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WO2003073066A2 - Reagents and methods for assaying bacillus anthracis lethal factor protease - Google Patents

Reagents and methods for assaying bacillus anthracis lethal factor protease Download PDF

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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
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labeled derivative
lysine
labeled
peptide
lethal factor
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WO2003073066A3 (en
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Barry R. Cunningham
Richard T. Cummings
Jeffrey D. Hermes
Scott P. Salowe
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Merck and Co Inc
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    • 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.

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Abstract

The present invention features Bacillus anthracis lethal factor substrate and assays employing the substrate to measure lethal factor activity and to screen for compounds affecting lethal factor activity (Figure 1). 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.

Description

TITLE OF THE INVENTION
REAGENTS AND METHODS FOR ASSAYING BACILLUS ANTHRACIS
LETHAL FACTOR PROTEASE
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to provisional application U.S. Serial No. 60/359,707, filed February 25, 2002, hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
The references cited throughout the present application are not admitted to be prior art to the claimed invention.
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"). (Mock et al, (2001) Annu. Rev. Microbiol. 55, 647-71.) Lethal factor is a zinc-dependent metalloprotease that appears to exert toxic affects by cleaving mitogen-activated protein kinase kinases (MKKs). (Vitale et al, (1998) Biochem. Biophys. Res. Commun. 248, 706-11, Vitale et al., (2000) Biochem. J. 352 Pt 3, 739-45, Duesbery et al, (1998) Science 280, 734-7, Duesbery et al, International Publication No. WO 99/50439, International Publication Date October 7, 1999.)
Vitale and co-workers have used microsequencing to identify the site in different MKKs that are cleaved by lethal factor. (See Table 1, Vitale et al, (2000) Biochem. J. 352 Pt 3, 739-45.) Lethal factor cleavage of different MKKs occurred within the N-terminal region preceding the kinase domain. Alignment of the sequences flanking the cleavage site revealed some consensus motifs: a hydrophobic residue in position P2 and PI', and at least one basic residue between P4 and P7. (Vitale et al, (2000) Biochem. J. 352 Pt 3, 739-45.)
Lethal factor has been indicated to cleave synthetic peptides in vitro. (Hammond et al., (1998) Infect. Immun. 66, 2374-8.) In vitro cleavage was inhibited by 1,10-phenanthroline or 10 mM EDTA, both of which chelate zinc. SUMMARY OF THE INVENTION
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.
Thus, a first aspect of the present inventions features a peptide consisting essentially of:
X1-X2-X^X4-X3-X6-X7-X8-z-X9-X10-X1 1-X12-X13-X14-X15-X16-X17-XI8-X19
wherein χl is either norleucine or a labeled derivative thereof; χ2 is either proline, lysine, or a labeled derivative thereof; χ3 is either lysine or a labeled derivative thereof; χ4 is either lysine or a labeled derivative thereof; χ5 is either lysine or a labeled derivative thereof; χ6 is either proline, valine, or a labeled derivative thereof; χ7 is either threonine, leucine, or a labeled derivative thereof; χ8 is either proline or a labeled derivative thereof; χ9 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; χl4 is either alanine or a labeled derivative thereof; χl5 is either proline, threonine, or a labeled derivative thereof; χl6 is either aspartic acid 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. Examples of 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.
Reference to 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.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the results of enzymatic cleavage experiments using fluoresceinated substrates [Nle']Mekl 6(KGG)-NH2 (SEQ ID NO: 1) and the MKK consensus sequence analog (Flu)NleKKKKVLPIQLNAATDKGG-NH2(SEQ ID NO: 2) as assessed by the HPLC cleavage assay. [Substrate] = 100 μM, [LF] = 1 μM, T = 37 °C with time points as indicated. [Nle1]Mekl 6(KGG)-NH2 (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 μM in 50 mM HEPES (pH 7.0V20 mM NaCl/10 mM MgCl2/100 μM CaCl2/100 μM ZnCl2/l mg/ml BSA/1 mM DTT were incubated with 100 nM lethal factor in a volume of 75 μl in a black 96-well plate. The increase in fluorescence (excitation 355 nm/emission 460 nm) was read on a Victor2 V plate reader. (Cou)Consensus(K(QSY-35)GG)-NH2 (SEQ ID NO: 3) (closed circles); (QSY-35)Consensus(K(Cou)GG)-NH2 (SEQ ID NO: 4) (open circles); (Cou)Consensus(K(DAB)GG)-NH2 (SEQ ID NO: 5) (closed squares); (DAB)Consensus(K(Cou)GG)-NH2 (SEQ ID NO: 6) (open squares).
DETAILED DESCRIPTION OF THE INVENTION
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 Substrate
Lethal factor substrates described herein consist essentially of:
X1-X2-X3-X4-X5-X6-X7-X8-z-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19
wherein χl is either norleucine or a labeled derivative thereof; χ2 is either proline, lysine, or a labeled derivative thereof; preferably, χ2 is either lysine or a labeled derivative thereof; χ3 is either lysine or a labeled derivative thereof; χ4 is either lysine or a labeled derivative thereof; χ5 is either lysine or a labeled derivative thereof; χ6 is either proline, valine, or a labeled derivative thereof; preferably χ6 is either valine or a labeled derivative thereof; χ7 is either threonine, leucine, or a labeled derivative thereof; preferably, X7 is either leucine or a labeled derivative thereof;
X8 is either proline or a labeled derivative thereof; χ9 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; χl8 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 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, 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 χ8 proline and χ isoleucine. In different embodiments 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. In a separate embodiment z is an amide linkage.
Table I provides examples of different lethal factor substrates. Table I
Figure imgf000008_0001
Flu = 5 , 6-carboxyf luoresceinyl
Cou = 7 -hydroxy-4-methyl-3 -acetylcoumarinyl
DAB = 4-dimethylaminoazobenzene-4 ' -carboxyl
Q-35 = N- ( { 4- [ ( 7 -nitro-2 , 1 , 3 -benzoxadiazol-4-yl ) amino ] phenyl } acetyl
Standard one and three letter codes for amino acids are as follows: Nle= norleucine; A=Ala=Alanine: C=Cys=Cysteine: D=Asp=Aspartic acid: E=Glu=Glutamic acid: F=Phe=Phenylalanine: G=Gly=Glycine: H=His=Histidine: I=Ile=Isoleucine: K=Lys=Lysine: L=Leu=Leucine: M=Met=Methionine: N=Asn=Asparagine: P=Pro=Proline: Q=Gln=Glutamine: R=Arg= Arginine: S=Ser=Serine: T=Thr=Threonine: V=Val= Valine: W=Trp=Tryptophan: and Y=Tyr=Tyrosine.
Assay Formats
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. In 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. In the absence of cleavage, 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.
A variety of different fluorophores and quenchers are well known in the art. Examples of 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). Examples of 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.)
With appropriately labeled substrates alternate technologies can be used to measure substrate cleavage. Examples of homogeneous formats would include fluorescence polarization, time resolved FRET, SPA™, FlashPlate™, and AlphaScreen™. Examples of heterogeneous formats would include DELFIA™, chemiluminescence plate based assays, HPLC, radioactive filter binding assays, absorbance assays, and fluorescence assays.
Measuring Activity
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.
EXAMPLES
Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
Example 1: Materials and Methods
This example describes different materials and methods that were employed to produce and study lethal factor substrates.
Substrate Synthesis Peptide synthesis was carried out on an ABI Model 433A Peptide
Synthesizer employing FastMoc™ chemistry with increased acylation times on a 250 μmol scale. All reagents, including amino acids, were from PE Applied Biosystems except for (Fmoc)-L-Nle (Bachem), (Fmoc)-L-Lys(Mtt) (Novabiochem) and PL-Rink Resin (Polymer Laboratories). All fluorophores and quenchers were from Molecular Probes, Inc. (Eugene, OR).
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 μmol 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 (CH2C12 with 2% TFA and 3% Triisopropylsilane, room temperature, 45 minutes), resin washing, and reaction with the label as described above.
After incorporation of appropriate reporters, the resin was washed with NMP, acetic acid, CH2C12, and methanol (3x each), dried briefly in vacuo and the peptides cleaved using 95% TFA/2.5% H2O/2.5% Triisopropylsilane for 90 minutes. After precipitation from cold diethyl ether, the crude peptides were purified on a Waters PrepLC 4000 system using a 25 x 400 mm 300 A DeltaPak C18 column and a CH3CN/H2O (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 (A2ι4).
Lethal Factor HPLC-Based Assay
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 μM with varying levels of lethal factor in 50 mM HEPES (pH 7.0)/20 mM NaCl/10 mM MgCl2/100 μM CaCl2/100 μM ZnCl2/l mg/ml BSA/1 mM DTT) for various times.
Prior to HPLC injection, samples were treated by precipitation of the protein materials by addition of a 10-fold excess volume of 60% CH3CN in H2O. 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 C18 column (HAIPEEK Targa C18 5 μm, 20 x 2.1 mm, Higgins Analytical) using a CH3CN/H2O (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.
Lethal Factor Plate-Based Assay
To each well of a 96-well black flat-bottomed plate (Packard) was added 25 μl of a 6 μM solution of the peptide substrate (Cou)Consensus(K(QSY- 35)GG)-NH2 (SEQ ID NO: 3) in assay buffer (20 mM HEPES (pH 7.0)/l mM CaCl2/0.1 mg/ml BSA/0.01% Tween-20). Test compounds in DMSO (1.5 μl) were added with additional assay buffer (18.5 μl) to the plate with a CyBi well dispenser. The enzymatic reaction was initiated with 30 μl of 10 nM lethal factor in assay buffer and terminated after 15 minutes at room temperature by the addition of 25 μl 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).
Example 2: Labeled Peptide MEKl-Peptide Substrate
The fluoresceinated amide analog (Flu)MPKKKPTPIQLNPAPD-NH2 (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.
Experiments were preformed using (Flu)MPKKKPTPIQLNPAPD- NH2 (SEQ ID NO: 7) incubated at 100 μM with various amounts of lethal factor in a metal ion supplemented buffer (see Example 1) similar to that found in Hammond et al, (1998) Infect Immun 66, 2374-8. Good levels of turnover (15-30%) were observed employing high levels of enzyme (~1 μM) for extended periods (2-4 hours at 30°C).
The anticipated products, those resulting from cleavage of the Pro8-Ile peptide bond, were detected by mass spectral analyses (data not shown). HPLC analysis was complicated by an unanticipated side reaction: a fluorescein-mediated oxidation of the methionine residue's sidechain to the corresponding sulfoxide. The product of this reaction (the Met(O) substrate) nearly co-eluted with the cleavage product, (Flu)MPKKKPTP(OH) (SEQ ID NO: 8), making analysis difficult. This oxidation occurred with peptide both as a solid and in solution, and was particularly rapid in solution in the presence of light with up to 50% conversion within one hour (data not shown). Example 3: Enhanced MEKl -Peptide Substrate
The peptide (Flu)NlePKKKPTPIQLNPAPDKGG-NH2 ("[Nle'jMekl,. ι6(KGG)-NH2") (SEQ ID NO: 1) was produced to eliminate the oxidation side reaction and to facilitate peptide labeling at the C-terminus. [Nle']Meklι.ι6(KGG)- NH2 (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.
[Nle1]Mekl,.16(KGG)-NH2 (SEQ ID NO: 1) was stable and showed no propensity towards oxidation. The peptide was cleaved by lethal factor between the Pro8-Ile9 bond (identified by mass spectral analysis) at a rate similar to the original peptide, indicating that the substitution of norleucine for methionine and the addition of three amino acids had no adverse effect on cleavage.
Example 4: Lethal Factor Substrate
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-NH2 (SEQ ID NO:
2) was designed to reflect the common elements present in lethal factor substrates and to allow incorporation of reporters in an orthogonal manner. (Flu)NleKKKKVLPIQLNAATDKGG-NH2 (SEQ ID NO: 2) differs from the [Nle']Mekl,.ι6(KGG)-NH2 (SEQ ID NO: l)substrate at five positions. The three proline residues at positions 6, 13, and 15 were converted to residues found in the other MKK substrates in order to reduce the possibility of alternate cleavages adjacent to prolyl residues and to minimize potential secondary structure. The proline at the second position was converted to lysine for these reasons and to reflect the tetrabasic component of the consensus sequence. The threonine at position seven was converted to leucine, a residue found in five out of the eight known sequences. The -KGG- functionality was retained at the C-terminus.
Using the HPLC assay (Flu)NleKKKKVLPIQLNAATDKGG-NH2 (SEQ ID NO: 2) was compared head-to-head with the [Nle1]Mekl 1-16(KGG)-NH2 (SEQ ID NO: 1) peptide. As shown in Figure 1,
(Flu)NleKKKKVLPIQLNAATDKGG-NH2 (SEQ ID NO: 2) was a much better substrate, with an improvement in turnover number of approximately 100-fold. Mass spectral analysis confirmed that the appropriate cleavage was taking place.
Example 5: FRET Peptide Labels
Different fluorophores and quenchers were chosen and examined for their ease of synthesis, degree of quenching, and substrate suitability. Preferred pairs were a coumarin fluorophore (7-hydroxy-4-mefhyl-3-acetylcoumarinyl; λex 386 nm, λem 448 nm) paired with either DABCYL (4-dimethylaminoazobenzene-4'-carboxyl; λmax 454 nm) or QSY-35® (N-({4-[(7-nitro-2,l,3-benzoxadiazol-4- yl)amino]phenyl}acetyl), λmax 475 nm) as the quencher.
Analogs of the (Flu)NleKKKKVLPIQLNAATDKGG-NH2 (SEQ ID NO: 2) were produced with the coumarin fluorophore at either the C- or N- terminus and one of the two quenchers at the distal end of the peptide. The analog reactivity with lethal factor was analyzed via the increase in fluorescence upon separation of fluorophore and quencher. As shown in Figure 2, all four peptides cleaved at approximately the same rate with similar signal/background (S/B) varying between 9 and 16 at 100% cleavage. The progress curves were first order, indicating that the Kms of the four substrates are »10 μM. The peptide with the largest S/B, denoted (Cou)Consensus(K(QSY-35)GG)-NH2, (SEQ ID NO: 3) was chosen for further studies.
The tested peptides included (Flu)NleKKKKVLPIQLNAATDK(QSY- 9)GG-NH2 (SEQ ID NO: 9) and (QSY-9)NleKKKKVLPIQLNAATDK(Flu)GG-NH2 (SEQ ID NO: 10) where Flu = fluorescein and QSY-9 is a quencher from Molecular Probes, Inc. These peptides did not produce a good signal.
Example 6: Assay optimization
A series of optimization experiments indicated that a simplified buffer consisting of only 20 mM HEPES (pH 7.0) and 1 mM CaCl2 improved the enzyme activity more than ten-fold relative to the more complex buffer used earlier (Example 2). BSA and Tween-20 were added at low levels (0.1 mg/ml and 0.01%, respectively) to prevent adsorptive losses of enzyme and facilitate automated liquid handling.
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. As described in Example 1, the assay is configured to run in 96-well plates with a 75 μl reaction volume followed by a 25 μl quench. While these volumes are convenient for both manual and automated liquid handling, there is room for miniaturization to higher density formats if desired.
An optimized protocol is described in Example 1, Lethal Factor Plate- Based Assay. Using the optimized protocol, the S/B was approximately 9 at 100% cleavage of 2 μM 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.
Since the reaction is first-order, 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.
As with other assays using a fluorophore at this wavelength, artifactual low signal (due to compound absorption) or high signal (due to compound fluorescence) can be encountered during screening. The use of longer wavelength fluorophore/quencher pairs should reduce such artifacts.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention.

Claims

WHAT IS CLAIMED IS:
A peptide consisting essentially of:
X1-X2-X3-X4-X5-X6-X7-X8-z-X9-XI0-X11-X12-X13-X14-X,5-X,6-X17-X18-X19
wherein Xl is either norleucine or a labeled derivative thereof; χ2 is either proline, lysine, or a labeled derivative thereof; χ3 is either lysine or a labeled derivative thereof; χ4 is either lysine or a labeled derivative thereof; χ5 is either lysine or a labeled derivative thereof; χ6 is either proline, valine, or a labeled derivative thereof; χ7 is either threonine, leucine, or a labeled derivative thereof; χ8 is either proline or a labeled derivative thereof; χ9 is either isoleucine or a labeled derivative thereof;
XlO is either glutamine or a labeled derivative thereof;
Xll is either leucine or a labeled derivative thereof; χl is either asparagine or a labeled derivative thereof;
X 3 is either proline, alanine, or a labeled derivative thereof; χl4 is either alanine or a labeled derivative thereof;
Xl5 is either proline, 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;
Xl 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.
2. The peptide of claim 1, wherein
X is either norleucine or a labeled derivative thereof; χ2 is either lysine or a labeled derivative thereof; χ3 is either lysine or a labeled derivative thereof; χ4 is either lysine or a labeled derivative thereof; χ5 is either lysine or a labeled derivative thereof; χ6 is either valine or a labeled derivative thereof; χ7 is either leucine or a labeled derivative thereof; χ8 is either proline or a labeled derivative thereof; χ 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; χl3 is either alanine or a labeled derivative thereof;
Xl4 is either alanine or a labeled derivative thereof; χl5 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;
Xl 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.
3. The peptide of claim 1, wherein said peptide consists of:
X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-XI 1-X12-X13-X14-X15-X16-X17-X18-X19
wherein Xl is a labeled derivative of norleucine; χ2 is lysine; χ is lysine; χ4 is lysine; χ is lysine; χ is valine; χ7 is leucine; χ is proline; χ is isoleucine;
XlO is glutamine; χl l is leucine; χl2 is asparagine; χ! is alanine; Xl4 is alanine; χl5 is threonine;
Xl6 is aspartic acid;
Xl7 is lysine or a labeled thereof; χl is glycine; and Xl9 is glycine-NH2.
4. The peptide of claim 3, wherein said Xl is norleucine labeled with 5, 6- carboxyfluoresceinyl and said Xl is lysine.
5. The peptide of claim 3, wherein either: a) said Xl is norleucine labeled with 7-hydroxy-4-methyl-3- acetylcoumarinyl; and said Xl is lysine labeled with either 4- dimethylaminoazobenzene-4'-carboxyl or N-({4-[(7-nitro-2,l,3-benzoxadiazol-4- yl)amino]phenyl}acetyl; or b) said Xl is norleucine labeled with either 4- dimethylaminoazobenzene-4'-carboxyl or N-({4-[(7-nitro-2,l,3-benzoxadiazol-4- yl)amino]phenyl}acetyl; and said χl7 is lysine labeled with 7-hydroxy-4-methyl-3- acetylcoumarinyl.
6. The peptide of claim 5, wherein said Xl is norleucine labeled with 7-hydroxy-4-methyl-3-acetylcoumarinyl and Xl7 is lysine labeled N-({4-[(7- nitro-2,l,3-benzoxadiazol-4-yl)amino]phenyl }acetyl.
7. A method of measuring Bacillus anthracis lethal factor activity comprising the steps of: a) incubating the peptide of any one of claims 1-6 with Bacillus anthracis lethal factor using an incubation medium wherein said Bacillus anthracis lethal factor is active; and b) measuring cleavage of said peptide.
8. A method of measuring the ability of a compound to affect Bacillus anthracis lethal factor activity comprising the steps of: a) incubating together said compound and the peptide of any one of claims 1-6 with Bacillus anthracis lethal factor using an incubation medium wherein said Bacillus anthracis lethal factor is active; and b) measuring cleavage of said peptide.
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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

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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
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