WO1991015577A1 - INTERLEUKIN 1'beta' PROTEASE - Google Patents

INTERLEUKIN 1'beta' PROTEASE Download PDF

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Publication number
WO1991015577A1
WO1991015577A1 PCT/US1991/002339 US9102339W WO9115577A1 WO 1991015577 A1 WO1991015577 A1 WO 1991015577A1 US 9102339 W US9102339 W US 9102339W WO 9115577 A1 WO9115577 A1 WO 9115577A1
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pro
asp
sequence
amino acid
nucleotide
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PCT/US1991/002339
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English (en)
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Corporation Immunex
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Black, Roy, A.
Sleath, Paul, R.
Kronheim, Shirley, R.
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Publication of WO1991015577A1 publication Critical patent/WO1991015577A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/545—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to an interleukin l ⁇ protease enzyme (IL-l ⁇ pro) having biological activity to cleave inactive precursor interleukin- l ⁇ QL-l ⁇ ) polypeptides into active mature IL-l ⁇ polypeptides. More specifically, the invention provides an isolated B - l ⁇ pro polypeptide and derivatives thereof that are capable of cleaving a particular amino acid sequence, including the amino acid sequence at the N-terminus of human IL- l ⁇ . The present invention further provides a group of compounds that can inhibit IL-l ⁇ pro activity and thereby function as IL-1 antagonists.
  • IL-l ⁇ pro interleukin l ⁇ protease enzyme
  • Interleukin l ⁇ is a 17.5 kDa polypeptide hormone synthesized and secreted by stimulated monocytes.
  • the initial translation product of IL-l ⁇ is a larger 31 kDa biologically inactive precursor polypeptide.
  • the N-terminus of biologically active, mature IL-l ⁇ derived from human activated monocytes has been characterized by an N-terminal amino acid sequence beginning with Ala-Pro. See, for example, European Patent Application EP-A-0165654 and March et al. Nature (London) 315: 641-47 (1985) for sequence information of human IL-l ⁇ .
  • the N-terminal Ala residue of human mature IL- l ⁇ is in the 117 position and an Asp residue is in the 116 position counting from the N-terminus of human precursor IL-l ⁇ polypeptide.
  • Mature IL-l ⁇ consists of the C-terminal 153 residues of the precursor polypeptide.
  • IL-l ⁇ is not associated with a membrane-bound compartment in monocytes. (Singer et al. J. Exp. Med. 167:389-407 (1988)). Most secretory proteins are characterized by the presence of a hydrophobic stretch of amino acids called a signal sequence. The signal sequence directs the translocation of the protein across the membrane of the endoplasmic reticulum during protein synthesis. The protein is subsequently ushered out of the cell via exocytosis.
  • IL-l ⁇ and IL-l ⁇ (March et al.) lack any region (either amino terminal or internal) with sufficient hydrophobicity and length to qualify as a signal sequence.
  • a further indication of an unusual maturation pathway for IL-l ⁇ is the absence of a pair of basic amino acids near the N-terminus of the mature polypeptide. The amino acid sequence Tyr-Val-His-Asp- precedes the N-terminal Ala-Pro of the mature human IL-l ⁇ polypeptide.
  • Kostura et al. Proc. Nat Acad Sci. USA 86:5227-31 refers to a protease with a similar cleavage pattern but "qualitatively different" from the protease described in Black et al. ⁇ .
  • the Kostura et al. protease is characterized as being located in cytosol of monocytic cells. However, Kostura et al. did not further define or isolate the responsible polypeptide.
  • Black et al. FEBSLett.247:386-90 (April, 1989) [Black et al. DTJ refers to a protease that generates mature IL-l ⁇ rom the precursor polypeptide and is characterized by being inhibited by iodoacetate and N-e ⁇ ylmaleimide.
  • Black et al. DI attempted to purify their protease approximately 500 fold by a process starting by freeze-thawing cell lysates from THP-1 cells (ATCC) four times. Black et al. DI centrifuged the lysates for 20 min at 36,590 X g.
  • the supernatant was applied to a DEAE- Sephacel column equilibrated with 10 mM Tris-HCl (pH 8.1) and 5 mM dithiothreitol.
  • the protease was eluted with 80- 140 mM NaCl.
  • the eluted material was diluted 1:5 with a buffer of 10 mM Tris-HCl (pH 8.1 ) and 5 mM dithiothreitol and applied to a procion red agarose column.
  • the protease was eluted with 0.5-0.8 M NaCl, concentrated 20-fold in a Centriprep-10 concentrator, and then subjected to gel filtration with Sephadex G-75.
  • the protease functions as an IL-1 agonist to increase IL-1 biological activity in vivo.
  • the isolated protease will be useful for improving wound healing, treating arthritis, and treating or preventing the onset of autoimmune diseases, such as insulin dependent diabetes melitus, lupus disorders, Graves' disease, Hashimotos disease, and the detrimental side effects of radiation treatment
  • isolation and characterization of the protease responsible for processing precursor IL-l ⁇ into its biologically active form will aid in designing inhibitors for IL-l ⁇ processing, because the availability of large quantities of IL-l ⁇ pro will serve as a useful screening vehicle for finding compounds having IL-1 antagonist activity.
  • Such IL- 1 antagonists or IL-l ⁇ pro inhibitors should be useful for treating inflammation and transplantation rejection.
  • protease inhibitors have been described (for example, U.S. Patents 4,644,055, 4,636,492 and 4,652,552).
  • protease inhibitors tend to be specific for the protease they inhibit by having a chemical structure that mimics the substrate of a protease.
  • none of the previously-described protease inhibitors would be expected to inhibit IL-l ⁇ pro activity.
  • the present invention is directed to an isolated polypeptide having proteolytic activity for a specific protease cleavage site, wherein the protease activity is specific for a substrate peptide having an amino acid sequence comprising:
  • Rl -Asp - R2 - R3 wherein Ri and R3 are independently any D or L isomer amino acid, R2 is Ala or Gly, and wherein the specific protease cleavage site is between Asp and R2.
  • the substrate peptide is at least eight amino acids in length.
  • the isolated polypeptide is called the interleukin l ⁇ protease (IL-l ⁇ pro) because it cleaves precursor IL-l ⁇ polypeptide to yield mature IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues. This region of precursor IL-l ⁇ corresponds to a species within the genus of protease cleavage sites described herein.
  • IL-l ⁇ pro is further characterized by a cDNA and amino acid sequence in Figure 1 (Sequence I.D. No. 2).
  • Full length (precursor) IL-l ⁇ pro comprises 404 amino acids.
  • Purified IL-l ⁇ pro begins with the Asn Pro Ala Met Pro sequence begiiining with amino acid 120. Based upon a molecular weight analysis, the approximate C-terminus of mature IL-l ⁇ pro is about amino acid 297. However, molecular weight determination indicates that the C-terminus of the mature IL-l ⁇ pro enzyme is from about amino acid 278 to about amino acid 315.
  • the present invention comprises an isolated IL-l ⁇ pro polypeptide or a derivative, analog, or allelic variant thereof displaying IL-l ⁇ pro biological activity comprising proteolytically cleaving human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues.
  • Figure 1 also shows a nucleotide sequence encoding a 404 amino acid polypeptide having IL-l ⁇ pro biological activity.
  • the present invention further comprises an isolated DNA sequence encoding IL-l ⁇ pro or a derivative, analog or allelic variant thereof displaying biological activity to proteolytically cleave human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues.
  • the isolated DNA sequence is selected from the group consisting of the nucleotide sequences in Figure 1 beginning at nucleotide 1 and extending to nucleotide 1232, beginning at nucleotide 374 and extending to nucleotide 1232, beginning at nucleotide 374 and extending to a nucleotide from about 851 to about 962, DNA sequences which detectably hybridize to the Figure 1 sequence from nucleotide 1 to nucleotide 1232 and encode a polypeptide displaying biological activity to proteolytically cleave human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues, and DNA sequences which, due to degeneracy of the genetic code, encode a mammalian IL-l ⁇ pro polypeptide encoded by any of the foregoing DNA inserts and sequences.
  • the present invention further comprises a recombinant expression vector comprising an isolated DNA sequence as described herein and a host cell which comprises the recombinant expression vector.
  • the present invention provides substituted peptide inhibitor compounds comprising an amino acid sequence of from 1 to about 5 amino acids, having an N-terminal protecting group and a C-terminal Asp residue connected to an electronegative leaving group.
  • the amino acid sequence corresponds to at least a portion of the amino acid sequence Ala-Tyr-Val-His-Asp.
  • the inhibitor compounds of the present invention have the formula: where Z is an N-terminal protecting group; Q2 is 0 to 4 amino acids such that the sequence 02-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing LD. No.2; and Qj comprises an electronegative leaving group.
  • Z is preferably Cj-C ⁇ alkylketone, benzyl, acetyl, alkoxycarbonyl, benzyloxycarbonyl or Cj-Cg alkylcarbonyl. More preferably, Z is t-butoxycarbonyl (t-
  • Boc acetyl carbonyl or benzyloxycarbonyl
  • Ql is preferably C1-C3 alkyl, an aldehyde, diazomethyl ketone or halomethyl ketone. More preferably, Qj is an aldehyde or fluoromethyl ketone.
  • the present invention further provides reversible and irreversible IL-l ⁇ pro inhibitors.
  • Irreversible inhibitors are inhibitor compounds comprising an amino acid sequence of from 1 to about 5 amino acids having an N-terminal protecting group and a C- terminal Asp residue connected to a diazomethyl ketone or a halomethyl ketone, wherein the amino acid sequence corresponds to at least a portion of the sequence Ala-Tyr- Val-His- Asp, residues 112 to 116 of sequence listing I.D. No. 1.
  • Reversible IL-l ⁇ inhibitors are compounds comprising an amino acid sequence of from 1 to about 5 amino acids having an N-terminal protecting group and a C-terminal Asp residue connected to an aldehyde moiety, wherein the amino acid sequence corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing I.D. No. 1.
  • the present invention also provides a method of inhibiting the physiological actions of interleukin l ⁇ in a mammal in need of such treatment, comprising administering to said mammal an effective amount of a compound of the formula:
  • Z is an N-terminal protecting group
  • Q2 is 0 to 4 amino acids such that the sequence Q2-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing I.D. No. 2
  • Qj comprises an electronegative leaving group
  • Qi is a fluoromethyl ketone and inhibition is irreversible.
  • Qi is an aldehyde moiety and inhibition is reversible.
  • the present invention still further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a physiologically acceptable carrier and a compound comprising an amino acid sequence of from 1 to about 5 amino acids having an N-terminal protecting group and a C- terminal Asp residue connected to an electronegative leaving group, wherein said amino acid sequence corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing I.D. No. 1.
  • the present invention still further provides a method of treating inflammation associated with autoimmune disease in a mammal in need of such treatment comprising administering to said mammal an effective anti-inflammatory amount of a compound comprising an amino acid sequence of from 1 to about 5 amino acids having an N-terminal protecting group and a C-terminal Asp residue connected to an electronegative leaving group, wherein said amino acid sequence corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing I.D. No. 1.
  • the present invention further comprises antisense oligonucleotides that correspond to a sequence or antisense sequence of at least 15 nucleotides selected from the nucleotide sequence of Sequence ID No. 2 ( Figure 1).
  • the present invention further comprises a method for treating arthritis, a method for treating an autoimmune disease in a susceptible individual, a method for improving wound healing, and a method for reducing the detrimental side effects of radiation treatment. All of the methods comprise administering a therapeutically effective amount of an isolated IL- l ⁇ protease or a biologically active derivative thereof in a suitable pharmaceutical carrier.
  • Figure 1 shows the DNA and corresponding amino acid sequence for a polypeptide having IL-l ⁇ pro biological activity and corresponding to human mature IL-l ⁇ pro or a biologically active fragment thereof.
  • PCR polymerase chain reaction
  • Rl -Asp - R2 - R3 wherein Ri and R3 are independently any D or L isomer amino acid, R2 is Ala or Gly, and wherein the specific protease cleavage site is between Asp and R2-
  • the substrate peptide is at least eight amino acids in length.
  • R2 is Gly.
  • Mammalian IL-l ⁇ pro is preferably a human IL-l ⁇ pro and has substrate specificity for a substrate peptide having the amino acid sequence described herein.
  • the human IL-l ⁇ pro polypeptide or derivative thereof is a polypeptide having biological activity that cleaves human precursor IL-l ⁇ polypeptide to yield human mature IL-l ⁇ polypeptide.
  • IL-l ⁇ pro is further characterized by the cDNA and amino acid sequence in Figure 1 (Sequence LD. No. 2).
  • Full length (precursor) IL-l ⁇ pro comprises 404 amino acids.
  • Purified IL- l ⁇ pro begins with the Asn Pro Ala Met Pro sequence begirining with amino acid 120. Based upon a molecular weight analysis, the approximate C-teiminus of mature IL-l ⁇ pro is about amino acid 297. However, the molecular weight determination indicates that the C-terminus of the mature enzyme is from about amino acid 278 to about amino acid 315.
  • the present invention comprises an isolated IL- l ⁇ pro polypeptide or a derivative, analog, or allelic variant thereof displaying biological activity comprising proteolytically cleaving human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues.
  • the present invention comprises an isolated IL-l ⁇ pro polypeptide or a derivative, analog, or allelic variant thereof displaying biological activity comprising proteolytically cleaving human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues.
  • the term "IL- l ⁇ pro” shall encompass the amino acid sequence shown in Figure 1, plus all allelic variants, derivatives, analogs and fragments of this sequence that display IL-l ⁇ pro biological activity.
  • IL-l ⁇ pro biological activity is determined, for example, by assaying for EL-1 activity with a precursor IL-l ⁇ polypeptide.
  • Precursor IL-l ⁇ is inactive, while mature IL- l ⁇ is an active IL-1 polypeptide.
  • a method for measuring IL-l ⁇ pro activity is described in Black et al. II. Briefly, this method provides approximately five microliters of precursor IL-l ⁇ (pIL-l ⁇ ) (10-50 ⁇ g ml prepared as described in Black et al. I) incubated with 10 ⁇ l of IL-l ⁇ pro polypeptide or another substance suspected of having IL-l ⁇ pro biological activity.
  • the incubation proceeds for approximately one hour at approximately 37°C and is terminated by addition of 15 ⁇ l of 2 X SDS sample buffer followed by boiling for five minutes.
  • the boiled sample is electrophoresed on a SDFS-polyaciylamide gel and placed onto a Western blot using an IL-l ⁇ C-terminal-specific monoclonal antibody, such as 16F5 described in Black et al. I.
  • Figure 1 also shows a nucleotide sequence encoding a 404 amino acid sequence having IL-l ⁇ pro biological activity.
  • the present invention further comprises an isolated DNA sequence encoding IL-l ⁇ pro or a derivative, analog or allelic variant thereof displaying biological activity to proteolytically cleave human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues.
  • the isolated DNA sequence is selected from the group consisting of the nucleotide sequences in Figure 1 beginning at nucleotide 1 and extending to nucleotide 1232, beginning at nucleotide 374 and extending to nucleotide 1232, beginning at nucleotide 374 and extending to a nucleotide from about 851 to about 962, DNA sequences which detectably hybridize to the Figure 1 sequence from nucleotide 1 to nucleotide 1232 and encode a polypeptide displaying biological activity to proteolytically cleave human precursor IL-l ⁇ polypeptide at a cleavage site between the Asp 116 and Ala 117 residues, and DNA sequences which, due to degeneracy of the genetic code, encode a mammalian IL-l ⁇ pro polypeptide encoded by any of the foregoing DNA inserts and sequences.
  • Inventive DNA sequences that detectably hybridize to the Figure 1 nucleotide sequence from nucleotide 1 to nucleotide 856 hybridize under conditions of high or severe stringency.
  • Severe or high stringency conditions comprise, for example, overnight hybridization at about 68"C in a 6 X SSC solution followed by washing at about 68°C in a 0.6 X SSC solution.
  • the inhibitor compounds of the present invention are substituted compounds comprising an amino acid sequence of from 1 to about 5 amino acid residues having an N- terminal blocking group and a C-terminal Asp residue connected to an electronegative leaving group, wherein the amino acid sequence corresponds to at least a portion of the sequence Ala- Tyr- Val-His-Asp, which sequence represents the sequence of residues 112 to 116 of precursor IL-l ⁇ .
  • precursor IL-l ⁇ The amino acid sequence of precursor IL-l ⁇ is presented in sequence listing I.D. No. 2 taken from March et al., Nature, 315:641-647 (1985). Mature IL-l ⁇ is represented by the C- terminal 153 amino acid residues of precursor IL-l ⁇ . Thus, the N-terminal of IL-l ⁇ is the Ala residue at position 117 of sequence listing ID. No. 1.
  • the naturally occiirring amino acids are the L isomers and are so indicated without any isomeric (e.g., L or D) designation.
  • the D isomers are so indicated.
  • the phrase "corresponds to" means that a particular sequence may diffe from the disclosed sequence by one or more conservative substitutions so long as such substitutions do not materially alter the inhibitory activity of the compounds of the present invention.
  • substitutions that do not materially alter inhibitory activity are replacement of the Ala at position 112 of sequence listing LD. No. 1 with Ser or Gly; replacement of the T at position 113 of sequence listing LD. No. 1 with Phe; replacement of the Val at position 114 of sequence listing LD. No. 1 with Leu, De or Met and replacement of the His at position 115 of sequence listing LD. No. 1 with Phe, Pro, a positively charged amino acid such as Lys, Arg, His or Tyr, or the use of D isomers.
  • the C-te ⁇ r nal amino acid residue of the compounds of the present invention is asparti acid (Asp).
  • Asp has a side chain of the formula C ⁇ 2-COOH
  • the Asp side chain carboxyl group is protected to facilitate synthesis of the compounds of the present invention.
  • Asp side chain protection groups include, for example, a benzyl, substituted benzyl, formyl methyl or t-butyl moiety.
  • the benzyl substituents increase the acid lability of the Asp side chain protecting moiety.
  • Exemplary substituted benzyls are 2,4,6-trimethyl benzyl and 4- methoxybenzyl.
  • the Asp side chain protecting moiety is connected to the As side chain via an ester linkage, which linkage is subject to cleavage by naturally occurring intraceUular esterase enzymes.
  • the protected Asp with high lipid solubility gains access to a cell and is cleaved by an esterase to yield a charged, water soluble deprotected Asp that remains in the cytoplasm where IL-l ⁇ pro is predominantly located.
  • N-terminal blocking group refers to chemical groups attached to the amino group of the N-terminal amino acid residue of the sequences of the pres invention. Such blocking groups are well known and readily apparent to those of skill in the art. The Peptides. ed. by Gross and Meienhofer, Academic Press, New York, pp. 3-81 (1981). N-terminal blocking groups have been utilized with other types of protease inhibitors See, e.g.. U.S. Patent Nos. 4,652,552 and 4,636,492.
  • electronegative leaving group refers to chemical groups susceptible to nucleophilic attack by an amino acid residue in the enzyme active site, thus modifying IL-l ⁇ pro such that D -l ⁇ pro cannot interact with and cleave precursor IL-l ⁇ .
  • inhibitor compounds of the present invention inhibit the catalytic activity of IL-l ⁇ pro in a reversible or an irreversible fashion.
  • irreversible means the formation of a covalent bond between the enzyme and the inhibitor.
  • the reversibility of IL- l ⁇ pro activity is a function of the electronegative leaving group
  • the inhibitor compounds of the present invention have the formula: where Z is an N-terminal blocking group; Q2 is 0 to about 4 amino acids such that the sequenc Q2-Asp corresponds to at least a portion of the sequence Ala-Tyr-Val-His-Asp, residues 112 t 116 of sequence listing LD. No. 1; and Q ⁇ is an electronegative leaving group.
  • Z is Cj-C6 alkyl, benzyl, acetyl, Cj-C6 alkoxycarbonyl, benzyloxycarbonyl or C1-C6 alkyl carbonyl.
  • alkyl refers to linear or branched chains having 1 to 6 carbon atoms, which may be optionally substituted as herein defined. Representative alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like.
  • Z is t-butoxycarbonyl (t-Boc), acetyl or benzyloxycarbonyl (Cbz).
  • Q2 is preferably 1 amino acid.
  • Q2 is His, Phe, Pro, or Tyr.
  • Q2 is His or Phe.
  • Ql is preferably an aldehyde, a diazoalkyl ketone or a haloalkyl ketone.
  • alkyl refers to linear or branched chain radical having 1 to 3 carbon atoms, which may be optionally substituted as herein defined.
  • alkyl groups include methyl, ethyl, propyl and the like. More preferably, Q is an aldehyde or fluoromethyl (CH2F) ketone.
  • the inhibitor compounds of the present invention are made by techniques generally corresponding to methods known and readily apparent to those of skill in the art. See, e.g.. Kettner, C.A. et al., Arch. Biochem. Biophvs.. 162:56 (1974); U.S. Patent No. 4,582,821; U.S. Patent No. 4,644,055; Kettner, C.A. et al., Arch. Biochem. Biophvs.. 165:739 (1974); Dakin, H.D. and West, R., J. Biol. Chem.. 78:91 (1928); Rasnick, D., Anal. Biochem.. 149:461 (1985).
  • Inhibitor compounds having a fluoromethyl electronegative leaving group are preferably synthesized by the Rasnick procedure.
  • Inhibitor compounds having a non-fluoro, haloalkyl ketone electronegative leaving group are synthesized in accordance with the Kettner procedure.
  • An N-blocked amino acid or peptide is reacted with N-methylmorpholine and an alkyl, non-fluoro haloformate to generate a peptide-acid anhydride.
  • the anhydride is then reacted with a diazoalkane in an inert, aprotonic solvent to form a peptide-diazomethane ketone.
  • the diazomethane ketone is then reacted with an anhydrous solution of HC1, HBr or HI to produce the desired N-blocked, C-te ⁇ ninal haloalkyl ketone peptide or amino acid.
  • Inhibitor compounds having a fluoroalkyl ketone electronegative leaving group are synthesized in accordance with a Rasnick procedure.
  • An N-blocked peptide is reacted with fluoroacetic anhydride and a trialkylamine in an organic solvent to form a peptide-anhydride.
  • the anhydride is then reacted with a catalyst such as 4-dimethylaminopyridine and the reaction mixture maintained at about 25° C for about two hours to allow for CO2 evolution.
  • the reaction mixture is then extracted with an organic solvent and the organic phase washed and dried. The organic solvent is removed to form an oil, which is then applied to a silica gel column.
  • N-blocked, fluoroalkyl ketone peptide is then eluted from the gel and purified.
  • Inhibitor compounds having a fluoroalkyl ketone electronegative leaving group can be extended in the N-terminus direction by removing the N-terminal blocking group and coupling the deprotected compound with other protected amino acids.
  • Bodanszky The Practice of Peptide Synthesis, Springer-Verlag, Berlin (1984).
  • deprotected compounds are acetylated to yield compounds having an N-terminal acetyl protecting group. Stewart et al., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL. (1984).
  • the inhibitor compounds of the present invention are useful in inhibiting the physiological actions of interleukin l ⁇ by preventing formation of biologicaUy active IL-l ⁇ .
  • the inhibition of IL-l ⁇ pro results in a decrease in active IL-l ⁇ levels and a concomitant increase in precursor IL-l ⁇ , which compound is biologicaUy inactive.
  • the inhibitor compounds of the present invention are also useful in treating dysfunctional states, such as autoimmune disease-associated inflammation, often mediated by increased IL-1 activity.
  • Mammals needing treatment for an iiiflammatory disorder or prevention of an autoimmune condition are administered effective amounts of the compounds of this invention either alone or in the form of a pharmaceutical composition.
  • compositions of the present invention comprise physiologically acceptable carriers and compounds comprising amino acid sequences of from 1 to about 5 amino acids having an N-terminal blocking group and a C-terminal Asp residue connected to a electronegative leaving group, wherein said amino acid sequence corresponds to the sequence Ala-Tyr-Val-His-Asp, residues 112 to 116 of sequence listing LD. No. 1.
  • Antisense oligonucleotides can be synthesized (by conventional phosphodiester techniques such as by Synthecell, Rockville, MD) that are complementary to unique species-specific coding regions of mRNA for IL-l ⁇ pro.
  • regions include regions of at least 18 bases at the initiation codon (TACCGGCTGTTCCAGGAC or TACCTATTCTGGGCTCGA) complementary to bases 18-36 and 168 to 196, respectively in Figure 1, at the N-terminus of mature IL-l ⁇ pro (TTGGGTCGATACGGGTGT) complementary to bases 374 to 392 in Figure 1, at the approximate C terminus after protease cleavage (CACCACACCAAATTTCTA) complementary to bases 890 to 908 in Figure 1, or at a region immediately 5' to the termination codon (ATGGAGAAGGGTCCTGTA) complementary to bases 1205 to 1229 in Figure 1.
  • the primary amino acid structure of IL- l ⁇ pro or its active fragment thereof may be modified by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, or by creating a ⁇ iino acid sequence mutants or derivatives.
  • Covalent derivatives of IL-l ⁇ pro are prepared by linking particular functional groups to IL-l ⁇ pro amino acid side chains or at the N-terminus or C- terminus of the IL-l ⁇ pro polypeptide.
  • the conjugated polypeptide may be a signal (or leader) polypeptide sequence at the N-terminal region of the IL-l ⁇ pro polypeptide which co- translationally or post-translationally directs transfer of the IL-l ⁇ pro polypeptide from its site of synthesis to a site inside or outside of the cell membrane or wall (e.g., the yeast ⁇ - factor leader).
  • IL-l ⁇ pro polypeptide fusions can comprise polypeptides added to facilitate purification and identification of IL-l ⁇ pro (e.g., poly-His). Further, the amino acid sequence of IL-l ⁇ pro can be linked to the peptide Asp-Tyr-Lys- Asp-Asp-Asp- Asp-Lys (Hopp et al. Bio/Technology 6:1204 1988), which is a highly antigenic sequence and provides an epitope reversibly bound by a specific monoclonal antibody to enable rapid assay and facile purification of the expressed recombinant polypeptide.
  • This specific leader sequence is cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Moreover, fusion polypeptides having this leader sequence at its N- terminal may be resistant to degradation in E. coli host ceUs.
  • the present invention further includes IL-l ⁇ pro polypeptides with or without associated native-pattern glycosylation.
  • IL-l ⁇ pro expressed in yeast or mammalian expression systems may be similar or significantly different in molecular weight and glycosylation pattern than the native human IL-l ⁇ pro polypeptide. This depends upon the choice of expression system.
  • Expression of IL-l ⁇ pro polypeptides in bacterial expression systems, such as E. coli provides non-glycosylated molecules.
  • Functional mutant analogs of human IL-l ⁇ pro can be synthesized, for example, with inactivated N-glycosylation sites by oligonucleotide synthesis and ligation or by site- specific mutagenesis techniques.
  • the D_-l ⁇ pro derivatives can be expressed in homogeneous, reduced carbohydrate form using yeast expression systems.
  • N- glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn- ⁇ - ⁇ where ⁇ is any amino acid except Pro and ⁇ is Ser or Thr. In this sequence, carbohydrate residues are covalently attached at the Asn side chain.
  • IL-l ⁇ pro analogs or derivatives may also be obtained by mutations of the IL-l ⁇ pro DNA sequence.
  • An IL-l ⁇ pro mutant derivative as referred to herein, is a polypeptide substantiaUy homologous to IL-l ⁇ pro but which has an amino acid sequence different from native IL-l ⁇ pro because of a deletion, insertion or substitution.
  • IL-l ⁇ pro is expressed from a mammalian gene, presumably encoded by one or more multi-exon genes.
  • the present invention further includes alternative mRNA constructs which can be attributed to different mRNA splicing events following transcription, and which share regions of identity or similarity with the cDNA's disclosed herein. Bioequivalent analogs of IL-l ⁇ pro polypeptides (defined as polypeptides having
  • IL-l ⁇ pro biological activity may be constructed, for example, by making various substitutions of amino acid residues or sequences, or by deleting terminal or internal residues or sequences not needed for biological activity.
  • Cys residues can be deleted or replaced with other amino acids to prevent formation of incorrect intramolecular disulfide bridges upon renaturation.
  • Other approaches to mutagenesis involve modification of dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. GeneraUy, substitutions are made conservatively by substituting an amino acid having phy siochemical characteristics resembling those of the replaced residue. Further substitutions may be made outside of the "core" sequence needed for IL-l ⁇ pro biological activity.
  • Subunits of IL-l ⁇ pro may be constructed by deleting terminal or internal residues or sequences.
  • the resulting polypeptide should have IL-l ⁇ pro biological activity as defined herein.
  • the terms "IL-l ⁇ pro", “human IL-l ⁇ pro” or “D -l ⁇ protease” include, but are not limited to, analogs or subunits of IL-l ⁇ pro which are substantially similar to human IL-l ⁇ pro and/or which exhibit the substrate-specific proteolytic biological activity associated with IL-l ⁇ pro as described herein.
  • substantially similar when used to describe amino acid sequences, means that a particular sequence may vary from a disclosed reference sequence by one or more substitutions, deletions, or additions.
  • a substantially similar human IL-l ⁇ pro polypeptide for example, can have a truncated sequence comprising a "core region" of a sequence of amino acids necessary for the specific protease biological activity characteristic of IL-l ⁇ pro.
  • Substantially similar IL-l ⁇ pro derivatives will be greater than about 30% similar to the corresponding sequence of human D -l ⁇ pro and have IL-l ⁇ pro biological activity.
  • Polypeptides having amino acid sequences of lesser degrees of similarity but comparable biological activity (including substrate specificity) are considered to be equivalents.
  • the derivative polypeptides will have greater than 80% amino acid sequence homology to human IL-l ⁇ pro polypeptide. Percent similarity may be determined, for example, by comparing sequence information using a GAP computer program, version 6.0, available from University of Wisconsin Genetics Computer Group. The GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol 48:443 1970), as revised by Smith and Waterman ⁇ Adv. Appl. Math 2:482 1981). Briefly, the GAP program defines similarity as the number of aligned symbols which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a weighted comparison matrix for amino acids (see, Schwartz and Dayhoff, eds. Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-58 1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Bioly active refers to IL-l ⁇ pro biological activity to cleave a particular amino acid sequence at the peptide bond between an Asp residue and an Ala or Gly residue.
  • Recombinant means that a polypeptide is derived from recombinant (e.g., microbial or mammalian) expression systems.
  • Microbial refers to bacterial or fungal (e.g., yeast) expression systems.
  • recombinant microbial defines a polypeptide produced in a microbial expression system which is substantially free of native endogenous substances.
  • Polypeptides expressed in most bacterial expression systems (e.g., E. coli) wiU be free of glycan.
  • Polypeptides expressed in yeast may have a glycosylation pattern different from that expressed in mammalian cells.
  • the IL-l ⁇ pro protease has a highly restricted substrate specificity.
  • Human precursor IL-l ⁇ polypeptide has an amino acid sequence His-Asp- Ala-Pro for residues 115-118.
  • Human D_-l ⁇ pro pro cleaves this sequence between residues 116 and 117 (Asp- Ala) to form human mature IL-l ⁇ polypeptide.
  • Changing Asp-116 to Ala in a human precursor IL-l ⁇ polypeptide by site-directed mutagenesis prevented cleavage of the mutant IL-l ⁇ polypeptide derivative.
  • Isolated human IL-l ⁇ pro was able to cleave at its specific substrate site even when the tertiary structure of the substrate precursor IL-l ⁇ polypeptide was altered by denaturing the substrate polypeptide in boiling water.
  • Precursor human IL-l ⁇ was denatured by boiling a solution of precursor IL-l ⁇ for fifteen minutes. Denaturation had little effect on the ability of human IL-l ⁇ pro to be able to cleave human precursor IL-l ⁇ into mature B-- l ⁇ .
  • the tertiary structure of the substrate polypeptide does not significantly contribute to the reaction with the enzyme IL- l ⁇ pro.
  • IL-l ⁇ pro biological activity was determined by a protease assay.
  • samples having a salt concentration greater than 50mM were initially desalted. Samples can be desalted, for example, by applying 100 ⁇ l of sample to a pre-spun 1 ml Biogel P-6DG (Bio-Rad) column, which was equilibrated in 10 mM Tris- HCl, 5 mM dithiothreitol, pH 8.1 , and centrifuging for 5 min at 1876 X g.
  • the assay was conducted by incubating a mixture of five ⁇ l (30 ng) of purified human IL-l ⁇ precursor and 10 ⁇ l of the sample to be tested for IL-l ⁇ protease biological activity for 60 min at 37°C.
  • a control sample was similarly incubated to check for endogenous IL-1.
  • the control sample mixture contained 5 ⁇ l of 10 mM Tris-HCl, pH 8.1 and 5 mM dithiothreitol instead of IL- l ⁇ precursor.
  • the control sample incubations were terminated by addition of SDS (sodium dodecyl sulfate) in sample buffer followed by five min of boiling.
  • THP-1 cells obtainable from the American Type Culture Collection (ATCC). Approximately 120 liters of cells were cultured and then stimulated for 16 hours with liposaccharide, hydroxyurea and silica as described in Matsushima Biochemistry 25:3424 1986. The cells were harvested by centrifugation, washed in Hanks balanced salt solution, and then recentrifuged. The cells were resuspended in 10 mM Tris-HCl, 5 mM dithiothreitol, pH 8.1 at a density of lO ⁇ /ml.
  • the suspended cells were frozen and thawed three times and the lysates stored at -80°C until further use. Prior to purification, the lysates were thawed and then centrifiiged for 20 min at 47,800 X g at 4°C. The supernatant was taken for further purification. The freeze- thawing procedure repeated four times released over 50% of the IL- l ⁇ pro activity into the supernatant Additional freeze-thaws did not increase the yield of soluble material.
  • the human IL-l ⁇ pro polypeptide was purified in a six-step process. All the chromatography steps were performed at 4°C using a Pharmacia FPLC System.
  • DEAE- Sephacel, Hydroxyapatite and Blue Agarose gels were pretreated with 0.1% Triton-X-100 and 10% bovine calf serum to prevent non-specific absorption of proteins to the gels. Further, the Blue Agarose column was washed with 8M urea to remove any noncovalently absorbed dye. 1. Approximately 500-600 ml of lysate supernatant was diluted 1:2 in lOmM Tris-
  • buffer A HCl and 5mM dithiothreitol, pH 8.1
  • pH was adjusted to 8.1.
  • the diluted lysate supernatant was applied to a DEAE- Sephacel column (20 x 4.4 cm, Pharmacia Fine Chemicals), equilibrated with buffer A. The flow rate was 120 ml/hr.
  • the column was washed with two column volumes of buffer A and then eluted with a linear gradient (3 column volumes) ranging from 0 to 300 mM NaCl in buffer A. Fifteen ml fractions were collected, analyzed for IL-l ⁇ pro activity and stored for further purification.
  • the D -l ⁇ pro activity was eluted with between 0.07 and 0.13 M NaCl. This step removed 79% of the contaminating proteins. The bulk of the contaminating proteins eluted between 0.15 and 0.25 M NaCl. This step was further useful in partially removing endogenous mature IL-l ⁇ , which eluted between 0.06 and 0.11 M NaCl, and endogenous precursor EL-l ⁇ which eluted between 0.12 and 0.18 M NaCl.
  • the pooled active fractions from the DEAE column were diluted in 50 mM potassium phosphate buffer, 5 mM dithiothreitol, pH 7.0 ("buffer B").
  • buffer B 50 mM potassium phosphate buffer, 5 mM dithiothreitol, pH 7.0
  • a 14 x 3 cm column of hydroxyapatite (HA Ultrogel, IBF Biotechnics) was equilibrated with buffer B.
  • the diluted fractions were applied to the equilibrated hydroxyapatite column at a flow rate of 60 ml/hr.
  • the column was washed with 2 column volumes of buffer B and then eluted with a linear gradient (4 column volumes) ranging from 50-200 mM potassium phosphate.
  • IL-l ⁇ pro eluted between 0.085 and 0.113 M potassium phosphate. Forty percent of the contaminating polypeptides eluted before the protease and 40% eluted later than the protease. Further, endogenous mature IL-l ⁇ eluted between 0.05 and 0.08 M potassium phosphate.
  • a 20 x 1.6 cm Blue Agarose column (Gibco-BRL) was equilibrated with buffer A. Fractions from the hydroxyapatite column with activity were diluted 1:3 in buffer A to reduce ionic strength to ⁇ 30 mM. This was necessary in order to aUow IL-l ⁇ pro to bind to the coliimn. Diluted fractions were applied to the Blue Agarose column at a 30 ml/hr rate. The column was washed with three column volumes of buffer A. The proteins were eluted with with five column volumes of a linear gradient ranging from 0.1 to 1 M Nad in buffer A.
  • IL-l ⁇ pro was eluted with 0.5 to 0.68 M NaCl. Eighty percent of the contaminating proteins were removed in this step, with 20% eluting earlier and the remaining 60% remaining bound to the column.
  • a 95 x 2.5 cm Sephadex G-75 column (Pharmacia Fine Chemicals) was equilibrated in buffer A and initially calibrated with ferritin (MW 400,000), ovalbumin (MW 43,000), soybean trypsin inhibitor (MW 20,000) and DNP-aspartic acid (MW 300).
  • the Blue Agarose column fractions containing protease activity were pooled and concentrated on a Centriprep-10 concentrator (Amicon) to a volume of approximately 2 ml and then applied to the Sephadex G-75 column. Proteins were eluted with buffer A at a flow rate of 20 ml/hr. Four ml fractions were collected and the fractions containing protease activity were pooled for further purification.
  • IL-l ⁇ pro activity was eluted with between 196 and 220 ml. This position is identical to the elution position of soybean trypsin inhibitor, which suggests that human IL-l ⁇ pro has a molecular weight of about 20,000 daltons. This step removed over 90% of the contaminating proteins from the preparation. Thus, through the Sephadex step, more than 99.8% of the starring protein contaminants have been separated from IL-l ⁇ pro. However, PAGE (polyacrylamide gel electrophoresis) analysis of the fractions still revealed several protein bands that did not correlate with IL-l ⁇ pro biological activity.
  • a Mono P5 20 EPLC chromatofocussing column (Pharmacia Fine Chemicals) was equilibrated with 25 mM Tris-acetate and 5 mM dithiothreitol, pH 8.3 buffer. The concentrated solution was mixed (1:1 v/v) with 500 ⁇ l of 25 mM Tris-acetate and 5 mM dithiothreitol, pH 8.3 and applied to the Mono P5/20 FPLC column. Proteins were eluted with Polybuffer 96:Polybuffer 74 (3:7) pH 5.0 (Pharmacia) at a 15 ml/hr flow rate. One ml fractions were coUected and analyzed for pH and biological protease activity.
  • This chromatofocusing step increased the purity of IL-l ⁇ pro a further 100 fold and allowed for the visualization of a single protein band that correlated with IL-l ⁇ pro biological activity.
  • IL-l ⁇ pro was eluted off the chromatofocusing column between pH 6.95 and 6.70. The fractions were concentrated on BSA-pretreated Centricon 10 Concentrators (Amicon) from 1 ⁇ l to 50 ⁇ l. 6. The fractions were subjected to electroph ⁇ resis on a polyacrylamide gel (PAGE), followed by electroblotting onto polyvinyl difluoride membrane paper (PVDF, Millipore Immobilin-P®) at 300 mA for 30 min. The PVDF membrane was stained with Coomassie Blue.
  • PVDF polyvinyl difluoride membrane paper
  • the 22,000 dalton band correlated with IL-l ⁇ pro activity and was sequenced.
  • the N-terminal sequence of the 22,000 dalton band yielded an amino acid sequence described herein.
  • a mature human IL- l ⁇ pro cDNA or an active fragment thereof was cloned using this N-terminal amino acid sequence and a three-stage polymerase chain reaction (PCR) procedure.
  • PCR polymerase chain reaction
  • fuUy degenerate PCR primers were designed and made from the N-terminal amino acid sequence.
  • the degenerate primers were used to amplify IL-l ⁇ pro-specific sequences from a cDNA library prepared from THP-1 cell mRNA.
  • a random primed first strand THP-1 cDNA library was constructed according to supplier instructions (Amersham).
  • a mixed oligonucleotide primed amplification was carried out according to the procedure described in Lee et al. "cDNA Cloning Using Degenerate Primers" in PCR Protocols (Innis, Gelfand, Sninsky and White eds.) Academic Press, Inc. New York pp. 46-53 1990.
  • Primer #1 was designed to cross-hybridize to IL-l ⁇ pro DNA (nucleotides 1-17) and contain an EcoRl restriction site.
  • Primer #1 (Sequence ID No. 3) had the sequence:
  • Primer #2 was designed to cross-hybridize to IL-l ⁇ pro DNA (complementary to nucleotides 31-47 and contain anXbal restriction site.
  • Primer #2 (Sequence ID No.4) had the sequence
  • PCR amplification was performed with thermus aquatius polymerase (Perkin-Elmer Cetus) in lOO ⁇ l of buffer for 30 cycles as described in Lee et al. infra. A 63 bp amplified fragment was obtained from PCR amplification. This amplified fragment was subcloned into a pGem-4 vector (Promega). DNA sequence analysis of 10 isolates indicated that this fragment encoded the first 16 amino acids of the N-terminus of IL-l ⁇ pro as determined by purification and N-terminal sequence analysis.
  • Primer #3 composed of nucleotides 1-17 ( Figure 1) and a Notl restriction site and Primer #4 containing 20 T residues and a Notl restriction site.
  • Primers #3 and #4 were added to the T ⁇ P-1 cDNA library described above and PCR amplified for 6 cycles at 94 * C for 1 min, 50°C for 1 min, and 72 * C for 1 min, and for 24 cycles at 94 * C for 1 min, 60 * C for 1 rnin and 72 * C for 1 min.
  • IL-l ⁇ pro clones were isolated from a cDNA library prepared from pheripheral blood neutrophils. We found that neutrophils expressed IL-l ⁇ pro mRNA. We isloated two clones (p48 and p214) with IL-l ⁇ pro specific inserts of 1367 and 1360 base pairs, respectively. The DNA sequence shown in Figure 1 is a composit of all the IL-l ⁇ pro clones. The amino acids encoded by all of the IL-l ⁇ pro clones we found were identical.
  • IL-l ⁇ pro cDNA is approximately 1373 base pairs in length, including a stretch of A nucleotides corresponding to the poly (A) tail of mRNA. These A residues are preceded by two polyadenylation signals, AATAA, at 1316 and 1335 base pair.
  • the sequence has an open reading frame of 404 amino acids, starting with an initiator Met codon at nucleotide 18 and ending with a termination codon at nucleotide 1230. Initiation of translation could also begin with an in-frame Met codon at nucleotide 66. Both initiator Met codons have consensus Kozak translation initiation sequences. Polypeptides initiated with the Met residue at position 51 also have biological activity.
  • IL-l ⁇ pro is a cytoplasmic enzyme.
  • N-terminal amino acid is Asn (120)
  • the protease undergoes N-terminal processing resulting in removal of 119 amino acids or 69 amino acids if the alternate initiator codon is used. Deletion analysis has indicated that at least 107 amino acids are removed from the C-terminus. However, it appears that the full C-terminus is necessary for proper folding of the protease before approximately 107 C-terminal amino acids can be removed to insure biological activity for the protease.
  • the 5' primer (5'- ATATCGGTACCGCCTCCAGCATGCCTCCGGCAATGCCCACATC-3') contains an Asp718 restriction site and an initiator Met residue fused to the N-terminus of the enzyme (nucleotides 1-20).
  • the 3' primer (5'--
  • CTGCTAGATCTGCCCGCAGACATTCATACAG-3 contains a.Bgl2 restriction site and is complementary to nucleotides 883-902 of Figure 1.
  • the PCR generated fragment was ligated into pDC303 mammalian vector, as described in Mosley et al. Cell 59:335-348 1989.
  • Human IL-l ⁇ pro is preferably produced by recombinant DNA techniques.
  • a recombinant DNA expression system inserts a clone encoding human IL-l ⁇ pro polypeptide or a derivative thereof with biological activity into an expression vector.
  • the expression vector is inserted into a host cell.
  • the host ceU's protein synthesis machinery synthesizes the recombinant human IL-l ⁇ pro polypeptide.
  • Suitable host cells for expression of mammalian IL-l ⁇ pro polypeptides or derivatives thereof include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli.
  • Suitable prokaryotic hosts cells for transformation include, for example, E. coli, Bacillus s btilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below.
  • IL-l ⁇ pro polypeptides or derivatives thereof could also be employed to produce mammalian IL- l ⁇ pro polypeptides or derivatives thereof using RNAs derived from the DNA constructs disclosed herein.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985.
  • the nucleotide sequence e.g., structural gene
  • the leader sequence enables improved extracellular secretion of translated polypeptide by a yeast host cell.
  • the IL-l ⁇ pro polypeptide or derivative thereof may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host cell.
  • the N-terminal Met may be cleaved from the expressed recombinant IL-l ⁇ pro polypeptide or derivative thereof.
  • prokaryotic host cells may be used for expression of IL-l ⁇ pro polypeptides or derivatives thereof that do not require extensive proteolytic and disulfide processing.
  • the recombinant expression vectors carrying the recombinant IL-l ⁇ pro structural gene nucleotide sequence or derivative thereof are transfected or transformed into a substantially homogeneous culture of a suitable host microorganism or mammalian cell line.
  • suitable host cells include bacteria such as E. coli, yeast such as S. cerevisiae, or a mammalian cell line such as Chinese Hamster Ovary (CHO) ceUs.
  • Transformed host cells are ceUs which have been transformed or transfected with IL-l ⁇ pro or a derivative thereof structural gene nucleotide sequences.
  • Expressed IL-l ⁇ pro polypeptides will be located within the host cell and/or secreted into culture supernatant, depending upon the nature of the host cell and the gene construct inserted into the host cell.
  • Expression vectors transfected into prokaryotic host cells generally comprise one or more phenotypic selectable markers.
  • a phenotypic selectable marker is, for example, a gene encoding proteins that confer antibiotic resistance or that supply an autotrophic requirement, and an origin of replication recognized by the host to ensure amplification within the host
  • Other useful expression vectors for prokaryotic host cells include a selectable marker of bacterial origin derived from commercially available plasmids. This selectable marker can comprise genetic elements of the cloning vector pBR322 (ATCC 37017).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. The pBR322
  • backbone sections are combined with an appropriate promoter and an IL-l ⁇ pro structural gene sequence.
  • Other commercially vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA).
  • Promoter sequences are commonly used for recombinant prokaryotic host cell expression vectors. Common promoter sequences include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (tip) promoter system (Goeddel et al., Nucl.
  • a particularly useful prokaryotic host cell expression system employs a phage ⁇ PL promoter and a cI857ts thermolabile repressor sequence.
  • Plasmid vectors available from the American Type Culture CoUection which incorporate derivatives of the ⁇ P promoter include plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (resident in E. coli RR1 (ATCC 53082)).
  • Human IL-l ⁇ pro polypeptides and derivative polypeptides may be expressed in yeast host ceUs, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces , may also be employed.
  • yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, and sequences for transcription termination.
  • yeast vectors include an origin of replication sequence and selectable marker.
  • Suitable promoter sequences for yeast vectors include promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., /. Biol. Chem.255:2073, 1980) or other glycolytic enzymes (Hess et al, /. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem.
  • enolase such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP-A-73,657.
  • Yeast vectors can be assembled, for example, using DNA sequences from pBR322 for selection and replication in E. coli (Amr gene and origin of replication).
  • Other yeast DNA sequences that can be included in a yeast expression construct include a glucose- repressible ADH2 promoter and ⁇ -factor secretion leader.
  • the ADH2 promoter has been described by Russell et al. (J. Biol. Chem.258:2674, 1982) and Beier et al. (Nature 300:724, 1982).
  • the yeast ⁇ -factor leader sequence directs secretion of heterologous polypeptides.
  • the ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence.
  • a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
  • Yeast transformation protocols are known to those of skill in the art.
  • One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.
  • the Hinnen et al. protocol selects for Trp + transf ⁇ rmants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g ml uracil.
  • Yeast host cells transformed by vectors containing ADH2 promoter sequence may be grown for inducing expression in a "rich" medium.
  • a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
  • Mammalian or insect host cell culture systems could also be employed to express recombinant IL-l ⁇ pro polypeptide or derivatives thereof.
  • suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells (Gluzman Cell 23:115, 1981), L ceUs, C127 cells, 3T3 ceUs, Chinese hamster ovary (CHO) cells, HeLa cells, and BHK cell lines.
  • Suitable mammalian expression vectors include nontranscribed elements such as an origin of replication, a promoter sequence, an enhancer linked to the structural gene, other 5 * or 3' flanking nontranscribed sequences, such as ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Transcriptional and translational control sequences in mammalian host cell expression vectors may be provided by viral sources.
  • mammalian cell promoter sequences and enhancer sequences are derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
  • DNA sequences derived from the S V40 viral genome for example, S V40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide the other genetic elements required for expression of a structural gene sequence in a mammalian host cell.
  • Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind HI site toward the Bgl I site located in the SV40 viral origin of replication site is included.
  • mammalian genomic IL-l ⁇ pro promoter, control and/or signal sequences may be utilized, provided such control sequences are compatible with the host cell chosen.
  • Exemplary vectors can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983).
  • Purified human IL-l ⁇ pro polypeptides or derivatives thereof are prepared by culturing transformed host ceUs under culture conditions necessary to express IL-l ⁇ pro polypeptides or derivatives thereof.
  • the expressed polypeptides are purified from culture media or cell extracts.
  • supernatants from cultured transformed host cells can secrete recombinant IL-l ⁇ pro polypeptide into culture media.
  • the IL-l ⁇ pro polypeptide or derivative thereof is concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit Following the concentration step, the concentrate can be applied to a purification matrix.
  • a suitable purification matrix is an IL-l ⁇ pro inhibitor or an antibody molecule specific for an IL-l ⁇ pro polypeptide or derivative thereof and bound to a suitable support.
  • an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, ceUulose or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
  • one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., sUica gel having pendant methyl or other ahphatic groups, can be employed to further purify an IL-l ⁇ pro polypeptide composition.
  • RP- HPLC reverse-phase high performance liquid chromatography
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
  • some or aU of the steps used in the purification procedure described herein can also be employed.
  • Recombinant polypeptide produced in bacterial culture is usuaUy isolated by initial disruption of the host cells, extraction from cell peUets if an insoluble polypeptide, or from the supernatant if a soluble polypeptide, foUowed by one or more concentration, salting- out, ion exchange or size exclusion chromatography steps. FinaUy, reverse phase high performance liquid chromatography (RP-HPLC) can be employed for final purification steps.
  • Microbial cells can be disrupted by any convenient method, including freeze-fhaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • Transformed yeast host ceUs wiU generally express IL-l ⁇ pro polypeptide as a secreted polypeptide. This simplifies purification.
  • Secreted recombinant polypeptide from a yeast host ceU fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog.296:111, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
  • IL-l ⁇ pro Polypeptide and Derivative Compositions The present invention provides methods of using therapeutic compositions comprising an effective amount of IL-l ⁇ pro polypeptides and derivatives thereof in a suitable dUuent and carrier.
  • a suitable dUuent and carrier for therapeutic use, purified IL-l ⁇ pro or a biologicaUy active derivative thereof is administered to a patient, preferably a human, for treatment in a manner appropriate to the indication.
  • IL-l ⁇ pro compositions administered to suppress autoimmunity can be given by bolus injection, continuous infusion, sustained release from implants, or other suitable technique.
  • an IL-l ⁇ pro therapeutic agent wiU be administered in the form of a pharmaceutical composition comprising purified polypeptide in conjunction with physiologicaUy acceptable carriers, excipients or dUuents.
  • Such carriers wUl be nontoxic to patients at the dosages and concentrations employed.
  • the preparation of such compositions entaUs combining IL-l ⁇ pro with buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrans, chelating agents such as EDTA, glutathione and other stabihzers and excipients.
  • Neutral buffered sahne or sahne mixed with conspecific serum albumin are exemplary appropriate dUuents.
  • compositions of the present invention include one or more of the IL- l ⁇ pro inhibitor compounds of this invention formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are coUectively referred to herein as carriers, for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, and the like.
  • compositions can be administered to humans and animals either oraUy, rectally, parenteraUy (intravenously, intramuscularly or subcutaneously), intracisternaUy, intravaginally, intraperitoneaUy, locaUy (powders, ointments or drops), or as a buccal or nasal spray.
  • compositions suitable for parenteral injection may comprise physiologicaUy acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oUs (such as oUve oU) and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants
  • compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like.
  • Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin.
  • the compounds can be incorporated into slow release or targeted deUvery systems such as polymer matrices, Uposomes, and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. SoUd dosage forms for oral administration include capsules, tablets, piUs, powders and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fiUers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol and siUcic acid, (b) binders, as for example, carboxymethylceUulose, alignates, gelatin, polyvinylpyrroUdone, sucrose and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex siUcates and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, and (b) bind
  • SoUd compositions of a simUar type may also be employed as fiUers in soft and hard- fiUed gelatin capsules using such excipients as lactose or milk sugar as weU as high molecular weight polyethyleneglycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pUls and granules can be prepared with coatings and shells, such as enteric coatings and others well known in this art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes. The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceuticaUy acceptable emulsions, solutions, suspensions, syrups and elixirs.
  • th liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubiUzing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3- butyleneglycol, dimethylformamide, oUs, in particular, cottonseed oU, groundnut oil, com germ oU, olive oil, castor oU and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • inert diluents commonly used in
  • composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalUne ceUulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • suspending agents as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalUne ceUulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component
  • Dosage forms for topical adminwtration of a compound of this invention include ointments, powders, sprays and inhalants.
  • the active component is admixed under sterile conditions with a physiologicaUy acceptable carrier and any needed preservatives, buffers or propeUants as may be required.
  • Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • the compounds of the present invention can also be administered in the form of Uposomes.
  • Uposomes are generaUy derived from phosphoUpids or other Upid substances, uposomes are formed by mono- or multi-lameUar hydrated Uquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologicaUy acceptable and metabolizable lipid capable of forming Uposomes can be used.
  • the present compositions i liposome form can contain, in addition to the IL-l ⁇ pro inhibiting compounds of the present invention, stabiUzers, preservatives, excipients, and the like.
  • the preferred Upids are the phosphoUpids and the phosphatidyl cho ⁇ nes (lecithins), both natural and synthetic.
  • Uposomes are known in the art. See, for example, Methods in CeU Biologv. Ed. by Prcscott, Volume XIV, Academic Press, New York, N.Y., p. 33 et seq., (1976).
  • compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
  • the selected dosage level therefore depends upon the desired therapeutic effect, on the route of administration, on the desired duration of treatment and other factors.
  • the total da y dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 0.1 mg to about 160.0 mg pe lrilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daUy dose. It will be understood, however, that the specific dose level for any particular patient wiU depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated. The foUowing examples are for the purposes of iUustration and not by way of limitation.
  • This example Ulustrates the range of substrate specificity of purified human IL-l ⁇ pro enzyme to cleave a group of amino acid sequences.
  • a variety of peptide substrates were prepared featuring changes in individual amino acids in the region corresponding to the cleavage site in human precursor IL-l ⁇ (Hisl 15 to Prol 18). The reactivity of the peptide substrates was expressed relative to the peptide corresponding to Alal 12 to Serl21 of the precursor IL-l ⁇ sequence.
  • Substrate peptides were synthesized by soUd phase method (Merrifield J. Amer.
  • the substrate peptide resin mixtures were washed with diethyl ether and extracted with 15% (w/v) acetic acid, lyophUized and purified on reverse phase high performance liquid chromatography (RP-HPLC) on a Vydac C18, 2.2cm x 25 cm column.
  • RP-HPLC reverse phase high performance liquid chromatography
  • Trifluoroacetic acid (0.1%) in water was solvent A and 0.1% trifluoroacetic acid in acetonitrile was solvent B for the mobile phases.
  • the purified substrate peptides were characterized by amino acid analysis using a Beckman 6300 system, RP-HPLC and mass spectrometry. Mass spectra were obtained by either fast atom bombardment on a VG Trio-2 system with xenon as the ionizing gas and glycerol/thioglycerol (1:1) as the sample matrix or by 2 2Qf plasma desorption mass spectrometry on a Bio-Ion 20 mass spectrometer (See Tsarbopoulos Peptide Res.2:258-66 1989). In each case, the mass of the observed peptide substrate corresponded with the theoretical value.
  • Peptide solutions of standard concentration were prepared by dissolving about 2-3 mg of peptide substrate in water, loading the solution onto a Waters Sep-Pak C18 cartridge and washing three times with 5 ml of water The peptide substrates were eluted with acetonitrile and then evaporated to dryness. Each substrate was standardized to 1 mM by amino acid analysis prior to use. Purified human EL-l ⁇ pro enzyme (lO ⁇ l), peptide substrate in water (lO ⁇ l), and 10 mM Tris buffer, pH 8.0 containing 25% v/v glycerol (lO ⁇ l) were mixed and the mixtures were incubated at 37°C for four hours.
  • the reaction was quenched with by adding 1 M glycine/HCl buffer pH 2.0 (lO ⁇ l).
  • the samples were then analyzed using RP-HPLC with a Vydac C18 column (0.46 cm x 25 cm) and eluting with a Unear gradient from 100% solvent A to 70% solvent A 30% solvent B over 30 min at a flow rate of 1 ml/min.
  • the effluent was monitored at 280 nm except with digestions of Ac- VHDAPV-NH2 and Ac- HDAP-NH2 (see Example 2) which were monitored at 220 nm.
  • Table 1 shows the relative reactivities of a series of eight peptide substrates that were subject to digestion by purified IL-l ⁇ pro enzyme. TABLE 1
  • the cleavage site can be described with the corresponding human precursor IL-l ⁇ amino acid residues as follows:
  • Peptides 7 and 8 represent changes to the P2 and P2' sites, respectively. Changing the proUne of peptide 1 to an alanine yielded a substrate which was still cleaved by human IL-l ⁇ pro but only half as efficiently as the peptide with human IL-l ⁇ native sequence. A simUar result was obtained when the histidine of peptide 1 was replaced with a phenylalanine.
  • This example Ulustrates the effect of substrate peptide length on the abiUty of human IL-l ⁇ pro enzyme to cleave peptide substrates.
  • the experiment was conducted as described in Example 1.
  • Five substrate peptides were made that correspond to the amino acid sequence of the IL-l ⁇ pro cleavage site of human precursor IL-l ⁇ . The results are shown in Table 2 below:
  • IL-l ⁇ pro has a minimum number of amino acid residues necessary for substrate peptide cleavage.
  • A. Synthesis of Boc-Asp-CH2F A suspension of Boc-Asp-OH (8.11 mmol) and fluoroacetic anhydride (16.2 mmol) in benzene (30 ml) was treated with triethylamine (16.2 mol) at room temperature. The catalyst dimethylaminopyridine (0.41 mmol) was added to the solution and the reaction stirred for abo 2 h at room temperature. About 100 ml benzene was added to the reaction mixture.
  • Boc-Asp-CH2F prepared in accordance with the method of Example 3A above may be dissolved in trifluoroacetic acid (TFA) and the mixture stirred for about 5 minutes at about 23"C.
  • Cold ether may then be added to the mixture.
  • the ether is evaporated and toluene adde to co-evaporate residual TFA.
  • the deprotected peptide (H-Asp-CH2F) is obtained as a TFA salt.
  • the deprotected peptide may then be coupled to a protected amino acid (i.e.
  • Boc-HisOH Boc-ProOH, Boc-TyrOH, Boc-PheOH using a standard symmetric anhydride procedure employing dicyclohexylcarbodiimide as a coupUng reagent Bodanszky, supra.
  • the Boc protecting group may be removed from the compounds made in accordance with the method of Example 3B using trifluoroacetic acid as described above. Each deprotect compound may then be acetylated with acetic anhydride and dUsopropylamine (DIAE) according to standard techniques. Stuart et al. supra.
  • DIAE dUsopropylamine
  • the Boc protecting group may be removed from Boc-Asp-CH2F prepared according t the method of Example 3A using TFA as described above.
  • Benzyloxycarbonyl-protected ami acids i.e. Cbz-His-OH, Cbz-Phe-OH, Cbz-Tyr-OH, Cbz-Pro-OH
  • commerci sources Bachem, Philadelphia, PA
  • Boc-Asp-CH2F was tested for its abUity to inhibit IL-l ⁇ pro catalyzed degradation of precursor DL-l ⁇ using an in vitro assay method.
  • IL-l ⁇ precursor IL-l ⁇ (about 50 ⁇ g ml in PBS) was mixed with 10 ⁇ l of purified IL- l ⁇ pro (15-75 ⁇ g ml in PBS) and incubated at 37 * C for 30 min. The incubation was terminate by placing the samples on dry ice or by the addition of SDS sample buffer. PMSF (phenylmethanesulfonyl) was then added to a concentration of 1 mM, and the samples were dialyzed against water. After dialysis, the samples were concentrated to dryness in a Speed- Va concentrator and dissolved in SDS sample buffer. SDS-PAGE was carried out with 12% polyacrylamide gels.
  • PMSF phenylmethanesulfonyl
  • the gels were placed in transfer buffer (0.192 M glycine. 0.025 M Tris-HCl (pH 8.3), 20% v/v methanol), and protein was then electrophoresed onto nitrocellulose (Sartorius) in a Hoeffer transfer apparatus (1 h at maximum voltage).
  • the nitrocellulose was subsequently placed in 20 mM sodium phosphate, pH 7.4 (PBS) containing 3% bovine serum albumin for at least 15 min at room temperature.
  • PBS sodium phosphate, pH 7.4
  • MAb monoclonal antibody specific for mature IL-l ⁇ to probe the blot. MAb was added to a concentration of 9 ⁇ g ml, and the incubation was continued for 30 min.
  • the blot was then rinsed three times with PBS and was developed with a solution obtained by mixing 6 mg of horseradish peroxidase developing reagent (Bio-Rad) dissolved in 2 ml methanol and hydrogen peroxide (60 ⁇ l dUuted into 10 ml of Tris-buffered sahne).
  • the data show that Boc-Asp-Ch2F completely inhibits the generation of mature IL- l ⁇ from precursor IL-l ⁇ at a concentration of 5 ⁇ M and partiaUy inhibits generation of mature IL-l ⁇ at a concentration of 1 ⁇ M.
  • EXAMPLE 5 This example iUustrates biological activity of IL- l ⁇ pro when transfected into COS ceUs.
  • a mammaUan cell expression vector pDC303.
  • This plasmid was co-transfected into COS-7 cells (monkey kidney) with a second mammalian expression plasmid containing a cDNA encoding precursor IL-l ⁇ .
  • ceUs were radiolabeled with 35s and IL-l ⁇ specific proteins were immunoprecipitated from cell lysates. The immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
  • transfected COS-7 ceUs can process precursor IL-l ⁇ to mature IL-l ⁇ only if the ceUs were co-transfected with a plasmid encoding IL-l ⁇ pro.
  • Cells co-transfected with a control plasmid or ceUs mock transfected did not show any processing of precursor IL-l ⁇ .
  • IL-l ⁇ pro lacing the N-terminal 119 amino acids enables ceUs to process precursor IL-l ⁇ to the mature form of this protein.
  • Gin Leu Arg lie Ser Asp His His Tyr Ser Lys Gly Phe Arg Gin Ala 50 55 60
  • Lys Lys Met Glu Lys Arg Phe Val Phe Asn Lys lie Glu lie Asn Asn 210 215 220
  • Lys Leu Glu Phe Glu Ser Ala Gin Phe Pro Asn Trp Tyr lie Ser Thr 225 230 235 240
  • GCACACCGCC CAGAGCACAA GTATATGAGG GCGGACCTCT GACAGCACGT TCCTGGTGTT 900
  • GAAAGCCCAC ATAGAGAAGA AACTAAATAG TTGAGATTTT ATCGCTTTCT GCTCTTCCAC 1260

Abstract

Polypeptide isolé et ses dérivés ayant une activité biologique de protéase pour le précurseur humain IL-1beta et pour un substrat comprenant : R1-Asp-R2-R3 dans lequel R1 et R3 représentent indépendamment n'importe quel amino-acide isomère, R2 représente Ala ou Gly, et dans lequel n'importe quel site de clivage de protéase spécifique se trouve entre Asp et R2. Sont également proposés des composés inhibiteurs, des compositions et des procédés servant à inhiber l'activité de Interleukin 1beta protéase. Les composés inhibiteurs comprennent une séquence amino-acide de 1 à environ 5 amino-acides ayant un groupe de blocage N-terminal et un reste Asp C-terminal connecté à un groupe partant électronégatif, dans lequel la séquence amino-acide correspond à la séquence Ala-Tyr-Val-His-Asp
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