Development of novel assays for proteolytic enzymes using rhodamine-based fluorogenic substrates

Components within synthetic chemical and natural product extract libraries often interfere with fluorescence-based assays. Fluorescence interference can result when the intrinsic spectral properties of colored compounds overlap with the fluorescent probes. Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. However, because many organic compounds absorb in the ultraviolet region, they can interfere with coumarin-based fluorescence assays. Red-shifted fluorescent dyes such as peptidyl rhodamine derivatives are useful because there is generally less interference from organic compounds outside the ultraviolet wavelengths. In this report, rhodamine-based fluorogenic substrates, such as bis-(Leu)(2)-Rhod110 and bis-(Ala-Pro)-Rhod110, were developed for leucine aminopeptidase and dipeptidyl aminopeptidase. Novel, tandem rhodamine substrates such as Ala-Pro-Rhod110-Leu were designed with 2 protease cleavage sites and used to assay 2 proteases in a multiplex format. General endpoint high-throughput screening (HTS) assays were also developed for leucine aminopeptidase, dipeptidyl aminopeptidase, and trypsin that incorporated both amidomethylcoumarin and rhodamine-based fluorogenic substrates into a single screening format. These dual-substrate assays allowed for the successful screening of the LOPAC trade mark collection and natural product extracts despite high levels of fluorescence interference.     

Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates

Two assays for the plasminogen activator urokinase using peptide fluorogenic substrates are described. N-carbobenzoxy-glycyl-glycyl-l-arginine-4-methoxy-β-naphthylamide (CBZ-Gly-Gly-Arg-4MβNA) can be used in a direct assay that is simple, rapid, and sensitive to as little as 0.5 IU/ml urokinase. Additional sensitivity, to 0.01 IU/ml urokinase, is obtained in a second method that uses plamsinogen as the primary substrate followed by a fluorogenic substrate assay employing N-carbobenzoxy-l-alanyl-l-alanyl-l-lysine-4-methoxy-β-naphthylamide (CBZ-Ala-Ala-Lys-4MβNA) as a specific substrate for the activated plasmin. These assays are as sensitive as the best assays presently in use and are simpler to perform. In addition, these assays can readily be used for kinetic analysis of the hydrolytic activity of urokinase or other plasminogen activators.     

Evaluation of the use of chromogenic and fluorogenic substrates in solid-phase enzyme linked immunosorbent assays (ELISA)

Fluorogenic and chromogenic substrates were used in direct and trapping enzyme-linked immunosorbent assays (ELISA) for the detection of mouse IgG and foot-and-mouth disease virus (FMDV). The detection limits for both antigens were compared using different combinations of enzymes and substrates. Various times and concentrations of chemicals were used to obtain maximum sensitivity for both systems. Similar sensitivities were found using fluorogenic and chromogenic substrates. Tetramethyl benzidine substrate for horse-radish peroxidase enzyme conjugates was found to attain the highest sensitivity levels for chromogenic assays (0.12 ng IgG/ml and 1.0 ng/ml FMDV respectively), after 10 min incubation. Of the two fluorogenic enzyme/substrates studied, B-galactosidase was the most sensitive but required extended incubation times (2-3 h) as compared with chromogenic systems. Special microplates for fluoro-immunoassay (FIA) were compared with conventional microplates and no advantage was found to justify their use. An alkaline phosphatase anti-guinea-pig conjugate was used to confirm the equivalence of fluorogenic and chromogenic substrates in terms of sensitivity. A comparison of the amount of signal generated using various concentrations of enzyme in the absence of antigen was made for two different alkaline phosphatase conjugates to obtain theoretical sensitivity limits. One possible advantage of fluorogenic substrates is that high binding ratio can improve the confidence in discrimination of positive results.     

DICER-ARGONAUTE2 Complex in Continuous Fluorogenic Assays of RNA Interference Enzymes

Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC) have been hindered by lack of methods for continuous monitoring of enzymatic activity. “Quencherless” fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS) and hypoxia-inducible factor 1-α subunit (HIF1A). Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (K _m=74 nM) with substrate inhibition kinetics (K _i=105 nM), demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER) bound in the active site of DICER may undergo direct transfer (as AGO2 substrate) to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29), suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a strong (~2800-fold) improvement in potency for mRNA knockdown. This study lays the foundation of a systematic biochemical approach to optimize nucleic acid-based therapeutics for Dicing and ARGONAUTE2-loading for improving efficacy.     

Recent advances in enzyme assays

Enzyme assays for high-throughput screening and enzyme engineering, which are often based on derivatives of coumarin, nitrophenol, fluorescein, nitrobenzofurazane or rhodamine dyes, can be divided into two categories: those that depend on labelled substrates, and those that depend on sensing the reactions of unmodified substrates. Labelled substrates include, for example, fluorogenic and chromogenic substrates that generate a reporter molecule by beta-elimination, fluorescence resonance energy transfer (FRET) substrates and isotopic labels for enantioselectivity screening. By contrast, endpoint sensing can be done using amine reagents, fluorescent affinity labels for phosphorylated proteins, or synthetic multifunctional pores. Sensing assays can also be done in real time by using, for example, aldehyde trapping to follow vinyl ester acylation in organic solvent or calcein-copper fluorescence for sensing amino acids. The current trend is to assemble many such assays in parallel for enzyme profiling and enzyme fingerprinting.     

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.     

Assay of apical membrane enzymes based on fluorogenic substrates.

A series of enzymatic rate assays are described. The assays are based on coumarin derivatives that are fluorogenic substrates for the enzymes dipeptidase IV, aminopeptidase N, alkaline phosphatase, and gamma-glutamyltransferase. These simple assays are rapid and offer improved sensitivity over established colorimetric methods. The substrates have apparent affinities for the enzymes of 5-250 microM. L-Glutamic acid gamma-(7-amido-4-methylcoumarin) is characterized as a substrate of gamma-glutamyltransferase on the basis of inhibition of enzymatic cleavage when the glycylglycine acceptor molecule is omitted and inhibition of the enzymatic reaction by addition of glycine. Assay conditions for the four enzymes are established such that less than 0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Intestinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays.     

High-throughput screening for biocatalysts

Recent progress in high-throughput enzyme assays includes new examples of fluorogenic and chromogenic substrates, fluorescence resonance energy transfer substrates, and applications of the pH and pM indicator methods. Recent developments of Horeau's pseudo-enantiomer derivatisation method to screen enantioselectivities in high-throughput have also been reported.     

Synthesis and fluorescence properties of intramolecularly quenched fluorogenic p-nitroanilides containing coumarin or quinolinone derivatives as fluorophores.

Nine model intramolecularly quenched fluorogenic substrates (IQFS) of the general structure F-Phe-NH-Np, containing coumarin or quinolinone derivatives as fluorophores (F) and the p-nitroanilide group (Np) as quencher, were synthesized. The study of the fluorescence properties of the substrates synthesized and the corresponding fluorophores showed that efficient quenching of fluorescence (>89%) was observed in all cases. The combination of 7-glutarylamido-4-methyl-coumarin (Mec-NH-Glt-OH) or 7-methoxy-4-coumaryl-acetic acid (Mca) with the p-nitroanilide group gave the best results (97.2 and 98.8% quenching, respectively). These fluorophores can be used to convert peptide p-nitroanilides into IQFS, which, retaining their chromogenic properties, may be applied in both fluorometric and colorimetric assays.     

A low background high-throughput screening (HTS) fluorescence assay for lipases and esterases using acyloxymethylethers of umbelliferone

Esters and acyloxymethyl ethers of umbelliferone were evaluated as fluorogenic substrates for lipases and esterases with respect to stability and resistance to non-specific hydrolysis. Isobutyryloxymethyl ether 1c and pivaloxymethyl ether 1d were found to be optimal substrates for enzyme assays, particularly with respect to HTS applications.     

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.     

A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers

A method to simultaneously determine the relative numbers of live and dead cells in culture by introducing a combination of two fluorogenic substrates or a fluorogenic and a luminogenic protease substrate into the sample is described. The method is based on detection of differential ubiquitous proteolytic activities associated with intact viable cells and cells that have lost membrane integrity. A cell-permeable peptide aminofluorocoumarin substrate detects protease activity restricted to intact viable cells. Upon cell death, the viable cell protease marker becomes inactive. An impermeable peptide rhodamine 110 (or aminoluciferin) conjugated substrate detects protease activity from nonviable cells that have lost membrane integrity. The multiplex assay can detect 200 dead cells in a population of 10,000 viable cells. The protease substrate reagents do not damage viable cells over the course of the assay, thus the method can be multiplexed further with other assays in a homogeneous format. Ratiometric measurement of viable and dead cells in the same sample provides an internal control that can be used to normalize data from other cell-based assays.     

Simple assay for sialyltransferase activity with a new fluorogenic substrate

A new fluorogenic acceptor for sialyltransferase, 2-[(2-pyridyl)amino]ethyl O-beta-D-galactopyranosyl-(1----4)-beta-D-glucopyranoside, was prepared from lactose as a starting material. Sialyltransferase activity was assayed by incubation of the enzyme with the acceptor and CMP-N-acetylneuraminic acid, separation of the fluorogenic sialylated product from the enzymatic reaction mixture by HPLC, and measurement of the product. Compared to assays so far reported that use radioactive substrates, this assay is simple and rapid. This method was used to assay sialyltransferase activity in human serum.     

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.     

Determination of the substrate specificity of tripeptidyl-peptidase I using combinatorial peptide libraries and development of improved fluorogenic substrates

Classical late-infantile neuronal ceroid lipofuscinosis is a fatal neurodegenerative disease caused by mutations in CLN2, the gene encoding the lysosomal protease tripeptidyl-peptidase I (TPP I). The natural substrates for TPP I and the pathophysiological processes associated with lysosomal storage and disease progression are not well understood. Detailed characterization of TPP I substrate specificity should provide insights into these issues and also aid in the development of improved clinical and biochemical assays. To this end, we constructed fluorogenic and standard combinatorial peptide libraries and analyzed them using fluorescence and mass spectrometry-based activity assays. The fluorogenic group 7-amino-4-carbamoylmethylcoumarin was incorporated into a series of 7-amino-4-carbamoylmethylcoumarin tripeptide libraries using a design strategy that allowed systematic evaluation of the P1, P2, and P3 positions. TPP I digestion of these substrates liberates the fluorescence group and results in a large increase in fluorescence that can be used to calculate kinetic parameters and to derive the substrate specificity constant kcat/KM. In addition, we implemented a mass spectrometry-based assay to measure the hydrolysis of individual peptides in peptide pools and thus expand the scope of the analysis. Nonfluorogenic tetrapeptide and pentapeptide libraries were synthesized and analyzed to evaluate P1' and P2' residues. Together, this analysis allowed us to predict the relative specificity of TPP I toward a wide range of potential biological substrates. In addition, we evaluated a variety of new fluorogenic peptides with a P3 Arg residue, and we demonstrated their superiority compared with the widely used substrate Ala-Ala-Phe-AMC for selectively measuring TPP I activity in biological specimens.     

Fluorogenic substrates for chymotrypsin with 2-quinolinone derivatives as leaving groups

Sensitive and convenient fluorometric assays for the determination of chymotrypsin have been developed by using the substrates Glt-Leu-Phe-NH-Meq, Glt-Phe-NH-FMeq and Glt-Leu-Phe-NH-FMeq, which have been synthesized utilizing the mixed anhydride method. The amidolytic activity of chymotrypsin was measured by the release of the highly fluorescent amine 7-amino-4-methyl-2-quinolinone (AMeq) or 7-amino-4-trifluoromethyl-2-quinolinone (AFMeq). The fluorescence properties of the synthesized substrates and the new fluorescent marker AFMeq were examined. Kinetic constants, as well as the maximum sensitivity for the hydrolysis of the new substrates, were determined. All new substrates permit a rapid and sensitive determination of chymotrypsin in a continuous assay system. As little as 0.7 ng of enzyme can be detected using the substrate Glt-Leu-Phe-NH-Meq, which is the most sensitive fluorogenic substrate thus far reported.     

Establishment of a suite of assays that support the discovery of proteasome stimulators

BackgroundThe proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing.MethodsThe cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates.ResultsTo demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold.ConclusionAssays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides.General significanceA suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.     

Effects of intrinsic fluorescence and quenching on fluorescence-based screening of natural products

To evaluate the effects of intrinsic (natural) fluorescence and quenching as confounding variables in fluorescence-based enzyme inhibition assays of natural products, we measured the fluorescence and quenching properties of 25 components of popular herbal products. The analyses were performed under conditions typically employed in drug-drug interaction studies that use c-DNA-derived P450 isoforms and surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin, quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere with these assays. Intrinsic fluorescence had a greater effect on these assays than quenching and for one compound, yangonin, was sufficient to mask inhibition and potentially produce a false negative result. Quenching had less of an effect on these assays, but was significant enough for one compound, quercetin, to mimic "weak" inhibition. Therefore, because intrinsic fluorescence or quenching could render some natural products unsuitable for testing in certain fluorometric assays, it would be prudent to include an evaluation of these properties in experimental protocols.