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WO2000061634A2 - Modulateurs variants de l'ecotine de genie genetique agissant sur des serines proteases - Google Patents

Modulateurs variants de l'ecotine de genie genetique agissant sur des serines proteases Download PDF

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Publication number
WO2000061634A2
WO2000061634A2 PCT/US2000/009790 US0009790W WO0061634A2 WO 2000061634 A2 WO2000061634 A2 WO 2000061634A2 US 0009790 W US0009790 W US 0009790W WO 0061634 A2 WO0061634 A2 WO 0061634A2
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ecotin
polypeptide
amino acids
sequence
seq
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PCT/US2000/009790
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WO2000061634A3 (fr
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Charles S. Craik
Robert J. Fletterick
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The Regents Of The University Of California
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Priority to AU46433/00A priority Critical patent/AU4643300A/en
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Publication of WO2000061634A3 publication Critical patent/WO2000061634A3/fr

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    • 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
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors

Definitions

  • This invention relates the field of protein engineering.
  • this invention teaches the production of a wide variety of protease binding proteins (e.g., activity modulators) based on the structure of the bacterial protease inhibitor ecotin.
  • protease binding proteins e.g., activity modulators
  • the serine proteases are a large family of enzymes involved in a wide variety of vital biological processes.
  • the crucial physiological functions of these enzymes in blood coagulation, fibrinolysis, complement pathways, viral maturation, apoptosis, and cancer make them important targets for efforts to design and engineer potent and specific inhibitors.
  • a highly selective protease inhibitor can serve as a powerful tool to block key proteolytic activities for dissecting proteolytic pathways and cascades and elucidating the in vivo roles of particular proteases in complex biological processes. Ultimately, this may lead to the development of innovative therapies for life-threatening diseases.
  • Macromolecular substrate-like serine protease inhibitors such as bovine pancreatic trypsin inhibitor (BPTI)
  • BPTI bovine pancreatic trypsin inhibitor
  • BPTI bovine pancreatic trypsin inhibitor
  • This invention provides a novel class of binding proteins that specifically recognize and bind to polypeptides characterized by the presence of a chymotrypsin fold.
  • the binding proteins are based on the structure of ecotin and thus referred to as ecotin- derived binding proteins or ecotin variants.
  • this invention exploits the structure of ecotin to act as a scaffold that orients the domains comprising a primary and secondary binding site that mediate ecotin/ecotin and ecotin/substrate interactions.
  • the domains can be varied according to the methods of this invention to produce new modulators (e.g. inhibitors) of serine proteases.
  • Preferred modified ecotin molecules can be represented by generic formula (I): T'i-X'-L ⁇ X'-L ⁇ -X'- ⁇ X'- 1005 ,,- ⁇ - ⁇ (I) where X 1 is a polypeptide having the sequence of amino acids 8 through 50 of native ecotin, X 2 is a polypeptide having the sequence of amino acids 56 through 65 of native ecotin, X 3 is a polypeptide having the sequence of amino acids 72 through 78 of native ecotin, X 4 is a polypeptide having the sequence of amino acids 88 through 106 of native ecotin, X 5 is a polypeptide having the sequence of amino acids 115 through 135 of native ecotin, L 50s , L 60s , L 80s , and, L 100s are independently an amino acid or a polypeptide consisting of 2 to about 15 amino acids, more preferably 2 to about 7 amino acids, T 1 and T 2 are independently an amino acid, or a polypeptide
  • the ecotin variants do not include variants disclosed or claimed in U.S. patent 5,719,041.
  • any one of aa 81 aa 82 aa 83 , aa 84 , aa 85 , aa 86 , aa 87 are not Ser, Thr, Met, Met, Ala, and Cys, respectively 5 (amino acids 81 through 87 of native ecotin) the remainder of the ecotin variant does not have the amino acid sequence of a native ecotin;
  • the ecotin variant can be represented by the formula:
  • aa 86 , aa 87 , aa 107 , aa 108 , aa 109 , aa 110 , aa 111 , aa 112 , aa 113 , aa 114 , aa 136 , aa 137 , aa 138 , aa 139 , aa 140 , aa 141 , and aa 142 are optionally present amino acids that, when present, are independently selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, thxeonine, tryptophan, tyrosine, and valine.
  • T ⁇ -X 1 is a polypeptide having the sequence of amino acids 1 through 50 of native ecotin and X 5 -T 2 is a polypeptide having the sequence of amino acids 115 through 142 of native ecotin thus, T 1 is a polypeptide having the sequence of amino acids 1 through 7 of a native ecotin, and T is a polypeptide having the sequence of amino acids 136 through 142 of a native ecotin.
  • -aa 51 -aa 52 -aa 53 -aa 54 -aa 55 is a polypeptide having the sequence of amino acids 51-55 of native ecotin.
  • - aa 66 -aa 7 -aa 68 - aa 69 -aa 70 -aa 71 - is a polypeptide having the sequence of amino acids 66-71 of native ecotin.
  • -aa 107 -aa 108 -aa 109 -aa 110 -aa 111 -aa 112 -aa 113 -aa 114 - is a polypeptide having the sequence of amino acids 107-114 of native ecotin.
  • Other ecotin variants include
  • ecotin variants include variants in which -aa 51 -aa 52 -aa 53 -aa 54 -aa 55 - is a polypeptide having the sequence of amino acids 51-55 of native ecotin -aa 66 -aa 67 -aa 68 - aa 69 -aa 70 -aa 71 - is a polypeptide having the sequence of amino acids 66-71 of native ecotin
  • T 1 is optionally a polypeptide having the sequence of amino acids 1 through 7 of a native ecotin
  • T 2 is optionally a polypeptide having the sequence of amino acids 136 through 142 of a native ecotin.
  • the ecotin variant specifically binds a polypeptide that is a serine protease including, but not limited to plasma kallikrein.
  • Factor Xlla, Factor XIa, Factor IXa, Factor Vila, Factor Xa, Factor Ila (thrombin), Factor Clr, Factor Cls, Factor D, Factor B, C3 convertase, trypsin, chymotrypsin, elastase, enterokinase, urokinase plasminogen activator, tissue plasminogen activator, plasmin, tissue kallikrein, acrosin, ⁇ -subunit nerve growth factor, ⁇ -subunit nerve growth factor, granulocyte elastase, cathepsin G, mast cell chymase, mast cell tryptase.
  • the binding protein reduces or eliminates the activity of a serine protea
  • the ecotin variants of this invention do not include the serine protease inhibitors disclosed or claimed in U.S. Patent 5,719,041 and/or native ecotin.
  • This invention also provides a (binding) protein library comprising a of the ecotin variants described herein.
  • the library preferably comprises at least 100, more preferably at least 1000, and most preferably at least 10,000 different polypeptide species.
  • the ecotin variants may be polypeptides displayed on the surface of bacteria or phage.
  • the phage-displayed polypeptides can be expressed as fusion proteins with the c-terminal domain of a filamentous phage minor coat protein.
  • This invention also provides methods of identifying a protein that specifically binds a polypeptide having a chymotrypsin fold.
  • the methods involve contacting the polypeptide with an ecotin variant binding protein library described herein; and selecting members of the protein binding library that specifically bind to the polypeptide having a chymotrypsin fold.
  • the polypeptide having a chymotrypsin fold is a serine protease.
  • This invention also provides nucleic acids that encode any of the binding proteins described herein.
  • nucleic acids libraries are provided that encode the binding protein libraries described herein.
  • this invention provides methods of reducing or eliminating the activity of a serine protease.
  • the methods involve contacting the serine protease with an ecotin variant described herein where the ecotin variant is not a native ecotin; and the ecotin variant specifically binds to and reduces or eliminates (inhibits) proteolytic activity of the serine protease.
  • Preferred serine proteases of this method include, but are not limited to plasma kallikrein, Factor Xlla, Factor XIa, Factor FXa, Factor Vila, Factor Xa, Factor Ila (thrombin), Factor Clr, Factor Cls, Factor D, Factor B, C3 convertase, trypsin, chymotrypsin, elastinase, enterokinase, urokinase plasminogen activator, tissue plasminogen activator, plasmin, tissue kallikrein, acrosin, ⁇ -subunit nerve growth factor, ⁇ - subunit nerve growth factor, granulocyte elastase, cathepsin G, mast cell chymase, mast cell tryptase.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a co ⁇ esponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • amino acid identified by name herein "e.g., arginine” or “arginine residue” as used herein refers to natural, synthetic, or version of the amino acids
  • an arginine can also include arginine analogs that offer the same or similar functionality as natural arginine with respect to their ability of be incorporated into a polypeptide, effect folding of that polypeptide and effect interactions of that polypeptide with other polypeptide(s).
  • nucleic acid encoding or “nucleic acid sequence encoding” refers to a nucleic acid that directs the expression of a specific protein or peptide.
  • the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
  • the nucleic acid sequences include both full-length nucleic acid sequences as well as shorter sequences derived from the full- length sequences. It is understood that a particular nucleic acid sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • the nucleic acid includes both the sense and antisense strands as either individual single strands or in the duplex form.
  • mutation when used in reference to a polypeptide refers to the change of one or more amino acid residues in a polypeptide to residues other than those found in the "native" or “reference (pre-mutation) form of that polypeptide. Mutations include amino acid substitutions as well as insertions and/or deletions. A mutation does not require that the particular amino acid substitution or deletion be made to an already formed polypeptide, but contemplates that the "mutated" polypeptide can be synthesized de novo, e.g. through chemical synthesis or recombinant means. It will be appreciated that the mutation can include replacement of a natural amino acid with an "unnatural" amino acid.
  • a “protease” is a polypeptide that cleaves another polypeptide at a particular site (amino acid sequence).
  • the protease can also be self-cleaving.
  • a protease is said to be "specific” for another polypeptide when it characteristically cleaves the other "substrate” polypeptide at a particular amino acid sequence.
  • the specificity can be absolute or partial (i.e., a preference for a particular amino acid or amino acid sequence).
  • binding preference e.g., affinity for the target molecule/sequence is at least 2 fold, more preferably at least 5 fold, and most preferably at least 10 or 20 fold over a nonspecific e.g. randomly generated molecule lacking the specifically recognized amino acid or amino acid sequence
  • chymotrypsin fold refers to the anti-parallel beta ba ⁇ el protein "fold" characteristic of trypsin, chymotrypsin, elastase, and related serine proteases (see, e.g., Branden and Tooze (1991) Introduction to Protein Structure, Garland Publishing, New York; Creighton (1993) Proteins, 2nd edition, W.H. Freeman & Co., New York; Schulz and Schirmer (1979) Principles of Protein Structure, Springer- Verlag, New York; Perutz (1992) Protein Structure - New Approaches to Disease and Therapy, W.H. Freeman & Co., New York; Fersht (1976) Enzyme Structure and Mechanism, 2nd ed., W.H.Freeman & Co., New York).
  • a “protease substrate” is a polypeptide that is specifically recognized and cleaved by a protease.
  • randomized when referring to a polypeptide indicates that a collection of polypeptides contains members differing in amino acid composition at the randomized site(s). When the polypeptide is fully randomized, the collection contains a representative polypeptide for every possible natural amino acid at each randomized site .
  • randomized when referring to a nucleic acid refers to a collection of nucleic acids that encode a randomized collection of polypeptides.
  • module when used with respect to protease activity refers to an alteration in the rate of reaction (protein hydrolysis) catalyzed by a protease.
  • An increase in protease activity results in an increase in the rate of substrate hydrolysis at a particular protease concentration and a protease modulator that produces such an increase in protease activity is refe ⁇ ed to as an "activator” or "protease agonist".
  • activator or "agonist” are thus used synonymously.
  • a decrease in protease activity refers to a decrease in the rate of substrate hydrolysis at a particular protease concentration. Such a decrease may involve total elimination of protease activity.
  • a protease modulator that produces a decrease in protease activity is refe ⁇ ed to as a "protease inhibitor". It will be appreciated that generally the increase or decrease is as compared to the protease absent the protease modulator.
  • phage when used in the context of polypeptide display, includes bacteriophage as well as other "infective viruses", e.g. viruses capable of infecting a mammalian, or other, cell.
  • detectable label refers to any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, any label useful in such methods can be applied to the present invention.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include magnetic beads (e.g.
  • DynabeadsTM DynabeadsTM
  • fluorescent dyes e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like
  • radiolabels e.g., 3 H, l I, 35 S, 14 C, or 32 P
  • enzymes e.g., LacZ, CAT, horse radish peroxidase, alkaline phosphatase and others, commonly used as detectable enzymes, either as marker gene products or in an ELISA
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent labels are not to be limited to single species organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like.
  • CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily derivatized for coupling to a biological molecule (Bruchez et al. (1998) Science, 281 : 2013-2016).
  • highly fluorescent quantum dots (zinc sulfide-capped cadmium selenide) have been covalently coupled to biomolecules for use in ultrasensitive biological detection (Warren and Nie (1998) Science, 281: 2016-2018).
  • the label may be coupled directly or indirectly to the ecotin variant to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on the sensitivity required, ease of conjugation of the compound, stability requirements, available instrumentation, and disposal provisions. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 illustrates the tetrameric complex of ecotin-trypsin.
  • the high- resolution crystal structure of rat anionic trypsin-ecotin complex illustrated a network of interactions between ecotin-ecotin monomers, between ecotin-trypsin at the primary binding site, and between ecotin-trypsin at the secondary binding site.
  • Figure 2(a) shows an SDS-P AGE gel of ecotin primary site and dimer Interface variants.
  • Figure 2(b) shows an SDS-P AGE gel of ecotin secondary site variants.
  • Figure 3 shows ⁇ K I plot of ecotin truncation variants. The K t 's of ecotin truncation variants are plotted in log scale along Y-axis.
  • Figure 4(a) shows K t data of ecotin 60s loop variants.
  • Figure 4(b) shows Kj data of ecotin 100s loop variants.
  • Figure 4(c) shows Kj data of ecotin 60s and 100s loop variants.
  • Figure 5 shows a comparison of liquid and plate amplification.
  • the total phage yields from the liquid and plate amplification procedures are compared.
  • the panning experiment is conducted with ecotin M84R+60A4 library panning against uPA for four rounds.
  • Figure 6 shows the modeled electrostatic interaction at the 60s loop of ecotin.
  • the crystal structure of uPA was superimposed to one trypsin molecule in the ecotin-trypsin tetrameric complex by matching the residues Serl95, His57, Aspl02, and Aspl89 of each enzyme using the program MidasPlus (Computer Graphic Laboratory, UCSF) with RMS deviation of 0.66 A.
  • MidasPlus Computer Graphic Laboratory, UCSF
  • Figure 7 shows the rate of hydrolysis of the peptide nitroanilide substrate by the parent human factor IXa.
  • Figure 8 shows the rate of hydrolysis of the peptide nitroanilide substrate by factor IXa passed over an antifactor XI immunoaffinity column.
  • Figure 9 shows the rate of hydrolysis of the peptide nitroanilide substrate by factor IXa absorbed to and eluted from an anti-factor IX immunoaffinity
  • binding proteins that specifically bind polypeptides having a chymotrypsin fold (e.g. serine proteases) and methods of making such binding proteins.
  • Preferred binding proteins of this invention act as serine protease modulators effecting either an activation or an inhibition of a "target" serine protease.
  • the crucial physiological functions of serine proteases e.g., in blood coagulation, fibrinolysis, complement pathways, viral maturation, apoptosis, and cancer make them important targets for efforts to design and engineer potent and specific activators or inhibitors.
  • a highly selective protease modulator can serve as a powerful tool to regulate key proteolytic activities, for dissecting proteolytic pathways and cascades, and for elucidating the in vivo roles of particular proteases in complex biological processes.
  • the present invention relies, in part, on the discovery that ecotin, a macromolecular serine protease inhibitor found in the periplasm of Escherichia coli, offers a unique platform to develop a wide variety of binding proteins.
  • the binding proteins are based on the structure of ecotin and thus referred to as ecotin-derived binding proteins or ecotin variants.
  • This invention exploits the structure of ecotin to act as a scaffold that orients the domains comprising a primary and secondary binding site that mediate ecotin/ecotin and ecotin/substrate interactions.
  • the ecotin backbone as a scaffold the domains can be varied according to the methods of this invention to produce new modulators (e.g. inhibitors) of serine proteases.
  • the binding proteins do not only bind serine proteases, but are capable of specifically binding polypeptides characterized by the presence of a chymotrypsin fold.
  • the binding proteins of this invention are created by modifying the ecotin- protease interactions, particularly those that are distal from the ecotin reactive site described by Chung et al. (1983) J. Biol. Chem., 258(18): 11032-11038. Five sites are important for binding activity and/or modulation: the N-terminus, the C-terminus, the reactive site (primary site), the secondary site, and the dimer interface.
  • Ecotin is a competitive serine protease inhibitor that strongly inhibits trypsin, chymotrypsin and elastase and many other serine proteases with comparable potencies (Chung et al. (1983) supra.).
  • the inhibitor was purified and its reactive site was determined to be Met 84 which lies within a disulfide bonded protein segment (McGrath et al. (1991) J. Biol. Chem., 266(10): 6620-6625).
  • the gene encoding ecotin was cloned and expressed recombinantly in E. coli. (McGrath et al (1991) supra.; McGrath et al. (1991) J. Mol.
  • the ecotin dimer binds to two trypsin molecules at opposite ends to form a heterotetramer with a two-fold symmetry axis.
  • the crystal structure also reveals a network of interactions between ecotin and trypsin.
  • the protein-protein interaction surface between the inhibitor and the protease consists of two distinct areas, each provided by one of the two ecotin molecules.
  • the first area known as the "primary binding site" involves the reactive site loop of ecotin, i.e. the 80s loop (residues 81-86), the 50s loop(residues 52-54), and the active site of trypsin.
  • the second area known as the "secondary binding site" 25 A away ( ⁇ 3-5 A depending upon the ligand), includes two surface loops of ecotin, the 60s loop(residues 66-70) and 100s loop(residues 108-113), and the C-terminal region of the protease (including part of the C-terminal helix and part of the 90s loop of trypsin).
  • the dimer interface primarily amino acids 130 to 142 is important for intersite binding interactions.
  • the N- and C-terminus may also bind to the proteinase.
  • the N terminus includes amino acids 1-7 (or an insertion therein) while the carboxyl terminus includes amino acids 132-142 (or an insertion therein).
  • ecotin's four loops form two interface regions between ecotin and the protease resulting in a combined surface area of 2800 A 2 .
  • the enormous buried interface area between ecotin and its target protease is far greater than that of most other protease-inhibitor complexes.
  • ecotin's unique secondary binding site plays a major role in determining the strength of interaction between ecotin and the protease.
  • ecotin can be randomized in one or more of the above-described five domains to generate a library of ecotin variants (ecotin-like molecules) that are specific inhibitors or agonists of serine proteases typically not targeted by native ecotin.
  • mutation e.g. via randomization
  • Such ecotin variants are useful as serine protease binding proteins and/or for modulating the activity of a wide variety of serine proteases.
  • ecotin was modified in the 60s loop to produce variants having a wide range of activity against rat trypsin and uPA.
  • a serine protease particular a serine protease characterized by a chymotrypsin fold, or analogous folds such as found in viral proteases of the NS3, 2C, and 3C classes (see, e.g., Bazan and Fletterick (1989) Virology, 171(2): 637-639, Bazan and Fletterick (1989) FEBS Letts., 249(1): 5-7, and Bazan and Fletterick (1988) Proc. Natl. Acad. Sci. USA, 85(21): 7872-7876).
  • the methods involve contacting the serine protease with native ecotin or an ecotin variant of this invention.
  • the ecotin variants of this invention can be used simply as binding proteins.
  • the ecotin variants act in a manner analogous to antibodies in that they specifically bind to a target molecule.
  • Preferred ecotin variants specifically bind to polypeptides characterized by the presence of a chymotrypsin fold. It will be appreciated that many serine proteases are characterized by a chymotrypsin fold (e.g. chymotrypsin, elastase, thrombin, urokinase type plasminogen activator, factor IXa, factor Xa, etc.).
  • polypeptides characterized by a chymotrypsin fold that are not serine proteases (e.g., the 3C viral protease that has a cysteine in place of the active site serine), often not even proteases, and yet are still specifically bound by the ecotin variant binding proteins of this invention.
  • serine proteases e.g., the 3C viral protease that has a cysteine in place of the active site serine
  • target polypeptides include, but are not limited to, the 3C proteases, e.g., the 3C proteases from poliovirus, rhinovirus, and encephalovirus.
  • the ecotin variants of this invention can be used as binding proteins in a wide variety of contexts analogous to the use of antibodies.
  • they can be labeled with a detectable label and used to probe for the target polypeptide(s) to which they specifically bind, they can be used as binding agents in "immunoassays" (e.g. sandwich assays, lateral flow assays, etc.), and they can be used as binding partners in purification systems to selectively isolate their target (cognate) polypeptide from a mixture of molecules.
  • the binding proteins of this invention are used as affinity chromatography reagents.
  • one or more ecotin variants of this invention is attached to a solid substrate or an isolatable label (e.g. a magnetic bead, fluorescent moiety separable in a FACs system, etc.).
  • the ecotin variant is contacted with the mixture from which the target polypeptide(s) is to be isolated under conditions that permit protein recognition and binding.
  • the ecotin variant and its bound protein are separated from the mixture and the bound protein is then optionally separated from the ecotin complex (e.g. by high salt, high pH, low pH, temperature change, organic solvents, chaotropic agent, denaturing agents (e.g. urea, guanadinium salts, etc.).
  • the ecotin variant can be attached to a regular or i ⁇ egular, planar or non-planar, solid or porous surface.
  • Such surfaces can include, but are not limited to the surfaces of beads, pores, planar surfaces, microchannels, capillaries, and the like.
  • the ecotin variant can be coupled to particles that are packed into a column permitting the sample mixture, buffers or other reagents to be flowed past the binding protein, or conversely, ecotin variant-bound particles can be suspended in a solution containing the polypeptide that is to be separated. After binding occurs, the bound particles can be separated from the mixture (e.g. via centrifugation, the use of magnetic particles, etc.).
  • Proteins contain a variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH2) groups, which are available for reaction with a suitable functional group on either the surface or on a linker attached to the surface. . Proteins, for example, may be joined to linkers or to functional groups coupling through their amino or carboxyl termini, or through side groups of various constituent amino acids. Thus, coupling through a disulfide linkage to a cystein is common. Generally linkers are either hetero- or homo-bifunctional molecules that contain two or more reactive sites that may each form a covalent bond with the respective binding partner (i.e. surface or ecotin variant). Linkers suitable for joining biological binding partners are well known to those of skill in the art.
  • a protein molecule may be linked by any of a variety of linkers including, but not limited to a peptide linker, a straight or branched chain carbon chain linker, or by a heterocyclic carbon linker.
  • linkers including, but not limited to a peptide linker, a straight or branched chain carbon chain linker, or by a heterocyclic carbon linker.
  • Heterobifunctional cross linking reagents such as active esters of N-ethylmaleimide have been widely used. See, for example, Lemer et al. (1981) Proc. Nat. Acad. Sci. (USA), 78: 3403-3407 and Kitagawa et al. (1976) J. Biochem., 79: 233-236, and Birch and Lennox (1995) Chapter 4 in Monoclonal Antibodies: Principles and Applications, Wiley-Liss, N.Y.).
  • the linker is itself a polypeptide
  • it can be expressed as a fusion with the ecotin variant.
  • the ecotin variant is expressed with a poly-Histidine (e.g. His 6 ) tag that in turn binds to a Ni-NTA substrate, e.g. a NiNTA-column).
  • the ecotin variant can be bonded to the surface by any of a variety of other well-known chemical procedures.
  • the linkage may be by way of heterobifunctional cross-linkers, e.g. SPDP, carbodiimide, glutaraldehyde, or the like.
  • the linkage is achieved using cyanogen bromide.
  • cyanogen bromide Virtually any surface that is resistant to reagents used in binding and/or eluting the captured polypeptide and that does not substantially interfere with the ecotin variant/target polypeptide binding interaction is suitable for use as a matrix (surface) material.
  • matrix materials include glass beads, controlled pore glass, magnetic beads, various membranes or rigid various polymeric resins such as polystyrene, polystyrene/latex, and other organic and inorganic polymers, both natural and synthetic.
  • Illustrative polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluoride (PNDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like.
  • Other materials which may be employed include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like.
  • substances that form gels such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used.
  • Polymers which form several aqueous phases such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.
  • a plurality of different materials may be employed, e.g., as laminates, to obtain various properties.
  • protein coatings such as gelatin can be used to avoid non specific binding, simplify covalent conjugation, enhance signal detection or the like.
  • the surface will usually be polyfunctional or be capable of being polyfunctionalized.
  • Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like.
  • Serine proteases are a family of enzymes that utilize a uniquely activates serine residue in the substrate binding site to catalytically hydrolyze peptide bonds.
  • the active site serine can be characterized by the i ⁇ eversible reaction of its side chain hydroxyl group with diisopropylfluorophosphate (DFP).
  • DFP diisopropylfluorophosphate
  • the target of the serine proteases are specific peptide bonds in proteins and often their substrates are other serine proteases that are activated from an inactive precursor form (a zymogen) by the catalytic cleavage of a specific peptide bond in their structure.
  • Serine proteases are implicated in a wide variety of physiological processes including, but not limited to blood coagulation, fibrinolysis, complement activation, fertilization, hormone production, tumor cell metastasis, emphysema, arthritis, thrombosis, and some forms of hemostasis.
  • the viral proteases In case of viral infection, the viral proteases have been identified in infected cells.
  • Such viral proteases include, for example, HIN protease associated with AIDS and ⁇ S3 protease associated with Hepatitis C, and the like. These viral proteases play a critical role in the virus life cycle.
  • Proteases have also been implicated in cancer metastasis. For example, increased synthesis of the protease urokinase has been co ⁇ elated with an increased ability to metastasize in many cancers.
  • Urokinase activates plasmin from plasminogen which is ubiquitously located in the extracellular space and its activation can cause the degradation of the proteins in the extracellular matrix through which the metastasizing tumor cells invade.
  • Plasmin can also activate the collagenases thus promoting the degradation of the collagen in the basement membrane su ⁇ ounding the capillaries and lymph system thereby allowing tumor cells to invade into the target tissues (Dano, et al. (1985) Adv. Cancer. Res., 44: 139).
  • a number of other pathological conditions are associated with altered serine protease regulation.
  • cerebral infarction (stroke), coronary infarction, thrombosis, and bleeding disorders are associated with abnormal regulation of plasma kallikrein, Factor XIIA, Factor XIa, Factor IXa, Factor Vila, Factor Xa and Factor Ila (thrombin).
  • Inflammation, rheumatoid arthritis and autoimmune disease are associated with abnormal regulation of Factor Clr, Factor Cls, Factor D, and Factor B.
  • Digestive disorders e.g., pancreatitis
  • enterokinase are associated with altered regulation of trypsin, chymotrypsin, elastase and enterokinase.
  • Clotting disorders are associated with urokinase plasminogen activator (uPA), tissue plasminogen activator, or plasmin.
  • Infertility is associated with abnormal regulation of acrosin.
  • Inflammation and allergic response is associated with granulocyte elastase activity, cathepsin G, mast cell chymases, and mast cell tryptases.
  • Tumor invasiveness is associated with urokinase plasminogen activator and elastase activity.
  • modulators e.g. activators or inhibitors
  • serine protease activity are expected to prove useful in the treatment and/or mitigation of symptoms associated with these conditions.
  • regulation of Factor IXa will be useful in the development of anti-thrombotics that are not hemoragic and which could be used for deep vein thromboses
  • modulation of coUagenases is expected to be useful in the treatment of metastatic disease and the retardation of tumor invasiveness.
  • native ecotin and ecotin variants can act as serine protease activators enhancing serine protease activity.
  • native bacterial ecotin whose typical cognate protease is presently unknown will act as a significant agonist on mammalian (e.g. human) Factor FXa.
  • Other ecotin variants e.g. M84R
  • the compounds of this invention can be used to increase or decrease (modulate) serine protease activity.
  • ecotin variants modifications of ecotin. As indicated above, it was a discovery of this invention that ecotin variants can be used to specifically target and regulate a wide variety of serine proteases. The ecotin variants in some instances can inhibit target serine proteases, while in other instances can act agonistically with serine proteases to increase activity and/or binding specificity or avidity. In a preferred embodiment, the ecotin variants of this invention substantially comprise a native ecotin backbone, but contain one or more mutations in particular regions.
  • the mutations are in one or more of the following regions: the primary binding site, including, but not limited to the 50s loop (amino acids 52- 54 of native ecotin) and the 80s loop (amino acids 81 to 86 of native ecotin), and the secondary binding site including, but not limited to, the 60s loop (amino acids 67-70 of native ecotin), and the 100s loop (amino acids 108-113 of native ecotin).
  • the primary binding site including, but not limited to the 50s loop (amino acids 52- 54 of native ecotin) and the 80s loop (amino acids 81 to 86 of native ecotin)
  • the secondary binding site including, but not limited to, the 60s loop (amino acids 67-70 of native ecotin), and the 100s loop (amino acids 108-113 of native ecotin).
  • 50s loop mutations include mutations in amino acids 50-56, more preferably 51-55 and most preferably 52-54
  • 80s loop mutations include mutations of amino acids 79-88, more preferably 80-87, and most preferably 81-86
  • 60s mutations include mutations of amino acids 65-72, more preferably 66-71, and most preferably 67-70
  • 100s loop mutations include mutations of amino acids 106-115, more preferably 107-114, and most preferably 108-113.
  • Additions and the C-terminus and C- terminus may also be effective in binding and modulating protease function. Alterations in the ecotin interface can be made to enhance or limit binding interactions as well.
  • Suitable mutations include replacement of one naturally occurring amino acid with another different naturally occurring amino acid, replacement of an amino acid with a non-naturally occurring amino acid (e.g. an amino acid analogue).
  • the mutations can also include deletions or insertions of one or more amino acids. Similar modifications can be made at the ecotin carboxyl and amino termini. Thus, for example, up to 10 amino acids, more preferably up to 8 amino acids, and most preferably up to 7 amino acids can be deleted from either or both termini.
  • X 1 is a polypeptide having the sequence of amino acids 8 through 50 of native ecotin (SEQ ID NO: 1)
  • X 2 is a polypeptide having the sequence of amino acids 56 through 65 of native ecotin
  • X 3 is a polypeptide having the sequence of amino acids 72 through 78 of native ecotin
  • X 4 is a polypeptide having the sequence of amino acids 88 through 106 of native ecotin
  • X 5 is a polypeptide having the sequence of amino acids 115 through 135 of native ecotin
  • L 50s , L 60s , L 80s , and, L I00s are independently an amino acid or a polypeptide
  • T and T are independently an amino acid or a polypeptide consisting of 2 to about 120 amino acids, more preferably 2 to about 50 amino acids, and most preferably 2 to about 15 amino acids, and i, j, k, m, n, and p are independently 0 or 1.
  • the ecotin variant can be represented by the formula:
  • T 1 is a polypeptide having the formula aa'-aa 2 -aa 3 -aa 4 -aa 5 -aa 6 -aa 7 -
  • T 2 is a polypeptide having the formula aa I36 -aa 137 -aa 138 -aa ,39 -aa I40 -aa 141 -aa 142 , and aa 1 , aa 2 , aa 3 , aa 4 , aa 7 , aa 6 , aa 7 , aa 51 , aa 52 , aa 53 , aa 54 , aa 55 , aa 66 , aa 67 , aa 68 , aa 69 , aa 70 , aa 71 , aa 79 , aa 80 , aa 81 , aa 82 , aa 83 , aa
  • the ecotin is a variant and not a native ecotin and is capable of said specifically binding to and altering the activity of a serine protease.
  • prefe ⁇ ed variants include ecotin having mutations in the following combinations of loops: 50s, 60s, 80s, 100s; 50s and 60s, 50s and 80s, 50s and 100s, 60s and 80s, 60s and 100s, 80s and 100s; 50s and 60s and 80s, 50s and 80s and 100s, 50s and 60s and 100s, 50s and 80s and 100s; and 50s and 60s and 80s and 100s. Any of these mutations can be combined with mutations in the amino or carboxyl terminus as indicated above.
  • an insertion into a loop (50s, 60s, 80s, or 100s) will typically comprise no more than about 15 amino acids, more preferably no more than about 8 amino acids, and most preferably no more than about 4 amino acids.
  • insertions at either terminus will typically comprise no more than about 120, preferably no more than about 50 amino acids, more preferably no more than about 10 amino acids and most preferably no more than about 4 amino acids.
  • a complete domain e.g. a fibronectin type III domain
  • a complete domain can be added to the N- or C-termini.
  • ecotin variants having a particular binding specificity and/or avidity and/or a particular modulatory activity are identified by providing a library (a collection) comprising a number of different ecotin variants.
  • the library is then screened against one or more target serine proteases and members of the library having the desired avidity and/or specificity and/or modulatory activity are selected.
  • Methods of screening polypeptide libraries to select members having particular binding specificity and/or avidity and/or modulatory activity are well known to those of skill in the art. For example, binding specificity and avidity can be determined using simple binding assays of the type generally used for measuring antibody binding avidity or specificity (e.g.
  • Modulatory activity assays involves contacting the ecotin or ecotin variants to be screened, with one or more "target" serine protease(s) under conditions in which the serine protease is normally capable of cleaving its substrate. The effect of the ecotin variant on the protease activity can then be assayed according to standard methods. Ecotin variant production and assays are described in more detail below.
  • Libraries of ecotin variants can be produced by any of a wide number of methods including, but not limited to chemical syntheses of each individual variant, combinatorial based syntheses, a ⁇ ay-based combinatorial syntheses, recombinant expression of each individual variant, recombinant expression of randomized libraries, bacterial display systems, and phage display systems.
  • Phage display The ability to express polypeptides on the surface of bacteria or of viruses that infect bacteria (bacteriophage or phage) makes it possible to isolate a single binding polypeptide or a single polypeptide having a particular activity from libraries of greater than 10 10 nonbinding clones.
  • phage display a nucleic acid encoding the polypeptide is inserted into the gene encoding a phage surface protein (e.g., pill) and the polypeptide-surface fusion protein is displayed on the phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res.
  • phage bearing binding polypeptides can be separated from non-binding phage by antigen affinity chromatography (McCafferty et al. (1990) Nature, 348: 552-554). Depending on the affinity of the antibody fragment, enrichment factors of 20 fold -
  • Phage display has been successfully applied to a wide range of peptides and proteins, including antibodies McCafferty et al. (1990) Nature, 348: 552-554), growth hormone (Bass et al. (1990) Proteins: Struct. Fund. Genet. 8(4): 309-314), DNA binding proteins (Jamieson et al. (1994) Biochem., 33(19): 5689-5695), enzymes (McCafferty et al. (1991) Protein Eng, 4(8): 955-961); Corey et al. (1993) Gene, 128(1): 129-134); Soumillion et al. (1994) J Mol.
  • uPA is a serine protease (often an activator of coUagenases) that plays an active role in extracellular proteolysis, cell migration, and tissue remodeling processes (Fazioli et al. (1994) Trends Pharmacol. Sci., 15(1): 25-29). Because of its implication in cancer metastasis and tumor invasion, uPA has become an important target for drug design and inhibitor development efforts.
  • a high affinity inhibitor of uPA can be isolated from a library of phage-displayed ecotin variants at the PI and PI' positions (Wang et al. (1995) supra.).
  • phage display techniques are described herein to modify ecotin's secondary and other sites and thereby modify potency and specificity against target proteases.
  • phage display libraries are created that express ecotin, but are "randomized", or contain deletions or insertions, in particular regions (e.g. the 50s loop, the 80s loop, the 60s loop or the 100s loop). Nucleic acids encoding all possible amino acid variants at particular sites, can be prepared and inserted into the vectors comprising the phage display library.
  • the "randomized" nucleic acids are made according to methods well known to those of skill in the art.
  • the nucleic acids can be chemically synthesized using "doped" nucleotide reagents during the coupling steps forming the "randomized" codons.
  • the randomized nucleic acids are created using amplification (e.g., PCR) cloning with degenerate primers.
  • degenerate primers are used to amplify ecotin templates where the primers are degenerate in regions expressing the domain of the ecotin it is desired to randomize.
  • such primers contain N at the desired position, or more preferably introduce codons of the form NNS, where N is A/C/G/ and S is C/G.
  • ecotin variants can also be expressed from nucleic acids that are modified by site-directed mutagenesis.
  • Methods of site directed mutagenesis are well known to those of skill in the art.
  • site- directed mutagenesis is performed by the method of Kunkel (1985) Proc. Natl. Acad. Sci. USA, 82(2): 488-492 as described in Example 1. 3) Array-based approaches.
  • ecotin variant libraries can be created using combinatorial-based polypeptide synthesis techniques.
  • Array-based combinatorial synthesis techniques are well known to those of skill in the art (see, e.g. See, e.g., U.S. Patent No. 5,143,854; PCT Publication Nos. WO 90/15070, WO 92/10092 and WO 93/09668; and Fodor et al. (1991) Science, 251, 767-77). Briefly, in this approach, photolithographic methods are used to selectively couple derivatized amino acids at discrete locations in a solid-phase synthesis system. Highly complex arrays can be produced in relatively few coupling steps (Id.).
  • the library is screened for the desired binding specificity and/or avidity, and/or serine protease modulatory activity. Screens for binding avidity and/or specificity and for effect on serine protease activity are well known to those of skill in the art.
  • Direct binding assays In direct binding assays the ability of one or more ecotin variants to bind to a serine protease (e.g. trypsin, uPA, etc.) is assayed. Simple binding assays are well known to those of skill in the art.
  • a serine protease e.g. trypsin, uPA, etc.
  • Simple binding assays are well known to those of skill in the art.
  • either the ecotin variant or the serine protease is labeled, the ecotin variant and the serine protease are contacted with each other and the association of the labeled moiety its respective binding partner is detected and/or quantified.
  • both the ecotin variant and the protease can both be labeled and the association of the labels then indicates binding.
  • Direct binding assays can also be performed in solid phase where either the ecotin variant or protease is immobilized on a solid support. When the ecotin variant is immobilized it is contacted with the protease (optionally labeled) and conversely where the protease is immobilized it is contacted with the ecotin variant(s) (optionally labeled).
  • FRET Fluorescence resonance energy transfer systems
  • ecotin variant nor protease need be labeled prior to the assay.
  • An "indirect" subsequently applied label e.g. a labeled antibody specific for the ecotin variant or protease
  • a labeled antibody specific for the ecotin variant or protease can be used to detect the ecotin variant or protease in the ecotin variant protease complex.
  • no label need be used.
  • the bound polypeptides e.g. bound phage
  • Selection for increased avidity involves measuring the affinity of an ecotin variant for one or more target serine proteases. Methods of making such measurements are well known to those of skill in the art.
  • the Kd of a ecotin variant and the kinetics of binding to a target protease inhibitor are measured using a BIAcore, a biosensor based on surface plasmon resonance.
  • BIAcore a biosensor based on surface plasmon resonance.
  • serine protease is coupled to a derivatized sensor chip capable of detecting changes in mass.
  • the ecotin variant When ecotin is passed over the sensor chip, particularly when additional protease is available in solution to form the tetrameric complex, the ecotin variant binds to the serine protease resulting in an increase in mass that is quantifiable. Measurement of the rate of association as a function of ecotin variant concentration can be used to calculate the association rate constant (k on ). After the association phase, buffer is passed over the chip and the rate of dissociation of ecotin variant (k of f) determined. The equilibrium constant K ⁇ j is then calculated as k off /k on and thus is typically measured in the range 10 "5 to 10 "12 . Affinities measured in this manner co ⁇ elate well with affinities measured in solution by fluorescence quench titration. 2) FRET assays.
  • fluorescent resonance energy transfer (FRET) systems can also be used to assay protein-protein interactions.
  • FRET-based assays both components (e.g. both the ecotin variant and the serine protease) are labeled with fluorescent labels.
  • the absorption and emission spectra of the labels are selected such that one label emits at a wavelength that the other absorbs.
  • FRET is a powerful technique for measuring protein-protein associations and has been used previously to measure the polymerization of monomeric actin into a polymer (Taylor et al. (1981) J. Cell Biol., 89: 362-367) and actin filament disassembly by severing (Yamamoto et al. (1982) J. Cell Biol., 95: 711-719).
  • binding of ecotin variant to the serine protease can be detected by the use of liquid crystals.
  • Liquid crystals have been used to amplify and transduce receptor-mediated binding of proteins at surfaces into optical outputs.
  • Spontaneously organized surfaces can be designed so that formation of the ecotin variant serine protease tetrahedral complex on these surfaces, trigger changes in the orientations of 1- to 20-micrometer-thick films of supported liquid crystals, thus co ⁇ esponding to a reorientation of ⁇ 10 5 to 10 6 mesogens per protein. Binding-induced changes in the intensity of light transmitted through the liquid crystal are easily seen with the naked eye and can be further amplified by using surfaces designed so that protein-ligand recognition causes twisted nematic liquid crystals to untwist (see, e.g., Gupta et al. (1998) Science, 279: 2077- 2080).
  • This approach to the detection of protein/protein interactions does not require labeling of the analyte, does not require the use of electroanalytical apparatus, provides a spatial resolution of micrometers, and is sufficiently simple that it is useful in biochemical assays and imaging of spatially resolved chemical libraries.
  • native ecotin, or the ecotin variants of this invention are be screened for enzymatic (e.g. protease inhibitory or agonistic) activity.
  • enzymatic e.g. protease inhibitory or agonistic
  • screens involve combining the ecotin or ecotin variant(s) together with a protease of interest and a substrate for that protease under conditions in which the protease typically has proteolytic activity and determining the effect of the presence absence or amount of ecotin or ecotin variant on the proteolytic activity of the protease.
  • protease activity is well known to those of skill in the art (see, e.g. Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Eugene OR). Typically the protease activity is assayed with respect to its "native" target polypeptide.
  • the protease activity can be assayed by using an "indicator" substrate that provides a signal indicative of hydrolysis (see, e.g., chromogenic substrate N ⁇ -benzyloxy-carbonyl-L-glyylproylarginine-7-amino-4- methylcoumarin (Z-GPR-AMC) available from Bachem Biosciences, Inc (King of Prussia, PA), chromogenic substrate Z- ⁇ -Glu( ⁇ -t-butoxy)-Gly-Arg-p-nitroanilide (Spectrozyme UK), etc.).
  • Z-GPR-AMC chromogenic substrate N ⁇ -benzyloxy-carbonyl-L-glyylproylarginine-7-amino-4- methylcoumarin
  • Z-GPR-AMC chromogenic substrate Z- ⁇ -Glu( ⁇ -t-butoxy)-Gly-Arg-p-nitroanilide
  • a large number of such indicators are well known to those of skill in the art and
  • the rate of hydrolysis of a target substrate by a particular protease is assayed.
  • the rate of hydrolysis of Z-GPR-AMC substrate by of rat and bovine trypsin was assayed (e.g. by the change in emission to 460 nm) in the presence of various concentrations of ecotin variants.
  • the resulting data can be fit to the equation derived for kinetics of reversible tight-biding inhibitors, e.g. by non-linear regression analysis to determine the values for apparent K ( and true K ⁇ (see, Examples 1 and 2).
  • protease The activity of native ecotin or ecotin variants can be determined against virtually any protease.
  • Serine proteases are prefe ⁇ ed. Suitable serine proteases include, but are not limited to plasma kallikrein, Factor Xlla, Factor XIa, Factor IXa, Factor Vila, Factor Xa, Factor Ila (thrombin), Factor Clr, Factor Cls, Factor D, Factor B, C3 convertase, trypsin, chymotrypsin, elastinase, enterokinase, urokinase plasminogen activator, tissue plasminogen activator, plasmin, tissue kallikrein, acrosin, ⁇ -subunit nerve growth factor, ⁇ -subunit nerve growth factor, granulocyte elastase, cathepsin G, mast cell chymase, mast cell tryptase
  • the assays of the present invention offer the advantage that many samples can be processed in a short period of time.
  • plates having 96 or as many wells as are commercially available can be used.
  • the serine protease or ecotin variants can be attached to solid supports and spatially a ⁇ anged to form distinct a ⁇ ays, such as rows of dots or squares, or lines.
  • This, coupled to sophisticated masking, assay and readout machines greatly increase the efficiency of performing each assay and detecting and quantifying the results. It is possible with cu ⁇ ent technologies to efficiently make vast numbers (10 6 or more) of peptides having specified sequences and a ⁇ ay them at distinct locations in a chip, and then to detect fluorescent associated with each position of the chip.
  • high throughput screening methods involve providing a library containing a large number of compounds (test compounds) potentially having the desired activity. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display the desired specificity, avidity or activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
  • U.S. Patent 5,559,410 discloses high throughput screening methods for proteins
  • U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in a ⁇ ays)
  • U.S. Patents 5,576,220 and 5,541,061 disclose methods of screening for ligand/antibody binding.
  • high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc., Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.) These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
  • These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various high throughput assays.
  • Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
  • each assay mixture can be utilized to assay the effect of a ecotin vari-mt or the effect on a single protease
  • multiple ecotin variants or target proteases can also be screened in a single assay mixture.
  • two or more, preferably 4 or more, more preferably 16 or more and most preferably 32, 64, 128, 256, or even 512 or more ecotin variants or target proteases are screened in a single assay reaction mixture.
  • a positive result in that assay indicates that one or more of the ecotin variants are modulators of the target protease, or that one or more of the target proteases are acted upon by the ecotin variant.
  • the method is repeated wherein the candidate agents are separated out to identify the modulator individually, or to verify that the agents work in conjunction to provide the difference in binding specificity, affinity, or avidity.
  • an assay originally run with 16 ecotin variants e.g. per well
  • test agents can be assayed together to identify ecotin variants that are additive or even synergistic in their effect on a protease, or conversely, to identify test agent(s) that are antagonistic in their effects on a protease.
  • the method further comprises the step of entering the identity of an ecotin variant that has been identified to modulate activity of a protease in accordance with the present invention into a database of therapeutic, diagnostic or bioagricultural lead compounds.
  • a database of therapeutic, diagnostic or bioagricultural lead compounds it may be desirable to perform further assays on the compounds which have been identified herein.
  • activity of the identified compounds can be further assessed in areas other than their ability to modulate protease activity. For example, their ability to affect growth or proliferation of cells, particularly tumor cells, etc. can be assessed.
  • IV. Production of identified ecotin variant is a database of therapeutic, diagnostic or bioagricultural lead compounds.
  • the native ecotin or ecotin variants of this invention can be isolated (purified) from bacterial sources or alternatively can be recombinantly expressed.
  • the polypeptides can be chemically synthesized according to standard methods.
  • the native ecotin or ecotin variants of this invention may be synthesized using standard chemical peptide synthesis techniques. Where the sequence is amenable, the molecule may be synthesized as a single contiguous polypeptide. Where larger molecules are desired, subsequences can be synthesized separately (in one or more units) and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond.
  • de novo chemical synthesis can be used to incorporate non-natural amino acids as well as natural amino acids into polypeptides.
  • Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the prefe ⁇ ed method for the chemical synthesis of the polypeptides of this invention.
  • Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in TTze Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. , Merrifield et al. (1963) J. Am. Chem. Soc, 85: 2149-2156, and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111. (1984).
  • the ecotin variants of this invention are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes polypeptide, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
  • DNA encoding native ecotin or ecotin variants of this invention may be prepared by any suitable method as described above, including, for example, cloning and restriction of appropriate sequences, amplification techniques, or direct chemical synthesis.
  • Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
  • a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.
  • subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
  • the ecotin or ecotin variants of this invention may be cloned using DNA amplification methods such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the nucleic acid sequence is PCR amplified, using a sense primer containing one restriction site (e.g., Ndel) and an antisense primer containing another restriction site (e.g., Hindlll).
  • a sense primer containing one restriction site e.g., Ndel
  • an antisense primer containing another restriction site e.g., Hindlll
  • This nucleic acid can then be easily ligated into a vector containing a nucleic acid encoding the second molecule and having the appropriate co ⁇ esponding restriction sites.
  • Suitable PCR primers are provided in Examples 1 and 2, and others can be determined by one of skill in the art using the sequence information provided in SEQ ID No: 1.
  • Typical vectors for use in this invention contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and . Smith (1979), Gene, 8:81-97; Roberts et al.
  • the nucleic acid sequences encoding the ecotin or ecotin variants may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines.
  • the recombinant protein gene will be operably linked to appropriate expression control sequences for each host (e.g., E. coli, or Staphylococcus). For E.
  • control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • the plasmids of the invention can be transfected into the chosen host cell by well-known methods such as calcium phosphate transfection, electroporation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, D ⁇ A ⁇ dextran, receptor-mediated endocytosis, electroporation, micro-injection of the DNA directly into the cells, infection with viral vectors, etc.
  • Cells transformed by the plasmids can be selected by resistance to antibiotics confe ⁇ ed by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
  • the recombinant ecotin or ecotin variant polypeptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Guider, Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y. (1990)). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most prefe ⁇ ed. Once purified, partially or to homogeneity as desired, the polypeptides may then be used (e.g., as protease modulators).
  • the ecotin or ecotin variant (s) may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the prefe ⁇ ed conformation. Methods of reducing and denaturing proteins and inducing re- folding are well known to those of skill in the art (see, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.
  • modifications can be made to the ecotin or ecotin variants without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or purification of the protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly-His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
  • Example 1 Detailed protocols for ecotin or ecotin variant expression and purification are provided in Example 1.
  • recombinant expression systems can be used to express polypeptides encoding unnatural amino acids.
  • Methods for the incorporation of non-natural amino acids are well known to those of skill in the art (see, e.g., Koh (1997) Biochem., 36(38): 11314-11322, Liu et al. (1997) Proc. Natl. Acad. Sci. USA, 94(19): 10092-10097, Liu et al. (1997) Chem. and Biol, 4(9): 685-691, Cload et al (1996) Chelm.
  • new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
  • Lead compounds thus provide a convenient starting point or baseline for developing second or third generation variants.
  • this invention provides the identification of characteristic motifs that enhance the activity of the ecotin variant against a particular serine protease. Typically this is accomplished by screening an ecotin variant library for inhibitory or agonistic activity on a particular serine protease. Members of the library that display the desired activity can be isolated and their sequence determined. Identification of a consensus sequence provides a good lead compound for the desired activity on the subject protease. Having identified a consensus sequence in one region of the ecotin variant, other regions can then be varied to provide new variants of potentially greater activity and/or specificity.
  • the ecotin variants of this invention can be combined with a pharmaceutically acceptable carrier (excipient) to form a pharmacological composition.
  • Pharmaceutically acceptable carriers can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase or decrease the absorption of the active agent.
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the protease modulator(s), or excipients or other stabilizers and or buffers.
  • physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • a pharmaceutically acceptable carrier including a physiologically acceptable compound depends, for example, on the rout of administration of the protease modulator and on the particular physio-chemical characteristics of the modulator.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges.
  • the protease modulators when administered orally, must be protected from digestion. This is typically accomplished either by complexing the modulator with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the modulator in an appropriately resistant carrier such as a liposome.
  • Means of protecting compounds from digestion are well known in the art (see, e.g., U.S. Patent 5,391,377 describing lipid compositions for oral delivery of therapeutic agents).
  • Effective systems for the sustained delivery of therapeutic proteins preferably involve (i) processing and formulating the protein and delivery system so that the protein's fragile conformation and biological activity are maintained throughout processing and during prolonged release in the body. In addition such systems control the protein release so that therapeutic levels are maintained for the desired time.
  • Sustained protein delivery can be achieved with a variety of microsphere delivery systems.
  • the ProLease biodegradable microsphere delivery system for proteins and peptides (Tracy (1998) Biotechnol. Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut. Res. 15, 357) a dry powder composed of biodegradable polymeric microspheres containing a protein in a polymer matrix that can be administered by injection in an aqueous diluent through a narrow-gauge needle.
  • the ProLease microsphere fabrication process was specifically designed to achieve a high protein encapsulation efficiency while maintaining protein integrity (Gombotz, et al.
  • the process consists of (i) preparation of freeze-dried protein particles from bulk protein by spray freeze-drying the drug solution with stabilizing excipients, (ii) preparation of a drug-polymer suspension followed by sonication or homogenization to reduce the drug particle size, (iii) production of frozen drug-polymer microspheres by atomization into liquid nitrogen, (iv) extraction of the polymer solvent with ethanol, and (v) filtration and vacuum drying to produce the final dry-powder product.
  • the resulting powder contains the solid form of the protein, which is homogeneously and rigidly dispersed within porous polymer particles.
  • the polymer most commonly used in the process poly(lactide-co-glycolide) (PLG), is both biocompatible and biodegradable.
  • Encapsulation can be achieved at low temperatures (e.g., -40°C).
  • the protein is maintained in the solid state in the absence of water, thus minimizing water-induced conformational mobility of the protein, preventing protein degradation reactions that include water as a reactant, and avoiding organic-aqueous interfaces where proteins may undergo denaturation.
  • a prefe ⁇ ed process uses solvents in which most proteins are insoluble, thus yielding high encapsulation efficiencies (e.g., greater than 95%).
  • Microspheres/Microparticles R. S. Cohen and H. Bernstein, Eds.(Dekker, New York, pp.l- 49).
  • the choice of one or more stabilizing agents is determined empirically.
  • One effective approach is to form a complex with a divalent metal cation before encapsulation.
  • Zinc has been employed in this manner to stabilize recombinant human growth hormone (rhGH) and recombinant ⁇ -interferon ( ⁇ -IFN) in microspheres (Tracy (1998) Biotechnol. Prog. 14: 108, Johnson et al (1997) Pharmacol. Res. 14: 730; U.S. Patent 5,711,968 (1998)).
  • protein stability in hydrated microspheres can be improved by using certain salts. For example, ammonium sulfate has been shown to stabilize erythropoietin during release (U.S. Patent 5,711,968 ).
  • the microsphere formulation should preferably display the release kinetics required to achieve a sustained therapeutic effect.
  • the encapsulated protein is released by a complex process involving hydration of the particles, dissolution of the drug, drug diffusion through water- filled pores within the particles, and polymer erosion (Langer and Folkman (1976) Nature 263: 797; Bawa et al, (1985) J. Controlled Release 1: 259; Saltzman and Langer (1989) Biophys. J. 55: 163).
  • Two primary considerations are minimizing how much protein is released immediately (called the burst) and achieving the desired duration and rate of protein release.
  • the duration of release is governed by the type of PLG polymer used and the addition of release modifying excipients such as zinc carbonate (Saltzman and Langer (1989) Biophys. J. 55: 163).
  • sustained delivery of proteins are likely to include improved patient compliance (by reducing the need for self-injection), potentially lower costs (by reducing the frequency of visits to a caregiver's office), greater usage of a drug (through new indications and ease of use), and improved safety and efficacy (by reducing variability inherent in frequent injections).
  • microsphere-based sustained delivery systems may be limited by the daily dose of protein needed for a therapeutic effect.
  • Alternative approaches for sustained delivery of therapeutic proteins are also known.
  • An implantable osmotic pump system delivers peptide drugs at a constant rate for up to 1 year (Wright, et al. (1997), Proc. Int. Symp.
  • Pulmonary delivery of proteins in the form of aerosols may provide a less invasive route of administration compared to injection (Wall (1995) Drug Delivery 2: 1). Injection frequency can also be decreased by increasing plasma half-life. For example, chemical modification with polyethylene glycol has been reported to extend the plasma half- life of therapeutic proteins such as ⁇ -IFN (Nieforth et al, (1996) Clin. Pharmacol. Ther. 59: 636) resulting in a reduced injection frequency.
  • microspheres might be engineered to provide pulsatile drug release in response to relevant biofeedback (Id.) or to normal cyclical rhythms of the body.
  • formulations that contain multiple drugs and whose release profiles are tailored to changing physiological needs as treatment progresses represent embodiment. Examples of these indications are the dynamic cascade associated with wound healing and the degeneration, apoptosis, and regeneration sequence that occurs following spinal cord injury. This is accomplished by blending microspheres with different proteins and release characteristics.
  • microelectronic chips can be interfaced with the injected polymer mass to provide programmed control of protein release, thus offering far greater moment-to-moment flexibility and precision in the release characteristics.
  • Kits for screening, treatment, or affinity chromatography are provided.
  • kits for screening for ecotin variants having a particular activity against one or more proteases.
  • the kits preferably include an ecotin variant library and/or a nucleic acid library encoding an ecotin variant library.
  • the library preferably includes any of the ecotin variants described herein.
  • the kits may optionally contain any of the buffers, reagents, and/or media that are useful for the practice of the methods of this invention.
  • kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
  • Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • Such media may include addresses to internet sites that provide such instructional materials.
  • kits include, but are not limited to a native ecotin and/or an ecotin variant of this invention or a pharmaceutical composition thereof.
  • the various protease modulators may be provided in separate containers for individual administration or for combination before administration. Alternatively the various compositions may be provided in a single container.
  • the kits may also include various devices, buffers, assay reagents and the like for practice of the methods of this invention.
  • the kits may contain instructional materials teaching the use of the ecotin or ecotin variant in the various methods of this invention (e.g., in the modulation of one or more serine proteases, in the prophylaxis and/or treatment of diseases, and the like).
  • kits for performing affinity chromatography to isolate one or more polypeptides having a chymotrypsin fold e.g. serine proteases.
  • the kits include, but are not limited to a container containing an affinity matrix that is a solid support (surface) (e.g. a resin or glass particle, a membrane, a surface of a slide or other solid object, a gel, a porous or non-porous bead, a magnetic particle, a surface bearing one or more channels or microchannels or capillaries, etc. ) having attached thereto one or more ecotin variants of this invention.
  • the affinity matrix can be further package (e.g. in a column or other flow-through device) to facilitate ease of use.
  • kits may also include various devices, buffers, labels, assay reagents and the like for practice of the affinity chromatography or other "immunoassay" methods of this invention.
  • the kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
  • Example 1 Ecotin, A Serine Protease Inhibitor with Two Distinct and Interacting Binding Sites
  • Serine protease inhibitors are one of the most diverse families of macromolecules that achieve similar biological functions with entirely different scaffolds. They have been studied extensively due to their ubiquitous presence in numerous biological functions. The rich mechanistic and structural database available on the interactions between serine protease inhibitors and their target enzymes make these inhibitors an excellent model system to investigate the fundamental biochemical and biophysical principles of protein-protein recognition. Serine protease inhibitors have been classified into at least twenty sub-families based on amino acid sequence and mechanism of interaction (Laskowski and Kato (1980) Annu. Rev. Biochem., 49(593): 593-626). Four common reaction mechanisms have been postulated to describe the different chemical and kinetic pathways of the inhibition.
  • the substrate-like standard mechanism accounts for the inhibition by a large number of canonical small macromolecular inhibitors with less than 200 amino acids.
  • a classic example of this type of inhibition is that achieved by the binding of Bovine Pancreatic Trypsin Inhibitor (BPTI) to trypsin.
  • BPTI Bovine Pancreatic Trypsin Inhibitor
  • the PI f residue of the inhibitor occupies the SI binding pocket of the protease and the amino acid residues flanking PI bind to the enzyme in a substrate-like conformation.
  • Serpins a family of serine protease inhibitors consisting of plasma proteins of more than 400 amino acids, inhibit their target enzymes through a loop insertion followed by a conformational change (Bode and Huber (1992) Eur. J. Biochem., 204(2): 433-451; Engh et al. (1995) Trends Biotechnol 13(12): 503-510).
  • the other two serine protease inhibition mechanisms are more dependent on steric occlusion of the enzyme substrate binding pocket by the inhibitor.
  • the non-specific "molecular trap" inhibition by ⁇ 2-macroglobulin involves breaking a thiol ester bond and "engulfe ent" of the target protease (Ba ⁇ ett et al. (1981) Meth. Enzymol, 80 Pt C, 737:754).
  • the inhibition of thrombin by hirudin involves an extended binding site that bridges the active site nucleophile of the enzyme and blocks it from productive substrate binding.
  • a common structural theme among these reaction mechanisms is that the protease and the inhibitor form a one-to-one complex.
  • the majority of these inhibitors bind to the target protease at a single site.
  • This binding site is usually well-defined structurally and confined to a specific surface of the inhibitor.
  • an extended surface loop of the inhibitor binds to the active site of the protease with a substrate-like conformation.
  • This so-called "reactive site loop” provides all the binding interactions between the inhibitor and protease.
  • the rest of the inhibitor plays the structural role of maintaining the proper conformation of the reactive site loop.
  • Ecotin a serine protease inhibitor found in the periplasm of Escherichia coli, is a noticeable exception to the examples outlined above.
  • Ecotin is a competitive inhibitor that strongly inhibits trypsin, chymotrypsin and elastase and many other serine proteases with comparable potencies (Chung et al (1983) 258(18): 11032-11038).
  • the inhibitor was purified and its reactive site was determined to be Met 84 which lies within a disulfide bonded protein segment (McGrath et al (1991) J. Biol. Chem., 266(10): 6620-6625).
  • the gene encoding ecotin was cloned and expressed recombinantly in E.
  • the ecotin dimer binds to two trypsin molecules at opposite ends to form a heterotetramer with a two-fold symmetry axis.
  • the crystal structure also reveals a network of interactions between ecotin and trypsin.
  • the protein-protein interaction surface between the inhibitor and the protease consists of two distinct areas, each provided by one of the two ecotin molecules.
  • the first area known as the "primary binding site" involves the reactive site loop of ecotin, i.e. the 80s loop (residues 81-86), the 50s loop(residues 52-54), and the active site of trypsin.
  • the second area known as the "secondary binding site" 25 A away ( ⁇ 3-5 A depending upon the ligand), includes two surface loops of ecotin, the 60s loop(residues 66-70) and 100s loop(residues 108-113), and the C-terminal region of the protease (including part of the C-terminal helix and part of the 90s loop of trypsin).
  • the heterotetramer formed by two ecotin monomers and two trypsins is unique among the known structures of serine protease and inhibitor complexes. From a mechanistic point of view, ecotin's mode of inhibition against its target protease shows novel characteristics in addition to a substrate-like mechanism.
  • ecotin's primary binding site loop and trypsin's active site follows the classic model.
  • the conformation of the 80s loops is superimposable on that of the reactive site loop of BPTI and other canonical macromolecular inhibitors.
  • the inhibitor displays competitive inhibition kinetics and biochemical characterization identified Met84 as the PI residue with direct evidence of bond cleavage by trypsin, chymotrypsin or elastase between Met84 and Met85.
  • the distinct secondary binding site observed in ecotin has not been observed in protease-inhibitor complexes.
  • the E. coli strain JM101, XL-1 Blue F and the VCSM13 helper phage were from Stratagene (La Jolla, CA).
  • the E. coli ecotin gene deletion strain IM ⁇ ecoJ was derived from JM101. Enzymes and reagents for the manipulation of DNA were purchased from Promega(Madison, WI) or New England Biolabs (Beverly, MA) and were used following the manufacturer's instructions.
  • Low molecular weight uPA (LMuPA) was obtained from American Diagnostica (Greenwich, CT).
  • Bovine trypsin was from Sigma (St. Louis, MO). Rat trypsin was expressed in E.
  • the chromogenic substrate Z- ⁇ -Glu( ⁇ -t-butoxy)- Gly-Arg-p-nitroanilide (Spectrozyme UK) used for LMuPA kinetic analysis was from American Diagnostica (Greenwich, CT). 4-Methylumbelliferyl j-guanidinobenzoate was from Sigma (St. Louis, MO).
  • DNA oligonucleotides were synthesized on a Perkin Elmer/ Applied Biosystems 391 DNA synthesizer (Foster City, CA) by using the phosphoramidite method and reagents from the same company. Synthetic oligonucleotides were purified with NENSORB cartridge from DuPont NEN (Boston, MA).
  • DNA sequencing was performed with a Sequenase 2.0 kit from U.S. Biochemical Corp. (Cleveland, OH). ⁇ - 35 S-dATP was from DuPont NEN (Boston, MA). Geneclean® was from Bio 101, Inc (La Jolla, CA). Spectra/Por® molecularporous dialysis membrane was from Spectrum (Laguna Hills, CA). Amicon Centriprep-10 and Centricon-10 were from Amicon (Beverly, MA). All other chemicals were of reagent grade or better and were used without further purification.
  • Ecotin WT ⁇ (133-142): The ecotin-ecotin interface was determined by using the program Insightll (Dayringer et al. (1986) Mol. Graph., 5: 82-87) to search for all ecotin residues in one monomer that were within 4.2 A of the other monomer .
  • the ecotin-ecotin dimer interface buries 3000 A 2 of surface area as calculated with the program Access using a 1.4 A probe size.
  • Ecotin 5OA3+8OA5 and Ecotin 6OA4+I OA 4 Residues in the ecotin-trypsin interface were determined from the 2.4 A X-ray structure of ecotin-D102N trypsin (McGrath et al. (1994) EMBO J, 13(7): 1502-1507) using Insightll by finding all residues from one molecule within 4.2 A of the other molecule and vice versa. Area buried was computed according to Lee and Richards as implemented by the program Access using a probe size of 1.4 A Lee and Richards (1971) J. Mol. Biol, 55(3): 379-400).
  • Candidate residues for mutation to alanine were all ecotin residues whose solvent accessible surface decreased 50% or more upon binding to trypsin in either the primary binding site (ecotin 5OA3+8OA5) or secondary binding site (ecotin 6OA 4 +IOOA 4 ) with the exception of Gly66 which had an unusual phi psi angle such that its changing to another residue would destroy the secondary structure. This resulted in 8 residues in the primary site which were changed to alanine. This group of residues is comprised of Leu52, His53, Arg54, Val81, Ser82, Thr83, Met84, Met85, and Ala86 (left unchanged). The resulting construct was designated ecotin 5OA3+8OA5.
  • Primer M84R 5*-GT TCC CCG GTT ACT ACT AGG ATG GCC TGC C-3' (SEQ ID NO. 2) (with a unique Seal site);
  • Primer M84K 5'-GT TCC CCG GTT ACT ACT AAG ATG GCC TGC C-3' (SEQ ID NO.3);
  • Primer M84F 5'-GT TCC CCG GTT AGT ACT TTC ATG GCC TGC C-3' (SEQ ID NO. 4);-and
  • Primer M84W 5'-GT TCC CCG GTT ACT ACT TGG ATG GCC TGC C-3' (SEQ ID NO. 5). Plasmids for the production of the C-terminal deletion variants WT ⁇ and M84R ⁇ pBS Ecotin and pBS Ecotin M84R were used as templates in a PCR reaction with oligonucleotides EcoN and EcoC to generate C-terminal deletion variants at residues 133-142. The amplified region was digested with BamHI and Hindlll, purified and ligated to the BamHI/Hindlll fragment of pTacTacEcotin . The PCR condition used were: 94°C for 1 min, 40°C for 2 min, 72°C for 3 min, 35 cycles. The sequences of the two primers were:
  • Primer EcoN 5'-AAATTAACCCTCACTAAAGGG-3' (SEQ ID NO. 6); and 2) Primer EcoC: 5'-TTGTCAATTTAAGCTTACGCCTTCCAGACGCGG-3' (SEQ ID NO. 7).
  • Plasmids for the production of multiple alanine substitutions at the secondary binding site Plasmids for the production of multiple alanine substitutions at the secondary binding site.
  • the DNA in pBS Ecotin was changed to code for alanines at positions 52-54, 67-70, 81-85, 108,110, 112-113 by site-directed mutagenesis using oligonucleotides 5OA3,
  • Primer 50A 3 5'-CTG GAA GTC GAT TGC AAT GCG GCT GCC TTG GGC GGG AAG CTG GAA AAC-3' (SEQ ID NO. 8) (unique Styl site);
  • Primer 60A4 5'-aac aaa acg ctg gaa ggg gcc gcc gcg gcc tat tat gtc ttt gat aaa gtc-3' (SEQ ID NO. 9) (unique Sfil site); and
  • Primer 80A5 5'- AAA GTC AGT TCC CCG GCT GC A GCG GCG GCG GC A TGC CCG GAT GGC AAG-3' (SEQ ID NO. 10) (unique Sphl site); 4) Primer 100A4: 5'-GC GAT GCT GGA ATG CTG GCT TAC GCT AGC GCG GCG CCG ATC GTG GTG TAT AC-3' (SEQ ID NO. 11) (unique Nhel site).
  • IM ⁇ ecoJ cells were transformed with pTacTac Ecotin and transformants were selected by plating on LB/ampicillin plates. A single colony was used to inoculate 3 ml of LB containing 60 ⁇ g/ml ampicillin. The cultures were grown at 37°C for 9 hours and diluted to 1 liter of LB/ampicillin. Following growth at 37°C for 1 hour, IPTG was added to the cultures to a final concentration of 0.2 mM, and continued to grow for 12 hours at 37°C. Cells were harvested and treated with lysozyme in a solution containing 25% sucrose/10 mM Tris, pH 8.0.
  • the periplasmic fraction was dialyzed against 10 mM Tris, pH 8.0 with a Spectra/Por molecularporous dialysis membrane of 12-14 K molecular weight cut-off. Following dialysis, the supernatant was acidified to pH 2.8 by addition of 1M HC1 to the sample. The sample was incubated on ice for 30 minutes and the acid-insoluble material was removed by centrifugation. The supernatant was neutralized to pH 7.4 with 1 M Tris, pH 8.0, and adjusted to a NaCl concentration of 0.3 M. The solution was heated at 100°C for 10 minutes, and then cooled to room temperature.
  • the precipitate was removed by centrifugation, and the supernatant was dialyzed against deionized distilled water overnight at 4°C.
  • the ecotin sample was concentrated with a 10 K molecular weight cut-off concentrator Amicon Centriprep-10).
  • the concentrated sample was loaded onto a Vydac C4 reverse-phase high performance liquid chromatography column (2.2 x 25 cm) that had been equilibrated with 0.1 % trifluoroacetic acid.
  • the column was washed and ecotin was eluted with a linear gradient of 34-37% acetonitrile/0.1 % trifluoroacetic acid at a flow rate of 10 ml/minute over 30 minutes.
  • Rat and bovine trypsin activity assays were using the same substrate and procedure. Trypsin was titrated with 4-methylumbelliferyl .-guanidinobenzoate to obtain an accurate concentration of the enzyme's active sites. Various concentrations of ecotin or ecotin variants were incubated with trypsin in a total volume of 990 ⁇ l of buffer containing 50 mM NaCl/50 mM Tris/10 mM CaCl 2) pH 8.0. Following a 10-minute equilibration at room temperature, 10 ⁇ l of 2.5 mM Z-GPR-AMC substrate was added and the rate of substrate hydrolysis was measured by monitoring the change of emission at 460 nm in a 2- minute period at 25°C.
  • LMuPA assays were performed as described (Wang et al. (1995) J Biol. Chem., 270(20): 12250-12256). The assays were repeated five to seven times under different inhibitor concentrations. All the data were fit to the equation derived for kinetics of reversible tight-binding inhibitors (Morrison et al. (1969) Biochim. Biophys. Acta, 185(2): 269-286) by nonlinear regression analysis using the program Kaleidagraph. The values for apparent K, and true K , as well as the standard deviations of the K, were determined. The equation is:
  • VJ V 0 is the ratio of the inhibited rate vs. the uninhibited rate
  • [E 0 ] is the total enzyme concentration
  • [I 0 ] is the total inhibitor concentration.
  • the [E 0 ] ranges from 50 pM to 500 pM for rat and bovine trypsin, 1 to 2 nM for LMuPA.
  • PI is the substrate (or inhibitor) residue before the scissile bond, where PI -PI' is the scissile bond, SI, S2, etc. are the co ⁇ esponding binding subsites on the enzyme.
  • ecotin variants with amino acid substitutions and deletions at the primary or secondary binding site were generated by site-directed mutagenesis (Kunkel, et al. (1985) Proc. Natl. Acad. Sci. USA, 82(2): 488-492), expressed to high-levels in E. coli and subsequently purified to homogeneity. The final yield ranged from 20 to 100 mg protein per liter of liquid culture.
  • the same expression system and purification protocol using heat treatment and reverse phase HPLC steps were applied to both primary and secondary site ecotin variants.
  • Activity assays were performed at each purification step to monitor for activity loss of the samples. All variants were expressed in E. coli at comparable levels and were stable under the purification conditions.
  • Table 1 is a summary of the nomenclature of ecotin variants that appear in this study.
  • the SDS-P AGE analysis of purified aliquots of ecotin variants are shown in Figures 2(a)and 2(b).
  • Ecotin WT and six variants at the PI position and dimer interface (WT, M84R, WT ⁇ , M84R ⁇ , M84K, M84F, M84W) are shown in Figure 2(a), while Figure 2(b) shows ecotin WT and seven variants at the secondary site loops (WT, 6OA4, IOOA 4 , 6OA 4 +IOOA 4 , 5OA3+8OA5, M84R+60A 4 , M84R+IOOA 4 , M84R+6OA4+IOOA 4 ).
  • the C-terminal dimer interface between the two ecotin monomers is a key structural element of the tetrameric complex as it forms one of the three types of protein- protein interfaces in the tetramer.
  • the other two contact regions are the primary and secondary binding sites that interact with the protease.
  • the dimerization of ecotin not only dramatically increases the contact region between the inhibitor and the protease, but also adapts to maintain the proper orientation of the two ecotins as a molecular "hinge" to permit binding to different proteases which have a chymotrypsin fold structure.
  • the combined surface areas of the primary and secondary sites of ecotin that become buried upon binding to trypsin exceed 2800 A 2 .
  • This area is substantially greater than most of the other protease- inhibitor interfaces (e.g. BPTI-trypsin interface is only 1390 A 2 ). If the hinge region is disrupted or destroyed, the relative positions of the primary and secondary binding sites may change since each binding site results from different surfaces of the two contralateral ecotin monomers ( Figure 1).
  • Figure 1 We investigated the role of the dimer interface on ecotin's unique broad specificities by truncating the C-termini of the ecotin monomer. Based on the crystal structure of the ecotin-trypsin complex, residues 133-
  • ecotin WT ⁇ The ecotin variants with residues 133-142 truncated was denoted ecotin WT ⁇ .
  • the Kjs of ecotin WT and several truncation variants are shown in Table 3.
  • the Kj of ecotin WT was close to the value of 390 ⁇ 150 nM measured previously (Seymour et al. (1994) supra.).
  • ecotin WT ⁇ and M84R ⁇ had K d values of only 2.5 and 3.7-fold higher respectively, suggesting that the truncation variants of ecotin still formed dimers, even though their dimer interactions were weaker, as indicated by the modest increase of the Kj's.
  • replacement of Met at position 84 with an Arg also increased the K slightly relative to WT ecotin (1.7-fold).
  • the subtle change at one surface loop is conveyed to the C-terminal of the same molecule, implying ecotin has extensive structural flexibility.
  • ecotin M84R ⁇ not only regained nanomolar potency against rat trypsin, but also became an even better inhibitor of this enzyme (90 pM vs. 930 pM for WT). This result suggests that the favorable electrostatic interaction at the PI position could compensate for the weakened interactions due to the C-terminal truncation.
  • Ki data of uPA inhibition revealed yet another pattern. In this case, the M84R substitution only partially compensated for the unfavorable interactions created by the C-terminal deletion.
  • the Ki of the double variant, ecotin M84R ⁇ against uPA remained at micromolar levels (2.7 ⁇ M).
  • the primary site group includes residues 81-86 and 52-54 located on two surface loops (50s and 80s loops).
  • the secondary site group consists of residues 67-70, 108, 110, 112, and 113, also from two surface loops (60s and 100s loops).
  • Auncomplex Solvent accessible surface area in uncomplexed ecotin dimer
  • Acomplex Solvent accessible surface area in ecotin-trypsin tetrameric complex
  • Hydrogen Bond Distance Putative hydrogen bind distance with closest side chain or main chain atoms in trypsin.
  • Variant 6OA4+IOOA4 also has eight alanine substitutions at residues 67-70, 108, 110, 112 and 113. These two multiple alanine substitution variants were investigated to define the roles of the primary and secondary binding sites of ecotin. Their inhibition constants against bovine and rat trypsins are shown in Table 6.
  • the Kj of ecotin 5OA3+8OA5 against rat trypsin increased 30,000-fold compared to that of WT ecotin, from 0.93 nM to 27.9 ⁇ M; its Kj against bovine trypsin also increased significantly (about 300-fold).
  • These results confirmed the essential role of the reactive site loop as a major determinant of the strength of protease inhibition.
  • ecotin 60A 4 +100A4's Kj for bovine trypsin increased slightly (about 5-fold) compared to that of the WT ecotin.
  • the remarkable potency of this variant demonstrated that ecotin remains competent as an inhibitor even after a significant part of the molecule was replaced with alanines.
  • ecotin 6OA4+IOOA 4 The dramatic contrast of the inhibition of ecotin 6OA4+IOOA 4 for bovine and rat trypsin highlighted the importance of the 60s and 100s loops for different proteases. To ensure that the loss of inhibition was not due to its intrinsic destablization, ecotin
  • 6OA4+IOOA 4 was incubated with rat and bovine trypsin at pH 5.2 and 8.0, 37°C for up to 16 hours at 1 to 600 enzyme : inhibitor concentration.
  • the reaction mixture was analyzed by SDS-P AGE (data not shown).
  • the inhibitor was stable to proteolysis for the length of time for the Ki measurement (20 minutes).
  • partial proteolysis of ecotin is eventually seen by rat trypsin.
  • ecotin 5OA3+8OA5 27.9 ⁇ M vs. rat trypsin and 98.3 nM for bovine trypsin
  • ecotin WT is table to proteolysis by both enzymes at both conditions. Therefore the results of the over digestion assays are consistent with the results of the Kj measurement.
  • the Role of the 60s Loop and 100s Loop As shown in the case of rat ⁇ , the secondary binding site in ecotin plays a major role in the inhibition of particular serine proteases.
  • the substitution of the eight residues in the 60s and 100s loops was sufficient to weaken the binding interactions between ecotin and one target protease by at least four orders of magnitude. Since these residues are located at two discontinuous regions of the surface loops, their contributions can be further dissected and analyzed.
  • 60 A4 multiple alanine substitutions at positions 67-70.
  • IOOA4 multiple alanine substitutions at positions 108, 110, 112, 113.
  • 6OA4+IOOA4 multiple alanine substitutions at positions 67-70, 108, 110, 112, 113.
  • Figures 4(a)and 4(b) and 4(c) provide an overview of the interactions among ecotin's two surface loops and various proteases.
  • the Kj plots were used both horizontally and vertically to evaluate either the impact of specific surface loops on different enzymes, or the inhibition of a particular enzyme by different variants. Similar to the results from the previous experiment, the inhibition of bovine trypsin by ecotin was not affected by drastic changes to the inhibitor. The K s of all variants were approximately 1 nM. Ecotin IOOA4 bound even slightly tighter than ecotin M84R (40 pM vs. 90 pM). Thus, the only dominant factor for bovine trypsin binding was the proper conformation of the primary site reactive loop.
  • the Kj plot of rat trypsin indicated yet another pattern of secondary site inhibition.
  • the 60s loop was responsible for providing most of the binding energy as indicated by the over 4,000-fold increase of Kj with ecotin 6OA4, while the effect of the 100s loop was minimal.
  • the 60s loop played a more predominant role in determining the strength of the inhibition at the secondary site.
  • the Kj data set of the three groups of ecotin variants allow us to analyze the inte ⁇ elationships among the key determinants of ecotin's potency and specificity. In particular, we determined whether the separate mutations are additive by comparing the K t values of ecotin 6OA4+IOOA 4 versus those of ecotin 60 A 4 or ecotin IOOA 4 against one particular protease. In the case of rat trypsin inhibition, the ecotin variant with alanine- substitutions at two loops bound more weakly than either of the two single alanine- substituted loop variants. For uPA binding, the 60s loop had a more significant effect. The Ki of ecotin 6OA4 against uPA was even higher than the Ki of the double mutant ecotin 6OA4+IOOA 4 . It is clear that the contributions of these two loops were not independent of each other.
  • the K, plot was effective in directly comparing the strength of inhibition by ecotin variants and illustrating the relationships between the PI mutation and the secondary site multiple alanine substitutions (or C-terminal truncation).
  • additivity as the absence of interaction energy term ⁇ Gi' in:
  • ⁇ G (A ,B)' ⁇ G° (A ) + ⁇ G° (B ) + ⁇ G ⁇ ° (1)
  • the perturbation of the dimer interface changed the positions and/or conformations of the secondary binding site relative to other parts of the molecule. Therefore, if rat trypsin (or uPA) required the secondary site for tight binding, and if the C- terminal truncation dislocated the secondary binding site, the interactions between ecotin WT ⁇ and rat trypsin (or uPA ) would be severely weakened. In the network of interactions between the protease and ecotin, ecotin's C-terminal hinge region may affect the binding by modulating the relative contributions from the primary and secondary binding sites. The dramatic increase of T,- values of ecotin WT ⁇ against rat trypsin and uPA offered indirect evidence of the critical roles of the secondary site surface loops.
  • the interaction of the protease with the secondary site of ecotin requires the formation of a stable tetrameric complex.
  • K d of ecotin's monomer-dimer equilibrium is much higher than Kj values against most target proteases, the K of ecotin dimerization could be dramatically perturbed in the presence of proteases.
  • Proteases may serve as templates to facilitate the dimerization of ecotin. If there are strong cooperative interactions upon the binding of the second inhibitor and the second enzyme molecule, the reaction pathway may proceed from I -> El -> EI2 -> E2I2, where the last two steps are rapid and drive the tetrameric complex formation to completion. Analytical centrifugation has been used to pursue this question.
  • Ecotin offers unique opportunities to study the complex network of interactions between serine proteases and bi-dentate macromolecular inhibitors. It is also an ideal scaffold to design and engineer protease inhibitors. Potency and specificity toward target serine proteases may be introduced through the secondary binding site, a special structural feature of ecotin that does not resemble any previously known binding motifs within the families of macromolecular serine protease inhibitors.
  • Streptomyces subtilisin inhibitor which uses a substrate-like competitive inhibition mechanism, forms a tetramer with subtilisin, each subtilisin is bound to one SSI in a "chain-like" configuration (Takeuchi et al. (1991) J. Mol.
  • This unique bi-dentate binding mode has two advantages for macromolecular recognition for proteases with a chymotrypsin fold structure. First, it creates a buried surface area that is nearly 50 percent larger than the hirudin/rhodniin/ornithodorin-thrombin interface (about 1900 A 2 ), allowing a large variety of interdependent factors to contribute to the formation of the ecotin-protease complex.
  • the macromolecular recognition is very sensitive to the specific residues located at the binding interface.
  • the tetrameric network of interactions creates greater flexibility to modulate the strength of inhibition by introducing new controlling elements such as the "hinge" region at the dimer interface.
  • ecotin-protease interaction is reminiscent of the antibody- anti gen interaction, in which the hyper-variable regions of the six CDR loops of immunoglobin provide all the possible surface landscapes to recognize any given antigen through an astronomical number of combinatorial side chain conformations.
  • Ecotin's four surface loops, the 50s, 60s, 80s, and 100s loops, have great potential to be tailored to provide a complementary fit with different protease surfaces that are in direct contact with both the primary and secondary binding site.
  • the dramatic K differences of ecotin variants containing multiple alanine substitutions at the secondary site against bovine trypsin, rat trypsin, and uPA highlight the novel structural and functional features of ecotin.
  • Rat and bovine trypsins are closely related enzymes with the same substrate specificity. Their crystal structures are very similar. Although rat trypsin is anionic, while bovine trypsin is cationic, it is still not clear whether the electrostatic interaction is solely responsible for the selective inhibition of rat trypsin by the ecotin 60s loop variants. Other crucial structural factors may contribute to the binding energy of complex formation such as hydrophobic packing of the interface, or the flexibility and rigidity of the surface loops. For example, structural studies suggest that the relative position of the two ecotin monomers can shift upon binding to rat trypsin when binding determinants in the 60's loop are removed (unpublished results).
  • the primary and secondary sites are adapted to the surface features by their intrinsic flexibility and by the C- terminal hinge which permits relative adjustments between the primary and secondary sites.
  • the discriminating power of the individual surface loops at the secondary site of ecotin suggests a novel opportunity to exploit the subtle difference among proteolytic enzymes with identical primary substrate specificity and to design selective and potent macromolecular inhibitors against these enzymes.
  • Example 2 Engineering Bidentate Macromolecular Inhibitors for Trypsin and Urokinase-tvpe Plasminogen Activator
  • Enzymes and reagents for the manipulation of DNA were purchased from Promega (Madison, WI) or New England Biolabs (Beverly, MA) and were used following the manufacturer's instructions.
  • the E. coli strain JM101, XL-1 Blue F' and the VCSM13 helper phage were from Stratagene (La Jolla, CA).
  • the E. coli ecotin gene deletion strain IM ⁇ ecoJ was derived from JM101.
  • Low molecular weight uPA (LMuPA) was obtained from American Diagnostica (Greenwich, CT). Rat trypsin was expressed in E.
  • Mutagenesis was performed by the method of Kunkel [Kunkel, 1985 #10] . All mutations have been confirmed at the DNA level by sequencing.
  • the vector pBS eco- glll was used to construct phage libraries 6OX4 and M84R+6OX4.
  • a deletion and frameshift mutation was introduced at residues 67-70 of ecotin by primer 5'-C AAA ACG CTG GAA GG TAT TAT GTC TTT GAT-3' (SEQ ID NO. 12) to make pBS eco-gIII ⁇ 60.
  • the ecotin phage display vector pBSeco-glll and expression vector pTacTacEcotin were mutated to carry an Aatll site by primer 5'-CA GAC AAT GTA GAC GTC AAG TAC CGC GTC-3' (SEQ ID NO.
  • ecotin variants obtained from panning experiments could be directly cloned into the expression vector pTacTacEcotin.
  • the mutagenesis reaction mixture was purified by Geneclean, redissolved in water, electroporated into F' XL-1 Blue cells, and grow in 100 ml of 2YT/Ampicillin for 1 to 2 hours. The culture was then divided into two portions. One portion was transfe ⁇ ed into fresh 2YT/Ampicillin medium and grown for 8-12 hours. The cells were harvested by centrifugation and the double strand plasmid was prepared using a Promega Midiprep Kit.
  • This DNA sample was the ecotin phage library stock.
  • This infected culture was grown for 6 hours at 37°C with rigorous shaking, and the phage were harvested as described in the section of ecotin phage preparation below.
  • plasmid DNAs were transformed into a male strain (F 1 ) of IM ⁇ ecoJ.
  • F 1 male strain
  • a single colony selected on ampicillin plates was grown in 3 ml 2YT medium containing 60 ⁇ g/ml ampicillin at 37°C for 8 hours.
  • the infected culture was allowed to grow at 37°C with shaking for approximately 6 hours.
  • Phage particles were harvested by precipitation with one fifth volume of 20 % polyethylene glycol 8000, 2.5 M NaCl at 4°C overnight, centrifugation at 6000 g for 40 min, and resuspended in 1 ml TE buffer. Phage stocks were stored at 4°C for up to six months. Phage titers typically ranged from lO ⁇ to lO ⁇ cfu/ ⁇ l culture and were stable within six months.
  • Polystyrene petri dishes 35 mm, Falcon 1008 were coated with 1 ml of 10 ⁇ g/ml bovine trypsin, or rat trypsin, or LMuPA in PBS (137 mM NaCl/2.7 mM KC1/10 mM Na 2 HPO 4 /1.8 mM K 2 HPO pH 7.5) overnight at 4°C, and excess binding sites were blocked with 5%. non-fat dry milk PBS solution for 2 hours. Phage were added to the dishes in buffer containing 1 ml PBS/0.5% Tween 20 and were incubated for 2 to 24 hours with gentle agitation at 4°C.
  • Equal volumes of three eluates were pooled for subsequent amplification and characterization. Two amplification protocols were used in this experiment.
  • 900 ⁇ l of phage elution pool was incubated with 9 ml of fresh grown IM ⁇ ecoJ lawn cells for 30 min at 37°C with gentle shaking.
  • the infected culture was grown for 6 to 10 hours at 37°C with shaking, then harvested and precipitated as described above.
  • the infected culture was grown for 6 hours at 37°C with shaking, and the phage were harvested as described above.
  • the ecotin phage display vector pBSeco-glll expresses the fusion protein of full length ecotin connected to the C-terminal domain of filamentous phage minor coat protein pill via a GlyGlyGly linker.
  • the ecotin- pill fusion protein is assembled onto phage particles. Phage carrying this fusion protein has ecotin activity and can bind to the immobilized protease on the solid surface.
  • the three enzymes, bovine trypsin, rat trypsin, and uPA were coated onto polystyrene petri dishes and remained active as monitored by -nitroanilide release of Z-GPR-jo -Na substrate after 30 min incubation at room temperature (25°C).
  • Two different ecotin libraries were designed, taking advantage of three- dimensional structure information to randomize key residues thereby permitting isolation of the optimal cognate inhibitor for a target protease.
  • the first library, 6OX4 had four residues randomized at positions 67-70 of the 60s loop. This library was used to pan against bovine trypsin and rat trypsin separately.
  • the second library, ecotin M84R+6OX4, combined a favorable PI Arg residue with the randomized 60s loop.
  • This library was designed to encode ecotin variants that inhibit uPA with high potency.
  • the Ki of ecotin M84R+60A4 against uPA is 1470 nM, several hundred fold higher than the K, of ecotin M84R, suggesting that four amino acid substitutions 25 A away from the active site were sufficient to induce a dramatic change in the strength of the interaction.
  • the ecotin 6OX4 library contained approximately 5 million individual clones; the ecotin M84R+60X4 library contained approximately 50 million individual clones. The completeness of the library was calculated using the equation:
  • N ln(l-p) / ln[l-(l/n)] (1)
  • N is the number of total individual clones in the library
  • n is the number of possible combinations
  • p is the probability that any clone can be found in the library given library size N.
  • the sizes of the final libraries indicated that these libraries were well over 99% complete in representing all the possible four amino acid sequence combinations at positions 67-70. Both libraries were characterized for completeness using the same procedure. Random individual clones were isolated from the libraries. Their plasmid DNA and phage were purified using standard procedures. A BarnHI/Hindlll restriction digest of the sample plasmid DNA was used to monitor the total size of the ecotin phage display vector and the size of the DNA fragment containing the ecotin-glll fusion.
  • a typical round of ecotin phage library panning with significant enrichment would yield 10° " to 10 ⁇ phage from the elution, given that the input phage was in the range of 10 0 to l ⁇ l 1.
  • an elevated recovery usually suggested an increase of positive clones in the pool of panning intermediates, other factors that were not directly related to the in vitro binding between ecotin and the immobilized protease might also lead to an artificially high recovery.
  • the ecotin 6OX4 library was panned against two serine proteases, bovine and rat trypsin, which were coated onto separate polystyrene plates. After the first round of panning, the elution fraction from the two plates were pooled and amplified separately on multiple LB/ampicillin plates to generate two intermediate libraries for the next round of panning. The two panning experiments were then carried out in parallel for the subsequent rounds. Careful precautions were taken to avoid cross-contamination between the bovine trypsin and rat trypsin panning. Four rounds of panning were completed before the final sequencing of the individual clones.
  • the kinetic data shows that for both trypsins, ecotin Y69F+D70P and ecotin Y69L+D70P bound tighter than WT ecotin.
  • Ecotin Y69F+D70P was the stronger binder with a lower Kj (bovine trypsin 30 pM; rat trypsin 80 pM).
  • Kj bovine trypsin 30 pM
  • rat trypsin 80 pM The similar increase in affinity by ecotin Y69F+D70P for both trypsins suggested a general optimization of the hydrophobic packing at the secondary binding site.
  • ecotin Y69F+D70P bound better (670 pM) than WT (2800 pM) and ecotin Y69L+D70P bound worse (20200 pM), implying that the inhibition against uPA was extremely sensitive to changes at the 60s loop and that the uPA-ecotin interface differed from the trypsin-ecotin interface at the secondary site.
  • high affinity ecotin variants modified at the secondary site can be selected from a phage display library. Table 10. Kj of ecotin variants from library ⁇ O j.
  • the plate amplification protocol was further refined to eliminate the nutrient selection pressure for phage growth in liquid culture.
  • a "nursing protocol” was developed to ensure that the small fraction of positive clones in the initial input library are not lost during amplification. This protocol used plate amplification to minimize the selective pressure for nutrients and short incubation time to limit growth, preventing certain clones from dominating the pool of selected variants.
  • the low density growth on solid media and limited propagation times were essential in the first round of library amplification for subsequent selection of desired variant clones. Specifically, E.
  • the ecotin M84R+60X4 library was panned against rat trypsin and uPA in parallel with intermediate plate amplification steps. Similar to the results from panning the ecotin 6OA4 library, a significant increase of phage recovery was observed in the eluates in each of the four rounds of panning for both ligands. Again, the panning results confirmed our observations from prior mutagenesis experiments. In rat trypsin binding, the dominant role of the electrostatic interaction with Arg84 completely masked the impact of the 60s loop. Thus panning against rat trypsin did not generate a consensus sequence (data not shown), even though in the final round of panning, the phage recovery from the acid elution exceeded 10 ⁇ .
  • nucleotide sequences of the 18 samples from uPA panning showed a mixture of codons encoding the selected amino acids such as Gly (14 GGG and 3 GGC), Pro (3 CCG and 1 CCC) and Arg (5 AGG and 3 CGG), strengthening the conclusion that these residues were selected based on their contributions to increase the affinity towards uPA.
  • Table 11 The consensus sequence from library M84R + 60X* panning against uPA.
  • Ecotin M84R+D70R and ecotin M84R+D70P were constructed by cloning the specific variant sequences from the phage clone intoihe expression vector pTacTacEcotin, taking advantage of a pair of common restriction sites BamHI/ Aatll that flanked 90% of the ecotin gene.
  • the two variants were purified to homogeneity via reverse phase HPLC. Their K's against uPA were determined and are listed in Table 12.
  • the Kj of ecotin M84R+D70R was lower (50 pM) than that of ecotin M84R+D70P (80 pM), mirroring their relative occu ⁇ ence in the consensus sequences.
  • the increased potency and specificity of ecotin M84R+D70R validates the strategy to optimize the affinity of ecotin towards uP A through a stepwise approach.
  • the inhibition of rat trypsin can be used as a benchmark to assess the effectiveness of this methodology.
  • Ecotin M84R+D70R with a Ki against rat trypsin of 220 pM, was a better inhibitor of uPA than rat trypsin. This result was in sharp contrast with the preference of WT ecotin for rat trypsin over uPA by over 3,000-fold.
  • Table 12 summarizes the ecotin Kj S towards rat trypsin and uPA generated through the combination of region-specific mutagenesis and phage display.
  • the specificity of ecotin has been successfully converted from one serine protease to the other with a significant increase in potency at the same time.
  • the overall specificity preference was 13,680-fold.
  • Preference value is calculated by dividing the ratio of Kj ( ⁇ PA/Kj (rat trypsin) of ecotin WT by that of ecotin M84R or M84R + D70R. This value reflects the fold change in preference of the variant ecotin compared to ecotin WT for a given protease.
  • Residue 234 is either Tyr (rat and bovine trypsin) or Phe (uPA); residue 237 is a conserved Tip.
  • the stacking of aromatic rings between ecotin and the protease provides a tightly-packed hydrophobic interface. Due to tryptophan's large side chain volume and buried surface area, substituting Trp67 of ecotin to any other amino acid might create an unfilled cavity that destabilizes the ecotin-protease complex. Thus Trp67 appears to be an integral part of the hydrophobic "core" of the secondary binding site.
  • Gly68 was also selected from both panning experiments. This residue probably plays a more structural role to maintain the proper flexibility and main chain conformation of the 60s loop. Since Trp67 and Gly68 were conserved at the 60s loop, the other two residues, Tyr69 and Asp70 were the only candidates to provide differential recognition towards target proteases.
  • the ecotin consensus sequence WG (F/L) P at positions 67-70 that resulted when the ecotin library was panned against rat trypsin represented an overall improvement of the hydrophobicity at positions 69 and 70. Both Phe and Leu are less apolar than Tyr, which has a hydroxyl group. In addition to its hydrophobicity, pro line was commonly found at various types of turns to lock the surface loops into stable conformations. Pro70 was presumably selected for this reason. The ten-fold increase in affinity of ecotin Y69F+D90P towards both bovine and rat trypsin illustrated the modest improvement of side chain packing in the vicinity of amino acids 69 and 70.
  • Residue 70 of ecotin is close to residue 93 of uPA in the model of the ecotin-uPA complex.
  • residue 93 was an Asp instead of Asn.
  • the consensus sequence Arg70 from the ecotin M84R+60X4 library panning against uPA was indeed an ideal choice to provide a counter charge to stabilize Asp93 by forming a salt bridge between these two side chains.
  • the relative free energy was the ⁇ G(M84R + IOOA ) - ⁇ (M84R)G difference between co ⁇ esponding variants. According to this scale, a 100-fold difference in Ki will be equal to -2.73 kcal/mol.
  • the outcome of the ecotin phage display experiment is summarized in Table 14 for the comparison between the free energy difference and the in vitro selection process.
  • the ⁇ G was - 5.02 kcal mol and a consensus sequence was observed when the ecotin 6OX4 library was panned against rat trypsin.
  • the ⁇ G was - 3.56 kcal/mol and a consensus sequence was observed when the ecotin M84R+60X4 library was panned against uPA.
  • thrombin a key enzyme that cleaves fibrinogen and forms fibrin clots in the blood coagulation pathway, is regulated by heparin, ⁇ 2-macroglobulin, antithrombin III, thrombomodulin (Stubbs et al. 1995 Trends in Biochem. Sci., 20(3): 131; Stubbs et al. (1995) Trends in Biochem. Sc , 20(1): 23-281) and monovalent ions such as
  • thrombin Through several cofactor interactions that are distal to the active site, thrombin achieves a high level of fine tuning and balance between its coagulation and anticoagulation activities in an intertwined web of biological pathways in haemostasis, platelet aggregation, tissue remodeling, mitosis and chemotaxis.
  • the serine protease domain can form a high-affinity complex with several key partners such as PAI-1, PAI-2, protease nexin-1, and ⁇ 2 - macroglobulin receptor (Fazioli et al.
  • Ecotin offers a unique platform to investigate and utilize the contribution from a binding region distal to the primary binding site for protease inhibition.
  • the dimeric macromolecular inhibition has special structural features for innovative methods of inhibitor design and engineering. By modulating the amino acid residues at the 60s loop, another level of control has been achieved in designing the specificity and potency of ecotin variants.
  • the secondary binding site of ecotin not only facilitated the fine-tuning of the molecular recognition towards many known homologous enzymes, but also provided additional side chain conformational flexibility to accommodate other serine proteases with similar scaffolds.
  • the first advantage is the electrostatic and hydrophobic surface diversity available in the contact regions between the ecotin dimer and two protease molecules.
  • the combinatorial approach of phage display makes it feasible and highly efficient to search and sort the large repertoires of ecotin surface loop variants.
  • the crystal structures of ecotin-protease complexes can serve as a framework for designing inhibitors against enzymes with unknown structures.
  • a combination of site-directed mutagenesis and phage display approaches were taken to study the interactions between ecotin and several serine proteases.
  • the secondary binding site of ecotin was shown to play a critical role for certain proteases.
  • Phage display libraries of ecotin variants were then made at these surface loops and used for panning against the target proteases.
  • a protocol was developed that permitted identification of two distinctive consensus sequences from panning the ecotin variant phage libraries with rat trypsin and uPA. In both cases, the consensus sequence encoded ecotin variants with higher affinity for the target protease.
  • This study provided a general strategy to engineer potency and specificity of a macromolecular serine protease inhibitor by modulating various components of the network of extended interactions between the inhibitor and the protease.
  • Preparation #4 used in the experiments described in Figure 9 was derived from the parent human factor IXa (Lot L0430) by adsorption to and subsequent elution from an anti-factor IX immunoaffinity column.
  • the various factor IXa preparations were reconstituted to a final concentration of 2.0 ⁇ M in 0.05 M Tris-0.1 M NaCL,-0.005 M CaCl 2 , pH 7.4 and kept on ice. A portion was taken from each of these solutions and diluted in the same buffer to a concentration of 0.2 ⁇ M for the assay reaction.
  • Ecotin and the M84R variant was obtained as described above.
  • the concentration of ecotin was 15.4 ⁇ M and the M84R variant was 5.6 ⁇ M.
  • Both proteins were in 0.05 M Tris-0.1 M NaCl-0.005 M CaCl 2 , pH 7.4.
  • Example 4 Use of Ecotin Variants to Bind Proteins other than Serine proteases: Purification of haptoglobin.
  • haptoglobin was purified using an ecotin M84R, M85R affinity column.
  • the ecotin variant(s) were coupled to an affigel matrix using standard conditions to produce an Ecotin M84R, M85R affinity column.
  • the column (4 ml) was then equilibrated with phosphate buffered saline (PBS) and then incubated with bovine serum (-35 mL) for 2 h to bind the haptoglobin that is present in the serum.
  • PBS phosphate buffered saline
  • bovine serum -35 mL
  • the column was then washed with PBS (50 mL) and the OD280nm was obtained to be sure that the column was clean of non-retained material.
  • the retained material was then eluted using 50 mM glycine, 150 mM NaCl, pH 3.0 (-30 mL) and was immediaately neutralized using 1 M Tris base (10 ⁇ L/1 mL of retained material). The OD 280 nm was monitored continuously to permit collection of the ecotin bound protein.
  • the retained material represented haptoglobin that was visualized by Coomassie brilliant blue staining.
  • Haptoglobin run as an approximately 83 kD band that is recognized by monoclonal antibodies raised against haptoglobin.
  • the material was dialized and concentrated from the ecotin affinity column and then applied to a small Mono-Q column (buffer A-20 mM Tris, buffer B - Buffer A + 1 M NaCl, gradient 0-1 M in -20 min) and the haptoblobin was eluted in approximately 35%- 38% NaCl.
  • the N terminal 10 amino acids of the purified haptoglobin were sequenced to verify its identity.
  • two unidentified proteases were also retained by the ecotin column. The proteolytic activity of these proteases was observed by running the samples eluted from the ecotin affinity column on an activity gel containing gelatin.
  • Example 5 Modification of carboxyl or amino termini.
  • the carboxyl and amino termini of ecotin or ecotin variants of this invention can be modified by insertions or deletions of one or more amino acids.
  • a 17 megadalton bacteriophage particle can be added to the c- terminus of ecotin without affecting its function.
  • adding a (His) 6 tag at the C-terminus does not affect its function.
  • deletions are made at the amino and at the carboxyl terminus without affecting the ecotin activity.

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Abstract

L'invention concerne des protéines de liaison de classe qui se lient spécifiquement à des polypeptides pourvus d'un pli de chymotrypsine (par exemple des sérines protéases). Lorsque les polypeptides cibles sont des sérines protéases, les protéines de liaison peuvent inhiber ou activer la sérine protéase. Les protéines de liaison sont basées sur la structure de l'écotine. Une modification de la terminaison amino ou carboxyle et/ou la randomisation d'au moins une boucle 50s, 60s, 80s ou 100s produiront une banque de variantes de l'écotine à partir de laquelle on peut sélectionner des molécules de liaison (par exemple des modulateurs de protéase) propres à presque toutes les sérines protéases. Selon la variante de l'écotine et la sérine protéase cible, le modulateur peut agir comme inhibiteur ou activateur de la sérine protéase. L'invention traite en outre d'agonistes spécifiques du Facteur IXa.
PCT/US2000/009790 1999-04-12 2000-04-12 Modulateurs variants de l'ecotine de genie genetique agissant sur des serines proteases WO2000061634A2 (fr)

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Cited By (3)

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WO2006044650A3 (fr) * 2004-10-15 2006-12-14 Genencor Int Criblage differentiel competitif
CN106226428A (zh) * 2016-07-20 2016-12-14 未名生物医药有限公司 一种快速检测cho细胞发酵液中重组人神经生长因子含量的方法
CN107135653A (zh) * 2014-10-01 2017-09-05 安迅生物制药公司 大肠杆菌素变体

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WANG CHENG-I ET AL: "Isolation of a High Affinity Inhibitor of Urokinase-type Plasminogen Activator by Phage Display of Ecotin" JOURNAL OF BIOLOGICAL CHEMISTRY,THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC.,,US, vol. 270, no. 20, 1995, pages 12250-12256, XP002146128 ISSN: 0021-9258 cited in the application *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006044650A3 (fr) * 2004-10-15 2006-12-14 Genencor Int Criblage differentiel competitif
JP2008517275A (ja) * 2004-10-15 2008-05-22 ジェネンコー・インターナショナル・インク 競合示差スクリーニング
CN107135653A (zh) * 2014-10-01 2017-09-05 安迅生物制药公司 大肠杆菌素变体
CN106226428A (zh) * 2016-07-20 2016-12-14 未名生物医药有限公司 一种快速检测cho细胞发酵液中重组人神经生长因子含量的方法
CN106226428B (zh) * 2016-07-20 2019-01-01 未名生物医药有限公司 一种快速检测cho细胞发酵液中重组人神经生长因子含量的方法

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