WO2014193237A2 - Protéase de c. difficile - Google Patents

Protéase de c. difficile Download PDF

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Publication number
WO2014193237A2
WO2014193237A2 PCT/NL2014/050349 NL2014050349W WO2014193237A2 WO 2014193237 A2 WO2014193237 A2 WO 2014193237A2 NL 2014050349 W NL2014050349 W NL 2014050349W WO 2014193237 A2 WO2014193237 A2 WO 2014193237A2
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Prior art keywords
peptide
difficile
protease
analog
cleavage
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PCT/NL2014/050349
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English (en)
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WO2014193237A3 (fr
Inventor
Hans Christiaan VAN LEEUWEN
Paul Jacob HENSBERGEN
Jan Wouter Drijfhout
Eduard KUIJPER
Jeroen CORVER
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Academisch Ziekenhuis Leiden H.O.D.N. Lumc
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Publication of WO2014193237A2 publication Critical patent/WO2014193237A2/fr
Publication of WO2014193237A3 publication Critical patent/WO2014193237A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • 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
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • the invention is in the field of determining or quantifying an activity of a proteolytic enzyme of Clostridium difficile in a sample and concerns substrates, to be used therefore and an assay process and an assay kit and device therefore.
  • the invention additionally is in the field of a pharmaceutical composition for treatment of an individual suffering from infection with C. difficile.
  • Clostridium difficile is an anaerobic, Gram-positive spore-forming bacterium. Human intake of spores occurs through the faecal-oral route. When conditions become favourable (after leaving the stomach) in the small intestine, the C.
  • enteric pathogens express factors that reduce competition, allow evasion of host immune responses and promote adhesion and/or invasion of tissues. These virulence factors, which communicate with the environment are located in the cell membrane/wall (controlling adhesion and protection) or are secreted
  • C. difficile In C. difficile, few examples are described that illustrate how the bacteria circumvent host defense mechanisms and efficiently adhere to epithelial cells. In response to attack by antimicrobial peptides, C. difficile expresses a set of genes that change the surface charge, thereby diminishing the interaction of cationic antimicrobial peptides on the bacterial surface (McBride and Sonenshein, 2011. Microbiology 157: 1457-1465). C. difficile cell membrane and cell wall proteins interact with the host and are most likely involved in adhesion and colonization. Footholds on the host cell surface proteins include extracellular matrix components fibronectin, laminin, collagen and fibrinogen (Lin et al., 2011.
  • C. difficile infections include enzyme immune-detection, nucleic acid amplification-based detection, and animal-assisted detection (Bomers et al., 2012. BMJ 345:e7396). Enzyme immune-detection has a low sensitivity and a relatively long turnaround time. Nucleic acid amplification-based test such as, for example, BD GeneOhm Cdiff assay (San Diego, CA) and the Prodesse ProGastro CD assay (W aukesha, WI) have an acceptable sensitivity and a turnaround time of about 4 hours. However, these tests are expensive and require specialized equipment and expertise.
  • a fast, sensitive method for monitoring CDI is essential for patient management and infection control.
  • the present invention provides a peptide or peptide analog comprising the amino acid sequence A/PjA/P-V/P/I/C/T-P or N-A/PjA/P- V/P/I/C/T-P, wherein the arrow denotes the cleavage site.
  • Said peptide is a substrate of a C. difficile-specific protease, CD2830. This protease is highly specific and important for virulence of C. difficile.
  • Said peptide comprising the amino acid sequence A/P jA/P-V/P/I/C/T-P or N-A/PjA/P-V/P/I/C/T-P, provides a highly sensitive, fast and direct detection method that can be performed on culture medium, colonies and a sample, preferably feces, of infected individuals, without purification of the enzyme.
  • peptide refers to a short chain of between 5 and at most 100 amino acid monomers, preferably of between 6 and at most 50 amino acid monomers, more preferred between about 7 and at most 30 amino acid monomers, more preferred at most 25 amino acid monomers, more preferred at most 20 amino acid monomers such as, for example, eight amino acid monomers, nine amino acid monomers, ten amino acid monomers, eleven amino acid monomers, twelve amino acid monomers, thirteen amino acid monomers, fourteen amino acid monomers, fifteen amino acid monomers, sixteen amino acid monomers, seventeen amino acid monomers, eighteen amino acid monomers, nineteen amino acid monomers, twenty amino acid monomers, twenty one amino acid monomers or twenty two amino acid monomers.
  • peptide includes a peptide in which one or more of the amino acid monomers have been modified, for example by acetylation, amidation and/or glycosylation.
  • peptide also includes a peptide that is directly or indirectly attached to a solid surface, for example a surface of a multiwell plate or a magnetic bead.
  • peptide analog includes peptide analogues or peptidomimetics which are or which comprise small protein-like chains such as peptoids and 6-peptides designed to mimic a peptide.
  • the altered chemical structure is preferably designed to adjust one or more properties such as, for example, stability, of a peptide.
  • ribotype 001 is the most common isolated toxinogenic isolate in clinical infections (13%), followed by ribotype 014 (9%). Ribotypes 002, 012, 017, 020, and 027 were each found in 6% of toxinogenic isolates, whereas ribotype 078 was found in 3% of toxinogenic isolates.
  • ribotype 027 (sometimes referred to as BI/NAPl/027) are likely to have suffered from a more severe disease and to have been treated with metronidazole or vancomycin, compared with patients infected by another PCR ribotype.
  • the C. difficile-specific protease, CD2830 was found to be present in all toxinogenic ribotypes.
  • Said peptide preferably comprises the amino acid sequence V/L/I/P-V/L/I- N- A/P jA/P-V/P/I/C/T-P-P, more preferred V/L/I- N-A/P
  • a preferred peptide according to the invention comprises the amino acid sequence AjA-V-P, AjA-P-P, AjA-I-P, AjA-C-P, AjA-T-P, PjA-V-P, PjA-P-P, PjA-I-P, PjA- C-P, P jA-T-P, AjP-V-P, AjP-P-P, AjP-I-P, AjP-C-P, AjP-T-P, P jP-V-P, P jP-P-P, PjP-I-P, PjP-C-P, PjP-T-P, N-AjA-V-P, N-AjA-P-P, N-AjA-I-P, N-AjA-C-P, N- A
  • PjP-V-P or PjP-P-P such as, preferably, N-P jP-P-P-D and/or N- PjP-P-P.
  • Methods for detecting proteolytic activity of C. difficile-specific protease, CD2830 are known in the art and include methods for detecting the enzyme itself, methods for detecting products of peptide hydrolysis or methods for detecting residual substrate peptide.
  • the methods available for detection and assay of proteolytic activity vary in their simplicity, rapidity, range of detection and sensitivity. Said methods comprise qualitative assays such as protein agar plate assay, radial diffusion and thin layer enzyme assay and, preferred, quantitative assays which provide a measure of the proteolytic activity of the enzyme.
  • the commonly used methods employ natural or synthetic substrates using techniques such as enzyme-linked immunosorbent assay-based assays (ELISA),
  • a preferred ELISA is a double-antibody-sandwich ELISA, or an inhibition ELISA (Clements et al. 1990. Appl Environ Microbiol 56: 1188-1190) which can detect small quantities of said protease.
  • a peptide or analog according to the invention is preferably labeled, for example with a chromogenic group, a (chemo)luminescent group, a radiolabel and/or, most preferred, a fluorescent group.
  • a labeled peptide is preferably used in
  • Said label is preferably present at a terminus of the peptide.
  • Said terminus is either the amino-terminus (N- terminus) or the carboxy-terminus (C-terminus).
  • the term "terminus” indicates that the label preferably is present on one or more of the first five amino acid monomers from the N-terminus, and/or on one or more of the last five amino acid monomers at the C-terminus.
  • Said label is preferably present at the N-terminal amino acid monomer, and/or at the C-terminal acid monomer.
  • said label can be indirectly coupled to the N- or C-terminus, for example through a linker that is attached to N-terminus and/or C-terminus.
  • Said linker preferably is an amino acid residue, for example a glutamic acid residue at the N-terminus, and/or an aspartic acid or a cysteine residue at the C- terminus.
  • a preferred chromogenic group is 2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), p- nitroanihde, paranitrophenol and/or 5-bromo-4-chloro-3-hydroxyindole.
  • ABTS o-ethylbenzothiazoline-6-sulfonic acid
  • OPD o-phenylenediamine
  • TMB 3,3',5,5'-tetramethylbenzidine
  • p- nitroanihde paranitrophenol and/or 5-bromo-4-chloro-3-hydroxyindole.
  • a preferred (chemo)luminescent group is a dioxetane derivative such as 1,2- dioxetanedione (C204), 3,3,4,4-tetramethyl-l,2-dioxetane and 3,3,4-trimethyl- 1,2 -dioxetane, 3-(4-Methoxyspiro[l,2-dioxetane-3,2'-tricyclo[3,3,l, 13,7]decan]-4- yl- )-l-aniline and luminol.
  • a preferred radiolabel is 35S and/or 14C.
  • the labeled proteolytic products are preferably separated from the unhydrolyzed substrate, for example by employing magnetic beads.
  • Said group preferably comprises one or more fluorescent group.
  • Fluorescent groups are known and have been described, for example, in U.S. Pat. No.
  • fluorescent groups include coumarin derivatives such as 7-amino-4-methylcoumarin (AMC), 7-acetoxy-4-methylcoumarin (7-AC-4-MC) and 7-hydroxycoumarin, fluorescein, tetramethylrhodamine, rhodamine B, lissamine, rhodamine X, Texas Red, cyanine dyes, Dabcyl, BODIPY dyes, alexa dyes, QSY 7 and QSY 9 dyes, and other fluorescent dyes commonly available from, for example, Invitrogen Corp (Carlsbad, Calif.). Other dyes known to those skilled in the art may also be used.
  • AMC 7-amino-4-methylcoumarin
  • 7-AC-4-MC 7-acetoxy-4-methylcoumarin
  • fluorescein fluorescein
  • tetramethylrhodamine rhodamine B
  • lissamine rhodamine X
  • Texas Red cyanine dyes
  • a preferred fluorescence-based protease assay is simple, inexpensive and sensitive.
  • Said protease assay preferably comprises a soluble fluorescein isothiocyanate (FITC)-labeled peptide, or a soluble Alexa 488-labeled peptide.
  • a peptide or analog according to the invention preferably comprises a quencher group, preferably a fluorescent group and a quencher group, whereby said fluorescent group is present at a first terminus and said quencher group is present at a second terminus of the peptide. The presence of a fluorescent group and a quencher group allows detection of CD2830 activity by a fluorescent peptide energy transfer assay.
  • said quencher group is in close proximity to the fluorescent group while the peptide remains intact. Little, if any, light is emitted when the fluorescent group is excited, and a low or no signal is measured.
  • the quencher is separated from the fluorescent group, which now emits light when excited, and a signal can be measured. The intensity of the signal is proportional to the amount of peptide that is cleaved, which in turn is proportional to the amount of active enzyme, as the peptide is in excess.
  • fluorescent groups (donor)/quencher group (acceptor) pairs include: fluorescein/tetramethylrhodamine,
  • a preferred peptide or analog according to the invention comprises N-[4-(4- dimethylamino)phenylazo]benzoic acid (DABCYL) at a first terminus, and 5 - [(2 - aminoethyl)amino]naphthalene - 1 - sulfonic acid (EDANS) at a second terminus, or comprises Alexa 488 (Alexa Fluor® 488 carboxylic acid, succinimidyl ester) at a first terminus and EDANS at a second terminus, or comprises FITC at a first terminus, and graphene oxide at a second terminus. Said graphene oxide is preferably covalently attached to said peptide.
  • the term "at a first terminus” refers to the N-terminus or C-terminus of the peptide.
  • the term “at a second terminus” refers to the N-terminus or C-terminus of the peptide that differs from the first terminus.
  • the invention further provides an aqueous composition comprising the peptide or analog according to the invention.
  • Said composition preferably comprises means for maintaining the pH of the aqueous composition at a pH of between 2 and 10, preferably between 3 and 9, preferably between 4 and 8, preferably between 5 and 7, preferably around 6.
  • Said composition preferably additionally comprises an inorganic salt, preferably NaCl and/or ZnC12.
  • a preferred composition according to the invention preferably comprises a protease inhibitor.
  • Said protease inhibitor is added to the composition to suppress the activity of other proteases that may be present in the sample.
  • Said other protease includes a protease such as, for example, a serine protease, a threonine protease, a cysteine protease, an aspartate protease, and/or a glutamic protease.
  • Said other protease may be trypsin, chemotrypsin, cathepsin, papain, kallikrein, plasmin, thrombin, pepsin, elastase, factor Xa, and/or subtilisin.
  • a preferred protease inhibitor is selected from aprotinin, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 4- amidinophenylmethanesulfonyl fluoride hydrochloride (APMSF),
  • protease inhibitor is a protease inhibitor cocktail, preferably a cocktail without EDTA, most preferred the protease inhibitor cocktail cOmplete, EDTA-free (Roche).
  • the invention further provides a kit of parts or a device comprising the peptide or analog according to the invention or the composition of the invention.
  • Said kit or device provides a highly specific and highly sensitive tool for monitoring C.
  • the term "individual”, as used herein, includes birds and mammals such as a pet, for example dog and cat; ungulates including pig, horse and ruminants such as sheep, cow; and goat; fowl, including chicken, duck, goose, and turkey, and primates, including human.
  • a preferred individual is a chicken, pig or human.
  • Said kit or device preferably comprises a holder comprising the peptide according to the invention, preferably in an aqueous composition comprising means for maintaining the pH of the aqueous composition at a pH of between 2 and 10.
  • Said kit or device preferably comprises one or more separate holders such as a receptacle, for example a multiwell plate, and a holder comprising a protease inhibitor. Said protease inhibitor may be added to the holder comprising the peptide prior to the start of an assay.
  • sample includes meat, such as beef, veal, pork, and poultry, of the birds and mammals such as a pet, for example dog and cat; ungulates including pig, horse and ruminants such as sheep, cow; and goat; fowl, including chicken, duck, goose, and turkey.
  • sample also includes a tissue sample, an amount of bodily fluid such a blood or urine and, preferably, stool from the birds and mammals such as a pet, for example dog and cat; ungulates including pig, horse and ruminants such as sheep, cow; and goat; fowl, including chicken, duck, goose, and turkey; and primates, including human.
  • the invention further provides a method for identifying an agent that is capable of modulating protease activity of CD2830, comprising (a) providing said agent to a testing system comprising CD2830 protease; (b) contacting said testing system with a peptide of any one of claims 1-6; and (c) detecting reduced or increased cleavage of the peptide, compared to a testing system to which the agent was not provided.
  • Said testing system comprising CD2830 protease further comprises means for determining the activity of the CD2830 protease, preferably a peptide according to the invention, preferably a labeled peptide.
  • Said agent preferably inhibits the protease activity of CD2830 protease.
  • the invention also provides an inhibitor of the CD2830 protease that is identified by the methods and means provided by this invention. Said inhibitor preferably is used in a method for the treatment of an individual suffering from infection with C. difficile.
  • Said agent preferably is a chemical compound.
  • Protease-targeted compound libraries are available, for example from Otava Ltd, Canada, TimTec LLC
  • the invention further provides an agent that is identified by the methods of the present invention, for use in a method of treatment an individual suffering from infection with C. difficile, whereby the agent inhibits the protease activity of CD2830.
  • the invention further provides an in vitro method for determining the presence of Clostridium difficile in a sample, comprising incubating the sample with the peptide of the invention, the composition of invention, or the kit or device of the invention.
  • Said sample preferably a stool sample, is preferably directly incubated with the peptide of the invention, the composition of invention, or the kit or device of the invention, without prior purification of the protease.
  • the C. difficile-specific protease, CD2830 was found to be thermostabile and remains active after incubation of the sample for several minutes at a
  • the sample is preferably heated prior to determining the presence of Clostridium difficile in the sample, preferably heated for 1-10 minutes, preferably for 2-5 minutes, at a temperature between 55 and 80 °C, more preferred at a temperature of about 65 °C.
  • the inventors have established that a mutant strain of Clostridium difficile, in which the coding sequence for the C. difficile-specific protease, CD2830, had been functionally rendered inactive by a so called "knock out" mutation, is less virulent compared to the parent strain of C. difficile that does not comprise the mutation.
  • a peptide that inhibits the activity of the C. difficile-specific protease, CD2830, might therefore be used to treat infections with C. difficile.
  • Said inhibitory peptide preferably is a modified peptide of the invention that is not hydrolizable by the protease
  • the invention therefore also provides a pharmaceutical composition comprising protease CD2830, or an activator of this protease.
  • a pharmaceutical composition comprising protease CD2830, or an activator of this protease.
  • the presence of active protease CD2830 in the gut is thought to prevent adherens of C. difficile, resulting in an effective flush from the gut.
  • Methods for providing a delayed release formulation of CD2830, or of an activator of this protease, for example by providing a tablet comprising the active protease or activator with an enteric coating, are known in the art.
  • the invention therefore also provides a pharmaceutical composition comprising a peptide according to the invention.
  • Said peptide preferably inhibits the protease activity of CD2830.
  • a preferred inhibitor peptide comprises a non-cleavable Pro- Pro mimetic such as, for example, a structure in which the proline rings have been linked together by an additional bridge. Such structures have been described,, for example, in Reuter et al., (2011). Chem. Eur. J. 17, 12037-12044.
  • a further preferred non-cleavable Pro-Pro mimetic comprises a vinylidene bridge between the PI and Pl'prolines to restrict cleavage (Hack et al., 2013. Angew Chem Int Ed Engl 52: 9539-9543).
  • Said inhibitor peptide is preferably modified to adjust some properties of the peptide such as, preferably, the stability of the peptide. Said modification comprises, for example, replacing the peptide bond at the cleavage site with an alkene dipeptide isostere. Said inhibitor preferably mimics the transition state during hydrolysis of a peptide by CD2830. Said inhibitor peptide preferably is a phosphinic peptide, in which one or more peptide bonds (CO-NH) are replaced by a phosphinic acid moiety, for example (P02-CH2) (Dive et al., 2004. CMLS 61: 2010-2019).
  • CO-NH peptide bonds
  • Said phosphinic acid moiety preferably replaces the peptide bond that is cleaved by CD2830.
  • Further preferred phosphinic acid moieties are (PO2- NH) and (P02-0).
  • a further preferred inhibitor peptide comprises a sulfurous acid moiety, in which the peptide bond that is cleaved by CD2830 is replaced by a sulfurous acid moiety preferably (S02-CH2), (S02-NH), or (S02-0). Inhibition of protease activity by these peptides can be easily monitored in, for example, a FRET fluorogenic assay. Methods to produce such peptides are known in the art.
  • a further preferred inhibitor peptide comprises either the unprimed amino acid sequence V/L/I- N-A/P, preferably V/L/I/P-V/L/I- N-A/P with a C-terminal chelating group, or the primed amino acid sequence A/P-V/P/I/C/T-P, preferably A/P-V-P, A/P-V/P/I/C/T-P-P or P-V-P-P, with an N-terminal chelating group.
  • Said chelating group is preferably selected from the group consisting of a thiolate group, a carboxylate group, a phosphinyl group and, preferably, a hydroxamate group.
  • Said inhibitor peptide is preferably for use in a method of treatment of an individual suffering from infection with C. difficile, whereby the inhibitor peptide inhibits the protease activity of CD2830.
  • Methods to produce such peptides are known in the art. For example, methyl, ethyl or N-hydroxy-succinimide ester precursors of a desired peptide are prepared by using classical peptide synthesis methodology. These precursors are reacted with excess hydroxylamine in either ethanol or ⁇ , ⁇ -dimethylformamide, generating a hydroxamic acid derivative of the peptide according to the invention.
  • Said unprimed amino acid sequence is preferably selected from V-N-A, V-N-P, L- N-A, L-N-P, I-N-A, I-N-P, G-N-L, V-V-N-A, V-V-N-P, V-L-N-A, V-L-N-P, V-I-N-A, V-I-N-P, L-V-N-A, L-V-N-P, L-L-N-A, L-L-N-P, L-I-N-A, L-I-N-P, I-V-N-A, I-V-N- P, I-L-N-A, I-L-N-P, I-I-N-A, I-I-N-P, P-V-N-A, P-V-N-A, P-V-N-P, P-L-N-A, P-V-N-P, P-L-N-A, P-V-N-P
  • a further preferred inhibitor peptide comprises the amino acid sequence DTIVINP or DTIVGNL, with a C-terminal chelating group selected from a thiolate group, a carboxylate group, a phosphinyl group and, preferably, a hydroxamate group.
  • Said primed amino acid sequence is preferably selected from A-V-P, P-V-P, A-P- P, P-P-P, A-I-P, P-I-P, A-C-P, P-C-P, A-T-P, P-T-P, A-V-P-P, P-V-P-P, P-P-P, A-I-P -P, P-I-P-P, A-C-P-P, P-C-P-P, A-T-P -P, and P-T-P-P.
  • the invention further provides an inhibitor peptide according to the invention, preferably a hydroxamic acid derivative, for use in a method of treatment of an individual suffering from infection with Clostridium difficile.
  • the invention further provides a method of treatment of an individual suffering from infection with Clostridium difficile, the method comprising administering a pharmaceutical composition comprising an inhibitor peptide according to the invention, preferably a hydroxamic acid derivative according to the invention.
  • Clostridium difficile CD2830 is a metalloprotease with a fold similar to the Anthrax Lethal Factor catalytic domain
  • A Amino acid sequence alignment of C. difficile CD2830 with Anthrax lethal factor (ALF) according to the Phyre2 protein structure prediction. Identical amino acids are shaded in dark grey and similar residues shaded in gray. Zinc coordinating residues are highlighted in green. ⁇ (triangle) point to ALF amino- acids involved in peptide substrate recognition. HEXXH metal binding site is indicated.
  • Lane 1 Caco-2 lysate incubated at 4 °C; Lane 2, Caco-2 lysate incubated at 37 °C; Lane 3, Caco-2 lysate plus rCD2830 at 37°C . Arrow points at cleavage product.
  • a synthetic peptide library was constructed where all 6 positions surrounding the CD2830 scissile bond were permutated to the 20 standard amino acids within a core synthetic peptide (KAAEEPNAAVPDEIK), resulting in a total set of 120 peptides. Peptides were individually incubated 16hr. with rCD2830 and measured by MALDI-ToF MS to determine whether efficient cleavage had occurred. The resulting CD2830 cleavage motif based on this peptide library screen is shown.
  • CD2830 cleavage motif based on the alignment of in total 13 CD2830 cleavage sites within CD2831 and CD3246.
  • Figure 4. Clostridium difficile adhesin CD2831 is efficiently cleaved by the CD2830 protease
  • FIG. 1 Schematic representation of the C. difficile adhesin CD2831 showing the putative collagen binding domains, collagen stalk and transmembrane domain.
  • rCD2831 his-tagged recombinant CD2831 (rCD2831) protein corresponding to aa 732-947 (arrow) was produced, containing all the CD2830 cleavage sites.
  • a synthetic peptide containing one of the CD2830 cleavage sites in CD2831 was incubated with the medium collected from cultures of C. difficile WT (WT secretome) and CD2830 knockout cells (CT::CD2830 secretome). After incubation for 16 hrs, samples were analysed by MALDI-ToF MS to determine substrate peptide cleavage. As a positive control, the substrate peptide was incubated with recombinant CD2830 (+ recombinant CD2830) and as a negative control the peptide was incubated alone (- CD2830).
  • CD2830 cleaves CD2831 from live/intact C. difficile cells
  • CT:CD2830 cells treated with recombinant CD2830 showing the presence of the CD2831 peptide PAPPNTDEPIVNP as demonstrated by the similarity in MS/MS spectrum and elution time as shown for the peptide from recombinant CD2831 (upper graph).
  • Y-axis presents the relative number of hve, adhering bacteria per 24 well (2cm2) cell, CT::CD2830/WT, either on confluent monolayer of Caco-2 cells or uniformly coated with Collagen type I. * indicates the statistical difference, of four independent experiments, P ⁇ 0.05.
  • CD2830 knockout (CT::CD2830,.i). Time from inoculation to endpoint (sacrifice) is indicated. * indicates the statistical difference, P ⁇ 0.05.
  • Figure 9 Optimal reaction conditions for CD2830 activity.
  • A Titration curve of recombinant CD2830 protein.
  • Predictions of signal sequences were carried out using the SignalP 4.1 Server, (http://www.cbs.dtu.dk/services/SignalP). Prediction of cell wall binding motifs, anchors and subcellular prediction were performed at
  • Clostridium difficile strains were grown anaerobically in a microaerobic cabinet (Don Whitley DG 250) at 37°C in pre-reduced 3% Bacto Tryptose (Difco), 2% Yeast extract (Difco) and 0.1% thioglycolate (pH 7.4) medium (TY) or Brain Heart Infusion broth (Oxoid) supplemented with 0.5% yeast extract and 0.01% L- cysteine (Sigma) (BHIS) (Bakker et al., 2012. PLoS. One. 7: e43247) or minimal medium broth (Cartman and Minton, 2010. Appl. Environ. Microbiol. 76: 1103- 1109).
  • BHIS Brain Heart Infusion broth
  • the broths were supplemented with appropriated antibiotics.
  • Mid-logarithmic growth phase pre-cultures (OD600 0.4-0.8) were used to inoculate pre-reduced TY broth to a starting OD600 of 0.05 ( ⁇ 0.01).
  • cell wall binding motifs or a lipid anchor can retain the secreted proteins at the surface of the bacteria.
  • SignalP SignalP
  • LipoP LipoP
  • PSORTB Server NCBI CDD
  • CD2830 One of the predicted genuinely secreted proteins, the uncharacterized protein CD2830 (Q183R7), seems highly expressed based on the number of identified unique peptides (12) and overall sequence coverage (50%).
  • the MS/MS data of the secreted CD2830 protein indicated that the signal sequence had been cleaved off at the predicted position 26 (at sequence AHA
  • the structure of ALF comprises four structural domains (I, II, III and IV) including an N-terminal domain that binds the membrane-translocating component (domain I, Figure IB). Structural similarity with CD2830 only extends to the C-terminal domain containing the metalloprotease domain
  • ALF is a metalloprotease that cleaves MAP kinase kinase enzymes and the structure of ALF in complex with this target peptide has been solved (Pannifer et al., 2001. Nature 414: 229-233; Turk et al., 2004. Nat Struct Mol Biol 11: 60-66).
  • ALF amino-acids making substrate specific contacts are assigned but none of these contacts are identical to CD2830 ( Figure 1A, arrows). Identical residues between CD2830 and ALF mostly correspond to internal, structural fold determining residues.
  • ALF contacts with the MAPKK peptide are also present in domain III that forms a recognition groove together with the metalloprotease catalytic domain.
  • CD2830 is a secreted functionally active zinc metalloprotease, with a fold similar to ALF, but with a different mechanism of action (lacks membrane translocating domain) and different targets.
  • Clostridium difficile we evaluated the genomic DNA of 30 strains, encompassing the 6 major lineages (Knetsch et al., 2012. Infect Genet Evol 12: 1577-1585), by PCR using specific primers for CD2830. We found that in all strains tested the cd2830 gene is present (data not shown), emphasizing the importance of this gene throughout the species. Table 1
  • Collagen I from Life technologies (cat. no. A1048301 ), Mucin from Sigma-Aldrich (cat.no. M3895), IgA from VWR international (cat.no. 401098), Fibrinogen from (EMD Millipore).
  • Peptides were synthesized at the LUMC-facility as described previously (Hiemstra et al., 1997. Proc Natl Acad Sci USA 94: 10313-10318).
  • the CD2830 sequence was amplified by PCR from C. difficile strain 630 genomic DNA, using specific primers, AGGGAATCATATGGATAGTACTACTATACAACAAAATAAAGACAC (forward) and TATTGGATCCCTATTTAGCTAAATTTTGCAAAAAGC (reverse).
  • the PCR products were digested with Ndel and BamHI and ligated into vector pET16b (Novagen) similarly digested with Ndel and BamHI. This resulted in the construction of CD2830 expression vectors containing 10 consecutive histidines at its N-terminus replacing the signal sequence.
  • the bacterial expression constructs were transformed into the Escherichia coli DE3 C43 strain (Lucigen, USA ) and a single colony cultivated on a rotary shaker (200 rpm) in 100 ml Luria Broth at 37°C until an OD600 of 0.5, after which the cultures were induced with 1 M isopropylthio-6-galactoside for 5 hours, using ampicillin (50pg/mL) as a selection marker.
  • the E. coli cells were collected by centrifugation at 6000 x g. We followed the protocol for the preparation of cleared E.
  • the lysates were sonicated 3x10 seconds at 22 kHz, the pH of the lysis buffer, wash and elution buffer were set at 7.4.
  • the E. coli lysates 50 mM sodium phosphate buffer, pH 7.4, 5 mM 2-mercaptoethanol, 0.1% NP40, 300 mM NaCl) containing histidine- tagged proteins were loaded on a 1 mL HisTrap HP column (GE Healthcare).
  • the column was washed with 20 mL wash buffer (50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 5 mM 2-mercaptoethanol, 5% glycerol, 20 mM Imidazol).
  • the His-tagged proteins eluted at 150 mM imidazole while using a 25 mL linear gradient ranging from 20 to 250 mM imidazole.
  • CD2830 proteolytic assays were performed at 37°C in phosphate buffered saline (PBS) [pH 7.4], 0.5 mM ZnC12, unless otherwise stated, for a duration of 16 hours. Reactions were stopped by the addition of Laemmli loading buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 5% 6-mercaptoethanol or 350 mM dithiothreitol and 0.01% bromophenol blue) and by heating the samples at 95°C.
  • PBS phosphate buffered saline
  • Proteolytic assays with the single targets were performed with 0.5 ⁇ g of rCD2830 and 5 ⁇ g of Fibrinogen, 5 ⁇ g of Mucin, 5 ⁇ g of IgA, 3 ⁇ g of HSP90 a and 3 ⁇ g of HSP90 6.
  • the proteolytic assay with a Caco-2 lysate the lysate was prepared as described in the manual of Pierce IP lysis buffer (87787) from Thermo scientific. 10 pg lysate was incubated with 1 ⁇ g of rCD2830. Protease activity was visualized by SDS-PAGE electrophoresis of the samples followed by Coomassie staining.
  • the peptide cleavage assays were performed with 20 pmol peptides and 0.1 ⁇ g of rCD2830. After overnight incubation at 37 °C, samples were analysed with MALDI-ToF-MS (Ultraflex II, Bruker Daltonics) using dihydroxybenzoic acid as a matrix. Identification of CD2830 cleavage products with direct infusion Q-ToF MS
  • the liquid chromatography was performed with piece-linear gradient (0- 10 min at 2% B, 10-25 min to 5% B, 25-165 min to 25% B, 165-175 min to 30% B, 175-190 min to 35% B, 190-195 min to 100% B, 195-210 min at 100% B, were B is 95% acetonitrile/0.1% formic acid).
  • ions were generated using a Captive Spray (Bruker Daltonics) at a spray voltage of 1.4 kV.
  • the temperature of the heated capillary was set to 180°C.
  • CD2830 The histidine tag containing recombinant CD2830 protein (rCD2830) was overexpressed in E.coli and purified using a nickel affinity column.
  • rCD2830 lacks a structure known to be associated with membrane translocation
  • HSP906 was cleaved by rCD2830.
  • HSP90a isoform, which has 93% sequence identity to HSP906.
  • purified HSP906 is cleaved by rCD2830, which correlates with the proteolytic assay with the Caco-2 lysate.
  • purified HSP90a was not cleaved after incubation with rCD2830 ( Figure 2B, right panel).
  • the cleavage product observed with HSP906, is approximately 3-5 kDa smaller compared to the intact protein ( Figure 2B, left panel). This product therefore must be derived from a cleavage event near either the N- or C-terminus of the protein.
  • CD2830 cleavage between the two alanine residues ( Figure 2D).
  • Figure 2D The other visible peaks within the spectrum correspond to the sodium and potassium adducts of the same cleavage products.
  • DDTSAAVTEEM see Figure 2C
  • CD2830 substrate cleavage sites are found in C. difficile LPXTG adhesion molecules
  • ScanProsite search (Castro et al., 2006. Nucleic Acids Res 34: W362-W365) of the human as well as the C. difficile proteome using the above described cleavage motif ([P/A] T
  • CD0515 D-alanyl- D-alanine carboxypeptidase
  • CD2831 Adhesin
  • CD3043 a putative neuropeptide
  • transglutaminase and CD3246 (surface protein, putative collagen binding).
  • CD2831 located next to CD2830 in the genome, and CD3246 contain six and seven consecutive cleavage sites, respectively (Figure 3B).
  • the region containing the cleavage sites seems conserved between CD2831 and CD3246 (shaded areas in Figure 3B).
  • CD2831 and CD3246 have features common to LPXTG cell wall anchored proteins and the protease cleavage sites are directly adjacent to this peptidoglycan anchor motif.
  • the multitude of target sites suggests an effective cleavage of these two cell surface proteins and based on the 13 potential cleavage sites in these two proteins a consensus motif was constructed (Figure 3C).
  • the retargeted intron was cloned using the restriction enzymes BsrGI and Hindlll into plasmids pMTL007C-E2 and transformed to E.coli CA434 and transferred by conjugation into the wild type strain 630AErm (Purdy et al., 2002. Mol Microbiol 46: 439-452; Hussain et al., 2005. J Med Microbiol 54: 137-141).
  • the selection of C. difficile transconjugants was done by subculturing on pre-reduced BHIS agar supplemented with thiamphenicol (Sigma; 10pg/mL) and C. difficile selective supplement (Oxoid).
  • the genotype of the disruption was confirmed with conventional PCRs using the primer oDB0029 [5'-ATGAGACCAAGTAAAAAATT] and the EBS universal primer (5'-CGAAATTAGAAACTTGCGTTCAGTAAAC) and with primer pairs oDB0029 and oDB0030 [CTATTTAGCTAAATTTTGC A] , flanking the ClosTron insertion site. Sequence analysis confirmed that the disruption was at the expected site in CD2830 gene (data not shown). In addition, Southern blot analysis using an ermB specific probe clearly confirmed a specific single insertion of the Group II intron in the genome (data not shown).
  • CD2830 protease is very active and specific in vitro.
  • C::CD2830 a knockout of CD2830 in Clostridium difficile strain 630AErm using the Clostron system (Heap et al., 2010. Methods Mol Biol 646: 165-182).
  • C::CD2830 a knockout of CD2830 in Clostridium difficile strain 630AErm using the Clostron system (Heap et al., 2010. Methods Mol Biol 646: 165-182).
  • For phenotypic characterisation we first determined the growth rates of the WT and CT::CD2830 strain in TY medium and observed no differences demonstrating that the CT::CD2830 strain has no growth defect in vitro ( Figure 10).
  • both the recombinant protein as well as activity in the WT secretome cleaves the peptide.
  • the knockout CT::CD2830 medium showed no peptide cleavage activity. This shows that the CD2830 protease is functionally secreted and is the only activity within the C. difficile secretome that can cleave a CD2831 peptide containing the CD2830 target site.
  • Clostridium difficile cells were collected by centrifugation 10 min. at 7000xg and washed 3 times in PBS containing complete, EDTA-free Protease
  • acetonitrile Prior to LC-MS/MS analysis, acetonitrile was evaporated in a vacuum concentrator. Similarly, 10 ml of conditioned minimal growth medium was desalted and eluted on a reverse phase cartridge for analysis of peptides in the medium.
  • CD2830 is actively secreted and one of its targets is CD2831, a cell surface located adhesion molecule anchored to the cell wall through a LPXTG motif
  • rCD2830 protease can actively remove this adhesion molecule from the surface of C. difficile cells.
  • CD2831 was incubated with rCD2830 protease and resulting peptides similarly purified.
  • the peptides were analysed by reversed phase LC-ion trap MS/MS. During these analyses, we focused our attention to one of the expected CD2831 product peptides
  • CD2831 product peptide PAPPNTDEPIVNP in the culture medium of WT cells but not in that of CT::CD2830 cells ( Figure 6B).
  • CD2831 was also observed on the gel from our secretome analysis (see Table I).
  • Caco-2 cells were grown in RPMI (GE healthcare), 10% fetal calf serum, Penicilin and Streptavidin in a humidified 5% CO2 / 95% air atmosphere at 37°C in a 75 mL culture flask until the cells were nearly confluent.
  • Collagen coated plates were pre-incubated with PBS, 0.05% Tween-20 for 2hrs in a microaerobic cabinet (Don Whitley DG 250) at 37°C. Confluent monolayers of Caco-2 cells were washed twice in PBS before transfer to the cabinet. To each well lxlOE6 bacteria were added in 200 ⁇ pre-reduced PBS. After 3 hrs incubations, wells were washed 5 times in PBS. Adherent bacteria were released in 100 ⁇ lxTrypsin (PAA laboratories, Cat. No. Ll l- 001) before counting cells.
  • CT::CD2830 cells would show enhanced adhesion, particularly to collagen.
  • C. difficile WT and CT::CD2830 cells to Collagen (type I) coated plates.
  • Adhesion of CT::CD2830 cells to collagen was indeed ⁇ 3 fold higher than the wild type strain ( Figure 7).
  • We observed no difference in adhesion of WT and CT::CD2830 to Caco-2 cells (Figure 7). Possibly, adhesion to Caco-2 cells involves additional C.
  • CD2831 and possibly CD3246, may not be highly expressed on Caco-2 cells.
  • Hamsters were monitored for signs of infection and euthanised when a predetermined end point was reached. Hamsters were monitored 4-5 times per day following infection and were assessed for several parameters including presence and severity of wet tail, weight loss, level of activity, piloerection, sunken eyes, hunched posture and response to stimulus. In order to quantify changes in the condition of the animal a scoring system based of the severity of changes observed (ranging from 0-3 for each parameter) was used, with the animals being euthanised when a pre-determined cumulative value was reached. Faecal pellets were collected daily and upon euthanasia a caecum sample was taken from each hamster. Samples were homogenised and plated to determine the presence of C.
  • PCR was performed to determine the genotype of each strain recovered from the hamsters. Samples were heat treated (55 °C for 30 minutes) and plated on to fructose agar (C. difficile agar base. Oxoid) supplemented with cycloserine (Oxiod), cefoxitin (Oxoid), taurocholate (Sigma) and amphotericin (Sigma) to select for C. difficile. The following primers were used to determine the genotypes of the recovered strains:
  • oDB-0030 5'-CTATTAGCTAAATTTTGCA.
  • AH incubations were performed at 37°C in ⁇ ⁇ phosphate buffer saline pH 7.4 (Braun Melsungen), 0.5 mM ZnC12 and 50 ⁇ fluorogenic peptide Dabcyl- EVNPPVPD-Edans in a ⁇ plate, black 96 well (Greiner Bio-one).
  • growth medium from WT cells and CD2830 knockout cells was incubated with the fluorogenic peptide. Progressive cleavage (y-axis) was monitored by measuring fluorescence at 485 nm in a Mithras LB 940 Multimode Microplate Reader (Berthold Technologies) after excitation at 350 nm.
  • FIGS. 11A and 11B A FRET (Fluorescence Energy Transfer) assay for in vitro enzymatic activity of CD2830.
  • a dose-dependent response curve was obtained with recombinant CD2830 protein ( Figure 11A). Cleavage was also obtained with endogenous CD2830 protease from WT cells secreted in the growth medium, while no cleavage was observed with growth medium derived from CD2830 knockout cells (CD2830KO) ( Figure 11B), demonstrating that CD2830 is the only activity secreted by C. difficile cells that can cleave the peptide.
  • CD2830KO growth medium derived from CD2830 knockout cells
  • a hydroxamic acid-based metalloprotease inhibitor was designed, which is predicted to block the active site of CD2830 by binding to the catalytic Zn2+ ion through a chelating hydroxamate.
  • the use of a chelating hydroxamate for inhibitors of metalloproteases has been described in Jialiang Hu et al., 2007. Nature Reviews Drug Discovery 6, 480-498.
  • the inhibitor comprises the CD2830 cleavage half site plus hydroxamate i.e. DTIVINP-hydroxamate for P4-P3-P2-P1-NHOH.
  • three mutated hydroxamate peptides were designed as controls for the specificity of the inhibitor. The half site is thought to target the inhibitor peptide to the active site where the hydroxamate will interact with the zinc ion (/displace H20) and block the active site.
  • the inhibitors used are:
  • DTIVGGP-hydroxamate and DTIVGGL-hydroxamate did not.
  • the DTIVGNL-hydroxamate, containing a Leucine at the PI position showed an even more efficient inhibition of the cleavage reaction.
  • the Leucine at the PI position might reflect a post-cleavage conformation.
  • comparison of DTIVGGL-hydroxamate (no inhibition) and DTIVGNL- hydroxamate (efficient inhibition) emphasizes the importance of Asn at P2.
  • Selectivity of inhibition by hydroxamates DTIVLNP and DTIVGNL was confirmed by showing a lack of inhibition on human matrix metalloproteinase-2 (gelatinase A) enzyme activity (data not shown).

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Abstract

La présente invention concerne un peptide contenant la séquence d'acides aminés A/P- A/P-V/P/I/C/T-P en tant que substrat pour la protéase CD2830 de Clostridium difficile. L'invention concerne, en outre, un nécessaire ou un dispositif comprenant ledit peptide, et ce, à des fins de détermination de la présence de Clostridium difficile dans un échantillon, ainsi que d'identification d'un agent capable de moduler l'activité de la protéase CD2830. L'invention concerne, en outre, une composition, de préférence une composition pharmaceutique, contenant un inhibiteur de l'activité de la protéase CD2830.
PCT/NL2014/050349 2013-05-30 2014-05-30 Protéase de c. difficile WO2014193237A2 (fr)

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