抑制剂在氨氧化微生物研究中的应用

在氨氧化微生物的相关研究中经常使用各类抑制剂,包括针对硝化作用的抑制剂和针对微生物生长的抑制剂。自发现氨氧化古菌以来,人们在氨氧化细菌抑制剂的基础上重新筛选和使用不同的抑制剂来满足氨氧化微生物研究的需求。抑制剂既可以加速氨氧化古菌的富集,也可以帮助研究者区分古菌与细菌对硝化作用的贡献以及它们自身合成代谢能力的差别。本文综述了各类抑制剂的使用浓度和抑制效果,包括双氰胺(DCD)、3,4-二甲基吡啶磷酸盐(DMPP)、丙烯基硫脲(ATU)等传统抑制剂,乙炔和辛炔等炔烃类抑制剂,一氧化氮清除剂以及抗生素等对氨氧化微生物的活性和生长有特异性或通用抑制能力的抑制剂。通过对氨氧化微生物抑制剂的归纳总结,可为氨氧化微生物研究过程中抑制剂的选择提供参考。     

Expanding the chemical diversity of CK2 inhibitors

None of the already described CK2 inhibitors did fulfill the requirements for successful clinical settings. In order to find innovative CK2 inhibitors based on new scaffolds, we have performed a high-throughput screening of diverse chemical libraries. We report here the identification and characterization of several classes of new inhibitors. Whereas some share characteristics of previously known CK2 inhibitors, others are chemically unrelated and may represent new opportunities for the development of better CK2 inhibitors. By combining structure-activity relationships with a docking procedure, we were able to determine the binding mode of these inhibitors. Interestingly, beside the identification of several nanomolar ATP-competitive inhibitors, one class of chemical inhibitors displays a non-ATP competitive mode of inhibition, a feature that suggests that CK2 possess distinct druggable binding sites. For the most promising inhibitors, selectivity profiling was performed. We also provide evidence that some chemical compounds are inhibiting CK2 in living cells. Finally, the collected data allowed us to draw the rules about the chemical requirements for CK2 inhibition both in vitro and in a cellular context.     

Substrate Binding Sites of Leuconostoc Dextransucrase Evaluated by Inhibition Kinetics

Graphical analysis of inhibition kinetics for dextransucrase from Leuconostoc mesenteroides was done with typical inhibitors, competitive and noncompetitive. Based on the plots of Yonetani-Theorell and Semenza-Balthazar, mutual competition between the pairs of inhibitors of identical kinetic type was observed, while combination of competitive and noncompetitive inhibitors gave no significant mutual interactions. By the procedure of Nitta et al., binding sites for competitive and noncompetitive inhibitors were shown to be distant from each other. Moreover, two noncompetitive inhibitors competed with each other for a single binding site on the enzyme. Although biphasic reciprocal plots may suggest rather complicated binding of various inhibitors, the results obtained by the three graphical methods are fully explained when competitive and noncompetitive inhibitors for substrate sucrose bind to the so-called donor- and acceptor-sites of dextransucrase, respectively.     

Dietary inhibitors of mutagenesis and carcinogenesis

Dietary inhibitors of mutagenesis and carcinogenesis are of particular interest because they may be useful for human cancer prevention. Several mutagenesis inhibitors have been demonstrated to be carcinogenesis inhibitors also, e.g., ellagic acid, palmitoleic acid, and N-acetylcysteine. This means that the search for mutagenesis inhibitors may be useful for discovering anticarcinogenic agents. Many mutagenesis inhibitors have been discovered by the use of short-term assays, particularly the Ames Salmonella test. This simple in vitro system has provided opportunities to elucidate the mechanisms of inhibition. The elucidation of the mechanism may allow us to infer the possible anticarcinogenic activity of the reagent. In this chapter, inhibitors of mutagenesis and carcinogenesis that can arise as components of diet have been reviewed. Most of the inhibitors have been demonstrated to be effective against a specific class of mutagens or carcinogens. Therefore, it may be argued that these inhibitors are antagonistic only to those particular agents. Here again, understanding of the mechanisms of these inhibitions is necessary for the assessment. Dietary inhibitors reviewed in this article include: (1) as inhibitors of mutagenesis: porphyllins, fatty acids, vitamins, polyphenols, and sulfhydryl compounds, (2) as inhibitors of carcinogenesis: vitamins A, E and C, ellagic acid, sulfhydryl compounds, fats, selenium, calcium, and fiber. Further studies in this area of science appear to help establish the recipe of a healthy diet.     

PARP inhibitors in ovarian cancer: Sensitivity prediction and resistance mechanisms

Poly (ADP‐ribose) polymerase (PARP) inhibitors have provided great clinical benefits to ovarian cancer patients. To date, three PARP inhibitors, namely, olaparib, rucaparib and niraparib have been approved for the treatment of ovarian cancer in the United States. Homologous recombination deficiency (HRD) and platinum sensitivity are prospective biomarkers for predicting the response to PARP inhibitors in ovarian cancers. Preclinical data have focused on identifying the gene aberrations that might generate HRD and induce sensitivity to PARP inhibitors in vitro in cancer cell lines or in vivo in patient‐derived xenografts. Clinical trials have focused on genomic scar analysis to identify biomarkers for predicting the response to PARP inhibitors. Additionally, researchers have aimed to investigate mechanisms of resistance to PARP inhibitors and strategies to overcome this resistance. Combining PARP inhibitors with HR pathway inhibitors to extend the utility of PARP inhibitors to BRCA‐proficient tumours is increasingly foreseeable. Identifying the population of patients with the greatest potential benefit from PARP inhibitor therapy and the circumstances under which patients are no longer suited for PARP inhibitor therapy are important. Further studies are required in order to propose better strategies for overcoming resistance to PARP inhibitor therapy in ovarian cancers.     

Reversed hydroxamate-bearing thermolysin inhibitors mimic a high-energy intermediate along the enzyme-catalyzed proteolytic reaction pathway

A series of inhibitors that bear a reversed hydroxamate moiety have been evaluated as transition state analogue inhibitors for thermolysin. A linear correlation is observed between the K(i) values of these inhibitors and the kinetic parameters (K(M)/k(cat)) of the parallel series of related substrates, satisfying the criterion stipulated for transition state analogue inhibitors by Bartlett and Marlowe. Furthermore, examination of the binding mode of a related reversed hydroxamate bearing thermolysin inhibitor, in comparison with a transition state postulated for the enzyme-catalyzed proteolytic reaction revealed that the inhibitors under study mimic the electronic as well as the geometric characteristics of the transition state. On the basis of these results it may be concluded that the hydroxamate-bearing zinc protease inhibitors are a new type of transition state analogue inhibitors.     

Tripeptidic BACE1 inhibitors devised by in-silico conformational structure-based design

Previously reported pentapeptidic BACE1 inhibitors, designed using a substrate-based approach, were used as lead compounds for the further design of non-peptidic BACE1 inhibitors. Although these peptidic and non-peptidic inhibitors, with a hydroxymethylcarbonyl isostere as a substrate transition-state mimic, exhibited potent BACE1 inhibitory activities, their molecular-sizes appeared a little too big (molecular weight of >600daltons) for developing practical anti-Alzheimer's disease drugs. To develop lower weight BACE1 inhibitors, a series of tripeptidic BACE1 inhibitors were devised using a design approach based on the conformation of a virtual inhibitor bound to the BACE1 active site, also called 'in-silico conformational structure-based design'. Although these tripeptidic BACE1 inhibitors contained some natural amino acid residues, they are expected to be useful as lead compounds for developing the next generation BACE1 inhibitors, due to their low molecular size and unique structural features compared with previously reported inhibitors.     

Microorganisms as a source of tyrosinase inhibitors: a review

Tyrosinase is the main enzyme responsible for enzymatic browning of fruits post-harvest and melanogenesis in mammals, an undesirable phenomenon. This encouraged researchers to seek potent tyrosinase inhibitors for application in the food and cosmetics industries. Despite an increased knowledge of tyrosinase inhibitors from plants and synthetic sources in the past few years, inhibitors of microbial origin are under-explored. Thus, this article surveys tyrosinase inhibitors produced by microorganisms and hence, serves as an updated database of tyrosinase inhibitors from microbial sources.     

Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

Small-molecule protein kinase inhibitors are widely used to elucidate cellular signaling pathways and are promising therapeutic agents. Owing to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments that use these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we used functional assays to profile the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases. Quantitative analysis revealed complex and often unexpected interactions between protein kinases and kinase inhibitors, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can identify multitargeted inhibitors of specific, diverse kinases. The results have implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments involving kinase inhibitors.     

Method for Extracting Viral Hemagglutination-Inhibiting Antibodies from the Nonspecific Inhibitors of Serum

Various methods are used to remove nonspecific inhibitors from sera before titering viral hemagglutination-inhibiting antibodies. These methods have several undesirable features; some are tedious and time-consuming, some remove antibody along with nonspecific inhibitors, and different techniques are usually required to remove the nonspecific inhibitors for different viruses. This communication describes a single method that uses diethylaminoethyl-Sephadex to extract the immunoglobulin G antibodies for several viruses from nonspecific inhibitors. The procedure is fast, simple to perform, and removed the nonspecific inhibitors for influenza, Western equine encephalitis, dengue-2, and rubella viruses.     

Molecular docking of inhibitors into monoamine oxidase B

Monoamine oxidase B (MAO-B) functions in the deamination of monoamines, including dopamine and norepinephrine. The search for MAO-B inhibitors increased following the discovery that the enzyme may be responsible for generating neurotoxins from various endogenous or exogenous compounds. Computational screening methods aid in the search for new inhibitors, but validation studies for specific software packages and receptors are necessary for effective application of these methods. In this study, DOCK 6.0.0 was used to dock a series of inhibitors to MAO-B. Included were studies of re-docking ligands into MAO-B crystal structures, after which a set of 30 compounds with known inhibition constants for MAO-B were docked, including 15 strong inhibitors and 15 weak inhibitors. Good agreement was observed between the top experimental inhibitors and the top ranked docking results, and key interactions between the ligands and receptor were identified.     

Molecular docking and pharmacophore model studies of Rho kinase inhibitors

Molecular docking and pharmacophore model approaches were used to characterise the binding features of four different series of Rho kinase (ROCK) inhibitors. Docking simulation of 20 inhibitors with ROCK was performed. The binding conformations and binding affinities of these inhibitors were obtained using AutoDock 4.0 software. The predicted binding affinities correlate well with the activities of these inhibitors (R ² = 0.904). 3D pharmacophore models were generated for ROCK based on highly active inhibitors implemented in Catalyst 4.11 program. The best pharmacophore model consists of one hydrogen bond acceptor feature and two hydrophobic features, and they all seemed to be essential for inhibitors in terms of their binding activities. It is anticipated that the findings reported in this paper may provide very useful information for designing new ROCK inhibitors.     

Small-molecule inhibitors of the cell cycle

The cell cycle remains an attractive target for the development of small-molecule inhibitors for use as both novel chemotherapeutics and research probes. Given the importance of cytoskeletal dynamics and cyclin-dependent kinases for cell-cycle progression, much interest has focused on the identification of anti-mitotic agents and kinase inhibitors. However recent advances in cell-based screening technologies and an increased interest in inhibitors with greater specificity are beginning to influence the search for novel cell-cycle inhibitors.     

Docking-based 3D-QSAR study for selectivity of DPP4, DPP8, and DPP9 inhibitors

In order to obtain information regarding the design of selective DPP4 inhibitors, a 3D-QSAR study was conducted using DPP4, DPP8, and DPP9 inhibitors including newly synthesized six- and seven-membered cyclic hydrazine derivatives (KR64300, KR64301), which were evaluated in vitro for their inhibition of DPP4, DPP8, and DPP9. In this study, a highly predictive CoMFA model based on the fast-docking for DPP4, DPP8, and DPP9 inhibitors was obtained. This reliable model showed leave-one-out cross-validation q(2) and conventional r(2) values of 0.68 and 0.96 for the DPP4 inhibitors, 0.58 and 0.98 for the DPP8 inhibitors, and 0.68 and 0.97 for the DPP9 inhibitors, respectively. The validation of the CoMFA model was confirmed by the compounds in the test set, including the synthesized six- and seven-membered cyclic hydrazines. According to this study, to obtain selective DPP4 inhibitors compared to their isozymes, the interaction of the inhibitors with the S3 site and S1' site in DPP4 must be considered. The proposed newly synthesized compounds, KR64300 and KR64301, interact well with the sites mentioned above, showing excellent selectivity.     

HIV进入抑制剂的研究进展

随着对HIV进入细胞过程的了解,各种进入抑制剂相继问世,目前主要有三大类:吸附抑制剂、辅助受体抑制剂和融合抑制剂.对其中具有代表性的进入抑制剂研究进展进行了介绍,一些进入抑制剂已经进入到了临床试验阶段,其中融合抑制剂T20在2003年便被FDA批准可同其他ARTs联合用于治疗HIV感染者,CCR5拮抗剂Maraviro...     

Genetic variants of protease inhibitors against fungal protease and α-chymotrypsin from hemolymph of the silkworm,Bombyx mori

Many electrophoretic variants of hemolymph inhibitors of proteases from Aspergillus melleus and pancreatic alpha-chymotrypsin were found using 126 silkworm strains. Six inhibitors of the fungal protease were detected and eight of chymotrypsin; the distribution of inhibitors among Japanese, Chinese, and European races was investigated. Comparison of electrophoretic patterns from F1 hybrids and parents showed that the offspring produce inhibitors of both parental types. Segregation in F2 and backcrossing suggest that the expression of each inhibitor is controlled in most cases by a pair of alleles which are responsible for strong and null bands. Two bands of fungal protease inhibitors C and D were controlled by codominant alleles. These results suggest that polymorphism of hemolymph protease inhibitors in the silkworm would be a useful experimental system for the study of the genetic control of protease inhibitors.     

Development and validation of high-throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors

Poly(ADP-ribose) polymerase-1 and -2 (PARP1/2) are two key facilitators of DNA repair and are implicated in the pathogenesis of cancers and several chronic diseases. Inhibitors of PARP1/2 have shown powerful therapeutic effects in the treatment of cancer, cerebral ischemia, and inflammation. In addition, evidence from several studies suggests unique functions for PARP2 in genome surveillance, spermatogenesis, adipogenesis, and T cell development, and PARP2-specific inhibitors might have many other applications. To acquire PARP1/2 inhibitors, many high-throughput screening (HTS) assays for PARP1 inhibitors have been developed. However, detailed screening assays for PARP2 inhibitors have not been reported. Herein, three HTS assays for PARP2 inhibitors were developed and validated with reference inhibitors in each case. The results suggest that the HTS assays for PARP2 inhibitors using chemical quantification of NAD⁺, biotin-based quantification of PAR, and ELISA quantification of PAR are sensitive, robust, and cost effective.     

Serine proteinase inhibitors of seminal plasma of teleost fish: distribution of activity, electrophoretic profiles and relation to proteinase inhibitors of blood

Anti-proteinase activity has been found in seminal plasma of eight teleost fish species: brown trout, rainbow trout, brook trout, lake whitefish, bream, northern pike, Danube salmon and burbot. This activity correlated with seminal plasma protein and sperm concentrations. Using a mammalian (bovine) trypsin for detecting proteinase inhibitors it was found for the first time that there are species-specific electrophoretic profiles of anti-proteinase activity. One to three bands could be identified by this method. However, additional proteinase inhibitors could be identified by using fish (cod) trypsin. These inhibitors were detected in seminal plasma of salmonids and coregonids and have a slow migration rate. Fast-migrating proteinase inhibitors were present in rainbow, brown and brook trout, northern pike, whitefish and burbot. These inhibitors could be detected in brook and brown trout by using either trypsins. However, they were detected only with bovine trypsin in rainbow trout, northern pike, whitefish and burbot. These results suggest that multiple forms of serine proteinase inhibitors exist in seminal plasma of teleost fish and they differ in their affinity toward serine proteinases. Seminal plasma serine proteinase inhibitors of rainbow trout migrated during electrophoresis similarly to blood plasma proteinase inhibitors, and suggests that the two inhibitors may be similar or the same. Anti-proteinase specific activity was similar in blood and seminal plasma. Proteinase inhibitors of fish seminal plasma seem to be an important part of sperm physiology, possibly related to protection of spermatozoa. Staining for detection of serine proteinase inhibitors also allowed detection of presence of nonspecific esterase in seminal plasma of most species.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Development of novel telomerase inhibitors based on a bisindole unit

Telomerase is the enzyme that elongates telomere repeat at the ends of a chromosome. As high telomerase activity is observed in most cancer cells, inhibitors of human telomerase have been expected as new chemotherapeutic agents for cancer. We describe here the discovery of novel inhibitors with IC50 values in the submicromolar range. The structure of the novel inhibitors will be useful as a scaffold for construction of the library in the search for telomerase inhibitors.