Fluorescence resonance energy transfer studies on the proximity relationship between the intrinsic metal ion and substrate binding sites of Escherichia coli RNA polymerase

DNA-dependent RNA polymerase from Escherichia coli contains 2 mol of zinc/mol of holoenzyme (alpha 2 beta beta' sigma) with one zinc each in the beta and beta' subunits. A new method to substitute selectively the zinc in the beta subunit was developed by the inactivation of RNA polymerase with 0.25 M NaNO3, 1 M NaCl, 1 mM diaminocyclohexane tetraacetic acid, and 0.1 mM dithiothreitol followed by reconstitution with Co(II), Cd(II), or Cu(II). The hybrid Co-Zn, Cd-Zn, or Cu-Zn RNA polymerase thus obtained retains, respectively, 91, 88, and 50% enzyme activity of the reconstituted Zn-Zn RNA polymerase. Co-Zn RNA polymerase exhibits absorption maxima at 395 and 465 nm, and Cu-Zn RNA polymerase at 637 nm (epsilon = 815 M-1 cm-1). 1-Aminonaphthalene-5-sulfonic acid (AmNS) derivatives of ATP, UTP, and dinucleoside monophosphates (diNMPs), UpA or ApU, were synthesized with AmNS attached to NTP via a gamma-phosphoamidate bond or to diNMPs via a 5'-secondary amine linkage. Since the fluorescence emission maxima of (5'-AmNS)UpA, (gamma-AmNS)ATP, and (gamma-AmNS)UTP at 445, 464, and 464 nm, respectively, when excited at 340 nm, overlap the 465-nm absorption band of Co-Zn RNA polymerase, the spatial relationship between fluorescence substrate analogs and the intrinsic Co(II) in Co-Zn RNA polymerase was studied by fluorescence resonance energy transfer technique. The fluorescence of the initiator, (5'-AmNS)UpA, and elongator, (gamma-AmNS)UTP, of the RNA chain, was quenched 20.3 and 7.1%, by the addition of saturation concentration of Zn-Zn RNA polymerase, and 21.3 and 14.7%, respectively, by the addition of template, poly(dA-dT). The fluorescence of (5'-AmNS)UpA and (gamma-AmNS)UTP was quenched 81.8 and 80.6%, respectively, by the addition of the saturation concentration of Co-Zn RNA polymerase in the absence of template, and 82.7 and 82.9% in the presence of template. On the basis of respective Ro values of 21.3 and 21.9 A for the (5'-AmNS)UpA-Co and (gamma-AmNS)UTP-Co pairs, the distances from Co(II) to the initiation site and to the elongation site were calculated to be 17.4 and 17.5 A, respectively, in the absence and 17.2 and 17.4 A in the presence of template.     

Experimental screening of dihydrofolate reductase yields a "test set" of 50,000 small molecules for a computational data-mining and docking competition

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use "real-world" HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a "training set", and computational chemists and data analysts were challenged to predict the activity of compounds in this second "test set". Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.     

Substrate properties of C′-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N-type conformation.     

A multifaceted approach to identify non-specific enzyme inhibition: Application to Mycobacterium tuberculosis shikimate kinase

Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, ¹HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation.     

Identification of novel Kv1.3 blockers using a fluorescent cell-based ion channel assay

A functional cell-based assay was developed using a generic proprietary assay protocol, based on a membrane-potential sensitive dye, for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel. A high-throughput screen (HTS) was subsequently performed with 20,000 compounds from the Evotec library, preselected using known small molecule antagonists for both sodium and potassium ion channels. Following data analysis, the hit rate was measured at 1.72%, and subsequent dose-response analysis of selected hits showed a high hit confirmation rate yielding approximately 50 compounds with an apparent IC50 value lower than 10 microM. Subsequent electrophysiological characterization of selected hits confirmed the initial activity and potency of the identified hits on the Kv1.3 target and also selectivity toward Kv1.3 through measurements on HERG as well as Kv1.3-expressing cell lines. Follow-up structure-activity relationship analysis revealed a variety of different clusters distributed throughout the library as well as several singlicates. In comparison to known Kv1.3 blockers, new chemical entities and scaffolds showing potency and selectivity against the Kv1.3 ion channel were detected. In addition, a screening strategy for ion channel drug discovery HTS, medicinal chemistry, and electrophysiology is presented.     

Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.     

"Plate cherry picking": a novel semi-sequential screening paradigm for cheaper, faster, information-rich compound selection

This work describes a novel semi-sequential technique for in silico enhancement of high-throughput screening (HTS) experiments now employed at Novartis. It is used in situations in which the size of the screen is limited by the readout (e.g., high-content screens) or the amount of reagents or tools (proteins or cells) available. By performing computational chemical diversity selection on a per plate basis (instead of a per compound basis), 25% of the 1,000,000-compound screening was optimized for general initial HTS. Statistical models are then generated from target-specific primary results (percentage inhibition data) to drive the cherry picking and testing from the entire collection. Using retrospective analysis of 11 HTS campaigns, the authors show that this method would have captured on average two thirds of the active compounds (IC(50) < 10 microM) and three fourths of the active Murcko scaffolds while decreasing screening expenditure by nearly 75%. This result is true for a wide variety of targets, including G-protein-coupled receptors, chemokine receptors, kinases, metalloproteinases, pathway screens, and protein-protein interactions. Unlike time-consuming "classic" sequential approaches that require multiple iterations of cherry picking, testing, and building statistical models, here individual compounds are cherry picked just once, based directly on primary screening data. Strikingly, the authors demonstrate that models built from primary data are as robust as models built from IC(50) data. This is true for all HTS campaigns analyzed, which represent a wide variety of target classes and assay types.     

Differential inhibitory effects of 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates and related nucleotides on DNA-dependent RNA polymerases I and II from the cherry salmon (Oncorhynchus masou)

Various 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6 microM) than the Km value for UTP (8 microM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 microM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 microM) than the Km value for UTP (4 microM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.     

以蛋白激酶B为靶点的新型抗结核药物高通量筛选模型的建立和应用

【目的】建立以结核分枝杆菌蛋白激酶B为靶点的高通量筛选模型,并运用此模型进行化合物的筛选。【方法】克隆和表达结核分枝杆菌蛋白激酶B,并以其为靶酶建立并优化PknB抑制剂高通量筛选模型,利用该模型对化合物样品进行筛选,并对筛选到的阳性化合物进行抗菌和抑酶活性评价。【结果】利用该模型筛选了化合物样品18 000个,得到具有抑酶活性的阳性化合物8个,其中3个化合物具有较好的对结核分枝杆菌、海分枝杆菌、耻垢分枝杆菌的抑菌活性。【结论】建立的以PknB为靶点的抗结核药物高通量筛选模型具有灵敏度高、稳定性强等优点,可成功用于化合物的高效筛选。筛选得到3个在抑酶水平和抗菌方面均具有良好活性的阳性化合物样品,值得进一步研究。     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

Automated multiple ligand screening by frontal affinity chromatography-mass spectrometry (FAC-MS)

High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.     

High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB alpha

Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the scintillation proximity assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited 相似文献   

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.     

A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-dependent RNA polymerase

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3'UTR-U(30) RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT(PPi), which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3'dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC(50) values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3'UTR-C(30) RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC(50) (inhibitory concentration 50%) of 0.87 microM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC(50) in the range of 1.8-2.6 microM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics.