[go: up one dir, main page]

WO2003073066A2 - Reactifs et procedes pour des dosages des protesase de facteur letal du bacillus anthracis - Google Patents

Reactifs et procedes pour des dosages des protesase de facteur letal du bacillus anthracis Download PDF

Info

Publication number
WO2003073066A2
WO2003073066A2 PCT/US2003/005553 US0305553W WO03073066A2 WO 2003073066 A2 WO2003073066 A2 WO 2003073066A2 US 0305553 W US0305553 W US 0305553W WO 03073066 A2 WO03073066 A2 WO 03073066A2
Authority
WO
WIPO (PCT)
Prior art keywords
labeled derivative
lysine
labeled
peptide
lethal factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2003/005553
Other languages
English (en)
Other versions
WO2003073066A3 (fr
Inventor
Barry R. Cunningham
Richard T. Cummings
Jeffrey D. Hermes
Scott P. Salowe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of WO2003073066A2 publication Critical patent/WO2003073066A2/fr
Anticipated expiration legal-status Critical
Publication of WO2003073066A3 publication Critical patent/WO2003073066A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)

Definitions

  • Anthrax is a bacterial infection produced by Bacillus anthracis. Bacillus anthracis endospores can enter the body through skin abrasions, inhalation, or ingestion. Bacillus anthracis produces an anthrax toxin that is often lethal. (Dixon et al., (1999) N. Engl. J. Med. 341, 815-26.)
  • Anthrax toxin consists of three proteins, a receptor-binding component designated protective antigen, and two enzymatic components termed edema factor and lethal factor ("LF").
  • Lethal factor is a zinc-dependent metalloprotease that appears to exert toxic affects by cleaving mitogen-activated protein kinase kinases (MKKs).
  • MKKs mitogen-activated protein kinase kinases
  • the present invention features a Bacillus anthracis lethal factor substrate and assays employing the substrate to measure lethal factor activity and to screen for compounds affecting lethal factor activity.
  • Preferred substrates contain one or more detectable labels and have a sufficiently high turnover rate to be suitable for use in a high throughput screen.
  • a first aspect of the present inventions features a peptide consisting essentially of:
  • XlO is either glutamine or a labeled derivative thereof
  • Xl 1 is either leucine or a labeled derivative thereof
  • Xl2 is either asparagine or a labeled derivative thereof
  • Xl7 is either lysine, cysteine, or a labeled derivative thereof
  • Xl8 is an optionally present amino acid that, if present, is either alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid, or a labeled derivative thereof;
  • Xl9 is an optionally present amino acid that, if present, is either alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid, or a labeled derivative thereof; and "z” is either an amide linkage or an ester linkage.
  • Reference to "consisting essentially” or “consist essentially” with respect to a peptide indicate that additional groups may be present that do not significantly prevent the peptide from acting as a lethal factor substrate.
  • additional groups include one more of the following: salts, additional amino acid(s) at the N-terminus, additional amino acid(s) at the C-terminus, and protecting group(s). Unless otherwise indicated, reference to an amino acid having a chiral center refers to the L-enantiomer; and amino acids are joined by peptide (amide) linkages.
  • a "labeled derivative" of an amino acid indicates that the amino acid is modified with a chemical moiety (also referred herein as a label) that can be detected or specifically bound by another molecule and which does not significantly prevent the peptide from acting as a lethal factor substrate. Detection can be by direct means, such as the moiety producing a detectable signal; or by indirect means such as by the moiety serving as binding site for another moiety that emits a signal.
  • Another aspect of the present invention features a method of measuring Bacillus anthracis lethal factor activity.
  • the method comprises the steps of: (a) incubating lethal factor and a lethal factor substrate using an incubation medium wherein the lethal factor is active; and (b) measuring cleavage of the peptide.
  • Another aspect of the present invention features a method of measuring the ability of a compound to affect Bacillus anthracis lethal factor activity.
  • the method comprises the steps of: (a) incubating the compound, lethal factor, and a lethal factor substrate using an incubation medium wherein the lethal factor is active; and (b) measuring cleavage of the peptide.
  • Figure 1 illustrates the results of enzymatic cleavage experiments using fluoresceinated substrates [Nle']Mekl 6 (KGG)-NH 2 (SEQ ID NO: 1) and the MKK consensus sequence analog (Flu)NleKKKKVLPIQLNAATDKGG-NH 2 (SEQ ID NO: 2) as assessed by the HPLC cleavage assay.
  • [Substrate] 100 ⁇ M
  • [LF] 1 ⁇ M
  • T 37 °C with time points as indicated.
  • [Nle 1 ]Mekl 6 (KGG)-NH 2 (SEQ ID NO: 1) substrate consumption is illustrated by closed circles.
  • MKK consensus sequence analog substrate consumption is illustrated by open circles. First-order rate constants were derived from curve fitting each set of data; comparison of these rate constants indicated that the turnover of the consensus sequence was ⁇ 100-fold faster than the MEK1 sequence.
  • Figure 2 illustrates results from enzymatic cleavage experiments of four FRET analogs of the MKK consensus sequence.
  • Peptides at 10 ⁇ M in 50 mM HEPES pH 7.0V20 mM NaCl/10 mM MgCl 2 /100 ⁇ M CaCl 2 /100 ⁇ M ZnCl 2 /l mg/ml BSA/1 mM DTT were incubated with 100 nM lethal factor in a volume of 75 ⁇ l in a black 96-well plate.
  • the increase in fluorescence (excitation 355 nm/emission 460 nm) was read on a Victor 2 V plate reader.
  • the present invention features a Bacillus anthracis lethal factor substrate and assays employing the substrate to measure lethal factor activity and to screen for compounds affecting such activity. Assaying lethal factor activity can be used to better characterize and study such activity and to obtain lethal factor inhibitory compounds.
  • Lethal factor inhibitor compounds can be used to further study lethal factor activity, and those inhibitory compounds having appropriate pharmacological properties can be used to help treat or prevent Anthrax. Appropriate pharmacological properties include efficacy, metabolism and absence of unacceptable side effects.
  • High throughput screening for lethal factor inhibitors can be used to screen large number of compounds to identify those affecting lethal factor activity. High throughput screening is facilitated by an assay that is readily automated and utilizes low levels of purified enzyme.
  • Preferred substrates contain one or more detectable labels and have a sufficiently high turnover rate and low background rate to be suitable for use in a high throughput screen.
  • Preferred substrates can be used with low nanomolar amounts (in the range of 1-10 nM) of enzyme with short incubation times (5-60 minutes), using a multi-well plate-based format.
  • Lethal factor substrates described herein consist essentially of:
  • X8 is either proline or a labeled derivative thereof;
  • ⁇ 9 is either isoleucine or a labeled derivative thereof;
  • XlO is either glutamine or a labeled derivative thereof;
  • Xl 1 is either leucine or a labeled derivative thereof;
  • Xl2 is either asparagine or a labeled derivative thereof
  • Xl3 is either proline, alanine, or a labeled derivative thereof; preferably, Xl3 is either alanine or a labeled derivative thereof;
  • Xl4 is either alanine or a labeled derivative thereof;
  • Xl5 is either proline, threonine, or a labeled derivative thereof; preferably, Xl5 is either threonine or a labeled derivative thereof;
  • Xl6 is either aspartic acid or a labeled derivative thereof
  • Xl7 is either lysine, cysteine, or a labeled derivative thereof; preferably, Xl7 is either lysine or a labeled derivative thereof; ⁇ l8 is an optionally present amino acid that, if present, is either alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid, or a labeled derivative thereof; preferably, Xl8, is glycine or a labeled derivative thereof;
  • Xl9 i an optionally present amino acid that, if present, is either alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid, or a labeled derivative thereof; preferably, Xl , is glycine or a labeled derivative thereof; and
  • z is either an amide linkage or an ester linkage.
  • Techniques for chemical synthesis of peptides are well known in the art. (See e.g., Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990, and the Example 1, infra.) Preferred positions for labeled derivatives depend upon the label.
  • Labels should be chosen so as not to significantly affect the ability of the substrate to be cleaved by lethal factor. Small labels such as radioactive labels are not expected to affect cleavage. Bulky labels at or near the cleavage site may affect cleavage. The effect of a particular label on cleavage can readily be evaluated by assaying for cleavage.
  • the cleavage site for the lethal factor substrates described herein is between ⁇ 8 proline and ⁇ isoleucine.
  • there is no label at a cleavage site amino acid there is no label within one amino acid from the cleavage site (i.e., no label at a cleavage site amino acid or at a neighboring amino acid), there is no label within two, three, four, five, six, or seven amino acids from the cleavage site; only Xl and/or Xl7 contain a label; and there is no label.
  • z is an amide linkage.
  • Table I provides examples of different lethal factor substrates. Table I
  • a variety of different assay formats can be employed to detect peptide cleavage.
  • Homogeneous assays detect the cleavage product without separation of the materials through a direct readout such as FRET or by an alternate detection such as an antibody raised to a cleavage fragment.
  • Heterogenous assays detect the cleavage product after separating the cleaved peptide fragments from one another through some mechanism such as avidin capture of a biotin labeled material or separation by physical properties such as occurs on an HPLC column.
  • Assays can be performed employing a variety of different labels such as a fluorescent moiety, a quencher moiety, a chemiluminescent moiety, a radioisotope, biotin, biotin derivatives, lanthanides, and antibody specific tags such as anti-phosphotyrosine, anti-phosphoserine, anti-dinitrophenyl, anti-FLAG, anti- fluorescein, anti-rhodamine, and anti-digoxigenin.
  • One or more labels which may be the same or different, can be present on one or both sides of the cleavage site.
  • Single side label(s) can be used in instances where substrates and products separate based on physical properties (such as in HPLC) or when substrate cleavage generates a site for specific recognition of a product, for example by an antibody.
  • Labels on opposite sides of the cleavage site can be used in multiple ways. In homogenous formats these labels can interact such that signal will increase or decrease upon substrate cleavage.
  • An example of a homogeneous signal increase format is an assay employing an internally quenched FRET pair.
  • heterogeneous formats the separation of a product from substrate by one label allows specific detection of a cleavage product by the other label.
  • An acceptor and donor FRET pair present on opposite sides of a cleavage site produce a different signal depending upon whether cleavage has taken place.
  • a donor excited by a suitable light source emits energy having the proper wavelength to absorbed by the acceptor.
  • the acceptor quenches signal production from the donor by either emitting light at a different wavelength (if the acceptor is another fluorophore) or by dissipating the energy to the environment (if the acceptor is a quencher).
  • FRET assays can be run assaying for the appearance of donor fluorescence if the acceptor is a quencher since upon cleavage donor and quencher are separated. Alternatively, if both donor and acceptor are fluorophores and the donor is being excited, FRET assays can be run assaying for the appearance of donor fluorescence or the disappearance of acceptor fluorescence.
  • fluorophores include dansyl and its derivatives, fluorescein and its derivatives, rhodamine and its derivatives, Texas Red, coumarin derivatives, Cy dyes, AlexaFluor dyes (Molecular Probes), and BODIPY dyes (Molecular Probes).
  • quencher includes the QSY series (Molecular Probes), Dabcyl, p- nitrophenyl derivatives, dinitrophenyl derivatives, and the Cy quencher dyes (Amersham-Pharmacia). Techniques and reagents for performing FRET are well known in the art. (For example, see, Selvin, (2000) Nat. Struct. Biol, 7(9), 730-4, Clegg, (1995) Curr. Opin. Biotechnol, 6(1), 103-10, 1995, and Wu et al, Anal. Biochem., 218(1), 1-13, 1994.)
  • substrates With appropriately labeled substrates alternate technologies can be used to measure substrate cleavage.
  • homogeneous formats would include fluorescence polarization, time resolved FRET, SPATM, FlashPlateTM, and AlphaScreenTM.
  • heterogeneous formats would include DELFIATM, chemiluminescence plate based assays, HPLC, radioactive filter binding assays, absorbance assays, and fluorescence assays.
  • Lethal factor substrates can be used in methods measuring Bacillus anthracis lethal factor activity and the effect of a compound on such activity. Such methods involve incubating a lethal factor substrate described herein with Bacillus anthracis lethal factor using an incubation medium where the Bacillus anthracis lethal factor is active, and can include the presence of a compound being tested. Cleavage of the substrate can be detected as a measure of Bacillus anthracis lethal factor activity or the effect of a compound on lethal factor activity. Measuring can be qualitative or quantitative.
  • This example describes different materials and methods that were employed to produce and study lethal factor substrates.
  • Peptide labeling with reporters was carried out on resin subsequent to peptide synthesis. Incorporation of materials at the N-terminus was accomplished by labeling with commercially available N-hydroxysuccinimidyl esters. Typical reactions were carried out on a 20-100 ⁇ mol resin scale using a 1-10 fold excess of the label in a minimal volume (1-3 mL) of N-methylpyrrolidinone overnight. Incorporation of the two reporters for the FRET substrates was accomplished by first labeling the N-terminus. The second reporter was then introduced by selectively removing the Lys(Mtt) protecting group (CH 2 C1 2 with 2% TFA and 3% Triisopropylsilane, room temperature, 45 minutes), resin washing, and reaction with the label as described above.
  • Lys(Mtt) protecting group CH 2 C1 2 with 2% TFA and 3% Triisopropylsilane, room temperature, 45 minutes
  • the resin was washed with NMP, acetic acid, CH 2 C1 2 , and methanol (3x each), dried briefly in vacuo and the peptides cleaved using 95% TFA/2.5% H 2 O/2.5% Triisopropylsilane for 90 minutes.
  • the crude peptides were purified on a Waters PrepLC 4000 system using a 25 x 400 mm 300 A DeltaPak C 18 column and a CH 3 CN/H 2 O (both with 0.1% TFA) gradient. Purified peptides were lyophilized and their molecular weight confirmed by mass spectral analysis. All peptides were >95% pure by reversed-phase HPLC (A 2 ⁇ 4 ).
  • Peptides were prepared as 1 mM stock solutions in doubly distilled water. As long term solution stability has not been evaluated these materials were protected from incident light and used within a week of preparation. Test peptides were incubated at 100 ⁇ M with varying levels of lethal factor in 50 mM HEPES (pH 7.0)/20 mM NaCl/10 mM MgCl 2 /100 ⁇ M CaCl 2 /100 ⁇ M ZnCl 2 /l mg/ml BSA/1 mM DTT) for various times.
  • HPLC samples Prior to HPLC injection, samples were treated by precipitation of the protein materials by addition of a 10-fold excess volume of 60% CH 3 CN in H 2 O. The samples were then vortexed, allowed to sit on ice for at least 15 minutes, centrifuged (2000g x 60 seconds), and the supernatant removed from the protein pellet. HPLC samples typically contained 150-300 ng (75-150 pmol) of labeled peptide.
  • Peptide substrates and products were separated on a C 18 column (HAIPEEK Targa C 18 5 ⁇ m, 20 x 2.1 mm, Higgins Analytical) using a CH 3 CN/H 2 O (both with 0.1% TFA) gradient on a Waters 625 LC system with in-line UV-Vis (Waters 996) and fluorescence (Hitachi F-1050) detection. Fluorescence signals (excitation 445 nm/emission 520 nm) were integrated using Water's Millennium software.
  • the enzymatic reaction was initiated with 30 ⁇ l of 10 nM lethal factor in assay buffer and terminated after 15 minutes at room temperature by the addition of 25 ⁇ l of 4 mM ortho-phenanthroline/40 mM EDTA, both steps being executed on a Tecan Genesis workstation.
  • the fluorescence was read on a Victor V plate reader using the umbelliferone protocol (excitation 355 nm/emission 460 nm).
  • the fluoresceinated amide analog (Flu)MPKKKPTPIQLNPAPD-NH 2 (SEQ ID NO: 7) was employed to evaluate the ability of labeled lethal factor peptide to be cleaved by lethal factor.
  • the N-terminal fluoresceinyl group provides a sensitive spectroscopic handle for HPLC analysis of the anticipated cleavage reaction.
  • the presence of the N-terminal fluoresceinyl group also provides a large chromophore on the peptide' s N-terminus allowing a determination as to whether large detectable labels are tolerated by lethal factor.
  • the peptide (Flu)NlePKKKPTPIQLNPAPDKGG-NH 2 (“[Nle'jMekl,. ⁇ 6 (KGG)-NH 2 ”) (SEQ ID NO: 1) was produced to eliminate the oxidation side reaction and to facilitate peptide labeling at the C-terminus.
  • [Nle']Mekl ⁇ . ⁇ 6 (KGG)- NH 2 (SEQ ID NO: 1) contains an N-terminus isostere norleucine rather than methionine; and incorporates -KGG- at its C-terminus with the lysyl residue being added as an Mtt protected amino acid.
  • the differentially protected lysine provides a site for introduction of specific reporters via its selective deprotection while still on the resin.
  • the glycyl residues were added in order to facilitate labeling by providing a spacer between the lysyl sidechain and the resin.
  • This example describes the production of a "consensus" lethal factor substrate containing shared elements between the reported natural substrates.
  • the consensus peptide had a significantly higher turnover rate than the MEKl -derived peptides described in Examples 2 and 3, facilitating its use in high throughput screening.
  • the MEKl -derived peptide as a substrate in an assay of lethal factor proteolytic activity required a high concentration of enzyme required, even with long reactions times.
  • the peptide (Flu)NleKKKKVLPIQLNAATDKGG-NH 2 (SEQ ID NO:
  • Preferred pairs were a coumarin fluorophore (7-hydroxy-4-mefhyl-3-acetylcoumarinyl; ⁇ ex 386 nm, ⁇ em 448 nm) paired with either DABCYL (4-dimethylaminoazobenzene-4'-carboxyl; ⁇ max 454 nm) or QSY-35 ® (N-( ⁇ 4-[(7-nitro-2,l,3-benzoxadiazol-4- yl)amino]phenyl ⁇ acetyl), ⁇ max 475 nm) as the quencher.
  • DABCYL dimethylaminoazobenzene-4'-carboxyl
  • QSY-35 ® N-( ⁇ 4-[(7-nitro-2,l,3-benzoxadiazol-4- yl)amino]phenyl ⁇ acetyl
  • Conversion of the continuous assay into a fixed time assay for plate based screening was achieved by terminating the reaction with 1 mM ortho- phenanthroline/10 mM EDTA.
  • the assay is configured to run in 96-well plates with a 75 ⁇ l reaction volume followed by a 25 ⁇ l quench. While these volumes are convenient for both manual and automated liquid handling, there is room for miniaturization to higher density formats if desired.
  • Example 1 Lethal Factor Plate- Based Assay.
  • S/B was approximately 9 at 100% cleavage of 2 ⁇ M substrate; and 4 nM lethal factor leads to 40-50% cleavage in 15 minutes at room temperature for a typical assay S/B of ⁇ 4.
  • the degree of inhibition by a test compound is a linear function of the reduction in the rate constant, not the reduction in the amount of product formed; however, for turnover ⁇ 50% the correction is small and the percent inhibition is well approximated by the percent decrease in the net fluorescent signal.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un substrat de facteur létal du bacillus anthracis et des dosages utilisant ledit substrat pour mesurer l'activité du facteur létal et pour cribler les composés affectant l'activité du facteur létal. Des substrats préférés contiennent un ou plusieurs marqueurs détectables et présentent un taux de rotation suffisamment élevé de manière à être utilisés dans un criblage à débit élevé.
PCT/US2003/005553 2002-02-25 2003-02-21 Reactifs et procedes pour des dosages des protesase de facteur letal du bacillus anthracis Ceased WO2003073066A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US35970702P 2002-02-25 2002-02-25
US60/359,707 2002-02-25

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/424,954 Continuation US6955891B2 (en) 2002-02-25 2003-04-28 Reagents for assaying Bacillus anthracis lethal factor protease

Publications (2)

Publication Number Publication Date
WO2003073066A2 true WO2003073066A2 (fr) 2003-09-04
WO2003073066A3 WO2003073066A3 (fr) 2004-12-02

Family

ID=27766128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2003/005553 Ceased WO2003073066A2 (fr) 2002-02-25 2003-02-21 Reactifs et procedes pour des dosages des protesase de facteur letal du bacillus anthracis

Country Status (1)

Country Link
WO (1) WO2003073066A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7504425B2 (en) 2002-05-29 2009-03-17 Merck & Co., Inc. Compounds useful in the treatment of anthrax and inhibiting lethal factor
US7579487B2 (en) 2004-05-11 2009-08-25 Merck & Co., Inc. Process for making N-sulfonated-amino acid derivatives
AU2007254461B2 (en) * 2006-02-15 2013-01-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of The Health And Human Services, Centers For Disease Control And Prevention Detection of anthrax pathogenicity factors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001059149A2 (fr) * 2000-02-11 2001-08-16 Cellomics, Inc. Biocapteurs peptidiques destines a la protease du charbon bacteridien

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7504425B2 (en) 2002-05-29 2009-03-17 Merck & Co., Inc. Compounds useful in the treatment of anthrax and inhibiting lethal factor
US7579487B2 (en) 2004-05-11 2009-08-25 Merck & Co., Inc. Process for making N-sulfonated-amino acid derivatives
AU2007254461B2 (en) * 2006-02-15 2013-01-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of The Health And Human Services, Centers For Disease Control And Prevention Detection of anthrax pathogenicity factors
US8663926B2 (en) 2006-02-15 2014-03-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention Detection of anthrax pathogenicity factors

Also Published As

Publication number Publication date
WO2003073066A3 (fr) 2004-12-02

Similar Documents

Publication Publication Date Title
EP0873417B1 (fr) Compositions pour la detection de proteases dans des echantillons biologiques, et procedes d'utilisation de ces compositions
EP0988394B1 (fr) Compositions de detection de l'activite enzymatique dans des echantillons biologiques et procedes d'utilisation de telles compositions
US6893868B2 (en) Homo-doubly labeled compositions for the detection of enzyme activity in biological samples
US6593454B2 (en) Methods for identifying inhibitors of methionine aminopeptidases
US7410769B2 (en) Peptide biosensors for anthrax protease
CA2029123A1 (fr) Substrats fluorogeniques pour la detection d'un enzyme proteolytique
PT770876E (pt) Processo de varrimento para a identificacao de ligandos para proteinas alvo
Wang et al. A continuous colorimetric assay for rhinovirus-14 3C protease using peptidep-nitroanilides as substrates
JPH10508749A (ja) Ｎ−アセチルガラクトサミニルトランスフェラーゼ活性用のシンチレーション近接アッセイ
US6955891B2 (en) Reagents for assaying Bacillus anthracis lethal factor protease
Devillers et al. (R)‐α‐Trifluoromethylalanine as a 19F NMR Probe for the Monitoring of Protease Digestion of Peptides
ES2297742T3 (es) Sustratos peptidicos reconocidos por la toxina botulinica de tipo a, bont/a y sus usos.
WO2003073066A2 (fr) Reactifs et procedes pour des dosages des protesase de facteur letal du bacillus anthracis
US20100075297A1 (en) Photosensitive polypeptide and methods of their production and use
EP3097124B1 (fr) Moyens et procédés de détection de malt1 activé
JPWO2006075429A1 (ja) 酵素活性検出用粒子及びそれを用いた酵素活性の検出方法並びに酵素活性検出具
US20090258381A1 (en) Methods for Determining the Cleavability of Substrates
Hopskins et al. Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay
US20250042943A1 (en) Fap detection
FR2869908A1 (fr) Substrat peptidique reconnu par la toxine botulique de type a, bont/a et son utilisation pour detecter et/ou doser ladite toxine ou des inhibiteurs de son activite
Liu et al. A novel fluorescence derivatization method combined with HPLC for determining the activities of endogenous caspase
Weber et al. Heldelberg (DE)
Juliano et al. Chromogenic and fluorogenic glycosylated and acetylglycosylated peptides as substrates for serine, thiol and aspartyl proteases
Diaz-Mochon et al. From 10,000 to 1: Selective synthesis and enzymatic evaluation of fluorescence resonance energy transfer peptides as specific substrates for chymopapain
JP2001019699A (ja) Ｈｃｖのｎｓ３プロテアーゼ活性の測定

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 10424954

Country of ref document: US

AK Designated states

Kind code of ref document: A2

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP