A solid phase assay for the protease of human immunodeficiency virus

A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.     

A simple, continuous fluorometric assay for HIV protease

Novel fluorogenic substrates for human immunodeficiency viral protease have been developed based on the principle of fluorescence energy transfer. Starting from a p24/p15 cleavage site-derived hexapeptide substrate. Ac-Thr-Ile-Nle-Nle-Gln-Arg-NH2, incorporation of 2-aminobenzoic acid in place of the acetyl group as the donor and p-NO2-Phe at the P1' position as acceptor gave the intramolecularly quenched fluorogenic substrate. Cleavage of the substrate by HIV protease released the fluorescent N-terminal tripeptide from its close apposition to the quenching nitrobenzyl group, resulting in enhanced fluorescence. An automated assay based on 96-well microtiter plates and a fluorometric plate reader have been developed, which allow high throughput of compounds in the search for HIV protease inhibitors.     

A simple solid phase assay for the detection of 2,4-D in soil

Contaminated soils are usually characterized using chemical analyses. However, these do not assess the bioavailability of pollutants, a factor which may be important in estimating the risks associated with contamination. Thus there is a need to support chemical analyses with information on biological effects to determine the potential risks a pollutant may pose in the soil. Although bacterial bioreporters have been used to detect the presence of contaminants in soils, in general these studies have been carried out in slurries or soil extracts rather than soil itself. The following study presents the development of a simple solid-phase bioassay for the direct detection of the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) in soil using Ralstonia eutropha JMP 134-32, a luxCDABE-based 2,4-D whole cell bioreporter. The bioreporter was spotted onto glass microfibre filter discs that allowed its retrieval and analysis after exposure to 2,4-D amended soils. These disc-fixed cells responded in a concentration dependent manner to 2,4-D in solution (0-25 mg/L) and in spiked soil (0-50 mg/kg). The influence of environmental factors on bioavailability was demonstrated in soil with a low moisture content which prevented 2,4-D-induced bioluminescence but which did not affect bioluminescence from already induced cells. This rapid and low cost bioassay provides a proof of concept demonstrating that retrievable disk-fixed cells can be induced in soil, thus providing a measure of solid-phase bioavailability. This method overcomes some of the limitations associated with the inoculation and monitoring of bioreporters directly in soil. Additionally, this simple system should be amenable to use with other bioreporters.     

A solid phase assay for topoisomerase I interfacial poisons and catalytic inhibitors

We report a mechanism-based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells, and retained DNA is detected by Pico Green fluorescence. Compounds that result in an increase in Pico Green staining represent potential topoisomerase interfacial poisons, whereas those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a "bimodal" readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. This method is rapid, robust, economical, and scalable for large library screens.     

日本脑炎病毒NS3蛋白酶活性检测及抑制剂高通量筛选方法的建立

日本脑炎病毒(Japanese encephalitis virus,JEV)是单股正链RNA病毒,全基因组仅含有一个开放阅读框,编码一条多聚蛋白前体,病毒编码的NS3蛋白酶在JEV多聚蛋白加工过程中起着重要作用,是重要的药物靶标。通过PCR扩增了NS2BH-NS3蛋白酶的编码区,构建了原核表达质粒并转化到大肠杆菌BL21(DE3),经IPTG诱导得到可溶性的NS3蛋白酶,用镍亲和层析方法进行了纯化。建立了基于荧光共振能量转移的NS3蛋白酶活性检测方法,并确定了最佳的反应条件,对113个化合物进行了筛选,发现其中两个化合物对JEV NS3蛋白酶具有一定的抑制活性。本研究为JEV NS3蛋白酶的活性研究及抑制剂筛选提供了一种操作方便、成本低廉的方法。     

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.     

A simple and safe method for single HLA-antigen-typing by a solid phase assay

A rapid solid phase assay for detection of single HLA-antigens on platelets was developed. The platelets were attached to the surface of polystyrene microtitre plate wells by means of a sodium carbonate buffer and centrifugation. Uncovered areas were blocked by a gelatin blocking buffer. After incubation with commercially available anti-HLA-sera the bound anti-HLA-specific antibodies directed against HLA-antigens present on the platelets were made visible by anti-IgG-coated indicator red cells and a brief centrifugation. A positive result, meaning the presence of an HLA-antigen, was indicated by a slight red cell adherence over the reaction surface. In the absence of the HLA-antigen no binding occurred and the indicator red cells formed a small red disc-like pellet.     

A simple set-and-mix assay for screening of protein kinase inhibitors in cell lysates

Here we developed a simple set-and-mix assay to perform high-throughput screening of protein kinase A (PKA) inhibitors from the LOPAC 1280 compound library. This assay is based on the color change of gold nanoparticles on aggregation induced by a cationic substrate peptide as coagulant. In spite of the simplicity of this assay system, this assay can be applied to drug screening based on cellular kinases. We successfully found several highly active inhibitors, including compounds that have not been reported before.     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

A sensitive diffusion plate assay for screening inhibitors of protease activity in plant cell fractions

Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.     

The one-bead two-compound assay for solid phase screening of combinatorial libraries

Fluorescent quenched substrate libraries are a very powerful tool for investigation of protease activity and specificity. Particularly, libraries where the fluorescent resonance energy transfer (FRET) pair is 3-nitrotyrosine and 2-amino-benzamide are easy to prepare by split and combine synthesis to yield a one-bead one-compound library format. The solid support is critical for the successful hydrolysis of the resin-bound substrates. For this purpose, a range of highly porous poly(ethylene glycol) (PEG)-based resins have been developed. Active substrates yield highly fluorescent beads and these are selected under a fluorescence microscope or isolated on a bead sorter. Edman sequence analysis yields the substrate sequence, the cleavage point, and the degree of conversion. The method gives a complete map of the substrate specificity, and substrates with high affinity for the active site can be selected. These may in turn be used as inhibition indicators in a second solid phase library assay for enzyme inhibition where each single bead is transformed into an assay container. The substrate is attached to temporarily shielded functional groups after completion of inhibitor library synthesis. By using a photolabile linker and ladder synthesis, the active inhibitors may be rapidly identified by mass spectrometry. In each bead, the putative inhibitor competes with the substrate attached for binding to the enzyme, and when the inhibitor binds strongly, the substrate remains intact and quenched. Thus dark beads indicate inhibitors, and these may be isolated using a bead-sorter and the structure determined by mass spectrometry. A selection of the best substrates and inhibitors should always be resynthesized for solution kinetics and confirmation of the results obtained on solid support. The inhibitor assay is almost free from false positives, which is a consequence of combining the binding of the protease to the inhibitor with observation of activity toward a FRET substrate. The K(i) of the identified inhibitors are typically in the nM range.     

A continuous assay for foot-and-mouth disease virus 3C protease activity

Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3C(pro), carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k(cat)/K(M) of 57.6+/-2.0M(-1) s(-1), was used in inhibition assays to further characterize the protease's activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3C(pro).     

A FRET-based assay for the discovery of West Nile Virus NS2B-NS3 protease inhibitors

The West Nile Virus (WNV) has been a worldwide epidemic since the early 1990s. Currently there are no therapeutic treatments for WNV infections. One particular avenue of treatment is inhibition of the NS2B-NS3 protease, an enzyme that is crucial for WNV replication. In our effort to increase the number of NS2B-NS3 protease inhibitors, we report a novel FRET-based high throughput assay for the discovery of WNV NS2B-NS3 protease inhibitors. For this assay, a FRET-based peptide substrate was synthesized and kinetically characterized with the NS2B-NS3 protease. The new substrate exhibits a K_m of 3.35 ± 0.31 μM, a k_cat of 0.0717 ± 0.0016 s^?1 and a k_cat/K_m of 21,400 ± 2000 M^?1 s^?1.     

Design,synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors

A class of tripeptidyl transition state inhibitors containing a P1 glutamine surrogate, a P2 leucine, and a P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays. An array of warheads, including aldehyde, α-ketoamide, bisulfite adduct, and α-hydroxyphosphonate transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E, suggesting a broad range of antiviral activities.     

A fluorescent assay suitable for inhibitor screening and vanin tissue quantification

Vanin-1 is a pantetheinase that catalyzes the hydrolysis of pantetheine to produce pantothenic acid (vitamin B5) and cysteamine. Reported here is a highly sensitive fluorescent assay using a novel fluorescently labeled pantothenate derivative. The assay has been used for characterization of a soluble version of human vanin-1 recombinant protein, identification and characterization of hits from high-throughput screening (HTS), and quantification of vanin pantothenase activity in cell lines and tissues. Under optimized assay conditions, we quantified vanin pantothenase activity in tissue lysate and found low activity in lung and liver but high activity in kidney. We demonstrated that the purified recombinant vanin-1 consisting of the extracellular portion without the glycosylphosphatidylinositol (GPI) linker was highly active with an apparent K_m of 28 μM for pantothenate-7-amino-4-methylcoumarin (pantothenate-AMC), which was converted to pantothenic acid and AMC based on liquid chromatography-mass spectrometry (LC-MS) analysis. The assay also performed well in a 384-well microplate format under initial rate conditions (10% conversion) with a signal-to-background ratio (S/B) of 7 and a Z factor of 0.75. Preliminary screening of a library of 1280 pharmaceutically active compounds identified inhibitors with novel chemical scaffolds. This assay will be a powerful tool for target validation and drug lead identification and characterization.     

A microplate assay for the screening of ADAMTS-4 inhibitors.

Aggrecanase plays a major role in cartilage proteoglycan degradation in rheumatic diseases such as osteoarthritis and rheumatoid arthritis. The search of new inhibitors of aggrecanase activity necessitates a robust assays in order to be able to screen large numbers of compounds. We present in this paper an assay based on the cleavage of His-tagged aggrecan interglobular domain by N- and C- terminus truncated, active aggrecanase-1/ADAMTS-4, with formation of the aggrecanase-specific ARGSV neoepitope. This is detected by anti-ARGSV antibody, in turn recognized by a fluorescent anti-IgG. Furthermore, the formation of the reaction products was confirmed by high-pressure capillary electrophoresis. This assay allows the rapid screening of aggrecanase inhibitors in a 96-well plate format, allowing an immediate transposition to high-throughput scale up.     

A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity.     

High-throughput screening for the discovery of inhibitors of fatty acid amide hydrolase using a microsome-based fluorescent assay

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.     

Development of potent inhibitors of the coxsackievirus 3C protease

Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.     

Novel,sulfonamide linked inhibitors of the hepatitis C virus NS3 protease

A sulfonamide replacement of the P2–P3 amide bond in the context of macrocyclic HCV NS3 protease inhibitors was investigated. These analogs displayed good inhibitory potency in the absence of any P3 capping group. The synthesis and preliminary SAR are described.