Fluorogenic ester substrates to assess proteolytic activity (original) (raw)
Related papers
Proceedings of the National Academy of Sciences, 2000
A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors.
Fluorescein-based amino acids for solid phase synthesis of fluorogenic protease substrates
Bioorganic & Medicinal Chemistry, 2006
An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with 3030% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a 302.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.
Assembly and selective “in synthesis” labeling of quenched fluorogenic protease substrates
Analytical Biochemistry, 2006
Because impaired cellular protease activities are linked to many diseases, such as cancer, inXammation, neurodegeneration, and infection, internally quenched Xuorescent peptides have recently been developed as tools for analyzing the speciWcities of these enzymes. Here we report convenient and cost-eVective approaches for the selective "in synthesis" assembly of such substrate peptides for protease assays. Fluorescein and Dabcyl groups were covalently and selectively attached during synthesis to -amino groups of internal lysines. Functionality was then tested by digestion with leucine aminopeptidase, chymotrypsin, and microsomal vesicles. All peptides proved to be appropriate substrates of the enzymes tested and of the endogenous peptidases in the microsomal vesicles. In summary, we describe an innovative and cheap method to develop completely functional quenched Xuorescent peptides that are usable in speciWc detection of individual proteases, in particular aminopeptidases, in both in vitro and in vivo systems.
Latent Fluorophores Based on a Self-Immolative Linker Strategy and Suitable for Protease Sensing
Bioconjugate Chemistry, 2008
The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzymeinitiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in Vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.
Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family
Russian Journal of Bioorganic Chemistry, 2008
A chemoenzymatic syntheses was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA ( I ) and Glp-Phe-Ala-Amc ( II ) (Abz, pNA, Glp, and Amc are i -aminobenzoyl, p -nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate ( I ) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate ( II ) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate ( I ) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate ( II ), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate ( II ), than for substrate ( I ). The lowest detectable papain concentrations were 2.4 × 10 -10 M for ( I ) and 1.2 × 10 -11 M for ( II ). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.
Measurement of Specific Protease Activity Utilizing Fluorescence Polarization
Analytical Biochemistry, 1997
substrates offers an alternative method that avoids in-A fluorescence polarization assay was designed to terference, but disposal and safety concerns make this measure proteolytic cleavage of a specific peptide subapproach less attractive. strate for human cytomegalovirus protease. The pep-We present an assay for site-specific proteolytic actide substrate was derivatized by biotinylation of a tivity utilizing fluorescence polarization. The substrate g-aminobutyric acid-modified amino-terminus and lais a protease-specific peptide derivatized by biotinylabeled with 5-(4,6-dichlorotriazinyl)aminofluorescein tion of the amino-terminus and coupling of a fluoroat the carboxy-terminus. Incubation of this substrate phore at the carboxy-terminal end. Proteolytic activity with recombinant human cytomegalovirus protease was quantitated from the total fluorescence polarizaand subsequent addition of egg white avidin produced tion of the mixture of cleaved and uncleaved peptide a polarization signal that was proportional to the relaafter incubation with the protease. Since the fluorestive amounts of cleaved and uncleaved substrate. The cence polarization value is the ratio of orthogonal fluouncleaved substrate produced a high polarization rescence intensities, it is not sensitive to absorptive value upon binding to avidin, whereas the cleaved, interferants. We demonstrate the assay robustness to low-molecular-weight fluorescently tagged peptide absorptive interferants by measuring the polarization that cannot bind to avidin produced a low polarization of a constant concentration of biotin-fluorescein, biovalue. The inhibitory activity of a 3,4-dichloroisocoumtin, and avidin in the presence of increasing concentraarin against the protease was evaluated by comparing tions of dyes that absorb where fluorescein either abthe change in polarization with a noninhibited consorbs or emits. trol. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, ho-MATERIALS AND METHODS mogenous assay for high throughput screening. ᭧ 1997 Chemicals and Reagents Academic Press Avidin, biotin-fluorescein, and 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) 1 were purchased from Molecular Probes (Eugene, OR). Bovine serum albumin The activity of proteases recognizing specific cleav-(fraction V; BSA), 3-[(3-chloramidopropyl)-dimethyage sites is usually measured using substrates conlammonio]-1-propane sulfonate (Chaps), g-aminobusisting of a specific peptide modified by the addition of tyric acid (Abu), 3,4-dichloroisocoumarin, mordant blue latently colorimetric or fluorescent moieties (1-6). The 3, eosin B, and biotin were purchased from Sigma (St. hydrolysis products of these synthetic substrates pos-Louis, MO). All other chemicals were analytical grade. sess spectral features that enable quantitative deter-Buffers were stored at 4ЊC after preparation in ultramination of substrate cleavage. Substrates of this type pure water (Millipore Milli-Q) and further filtered are widely used in the characterization of many differthrough 0.2-mm filters. Recombinant human cytomegaent proteases. However, evaluation of potential inhibitors in complex mixtures such as natural products ex-1 Abbreviations used: DTAF, 5-(4,6-dichlorotriazinyl)aminofluortracts can be severely limited because these mixtures escein; BSA, bovine serum albumin; Chaps, 3-[(3-chloramidopropyl)often contain other components that interfere with dimethylammonio]-1-propane sulfonate; PBS, phosphate-buffered saline; HCMV, human cytomegalovirus; Abu, g-aminobutyric acid.
Journal of Medicinal Chemistry, 1992
A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2'-aminoethyl)aminolnaphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety that can be incorporated near the C-terminus of the peptide substrate. The corresponding fluorescent acceptor group containing a 4 4 [4-(dimethylamino)phenyl]am] benzoic acid (DABCYL) can then be attached (16) Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. A. Pharmacokinetic Analy~is Program (Multi) for Microcomputer. J. P h r m .
Analytical Biochemistry, 2005
New peptide substrates containing benzoxazol-5-yl-alanine derivatives for kinetic assay of cysteine proteases have been synthesized and characterized. The substrates are peptides internally quenched by the intramolecular fluorescence resonance energy transfer. The results demonstrate that the kind of donor-acceptor pair (D-A) significantly affects the kinetic parameters of the enzymatic process. The three longest peptides, Box-Lys-Phe-Gly-Gly-Ala-Ala-Tyr(NO 2 ) containing Box-alanine derivative as a donor and nitro-tyrosine as an acceptor, show two times greater affinity to papain than does the one peptide possessing Dabcyl-Edans as a D-A pair. Kinetic parameters for the best papain substrate, Lys-Box(benzfur)-Gly-Gly-Ala-Ala-Tyr(NO 2 ), are K m = 6.85 ± 0.59 lM, k cat = 19.51 s À1 , and k cat /K m = 2.85 lM À1 s À1 . It was found that the peptides Box(benzfur)-Lys-Phe-Gly-Gly-Tyr(NO 2 ) and Box(benzfur)-Phe-Gly-Gly-Tyr(NO 2 ) were also hydrolyzed by cathepsin B with the highest speed of hydrolysis as a result of caboxypeptidase activity of this enzyme. Moreover, these substrates show high affinity and selectivity to this enzyme.
Scientific Reports, 2017
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/ Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/ specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. The irreversible peptide bond hydrolysis of proteins and polypeptides is the most conserved post-translational modification occurring in biochemical pathways in all living organisms 1,2. This reaction is catalyzed by proteases, which specifically recognize protein targets to control numerous significant biological processes, including cell survival and cell death and the immune response to various pathogens 3. The selectivity of proteases for binding and subsequently hydrolyzing a selected group of peptides or proteins is termed substrate specificity 4,5. The increasing number of chemical tools for substrate specificity profiling allows the development of new, more efficient and more selective small molecule substrates 6,7 , inhibitors 8 , and chemical probes 9 , which are useful for the determination of protease activity and the dissection of their physiological functions. Internally quenched fluorescent (IQF) peptide substrates constitute a convenient tool for examining the specificity of the largest group of proteases-endopeptidases 10. These substrates contain a paired fluorophore (donor) and quencher (acceptor), which are located on opposite sides of the scissile peptide bond 11,12. If the fluorophore
Analytical Biochemistry, 2007
Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as k cat /K m values, which ranged from 0.2 to 80 · 10 6 M À1 min À1 . The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.