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Interleukin 1beta protease and interleukin 1beta protease inhibitors

a protease inhibitor and interleukin 1beta technology, applied in the direction of peptides, enzymology, peptide/protein ingredients, etc., can solve the problems of not further defining or isolating the responsible polypeptide, and none of the previously described protease inhibitors are effective in inhibiting the activity of interleukin 1 proteas

Inactive Publication Date: 2006-08-24
VERTEX PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The solution enables the effective processing of precursor IL-1β into mature IL-1β for therapeutic applications, including treating inflammation and autoimmune diseases, by specifically inhibiting IL-1β pro activity, thus providing a targeted approach to modulate IL-1β levels and reduce inflammatory responses.

Problems solved by technology

However, Kostura et al. did not further define or isolate the responsible polypeptide.
None of the previously described protease inhibitors are effective in inhibiting the activity of interleukin 1β protease.

Method used

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  • Interleukin 1beta protease and interleukin 1beta protease inhibitors
  • Interleukin 1beta protease and interleukin 1beta protease inhibitors
  • Interleukin 1beta protease and interleukin 1beta protease inhibitors

Examples

Experimental program
Comparison scheme
Effect test

example 1

Substrate Specificity of IL-1β pro

[0145] This example illustrates the range of substrate specificity of purified human IL-1β 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-1β (His 115 to Pro 118). The reactivity of the peptide substrates was expressed relative to the peptide corresponding to Ala 112 to Ser 121 of the precursor IL-1β sequence.

[0146] Substrate peptides were synthesized by solid phase method [Merrifield, J. Amer. Chem. Soc., 86:304-05 (1964)] using either an Applied Biosystems 430A peptide synthesizer or by the manual T-bag approach of Houghten [Proc. Nat. Acad. Sci. USA, 82:5131-35 (1985)]. 4-Methyl benzhydrylamine resin was used. The substrate peptides were acteylated prior to cleavage from resin, by liquid HF (0° C., 1 hr) in the presence of anisole as scavenger (HF:anisole 9:1). After evaporation o...

example 2

Effect of Substrate Length

[0154] This example illustrates the effect of substrate peptide length on the ability of human IL-1β 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-1β pro cleavage site of human precursor IL-1β. The results are shown in Table 2 below:

TABLE 2+HX,1 / 32 !? ? Reactivity? ? !?? Relative? !Peptide? Sequence? To Peptide 11Ala-Tyr-Val-His-1.00Asp-Ala-Pro-Val-Arg-Ser-(Seq. I.D. No. 13)9Glu-Ala-Tyr-Val-0.74His-Asp-Ala-Pro-(Seq. I.D. No. 21)10Tyr-Val-His-Asp-2.40Ala-Pro-Val-Arg-(Seq. I.D. No. 22)11Val-His-Asp-Ala-Not cleavedPro-Val-(Seq. I.D. No. 23)12His-Asp-Ala-Pro-Not cleaved(Seq. I.D. No. 24)

The eight amino acid peptide (Ac-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-NH2) is cleaved most efficiently while the four and six amino acid peptides are not cleaved. Thus, IL-1β pro has a minimum number of amino acid residues necessary for substra...

example 3

Synthesis of IL-1β Protease Inhibitors

[0155] A. Synthesis of Boc-Asp-CH2F.

[0156] 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 4-dimethylaminopyridine (0.41 mmol) was added to the solution and the reaction stirred for about 2 h at room-temperature. About 100 ml benzene was added to the reaction mixture. The organic solution was washed with 1N HCl (2×50 ml), saturated NaHCO3 (2×50 ml), and saturated NaCl (2×50 ml), followed by drying over anhydrous MgSO4. The solvent was then removed by evaporation under reduced pressure. The resulting oil was applied to a 2.5×80 cm column of silica gel (60-200 mesh). The title compound was eluded with 2% methanol in chloroform.

[0157]

[0158] B. Synthesis of Boc-His-Asp-CH2F, Boc-Tyr-Asp-CHF and Boc-Phe-Asp-CH2F.

[0159] Boc-Asp-CH2F prepared in accordance with the method of Example 3A above may be dissolved in trifluoroaceti...

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Abstract

There is disclosed an isolated polypeptide and derivatives thereof having protease biological activity for human precursor IL-1β and for a substrate comprising: R1-Asp-R2-R3 wherein R1 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. Inhibitor compounds, compositions and methods for inhibiting Interleukin 1β protease activity are also disclosed. The inhibitor compounds comprise an amino acid sequence of from 1 to about 5 amino acids 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 the sequence Ala-Tyr-Val-His-Asp.

Description

TECHNICAL FIELD OF THE INVENTION [0001] This invention relates to an interleukin 1β protease enzyme (IL-1β pro) having biological activity to cleave inactive precursor interleukin-1β (IL-1β) polypeptides into active mature IL-1β polypeptides, More specifically, the invention provides an isolated IL-1β 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-1β. The present invention further provides a group of compounds that can inhibit IL-1β pro activity; and thereby function as IL-1 antagonists. BACKGROUND OF THE INVENTION [0002] Interleukin 1β (IL-1β) is a 17.5 kDa polypeptide hormone synthesized and secreted by stimulated nonocytes. The initial translation product of IL-1β is a larger 31 kDa biologically inactive precursor polypeptide. The N-terminus of biologically active, mature IL-1β derived from human activated monocytes has been characterized by an N-terminal amino ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/20C07K14/545C07H21/04C12P21/04
CPCA61K38/00C12N9/6421C12N9/6475
Inventor SLEATH, PAULBLACK, ROYKRONHEIM, SHIRLEY
Owner VERTEX PHARMA INC
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