Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/8146—Metalloprotease (E.C. 3.4.24) inhibitors, e.g. tissue inhibitor of metallo proteinase, TIMP
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/02—Antidotes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
• • 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
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
  • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
  • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
  • G01N2333/96419—Metalloendopeptidases (3.4.24)

Definitions

• This invention relates to peptides and proteins inhibiting protease activity. More specifically, the invention relates to peptides exhibiting activity against a spec- trum of proteases of microbial or fungal origin, in particular against the metzincin family including thermolysin, anthrax neutral protease 599 (npr599), pseudolysin, and aureolysin.
• the invention relates further to methods for producing the peptides, to pharmaceutical compositions comprising the peptides or proteins, and to methods of using the peptides or proteins to prevent and/or treat bacterial or fungal infections and their symptoms, in particular to reduce the toxic effects of secreted or membrane bound bacterial proteases such as aureolysin, bacillolysin, pseudolysin, vibriolysin, and anthrax npr599 by inhibiting their respective proteolytic activity.
• bacterial proteases such as aureolysin, bacillolysin, pseudolysin, vibriolysin, and anthrax npr599
• Antibiotics are the actual standard treatment for bacterial infections.
• the widespread and sometimes inadequate use of antibiotics, however, has led to pathogens developing resistance against one or even multiple antibiotics.
• Multidrug resistant bacteria pose an increasing and serious threat to infected patients. Therefore the search for new companion compounds dampening the bacterial virulence has become an urgent need during the last years.
• a a rapid effect of compounds against dangerous or life threatening symptoms of bacterial infection is desirable to protect the patient immediately against these symptoms even before antibiotics or alternative compounds can kill the microbes.
• AMP antimicrobial peptides
• the degradation of immunity-related defense molecules of the host including antimicrobial peptides (AMP) (Otto 2008), and the acquisition of nutrients are important functions for the pathogen to survive.
• pathogen-associated proteolytic enzymes can serve as virulence factors that cannot or can only partially be inactivated by corresponding, endogenous host proteinase inhibitors.
• Virulence factors usually complement each other in degrading host peptides and proteins during infection. Consequently, pathogen-derived proteinases without corresponding endogenous inhibitors in the host will be evolutionarily selected because pathogens would otherwise waste their limited resources (Vilcinskas 2010).
• Thermolysin-like metalloproteinases encompass many prominent toxins produced by human pathogens, including aureolysin, bacillolysin, pseudolysin, vibriolysin, and anthrax npr599, which are presumably at the origin of many pathological symptoms associated with severe infections such as septicemia, hemorrhagic tissue bleeding, necrosis and enhancement of vascular permeability (Chung et al. 2006). Severe diseases like gastric and peptic ulcers and gastric carcinoma originate at least partly from the effect of metalloproteinases of the M4 family from pathogens (Schmidtchen et al. 2003, Smith et al. 1994).
• anthrax toxin leads to the destruction of the host macrophages, thereby enabling the bacteria to spread in the host tissue without encountering host defense.
• the anthrax toxin is a holoenzyme containing three different proteins, each of which is present in multiple copies. They are called protective antigen (PA), edema factor (EF), and lethal factor (LF). To be become active, it is required that at least PA and EF or PA and LF are combined. Just a single protein or just EF and LF without PA are not active in laboratory animals.
• gene BA0599 from bacillus anthracis codes for a protease called neutral protease 599 (npr599), which belongs to the M4 family of proteases and is highly homologous to bacillolysins from other bacillus species (Popov et al . 2005, Chung et al. 2006, Chung et al. 2008).
• anthrax can further be treated with antibiotics in case of an acute infection.
• the toxins shed by the bacteria continue to cause irreversible damage to tissue before the number of bacteria is sufficiently reduced by antibiotics. It is even likely that exposure to antibiotics stimulates bacteria to temporarily shed increased amounts of virulence factors, causing accelerated and irreversible damage of patient tissue.
• manmade synthetic or recombinant versions of the natural bacterial toxins may be inhaled by humans without bacteria being present, in which cases an antibiotic would have no curing effect at all.
• Nrp599 can be inhibited with EDTA and 1,10- phenanthroline (Chung et al. 2006), but further, therapeutically acceptable Nrp599 inhibitors are not published up to now.
• thermolysin family of inhibitors of other members of the thermolysin family were described earlier.
• Phosphoramidon is produced by the Bacterium Streptomyces tanashiensis, and Talopeptin is derived from it.
• Phosphonomadites termed ZG P LL and ZF P LA display inhibitory constants in the low nanomolar range (Holden et al . 1987) with respect to thermolysin.
• Khan et al described ⁇ -d-arabinofuranosyl-/V 4 -lauroylcytosine as potent inhibitor of thermolysin out of set of 12 inhibitors found in virtual screening run (Khan et al . 2009); and 3-Phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one as most potent inhibitor out of a set of novel 38 quinazolin-4(3H)-ones (Khan et al. 2010).
• thermolysin activity by administering non-specific met- alloproteinase inhibitors, however, is biased by negative side effects induced by collateral inhibition of essential host enzymes such as its matrix metalloproteinases.
• Phosphoramidon for example, is inhibiting the endogenous protein endothelin converting enzyme (ECE).
• ECE endogenous protein endothelin converting enzyme
• some of these compounds are toxic, while for others the toxicity is obviously unknown. Consequently, a strong demand for molecules capable of specifically inhibiting one microbial enzyme (Adekoya et al. 2009) or a corresponding enzyme family persists.
• Streptomyces metalloproteinase inhibitor (Oda et al . 1979) is a known proteinaceous inhibitior of thermolysin. SMPI consists of 102 amino acids and exhibits two cystein bridges exhibiting an IC50 of 0.6 nM for Thermolysin (Hiraga et al. 1999). It was discovered, however, that SMPI is degraded by other bacterial proteases (Tsuru et al.1992), disqualifying SMPI for use as a compound for treating bacterial infections. Furthermore, its low molecular weight of ca. 12 kD means that it is rapidly cleared from the bloodstream by renal filtration.
• thermolysin The only other peptide inhibitor of thermolysin reported to date which specifically inhibits thermolysin-like enzymes is the insect metalloproteinase inhibitor IMPIa . It was originally discovered in and purified from the hemolymph of immunized G. mellonella larvae (Wedde et al . 1998). Its active moiety comprises 69 amino acids including intramolecular cystein bonds, and a molecular weight of 7667.7 Da. The molecule has a reported IC50 of 0.62, 0.86 and only 81.66 nM for thermolysin, bacillolysin and pseudolysin, respectively, all of which are enzymes belonging to the M4 protease family (Clermont et al.2004).
• IMPIa was tested against human metallo-matrix proteases MMP1,2,3,7,8, and -9, of which only MMP1 and MMP2 showed a negligible inhibition.
• IMPIa was recombinantly produced in Schneider cells (Clermont et al. 2004) and later in E.coli. Although the structure of IMPI was not known until recently (Gomes-Rueth . 2011), it was suggested by homology searches that IMPIa should belong to the 18 or Ascaris fami- ly of serin protease inhibitors, despite the fact that IMPIa inhibits a metalloprotease and not a serin protease (Wedde et al., 2007).
• Tfhe polypeptide is a polypeptide having at least 70% homology, in particular 80%, 90% or 95% homology to the polypeptide of SEQ ID No 2 representing the wild-type of the protein insect metalloproteinase inhibitor IMPIa and having at least one mutation at position 35, 36 and/or 39 of the amino acid sequence of IMPIa wherein
• the nonpolar amino acid isoleucine at position 35 of IMPIa is replaced either by a nonpolar amino acid selected from the group consisting of leucine, methionine and phenylalanine or by polar amino acid selected from the group consisting of cysteine, asparagine, glutamine, histidine, lysine and arginine; and/or
• the nonpolar amino acid isoleucine at position 36 of IMPIa is replaced either by a nonpolar amino acid selected from the group consisting of valine, phenylalanine and tryptophan or by polar amino acid selected from the group consisting of tyrosine, serine, threonine, asparagine, glutamine, histidine, lysine and arginine; and/or the polar amino acid position 39 of IMPIa is replaced either by the nonpolar amino acid valine or by the polar amino acids histidine or lysine.
• the polypeptide of of the invention shows in particular an IC 50 value to thermo- lysine of less than the IC 50 value of IMPIa .
• the polypeptide of the invention has the amino acid sequences of SEQ ID numbers 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84.
• the polypeptide of the invention may comprise chemical modifications in the side chain or at the N and/or C terminal for improving biological or chemical properties such as bio availability, stability, effectivity.
• the modification may also provide for a detectable label, for example a chemiluminescent structural element, one or more radioactive isotopes in one or more side chains of an amino acid in the polypeptide, an enzyme which is able to generate a colour reaction and the like.
• Subject matter of the present invention is also a fusion polypeptide comprising the polypeptide of the invention and at least one polypeptide having a physiological function in particular an antibody or antibody fragment, scaffolds such as lipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin, albumin, uteroglobin, or protein A, functional peptides such as transferrin, peptides useful for diagnostic applications, such as green fluorescent protein (GFP), or peptide tags enabling immobilization on technical surfaces, such as hexahistidine, or glu- tathione-S-transferase (GST).
• scaffolds such as lipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin, albumin, uteroglobin, or protein A
• functional peptides such as transferrin, peptides useful for diagnostic applications, such as green fluorescent protein (GFP), or peptide tags
• polypeptide of the invention has the amino acid sequences of SEQ ID numbers ,6,8, 12, 86, 88, 90, 92.
• Cytosolic GSTs are divided into 13 classes based upon their structure: alpha, beta, delta, epsilon, zeta, theta, mu, nu, pi, sigma, tau, phi, and omega. Mitochondrial GSTs are in class kappa.
• the MAPEG superfamily of microsomal GSTs consists of subgroups designated I-IV, between which amino acid sequences share less than 20% identity. Human cytosolic GSTs belong to the alpha, zeta, theta, mu, pi, sigma, and omega classes, while six isozymes belonging to classes I, II, and IV of the MAPEG superfamily are known to exist:
• the fusion polypeptide of the invention is linked by a peptide of 1 to 100 amino acids in length to the the polypeptide of the invention.
• polypeptide of the invention can be conjugated by a chemical linking group with a polypeptide having a physiological function to yield the fusion polypeptide of the invention.
• Polypeptides or proteins can be manufactured by chemical methods, such as solid phase syntheses or by means of genetic engineering by means of utilizing coding polynucletides in a suitable expression system.
• Subject matter of the present invention is also a polynucleotide coding for the polypeptide of the invention or the fusion polypeptide of the invention.
• the polynucleotide of the invention may be operably linked to a heterologous promoter.
• Subject matter of the present invention is also a pharmaceutical composition
• a pharmaceutical composition comprising the polypeptide of the invention and/or the fusion polypeptide of the invention or their derivatives and/or a nucleic acid comprising a section coding for the polypeptide of invention.
• polypeptide of the invention or the fusion polypeptide of the invention can be used in the treatment of an animal or human infected by microorganisms secret- ing bacterial toxins of the M4 or Metzincin family of metalloproteinases, in particular thermolysine, aureolysin, bacillolysin, pseudolysin, vibriolysin or anthrax npr599.
• polypeptide and/or the fusion polypeptide of the invention can be used for detection of the presence or activity of proteases belonging to the M4 family in a sample.
• the invention is based on the result that mutations between amino acid 35 and 39 of IMPIa (SEQ ID NO: 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84) could be used to alter systematically the specificity profile and potency related to specifically inhibiting members of the thermolysin M4 protease family.
• the polypeptide IMPIa of the invention inhibit further proteases of the thermolysinM4 protease family, particular thermolysine, aureolysin, bacillolysin, pseudolysin, vibriolysin or anthrax npr599, and that the mutein IMPIa exhibit different inhibitory constants with respect to the various proteases.
• Proteins or peptides containing many cystein bridges are usually considered as difficult or impossible to express in any bacteria, including E.coli., and published protocols reveal low production efficacy and requirement for cooling (see, for example, Comis Ruets, 2011) while according to the method of invention wtlMPIa and the polypeptide of the invention can be produced without requiring cooling of the fermenter during the protein expression phase, and without requiring protease inhibitors to be added to protect the protein product from degradation.
• wtlMPIa refers to a protein with an amino acid sequence as in SEQ ID NO: 2 and consists of the N-terminal fragment of the full length IMPI molecule which is endogenously cleaved from the larger precursor molecule IMPI.
• ⁇ refers to a protein with an amino acid sequence as in SEQ ID NO:4 and consists of the C-terminal fragment of the full length IMPI molecule which is endogenously cleaved from the larger precursor molecule IMPI.
• loop mutein IMPIa comprises all proteins comprising an amino acid sequence identical to SEQ ID NO: 2 (wtlMPIa), with the exception of the amino acid stretch 35 to 39 , and in particular with exception of the amino acid stretch 35, 36, or 39, in which at least one amino acid is exchanged, added, or deleted.
• Loop mutein IMPIa includes, for example, amino acid sequence SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, in which one amino acid at position 35,36, or 39 is exchanged.
• SEQ ID NO 10 two additional amino acids at positions 37 and 38 are exchanged .
• mtlMPIa or “Mutein IMPIa” is the polypeptide of the invention and includes all recombinant or synthetic proteins with an amino acid sequence which is at least 70%, especially 80% 90%, 95% homologous, but not identical to SEQ ID NO: 2 and which inhibits thermoiysin or another protease of the M4 protease family with an IC 50 ⁇ 1000 nM, especially IC 50 ⁇ 100 nM, in particular those which can be expressed in E.coli and especially those including proteins of the group "loop mutein IMPIa” and "structure variants of IMPIa".
• mtlMPIa include inserted, substituted or deleted residues, including N-terminal and C-terminal and intrasequence deletions, provided the selected mutations preserve the biological activity of the Mutein IMPIa.
• biologically active refers to wtlMPIa or the polypeptide of the invention demonstrating inhibition of thermoiysin or another protease of the M4 protease family with an IC 5 o ⁇ 1000 nM especially IC 5 o ⁇ 100 nM or better.
• IMPIa family alone shall include biologically active mtlMPIa and wtlMPIa BRIEF DESCRIPTION OF THE FIGURES
• Fig. l Vectormap of the hybridplasmid pET41IMPIa .
• Total mRNA was isolated from immunchallenged Galleria melonella und subsequently used as template for the amplification of DNA coding for IMPIa .
• the amplicon was then cloned into the PshAl linearized pET-41A vector (Novagen) resulting in a GST (Glutathion-S-transferase) fusionprotein .
• Accurate in frame cloning of IMPIa into the vector was verified by sequencing .
• Fig. 2 SDS-PAGE of the soluble cytoplasmatic fraction of Rosetta garni 2 three hours after induction of the T7-Expression system.
• the overex- pressed fusionprotein is marked by an arrow.
• the corresponding controls (empty pET-41 and the uninduced T7-Expression system) show no corresponding bands.
• FIG. 3 HPLC UV detector data from purification run of GST-tagged IMPIa .
• the purification has been carried out by affinity chromatography on a HPLC, using a resin with immobilized glutathion at room temperature.
• the purification has been carried out by affinity chromatography on a HPLC, using a resin with immobilized glutathion at room temperature.
• Fig. 4a SDS-PAGE of the purified GST-tagged IMPIa
• mutein GST Bind Fractogel Cartriges Merck, Darmstadt
• Fig. 4b SDS-PAGE after the specific cleavage of the n-terminal GST-tagged
• IMPI alpha by recombinant enterokinase (Merck, Darmstadt). The cleavage was carried out for 16 hours at room temperature. Untagged IMPIa is marked by an arrow.
• Fig. 5 Sucessfull downstream processing was verfied by mass spectroscopy on a micrOTOF-Q mass spectrometer (Bruker Daltonics). Analysis of samples was achieved by chromatography on a reverse-phase column (Acclaim 120, C8, 3 prn, 120 A, 2.1 x 150 mm; Dionex ) by applying a gradient of 1-80 % (1,63 %/min) acetonitril in water containing 0.1 percent formic acid. By-product (peak termed 1 and 3) and main product (peak 1) show the correct mass of 7,677 kDa.
• Fig. 6 wild type protein assembly of full length IMPI, including IMPIa and ⁇ in G. melonella.
• Fig. 7 inhibitory plot of wtlMPIa and phenanthronin vs. the anthrax protein nrp599 and InhA
• Fig. 8a Multiple alignement of IMPIa homologues of Galleria melonella.
• Fig . 8b Multiple alignment of IMPI ' s found in the transcriptome of other lepi- doptera species (Sequences unbublished).
• Fig. 8c Pairwise alignment of the M4-metalloprotease thermolysin from Bacillus thermoproteolyticus with the metalloprotease npr599 from Bacillus anthracis (strain A0248)
• Fig. 9 The binding of the peptidic inhibitor IMPIa to the M4-metalloprotease thermolysin ⁇ Bacillus thermoproteolyticus) is depicted. Red areas represent Van der Waals contact areas between inhibitor and protease.
• the isoleucin (PI ' )(purple) fits perfectly into the SI ' pocket which is responsible for specificity of the metalloptotease hence the inhibitor is binding in a lock and key kind of fashion
• Fig. 10 Schematic drawing of a lateral flow device for detecting protease specific activity. The principle is based on thin layer chromatography. The eluent carries ichor collected by a cotton swap via tangential flow through the device. M4-metalloproteases produced by pathogens therefore also migrate through the device, until they get bound to IMPI muteins exhibiting certain specificity for different metalloproteases. The different muteins are bound at concentrated spots on the solid phase of the device, so that different metalloproteases can be clearly identified. Spots are made visible afterwards by detection antibodies present in the mobile phase.
• Thermolysine and Pseudolysine, and respective ratio of IC 50 values mean that an inhibitor with designed ratio of IC 50 s with respect to different proteases can be created.
• biologically active loop polypeptide of the invention polypeptides which exhibit a specificity profile different from the wtlMPIa profile for inhibiting members of the M4 protease family.
• the fact that the loop polypeptide of the invention are biologically active at all is surprising because the loop amino acids were particularly well conserved during evolution. It is even more surprising that altering a specific loop amino acid could increase the binding constant for thermolysin or pseudolysin, or even both.
• Loop the polypeptide of the invention molecules exhibiting improved inhibition of Thermolysin comprise I35L, I36F, K, R, V, Y, Q, D, H, M, T, or W, R39K or V.
• Loop the polypeptide of the invention molecules exhibiting improved inhibition of Pseudolysin comprise I35L or F, M, W or Y, I36R, Q, or M, R39K and A.
• Loop polypeptide of the invention molecules exhibiting improved inhibition of both, Thermolysin and Pseudolysin in parallel comprise I35L, I36R, Q, or M, R39K.
• Loop polypeptide of the invention exhibiting equal potency against both, Thermolysin and Pseudolysin, comprise I35L, or W.
• the polypeptide of the invention may be obtained by known techniques for directed evolution as described by Arnold et al., 2003a and Arnold et al., 2003b, or may further be obtained by knowledge based engineering of the respective amino acid sequence, including application of rules for conservative or non- conservative amino acid replacement considering size, charge, polarity and other biochemical parameters of the amino acids as know by one of ordinary skill in the art by the teaching and principles disclosed in US Pat. Nos.
• Loop Mutein IMPIa for example, SEQ ID: NO 10, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70 , 72 , 74, 76, 78, 80 , 82 , 84 also exhibit a strong, undexpected thermolysin inhibiting activity.
• wtlMPIa and Mutein IMPIa, and in particular Loop polypeptide of the invention can be recombinantly produced in bacterial cells under conditions identified by the in- ventors, especially at room temperature and up to 37°C, under conditions which do not require cooling of the fermenter or refolding of the protein produced .
• the method according to the invention, equipment and reagents used are described in detail in example 2 below. It should be understood, however, that modifica- tions in concentrations of reagents used, and modifications in the genetic configuration of the producer organism (E.coli) could be made without affecting the output of correctly folded protein.
• E.coli could be replaced by other bacteria as production host, additional plasmids or gene stretches with additional function could be added to the producer bacteria.
• medium compo- nents could be provided in different order or concentrations, and incubation times could be varied. It should be understood that those modifications which influence the production efficacy of biologically active Mutein or wtlMPIa negatively by 50% or less or even positively are considered within the scope of the invention.
• the invention contemplates further modifications of elements of the expression system inasmuch as the expression of a functional Mutein or wtlMPIa without the need for refolding is maintained to at least 50% compared to the system disclosed in Example 3 and 4, and no cooling of the fermenter is required.
• other expression vectors than pET-41a( + ) may be used, which may not be linked to a fusion tag, or may be linked to a fusion tag other than GST, His- tag; and other promoters than 17, and may also include further regulators or other fusion elements.
• IPTG or other inducers may be employed .
• restriction sites than PshAI may be used in pET- 41a( + ) or in other vectors, for example Xhol, EcoR I, BamH 1.
• Other E.coli strains than Rosetta-gami 2 pLysS may be used, with or without additional t-RNA genes or ahp C mutation, and other bacteria than E.coli may also be employed .
• downstream process steps which are known to the one skilled in the art, including but not limited, to incubation times, reagents and their concentrations and disposables used, for example capture resins and chromatography equipment and separation media for purification.
• Recombinant production of Mutein or wtlMPIa in bacteria is very cost effective compared to all other production methods for proteins. Cloning and the subsequent development of a cell line producing the peptide are faster and less costly with bacteria as, for example, with mammalian or insect cells. During production, the amount of protein produced per volume fermentation broth is much higher compared to eukaryotic cell cultures. Consequently there is a strong demand for methods of recombinant protein producing in bacteria, especially in E.coli since they are a proven host frequently used for protein production. It is known that wtlMPIa retains its biological activity after deglycosylation (Wedde et al. 1998); therefore bacterially produced Mutein or wtlMPIa lacking glycosylation are biologically active once folded correctly.
• Mutein or wtlMPIa and in particular of wtlMPIa is provided for inhibition of the anthrax protease nrp599.
• Current approaches to treat Anthrax include vaccination such as BioThrax (Emergent Biosolutions, Rockville MD, U .S. A) for disease prevention, or antibiotics such as Ciprofloxacin or Doxycyclin (http://www.nationalterroralert.com/anthraxtreatment/). While the vaccine is only approved for preventive use, it takes several days until antibiotics exert effects on bacteria to an extent that the symptoms caused by their virulence factors are reduced.
• nrp599 is one of the major anthrax virulence factors inducing hemorrhagic bleeding, for example (Chung et al., 2006) by targeting the fibrinolytic system (Chung et al, 2011), and that it also degrades the van Willebrand-Factor and thereby interferes with the coagulation cascade (Chung et al ., 2008).
• US 2004/018193 teaches the concurrent or offset administration of both, antibiotics and antigenic compounds, in particular to treat anthrax infected humans.
• the authors provide several examples of protease inhibitors including hypothetical antibodies, but they do not disclose nrp599 as a target.
• polypeptides of the invention and in particular wt IMPIa is a potent and specific inhibitor of nrp599, despite the fact that bacillus anthracis is not known to infect insects. It was found that wtlMPIa inhibits npr599 at nanomolar concentrations.
• the polypeptide of the invention and in particular wt IMPIa may be combined with antibiotics, in particular Ciprofloxacin or Doxycyclin, with antimicrobial peptides, with other inhibitors of bacterial proteases, or with compounds improving the physiological state of the infected patient.
• antibiotics in particular Ciprofloxacin or Doxycyclin
• antimicrobial peptides with other inhibitors of bacterial proteases, or with compounds improving the physiological state of the infected patient.
• polypeptide of the invention may also be produced effectively in recombinant cell lines and tissue, including mammalian and insect expression systems, plant and bacterial systems; or synthetically.
• Mutein and wtlMPIa each may typically be isolated and purified to be free of oth- er proteins and protein fragments.
• Mutein and wtlMPIa is about 80% free of other proteins which may be present due to the production technique used in the manufacturing process.
• the polypeptide of the invention or wtlMPIa is about 90% free of other proteins, particularly preferably about 95% free of other proteins, and most preferably about >98% free of other proteins.
• the desired protein may be combined with other active ingredients, chemical compositions and/or suitable pharmaceutical formulation materials prior to administration as medication, as described in further detail below.
• the biological activity of Mutein or wtlMPIa can be assessed in vitro by means of assays, such as measuring binding of the polypeptide of the invention or wtlMPIa to a protease immobilized on coated glass carrier used for Plasmon resonance spectroscopy.
• assays such as measuring binding of the polypeptide of the invention or wtlMPIa to a protease immobilized on coated glass carrier used for Plasmon resonance spectroscopy.
• the polypeptide of the invention or wtlMPIa can be immobilized on the glass carrier and the protease could be added to start the assay. This assay is able to measure on- and off-rates for the respective interaction.
• the biological activity can also be tested by means of an Parallel Line assay, for example in a microtiter plate format, and by detecting a coloration effect or by measuring changes in the fluorescence emitted upon excitation with a suitable light source.
• Amino acid sequence additions may further include N- or C-terminal fusions ranging in length from one residue to 150 or more residues, as well as internal intrasequence insertions of single or multiple amino acid residues into Mutein or wtlMPIa ranging typically from about 1 to 10 amino acids, more typically from 1 to 5 amino acids, and most typical from 1 to 3 amino acids.
• N-terminal additions include the addition of a methionine or an additional amino acid residue or sequence.
• Another example of N-terminal addition includes the fusion of a signal sequence - provided the signal sequence SEQ ID NO: 94 is not bluntly added to wtlMPIa of SEQ ID NO : 2.
• Mutein or wtlMPIa comprise fusions with antibodies (Abs) or antibody fragments.
• Ab fragments may comprise for example scFAb, scFv fragments, or single domain antibodies, or antibodies of camelide (hcIG) or shark (IgNAR) origin, or their respective VHH domains.
• the fusion may serve to provide a targeting moiety (the CDR domains) fused to to Mutein or wtlMPIa so that the fusion protein could be accumulated and enriched at surface displaying antigens specific for the antibody fragments.
• Mutein or wtlMPIa may be to benefit from the dimerization properties of the constant antibody regions to obtain a bivalent form of Mutein or wtlMPIa, in a way similar to Etanercept (Enbrel) in which 2 TNF receptor fragments are fused to the constant domains of an antibody.
• Etanercept Enbrel
• bivalent antibody chimera is sufficiently large to escape from rapid renal clearance, for which a size exclusion barrier of roughly 65 kDa exists, so that the lifetime of the fusion protein in the patient body is strongly prolonged.
• Additions may also comprise other scaffolds derived, for example, from lipocalin, ankyrin, fibronectin, transferrin, tetranectin, adnectin, albumin, uteroglobin, or protein A.
• Other additions contemplated comprise functional peptides, for example transferrin enabling the fusion protein to cross the blood brain barrier, a property poten- tially useful for inhibiting bacterial toxins accumulated in the brain or spinal cord, or additions useful for diagnostic applications, such as green fluorescent protein (GFP) enabling fluorescence detection, or peptide tags enabling immobilization on technical surfaces
• GFP green fluorescent protein
• polypeptide of the invention described herein may involve addition or deletion of or substitution with a non-native amino acid at the N- or C-terminus or at any site of the protein that is modified by the addition of an N-linked or O- linked carbohydrate.
• a cystein for example, may be added for linking a water soluble polymer such as polyethylene glycol, or other amino acids like lysine, cysteine, histidine, arginine, asparaginic acid, glutamic acid, serine, threonine, or tyrosin could also be used for coupling polymers to the peptide..
• Suitable, clinically acceptable, water soluble polymers include polyethylenglycol (PEG) and polysialic acid (PSA).
• tags in particular peptide tags, such as hexa-histidine which can be used to facilitate binding, for example, to a moiety of a purification column.
• tags may comprise a peptide sequence serving as a substrate for a protease, so that the tag can be cleaved in purpose once it is not required anymore.
• Mutein or wtlMPIa to which chemical compounds or nanoparticles are added .
• This can be achieved for example by employing the enzyme O-6-Alkylguanin-alkyltransferase (AGT) or derivatives thereof to Mutein or wtlMPIa, or to a peptide tag .
• Other examples comprise conjugation to a cysteine being introduced by a mutation (Jagath et al . 2008), or to carbohydrate moieties (Fischer-Durand et al. 2010).
• Chemical compounds may include for example labeling moieties, in particular for in vivo imaging applications, targeting moieties such as receptor ligands to enrich the conjugated IMPIa family members in specific organs.
• biotin Another compound suited to immobilize conjugated IMPIa family members on a surface or particle is biotin, for example for diagnostic purposes.
• Nanoparticles such as quantum dots may be added for in vivo detection or imaging applications.
• Dendrimers or others particles increasing the size of the conjugate may be added to prevent the conjugate from cleared rapidly from the blood stream by the kidneys.
• Modifications to the peptide backbone are also contemplated under the invention, such as beta peptides, where the amino group is attached to the beta carbon of the respective amino acid instead of the alpha carbon, rendering them invulnerable to proteases.
• Other modifications of the peptide backbone are possible, such as alkylation of the amid nitrogen and bioisosteric replacement of amide groups.
• the present invention further provides polynucleotides which contain nucleotide sequences encoding Mutein or wtlMPIa, provided the polynucleotide is not identical to the complete wild type sequence SEQ ID NO: l, or SEQ ID NO: l directly followed by SEQ ID NO: 3.
• nucleotide sequences in which oligonucleotide stretches are inserted which do not code for wtlMPIa .
• nucleic acid sequences which encode Mutein or wtlMPIa.
• Presently preferred nucleic acid sequences include those encoding SEQ ID NO: 9, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83.
• E.coli for producing Mutein or wtlMPIa with high quality and efficiency
• other hosts may also be used for recombinantly producing Mutein or wtlMPIa, including other strains of E.coli, other bacteria like bacillus megaterium, fungal systems like yeast, pichia, or hansenula, eukaryotic cells like protozoa, insect cell lines like Schneider T7 or Sf9 mammalian animal cell lines like CHO, human cell lines like HEK293, or transgenic animals like goats, or plants like tobacco.
• Mutein or wtlMPIa or modified versions thereof may be combined with ingredients to form a pharmaceutical composition.
• the pharmaceutical composition may include water and salts at physiological concentrations, solubilizing or dispersing agents, or anti-oxidant, or particles forming micelles, such as liposomes. Lipo- somes may also contain balMPIa or modified versions thereof internally. This pharmaceutical composition may be filled in a glass or plastic vial, or in a syringe. The pharmaceutical composition may also contain additives supporting drying or freeze-drying of the pharmaceutical composition, for example cyclodextrins or saccharides, in particular disaccharides.
• Mutein or wtlMPIa or modified versions thereof may be used as a medicine, in particular as a medicine for parenteral injection, to treat diseases in an animal, in particular in a mammal or bird, or in a human, and which are related to the activity of proteases from the M4 family.
• Such disease could be, for example, a bacterial infection, in particular infection by staphylococcus aureus, in particular multi-resistant staphylococcus aureus, bacillus anthracis, pseudomonas aeruginosa, helicobacter pylori, vibrio cholerae, or legionella pneumophilia.
• Such a disease could further be systemic inflammatory response syndrome (SIRS), or sepsis
• Mutein or wtlMPIa or modified versions thereof may be administered parenterally, orally, or topically using suitable pharmaceutical compositions, or attached to a patch or wound debridement from where the medication elutes into a wound of the patient.
• Mutein or wtlMPIa or modified versions thereof may be administered in combination with other treatments, in parallel or staggered, in particular with other inhib- itors of virulence factors, antibiotics, or antimicrobial peptides.
• RNA molecules whose sequences comprise RNA sequences of Mutein or wtlMPIa, in particular sequences like SEQ ID 1, 5, 9, 11, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, and which can be transcribed into Mutein or wtlMPIa protein in the target organism.
• Such RNA molecules may be administered in combination with transfection agent like liposomes or PEL
• the invention also contemplates the application of Mutein or wtlMPIa in devices for diagnostic purposes. In several cases it is beneficial to determine the prote- ase activity in biological fluids, and in particular the protease activity profile if several of them are present.
• protease activity would provide information not only about the presence of bacteria, but also of their ac- tivity and relative concentrations.
• protease activity profile In wounds, and especially in slowly healing or chronic wounds, determining the protease activity profile also is of interest since the physiological stages of wound healing are characterized also by a specific evolution of protease activity during healing . It is even more important to distinguish between the activity of endoge- nous host proteases and the activity of bacterial proteases.
• PCR or real time PCR assays do not provide sufficient information because these tests quantify the concentration of particular bacteria present, but they are not suited to test for the activity of the bacteria.
• a diagnostic method solves this issue.
• Mutein or wtlMPIa is used therein to bind M4 proteases specifically. Different concentrations of proteases can be determined when more than one type of Mutein or wtlMPIa is used. The difference in binding strength between the polypeptide of the invention or wtlMPIa is then used to calculate differences in concentrations of Mutein or wtlMPIa.
• a preferred embodiment of the diagnostic method uses a device similar to Lateral Flow tests known in the state of the art; in which a membrane strip (suagfahig) is used to take up the fluid sample and to transport the sample across the mem- brane by capillary forces.
• the membrane consists of nitrocellulose or nitrocellulose endorsed by nylon fibres or tissue.
• Mutein or wtlMPIa are immobilized so that at least part of the sample fluid passes across this location during flow. Proteases present in the sample are captured by the immobilized Mutein or wtlMPIa .
• a second step one further solution containing detection capture molecules targeted against different binding epitopes of the proteases are added.
• detection capture molecules are prepared to deliver a detection signal, which can be, for example, chromophoric, fluorescent, or electrochemical by means of a direct or enzymatic label. It is possible to increase signal strength by secondary capture molecules targeting the detection capture molecule.
• at least one kind of capture molecule is an antibody.
• a particular advantage of the diagnostic method according to the invention is that several proteases can be distin- guished by means of specific binding to speiific locations where different Mutein or wtlMPIa are immobilized, while only a single sort of antibody is required .
• a control area is present at a different location on the membrane strip where detection capture molecules or secondary capture molecules bind to even if no proteases are present in the sample.
• the cap- ture functionality in the control area is provided by immobilized proteases or immobilized detection capture molecules.
• capture molecules other than Mutein or wtlMPIa in particular antibodies are immobilized on the membrane strip and the proteases are added as detection capture molecule, in particular in combination with sec- ondary capture molecules.
• This embodiment also requires a contral area containing immobilized proteases or Mutein or wtlMPIa.
• the assessment of the protease content is made by visual inpection or a sensor device, including CMOS based imaging sensor or scanners.
• a software can be employed to calculate the true concentration ratios of the proteases from potentially overlapping binding profiles of the polypeptide of the invention or wtlMPIa if the individual binding constants or other binding parameters are known.
• such software may use a linear equation system to calculate the individual concentrations.
• the binding kinetics are sampled, in particular by using an imaging sensor, to determine concentrations more accurately.
• the sample may be added, for example, by a pipetting device, a paper strip, a cotton swab or a needle.
• the device may dispose of a seal preventing uncontrolled flow across the membrane strip.
• Last instar larvae were used for immunization using the following solutions: (4.05*10 4 cfu/mlj Escherichia coli strain BL21 (DE3) (Invitrogen, Carlsbad, CA) and (0.7*10 4 cfu/ml) Micrococcus luteus (DMSZ Reference number 495).
• Ten microliters of sample volume from each solution was injected dorsolaterally into the hemocoel using 1-ml disposable syringes and 0.4-mm needles mounted on a microapplicator.
• Larvae were homogenized at 8 h postinjection for total RNA isolation using the TRIzol reagent (Invitrogen, Germany) according to manufacturer ' s instructions.
• cDNA was synthesized using the OneStep RT-PCR System (Qiagen, Germany) according to the manufacturer ' s instructions.
• the T7-based expression system pET-41a(+), which allows protein expression upon induction of the T7-polymerase in Escherichia coli cells by IPTG, was chosen to obtain strong and reproducible expression of the recombinant protein.
• the sequence of the mature IMPIa was amplified using the forward primer (5 ' - GATAGTCCTAATTTGTAACGGTGGACAC-3 ' ) and the reverse primer (5 ' - CTACGAACGTATTTTAGGACAGTCTTTTATCG-3 ' ) .
• the fragment was cloned into the PshAI site of vector pET-41a(+), which was then designated p41IMPIa and transformed into E.coli Rosetta-gami 2 pLysS (Novagen, Germany). The resulting clones were verified via sequencing (Eurofins MWG Op- eron, Germany)
• LB-medium containing 1% (w/v) Glucose, Cm 34 , Kan 50 und Tci 2 ,5 was inoculated with cell material from a colony and cultivation started on an orbital shaker at 32°C up to a OD 60 o of 1. The preparatory culture was subsequently stored at 4°C in a refrigerator.
• Main culture LB-Medium containg 1% (w/v) Glucose, Cm 34 , Kan 50 , was inoculated with 3 %(v/v) of the preparatory culture and cultivation started on an orbital shaker at 32°C up to a OD 60 o of 1. Upon reaching an OD 6 oo of 1 the expression of the recombinant protein was induced with 1 mM IPTG. The main culture was subsequently incubated for further 3 hours at 32°C on an orbital shaker.
• iii Cells were harvested from liquid culture by centrifugation at 10,000 g for 10 min using a centrifuge tube of known weight. The pellet was decanted and allowed to drain, removing a maximum amount of liquid before the wet weight of the pellet was determined.
• Main culture LB-Medium containg 1% (w/v) Glucose, Cm 34 , Kan 50 was inoculated with 3 %(v/v) of the preparatory culture and cultivation started on an orbital shaker at 32°C until an OD 60 o of 1 was reached . At 1 OD the expression of the recombinant protein was induced with 1 mM IPTG. The main culture was subsequently incubated for further 3 hours at 32°C on an orbital shaker.
• Protein motifs were identified using SMART (http ://smart.embl-heidelberg .de/) and the conserveed Domain Database from NCBI (http ://ncbi : nlm. nih.gov/Structure/cdd/wrpsb.cgi).
• the signal peptide was pre- dieted using SignalP (http ://www.cbs.dtu.dk/services /SignalP/), and the theoretical isolelectric point and molecular weight were predicted using Compute pI/MW (http ://expasy.org/tools/protparam.html).
• Cysteine disulfide bonding state and connectivity prediction was done by using DISULFIND ( http ://disulfind .dsi.unifi.it/). Sequence similarity was analysed by BLAST from NCBI (http ://www.ncbi.nlm.nih.gov/BLAST/). Multi-sequence alignment was generated using Vector NTI 9.0 (Invitrogen, Germany). Homology modeling was performed by the CPHmodels 3.0 server
• the subsequent table lists the sequences printed in the ensuing sequenc proto ⁇ col.
• the leading number denotes the SEQ ID Nr. for the nucleotide sequence
• the subsequent even number mssing number would denote the SEQ ID of the re ⁇ spective peptide sequence.
• ctt aac eta act tea cca tgc ata cca att tgc gat tgt cca caa atg 1056 Leu Asn Leu Thr Ser Pro Cys He Pro He Cys Asp Cys Pro Gin Met
• Cys Pro lie lie Asn lie Arg Cys Asn Asp Lys Cys Tyr Cys Glu Asp
• Cys Pro lie lie Asn lie Arg Cys Asn Asp Lys Cys Tyr Cys Glu Asp
• n is a, c, g, . or t
• the 'Xaa' at location 62 stands for Lys, Arg, Thr, or lie

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