)(2)(μ-S-RS)(3)](+) (8(+)), respectively. Coordination of Ru to the S atom in 7 allows formation of a strong H-bond (2.02 ?) between the en-NH and the carbonyl oxygen. To assess the possible effect of ruthenium coordination on the redox regulation of PTP1B, reactions of these thiolato products with H(2)O(2) and/or GSH were then investigated, demonstrating that coordination to Ru largely retards both the oxidation (deactivation) of the thiol in compound 2 by H(2)O(2) and the subsequent reduction (reactivation) of the sulfenyl-amide by GSH, implying that the inhibition of complex 1 on PTP1B (IC(50) of 19 μM) may be attributed to coordination to its catalytic cysteine.     

Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase

In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.     

Pt对负载型Ni-B非晶态合金苯加氢活性及稳定性的影响

以拟薄水铝石、丝光沸石为原料制备载体AM,采用浸渍-化学还原法制备了不同Pt含量的Ni-Pt-B/AM非晶态合金催化剂,并运用XRD、ESEM、BET、ICP、XPS和H2 -TPR等手段对催化剂进行表征.以苯加氢制环己烷反应为探针,考察了微量Pt对Ni-B/AM非晶态合金催化剂加氢活性及稳定性的影响.结果表明,Ni-...     

Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy‐Resistant L858R/T790M/C797S Mutant

The treatment of non‐small‐cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations. Third‐generation EGFR inhibitors have been designed to overcome resistance through covalent binding to the Cys 797 residue of the enzyme, and these inhibitors are effective against most clinically relevant EGFR mutants. However, the high dependence of these recent EGFR inhibitors on this particular interaction means that additional mutation of Cys 797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients. A new generation of irreversible and reversible mutant EGFR inhibitors was developed with strong noncovalent binding properties, and these compounds show high inhibitory activities against the cysteine‐mutated L858R/T790M/C797S EGFR.     

Structural insight into exosite binding and discovery of novel exosite inhibitors of botulinum neurotoxin serotype A through in silico screening

Botulinum neurotoxin serotype A (BoNT/A) is the most lethal toxin among the Tier 1 Select Agents. Development of potent and selective small molecule inhibitors against BoNT/A zinc metalloprotease remains a challenging problem due to its exceptionally large substrate binding surface and conformational plasticity. The exosites of the catalytic domain of BoNT/A are intriguing alternative sites for small molecule intervention, but their suitability for inhibitor design remains largely unexplored. In this study, we employed two recently identified exosite inhibitors, D-chicoric acid and lomofungin, to probe the structural features of the exosites and molecular mechanisms of synergistic inhibition. The results showed that D-chicoric acid favors binding at the α-exosite, whereas lomofungin preferentially binds at the β-exosite by mimicking the substrate β-sheet binding interaction. Molecular dynamics simulations and binding interaction analysis of the exosite inhibitors with BoNT/A revealed key elements and hotspots that likely contribute to the inhibitor binding and synergistic inhibition. Finally, we performed database virtual screening for novel inhibitors of BoNT/A targeting the exosites. Hits C1 and C2 showed non-competitive inhibition and likely target the α- and β-exosites, respectively. The identified exosite inhibitors may provide novel candidates for structure-based development of therapeutics against BoNT/A intoxication.     

β-二酮锆/AlEt2Cl/MAO催化乙烯原位共聚合成支化聚乙烯

以β-二酮锆为唯一主催化剂, 以AlEt2Cl和MAO为助催化剂, 使之分别与主催化剂作用形成两种不同功能的催化活性中心, 考察乙烯原位共聚合成支化聚乙烯.     

Kinetic study of the effects of inhibitors on the catalyzed dehydration of HCO3- by copper(II) complexes [TpPh]CuX (X- = OH-, N3-, NCS-)

A series of half-sandwich copper(II) complexes [TpPh]CuX ([TpPh] = hydrotris(3-phenyl-pyrazolyl)borate; X- = OH- (1), N3- (2), NCS- (3)) have been synthesized as models for carbonic anhydrase. The structure of 3 was determined by X-ray diffraction analysis. Crystals of 3 (C37H30BCuN9S) are triclinic, space group P1 with a = 11.997(3) A, b = 12.116(3) A, c = 13.384(4) A, alpha = 81.088(5) degrees, beta = 79.289(6) degrees, gamma = 68.668(5) degrees, V = 1772.4(8) A3, and Z = 2. The dehydration kinetic measurements of HCO3- are performed by the stopped-flow techniques at pH < 7.9. The apparent dehydration rate constant kdobs varies linearly with total Cu(II) concentration, and the catalytic activity of the model complexes decreases in the order 1 > 2 > 3. The catalytic activity decreases with increasing pH indicating that the aqua model complex must be the reactive catalytic species in the catalyzed dehydration reaction and the rate-determining step is the substitution of the labile water molecule by HCO3-. The kdobs values increase with increasing reaction temperature, and the apparent activation energies of the model complexes with inhibitors are remarkably higher than that of the complex with no inhibitors, this being the origin of inhibition. The large negative entropy of activation also indicates an associative mode of activation in the rate-determining step. The inhibition ability of the inhibitor NCS- is stronger than that of the inhibitor N3-, which can be rationalized by the decrease in effective atomic charges of the Cu(II) ions as revealed by the theoretical calculations.     

Structural Insights into the Molecular Recognition Mechanism of the Cancer and Pathogenic Epitope,LacdiNAc by Immune-Related Lectins

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we investigated the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAcβ1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Galβ1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions was observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.     

Immobilized Fullerene C60‐Enzyme‐Based Electrochemical Glucose Sensor

A mixed‐valence cluster of cobalt(II) hexacyanoferrate and fullerene C60‐enzyme‐based electrochemical glucose sensor was developed. A water insoluble fullerene C60‐glucose oxidase (C60‐GOD) was prepared and applied as an immobilized enzyme on a glassy carbon electrode with cobalt(II) hexacyanoferrate for analysis of glucose. The glucose in 0.1 M KCl/phosphate buffer solution at pH = 6 was measured with an applied electrode potential at 0.0 mV (vs Ag/AgCl reference electrode). The C60‐GOD‐based electrochemical glucose sensor exhibited efficient electro‐catalytic activity toward the liberated hydrogen peroxide and allowed cathodic detection of glucose. The C60‐GOD electrochemical glucose sensor also showed quite good selectivity to glucose with no interference from easily oxidizable biospecies, e.g. uric acid, ascorbic acid, cysteine, tyrosine, acetaminophen and galactose. The current of H₂O₂ reduced by cobalt(II) hexacyanoferrate was found to be proportional to the concentration of glucose in aqueous solutions. The immobilized C60‐GOD enzyme‐based glucose sensor exhibited a good linear response up to 8 mM glucose with a sensitivity of 5.60 × 10² nA/mM and a quite short response time of 5 sec. The C60‐GOD‐based glucose sensor also showed a good sensitivity with a detection limit of 1.6 × 10^‐6 M and a high reproducibility with a relative standard deviation (RSD) of 4.26%. Effects of pH and temperature on the responses of the immobilized C60‐GOD/cobalt(II) hexacyanoferrate‐based electrochemical glucose sensor were also studied and discussed.     

一种基于多羧基荧光共轭聚电解质的氨基酸检测新方法

基于多羧基荧光共轭聚电解质PPE-(COOK)₄, 发展了一种检测半胱氨酸(Cys)和组氨酸(His)的简便方法. 该方法以PPE-(COOK)₄与Cu²⁺所构成的猝灭体系为基础, 当体系中存在半胱氨酸或组氨酸时, PPE-(COOK)₄/Cu²⁺体系的荧光恢复, 从而实现对上述氨基酸的测定. 由于PPE-(COOK)₄具有类似四乙酸乙二胺(EDTA)的螯状侧链结构, 使其在磷酸盐缓冲液(PBS, 10 mmol/L, pH=7.4)中对Cu²⁺具有超高的灵敏度和选择性, 因此可以借助各种金属掩蔽离子来提高氨基酸检测的特异性. 实验结果表明, 在PPE-(COOK)₄/Cu²⁺体系中引入Ni²⁺或Hg²⁺后, 可分别实现对半胱氨酸和组氨酸的特异性检测, 线性范围分别为1~5和0.5~2.5 μmol/L, 检出限分别为0.15和0.04 μmol/L.     

单茂钛催化剂的丙烯无规聚合反应及其动力学研究

比较了不同钛化合物／甲基铝氧烷（ＭＡＯ）催化体系的丙烯无规聚合，催化活性次序为ＣｐＴｉ（ＯＲ）３＞ＣｐＴｉ（ＯＰｈ）３＞ＣｐＴｉＣｌ３＞Ｃｐ２ＴｉＣｌ２＞Ｔｉ（ＯＢｕ）４＞ＴｉＣｌ４＞Ｔｉ（ＯＢｕ）２Ｃｌ２，所得聚丙烯用沸庚烷抽提８ｈ，溶解９５％以上，可溶部分经１３Ｃ ＮＭＲ、ＷＡＤＸ、ＦＴＩＲ等分析证明为无规聚丙烯（ａＰＰ），是没有结晶性的弹性体．ＧＰＣ测出其分子量Ｍｗ＝８．０～１０．０×１０４，Ｍｗ／Ｍｎ≈２．０．探索了催化体系ＣｐＴｉ（Ｏ ｎ Ｐｒ）３／ＭＡＯ中钛的浓度、［Ａｌ］／［Ｔｉ］摩尔比，丙烯聚合压力，聚合温度和时间对丙烯聚合反应的影响．研究了该催化体系丙烯聚合反应动力学规律，表观聚合反应速率对催化剂浓度和单体压力（浓度）都呈一级反应关系，表观聚合速率常数ＫＰ＝２９２×１０５ｍｏｌ／Ｌ·ｈ（４０℃）．活化能ΔＥ＝－７．８８×１０３Ｊ·ｍｏｌ－１，碰撞因子Ａ＝１．４１×１０－４ｍｏｌ／Ｌ·ｈ．     

Chelation of cadmium ions by phytochelatin synthase: role of the cysteine-rich C-terminal.

The interactions between Cd(2+) and the C-terminal region of phytochelatin (PC) synthase using recombinant wild-type and mutant PC synthase were studied. We show that site-directed mutagenesis of Cys residues at C(358)C(359)XXXC(363)XXC(366) motif decreases the number of Cd(2+) and other heavy metal ions interacting with the enzyme, and that the motif binds the metals discriminatingly. The optimum binding ratio of PC synthase to Cd(2+) was also determined. The findings indicate that Cys exists as a free SH residue and that it is involved in the regulation of PC enzyme activity by transferring the metals into closer proximity with the catalytic domain. These results are important in understanding heavy metal detoxification mechanisms in higher plants, a step towards phytoremediated-applications.     

Synthesis and Characterization of Chromium Hexacyanoferrate/Multiwalled Carbon Nanotube Composite and Its Biosensing for L‐Cysteine

This work describes the electrochemical properties of glassy carbon electrodes (GCE) modified with chromium(III) hexacyanoferrate(II) (Crhf) nanoparticles attached multiwalled carbon nanotube (MWNTs). The morphological characterization of Crhf/MWNTs nanocomposites was examined by scanning electron microscopy (SEM), UV‐vis spectroscopy, and Fourier transform infrared spectrometry (FT‐IR). The electrocatalytic activity of these nanocomposites was investigated and showed a good electrocatalytic effect for oxidation of L ‐cysteine (L ‐Cys) in 0.1 M phosphate buffer solution (pH 3.0). Under optimum conditions linear calibration graphs were obtained over the L ‐Cys concentration range 5.0×10^?7 to 6.0×10^?5 M with a correlation coefficient of 0.9998 and a detection limit (signal‐to‐noise ratio was 3) of 1.0×10^?8 M. The proposed method is simple and it also showed excellent sensitivity and stability. The excellent electrocatalytic ability of the modified electrode towards L ‐Cys manifests that the Crhf/MWNTs can provide a new platform for biosensors and other biology.     

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patients

A LC‐MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200 μL of whole blood was precipitated with 10 mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5 mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1×50 mm, 1.7 μm using gradient LC‐MS/MS analysis in positive electrospray mode. Time of analysis was 5 min. Linearity in blood was 5–2000 μg/L for CsA, AM1, and AM9; 2–500 μg/L for AM4N; and 2–500 μg/L for all substances in lymphocytes. Coefficient of variations was 1.8–9.8% and recovery was 92.0–110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested.     

Identification of Non-Macrocyclic Small Molecule Inhibitors against the NS3/4A Serine Protease of Hepatitis C Virus through in Silico Screening

Drug discovery and design for inhibition of the Hepatitis C Virus (HCV) NS3/4A serine protease is a major challenge. The broad, shallow, and generally featureless nature of the active site makes it a difficult target for "hit" selection especially using standard docking programs. There are several macrocyclic NS3/4A protease inhibitors that have been approved or are in clinical trials to treat chronic HCV (alone or as combination therapy), but most of the current therapies for HCV infection have untoward side effects, indicating a continuing medical need for the discovery of novel therapeutics with improved efficacy. In this study, we designed and implemented a two-tiered and progressive docking regime that successfully identified five non-macrocyclic small molecules that show inhibitory activity in the low micromolar range. Of these, four compounds show varying inhibition against HCV subgenotypes 1b, 1a, 2a, and 4d. The top inhibitor (3) has an IC(50) value of 15 μM against both subgenotypes 1b and 2a of the NS3/4A protease enzyme. Another inhibitor, 1, inhibits all four subgenotypes with moderate activity, showing highest activity for genotype 2a (24 μM). The five inhibitors presented in this study could be valuable candidates for future hit to lead optimization. Additionally, enzyme-inhibitor interaction models presented herein provide key information regarding structural differences between the active sites of the NS3/4A protease of the HCV subgenotype 1a and 1b that might explain the variable inhibitory activity between subgenotypes of the small molecule inhibitors identified here.     

Cu(PPh3)2BH4作还原剂制备PtCu/C催化剂催化烯烃硅氢 加成反应研究

采用湿法浸渍, Cu(PPh3)2BH4作为还原剂制备了负载型PtCu/C双金属催化剂, Pt∶Cu原子比为0.5～2.0. 用于苯乙烯与三乙氧基硅烷加成反应, Cu元素的引入使Pt/C催化剂的催化性能得到提高, 表现出比Speier催化剂、Karestedt催化剂和Pt/C催化剂更好的β-加成产物选择性. 催化直链烯烃与三乙氧基硅烷加成反应表现出优良的催化性能, 并且催化剂可重复使用.