U.S. patent application number 11/213530 was filed with the patent office on 2006-08-24 for interleukin 1beta protease and interleukin 1beta protease inhibitors.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. Invention is credited to Roy A. Black, Shirley R. Kronheim, Paul R. Sleath.
Application Number | 20060188474 11/213530 |
Document ID | / |
Family ID | 35734207 |
Filed Date | 2006-08-24 |
United States Patent
Application |
20060188474 |
Kind Code |
A1 |
Sleath; Paul R. ; et
al. |
August 24, 2006 |
Interleukin 1beta protease and interleukin 1beta protease
inhibitors
Abstract
There is disclosed an isolated polypeptide and derivatives
thereof having protease biological activity for human precursor
IL-1.beta. and for a substrate comprising:
R.sub.1-Asp-R.sub.2-R.sub.3 wherein R.sub.1 and R.sub.3 are
independently any D or L isomer amino acid, R.sub.2 is Ala or Gly,
and wherein the specific protease cleavage site is between Asp and
R.sub.2. Inhibitor compounds, compositions and methods for
inhibiting Interleukin 1.beta. 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.
Inventors: |
Sleath; Paul R.; (Seattle,
WA) ; Black; Roy A.; (Seattle, WA) ; Kronheim;
Shirley R.; (Seattle, WA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
1251 AVENUE OF THE AMERICAS FL C3
NEW YORK
NY
10020-1105
US
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Cambridge
MA
|
Family ID: |
35734207 |
Appl. No.: |
11/213530 |
Filed: |
August 25, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09670106 |
Sep 26, 2000 |
6995141 |
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11213530 |
Aug 25, 2005 |
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09039657 |
Mar 16, 1998 |
6136787 |
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09670106 |
Sep 26, 2000 |
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08440179 |
May 12, 1995 |
5756465 |
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09039657 |
Mar 16, 1998 |
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08203716 |
Feb 28, 1994 |
5416013 |
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08440179 |
May 12, 1995 |
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07750644 |
Aug 30, 1991 |
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08203716 |
Feb 28, 1994 |
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07505298 |
Apr 4, 1990 |
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07750644 |
Aug 30, 1991 |
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07656759 |
Feb 13, 1991 |
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07750644 |
Aug 30, 1991 |
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Current U.S.
Class: |
424/85.2 ;
435/320.1; 435/325; 435/69.52; 530/351; 536/23.5 |
Current CPC
Class: |
C12N 9/6475 20130101;
C12N 9/6421 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/085.2 ;
435/069.52; 435/320.1; 435/325; 530/351; 536/023.5 |
International
Class: |
A61K 38/20 20060101
A61K038/20; C07K 14/545 20060101 C07K014/545; C07H 21/04 20060101
C07H021/04; C12P 21/04 20060101 C12P021/04 |
Claims
1. An isolated polypeptide having protease activity for a specific
protease cleavage site, wherein the protease activity is specific
for a substrate peptide having an amino acid sequence comprising:
R.sub.1-Asp-R.sub.2-R.sub.3 wherein R.sub.1 and R.sub.3 are
independently any D or L isomer amino acid, R.sub.2 is Ala or Gly,
and wherein the specific protease cleavage site is between Asp and
R.sub.2.
2. The isolated polypeptide of claim 1 wherein the substrate
peptide is at least eight amino acids in length.
3-13. (canceled)
14. A method for improving wound healing at a wound site comprising
administering a pharmaceutical composition to the wound site
comprising the isolated polypeptide of claim 1 in a suitable
pharmaceutical carrier.
15. A method for treating arthritis comprising administering a
pharmaceutical composition comprising the isolated polypeptide of
claim 1 in a suitable pharmaceutical carrier.
16. A method for treating an autoimmune disease in a susceptible
individual comprising administering a pharmaceutical composition
comprising the isolated polypeptide of claim 1 in a suitable
pharmaceutical carrier.
17. The method of claim 16 wherein the autoimmunedisease is
selected from the group consisting of Insulin-dependent diabetes
melitus, Graves' disease, Hashimotos disease and a lupus
disease.
18. A method for reducing the detrimental side effects of radiation
treatment comprising administering a pharmaceutical composition
comprising the isolated polypeptide of claim 1 in a suitable
pharmaceutical carrier.
19-49. (canceled)
Description
TECHNICAL FIELD OF THE INVENTION
[0001] This invention relates to an interleukin 1.beta. protease
enzyme (IL-1.beta. pro) having biological activity to cleave
inactive precursor interleukin-1.beta. (IL-1.beta.) polypeptides
into active mature IL-1.beta. polypeptides, More specifically, the
invention provides an isolated IL-1.beta. 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-1.beta.. The present invention further provides a group
of compounds that can inhibit IL-1.beta. pro activity; and thereby
function as IL-1 antagonists.
BACKGROUND OF THE INVENTION
[0002] Interleukin 1.beta. (IL-1.beta.) is a 17.5 kDa polypeptide
hormone synthesized and secreted by stimulated nonocytes. The
initial translation product of IL-1.beta. is a larger 31 kDa
biologically inactive precursor polypeptide. The N-terminus of
biologically active, mature IL-1.beta. 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-1.beta..
[0003] Many physlologlcal actions and biological activities of IL-1
have been identified. IL-I biological activity is often determined
by assaying for stimulation of thymocyte proliferation or by
measuring interleukin-2 (IL-2) biological activity. IL-I activities
include stimulation of B-lymphocyte maturation, lymphocyte
proliferation, stimulation of fibroblast growth and induction of
acute-phase protein synthesis by hepatocytes.
[0004] Other biological activities have been attributed to IL-1
polypeptides. These include control of differentiation and
activation of lymphocytes, stimulation of lymphokine and
prostaglandin production, promotion of inflammation, induction of
acute phase proteins, stimulation of bone resorption, and
alteration of the level of iron and zinc in blood. Moreover, it has
recently been found that IL-1 can stimulate the
hypothalamus-pituitary-adrenal axis, suggesting that IL-1 is
integrated in the complex neuroendocrine network that controls
homeostasis. This is further supported by the finding that
administration of low doses of IL-I to normal mice results in both
a several-fold elevation of glucocorticoid output and in a
long-lasting blood glucose concentration increase.
[0005] The N-terminal Ala residue of human mature IL-1.beta. is in
the 117 position and an Asp residue is in the 116 position counting
from the N-terminus of human precursor IL-1.beta. polypeptide.
Mature IL-1.beta. consists of the C-terminal 153 residues of the
precursor polypeptide.
[0006] Maturation and release of mature IL-1.beta. from macrophages
does not proceed by conventional means normally associated with
most secretory proteins because the precursor IL-1.beta.
polypeptide lacks a hydrophobic signal sequence. Further,
IL-1.beta. 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.
Most secreted proteins have a signal sequence at the amino terminal
that is removed upon translocation. Other proteins, such as
ovalbumin, have an internal signal sequence that is not removed
upon translocation. Both precursor forms of IL-1.alpha. and
IL-1.beta. (March et al.) lack any region (either amino terminal or
internal) with sufficient hydrophobicity and length to qualify as a
signal sequence.
[0007] A further indication of an unusual maturation pathway for
IL-1.beta. 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- 1.beta. polypeptide. Moreover, Young et al., J. Cell Bio.,
107:447-56 (1988) found that fibroblasts transfected with cDNA
coding for precursor IL-1.beta. were unable to process the
precursor polypeptide into mature IL-1.beta.. Instead, the
transfected fibroblasts produced high levels of inactive precursor
polypeptide. The results reported by Young et al. are consistent
with other reports for other cell types, including T cells,
epidermal cells and B cells.
[0008] Hazuda et al., J. Biol. Chem., 263:8473-79 (1988) have
reported that both the precursor and mature forms of IL-1.beta.
appear in the supernatants of activated monocytes with little or no
preference. Hazuda et al. suggest that IL-1.beta. processing is
"intimately coordinated" with secretion.
[0009] There have been several attempts to characterize or isolate
the system responsible for processing IL-1.beta. from its
translated precursor form to its active mature form. Black et al.,
J. Biol. Chem., 263:9437-42 (1988) [Black et al. I] suggest that
the cleavage pattern of precursor IL-1.beta. is affected by myeloid
cell membranes and results from the action of a plurality of
proteases which act as an IL-1.beta. processing system. A
subsequent article by Black et al., J. Biol. Chem., 264:5323-26
(1989) [Black et al. II] describes a single protease that cleaves
IL-1.beta. between His.sup.115 and Asp.sup.16, one residue upstream
form the N-terminal Ala.sup.117 of mature IL-1.beta.. Thus, the
protease described in Black et al. II generates a form of
IL-1.beta. one amino acid longer than the mature IL-1.beta.
purified from monocyte cultures. Black et al. II suggests that
there may be an aminopeptidase in human blood that removes the
N-terminal asparate residue to complete the processing.
[0010] Kostura et al., Proc. Nat Acad Sci. USA, 86:5227-31 (1989)
refers to a protease with a similar cleavage pattern but
"qualitatively different" from the protease described in Black et
al. II. 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.
[0011] Finally, Black et al., FEBS Lett., 247:386-90 (1989) [Black
et al. III] refer to a protease that generates mature IL-1.beta.
from the precursor polypeptide and is characterized by being
inhibited by iodoacetate and N-ethylmaleimide. Black et al. III
attempted to purify their protease approximately 500 fold by a
process starting by freeze-thawing cell lysates from THP-1 cells
(ATTC) four times. Black et al. III centrifuged the lysates for 20
minutes at 36,590.times.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 get filtration with Sephadex G-75. This procedure
described in Black et al. III failed to demonstrate whether the
protease activity was due to a single polypeptide or a group of
processing enzymes.
[0012] Therefore, there is a need in the art to obtain the isolated
system or single protease polypeptide responsible for processing
precursor IL-1.beta. into its mature and biologically active form.
The protease functions as an IL-1 agonist to increase IL-1
biological activity in vivo. Moreover, the isolated protease is
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.
[0013] Further, isolation and characterization of the protease
responsible for processing precursor IL-1.beta. into its
biologically active form aids in designing inhibitors for
IL-1.beta. processing, because the availability of large quantities
of IL-1.beta. pro serves as a useful screening vehicle for finding
compounds having IL-1 antagonist activity. Such IL-1 antagonists or
IL-1.beta. pro inhibitors are useful for treating inflammation and
transplantation rejection.
[0014] A number of protease inhibitors specific for other protease
activities have been described and reported in the literature. See.
e.g, U.S. Pat. Nos. 4,644,055, 4,636,492 and 4,652,552. None of
these previously reported protease inhibitors are specific for
interleukin 1.beta. protease activity. None of the previously
described protease inhibitors are effective in inhibiting the
activity of interleukin 1.beta. protease. Therefore, there is a
need for a specific IL-1.beta. pro inhibitor that can prevent the
cleavage of preIL-1.beta. into biologically active IL-1.beta.. Such
an inhibitor can function as an IL-I antagonist.
[0015] This invention provides IL-I antagonists that prevent the
formation of biologically active IL-1.beta..
SUMMARY OF THE INVENTION
[0016] 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:
R.sub.1-Asp-R.sub.2-R.sub.3 wherein R.sub.1 and R.sub.3 are
independently any D or L isomer amino acid, R.sub.2 is Ala or Gly,
and wherein the specific protease cleavage site is between Asp and
R.sub.2. Preferably, the substrate peptide is at least eight amino
acids in length. The isolated polypeptide is called the interleukin
1 protease (IL-l.beta. pro) because it cleaves precursor IL-1.beta.
polypeptide to yield mature IL-1.beta. polypeptide at a cleavage
site between the Asp 116 and Ala 117 residues. This region of
precursor IL-1.beta. corresponds to a species within the genus of
protease cleavage sites described herein.
[0017] IL-1.beta. pro is further characterized by a cDNA (Seq. I.D.
No. 1) and amino acid sequence (Seq. I.D. No. 2) in FIG. 1. Full
length (precursor) IL-1.beta. pro comprises 404 amino acids.
Purified IL-1.beta. pro begins with the Asn-Pro-Ala-Met-Pro
sequence beginning with amino acid 120. Based upon a molecular
weight analysis, the approximate C-terminus of mature IL-1.beta.
pro is about amino acid 297. However, molecular weight
determination indicates that the C-terminus of the mature
IL-1.beta. pro enzyme is from about amino acid 278 to about amino
acid 315. The present invention comprises an isolated IL-1.beta.
pro polypeptide or a derivative, analog, or allelic variant thereof
displaying biological activity to proteolytically cleave human
precursor IL-1.beta. polypeptide at a cleavage site between the Asp
116 and Ala 117 residues.
[0018] FIG. 1 also shows a nucleotide sequence encoding a 404 amino
acid polypeptide having IL-1.beta. pro biological activity. The
present invention further comprises an isolated. DNA sequence
encoding IL-1.beta. pro or a derivative, analog or allelic variant
thereof displaying biological activity to proteolytically cleave
human precursor IL-1.beta. 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
FIG. 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 FIG. 1 sequence from nucleotide 1 to nucleotide 1232 and encode
a polypeptide displaying biological activity to proteolytically
cleave human precursor IL-1.beta. 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-1.beta. pro polypeptide encoded by any of the foregoing DNA
inserts and sequences.
[0019] 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.
[0020] The present invention also 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. Preferably, the amino acid sequence corresponds to at least
a portion of the amino acid sequence Ala-Tyr-Val-His-Asp.
[0021] The inhibitor compounds of the present invention have the
formula: Z-Q.sub.2-Asp-Q.sub.1 Where Z is an N-terminal protecting
group; Q.sub.2 is 0 to 4 amino acids such that the sequence
Q.sub.2-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. 3; and Q.sub.1 comprises an electronegative leaving group. Z is
preferably C.sub.1-C.sub.6 alkylketone, benzyl, acetyl,
alkoxycarbonyl, benzloxycarbonyl or C.sub.1-C.sub.6 alkylcarbonyl.
More preferably, Z is t-butoxycarbonly (t-Boc), acetyl carbonyl or
benzyloxycarbonyl (Cbz).
[0022] Q.sub.1 is preferably C.sub.1-C.sub.3 alkyl, an aldehyde
diazomethyl ketone or halomethyl ketone. More preferably, Q.sub.1
is an aldehyde or fluoromethyl ketone.
[0023] The present invention further provides reversible and
irreversible IL-1.beta. 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 Seq. I.D. No. 3.
[0024] Reversible IL-1.beta. 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 Seq. I.D. No. 3.
[0025] The present invention also provides a method of inhibiting
the physiological actions of interleukin 1.beta. in a mammal in
need of such treatment, comprising administering to said mammal an
effective amount of a compound of the formula:
Z-Q.sub.2-ASP-Q.sub.1 where Z is an N-terminal protecting group;
Q.sub.2 is 0 to 4 amino acids such that the sequence Q.sub.2-Asp
corresponds to at least a portion of the sequence
Ala-Tyr-Val-His-Asp, residues 112 to 116 of Seq. I.D. No. 3; and
Q.sub.1 comprises an electronegative leaving group.
[0026] In a preferred embodiment, Q.sub.1 is a fluoromethyl ketone
and inhibition is irreversible. In another preferred embodiment,
Q.sub.1 is an aldehyde moiety and inhibition is reversible.
[0027] The present invention still further provides 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
Seq. I.D. No. 3.
[0028] 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 groups 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 Seq. I.D. No. 3.
[0029] 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-1.beta. protease
or a biologically active derivative thereof in a suitable
pharmaceutical carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] In the drawings forming a portion of this disclosure:
[0031] FIG. 1 shows the DNA (Seg. I.D. No. 1) and corresponding
amino acid sequence (Seq. I.D. No. 2) for a polypeptide having
IL-1.beta. pro biological activity and corresponding to human
mature IL-1.beta. pro or a biologically active fragment
thereof.
[0032] FIG. 2 is the amino acid sequence of preIL-1.beta. (Seq.
I.D. No. 3), as published by March et al., Nature (London),
315(6021):641-647 (1985). The amino acid residues are numbered
(underneath the sequence) beginning with the initiator
methionine.
[0033] FIG. 3 shows a Western Blot analysis of products generated
by IL-1.beta. pro in the presence and absence of the IL-1.beta. pro
inhibitor Boc-Asp-CH.sub.2F. Products were subjected to SDS-PAGE,
transferred to nitrocellulose, and probed with an antibody raised
against the COOH terminus of mature IL-1. Lane 3. preIL-1.beta.
incubated with 25 .mu.M of inhibitor; lane 4. preIL-1.beta.
incubated with 10 .mu.M of inhibitor; lane 5. preIL-1.beta.
incubated with 5 .mu.M inhibitor, lane 6. preIL-1.beta. incubated
with 1 .mu.M inhibitor.
DETAILED DESCRIPTION OF TEE INVENTION
[0034] I. Interleukin 1.beta. Protease
[0035] Utilizing polymerase chain reaction (PCR) procedures and
other techniques, we have isolated, purified, characterized, and
expressed a mammalian IL-1.beta. pro polypeptide and active
fragments thereof.
[0036] The availability of abundant quantities of a recombinant
IL-1.beta. pro enzyme has further allowed us to find inhibitor
compounds capable of inhibiting IL-1.beta. pro activity and thereby
function as IL-1 antagonists. Further, use of IL-1.beta. pro has
IL-1 agonist activity. Thus, the invention relates to mammalian
IL-1.beta. pro polypeptides, derivatives, analogs and allelic
variants thereof having proteolytic activity for a substrate
peptide having an amino acid sequence comprising:
R.sub.1-Asp-R.sub.2-R.sub.3 wherein R.sub.1 and R.sub.3 are
independently any D or L isomer amino acid, R.sub.2 is Ala or Gly,
and wherein the specific protease cleavage site is between Asp and
R.sub.2. Preferably, the substrate peptide is at least eight amino
acids in length. Most preferably, R.sub.2 is Gly. Mammalian
IL-1.beta. pro is preferably a human IL-1.beta. pro and has
substrate specificity for a substrate peptide having the amino acid
sequence described herein. Preferably, the human IL-1.beta. pro
polypeptide or derivative thereof is a polypeptide having
biological activity that cleaves human precursor IL-1.beta.
polypeptide to yield human mature IL-1.beta. polypeptide.
[0037] IL-1.beta. pro is further characterized by the cDNA (Seq.
I.D. No. 1) and amino acid sequence (Seq. I.D. No. 2) in FIG. 1.
Full length (precursor) IL-1.beta. pro comprises 404 amino acids.
Purified IL-1.beta. pro begins with the Asn-Pro-Ala-Met-Pro
sequence beginning with amino acid 120. Based upon a molecular
weight analysis, the approximate C-terminus of mature IL-1.beta.
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-1.beta. pro polypeptide or a
derivative, analog, or allelic variant thereof displaying
biological activity to proteolytically cleave a human precursor
IL-1.beta. polypeptide at a cleavage site between the Asp 116 and
Ala 117 residues. For the purposes of this application, the term
"IL-1.beta. pro" shall encompass the amino acid sequence shown in
FIG. 1, plus all allelic variants, derivatives, analogs and
fragments of this sequence that display IL-1.beta. pro biological
activity.
[0038] IL-1.beta. pro biological activity is determined, for
example, by assaying for IL-1 activity with a precursor IL-1.beta.
polypeptide. Precursor IL-1.beta. is inactive, while mature
IL-1.beta. is an active IL-1 polypeptide. A method for measuring
IL-1.beta. pro activity is described in Black et al. II. Briefly,
this method provides approximately five microliters of precursor
IL-1.beta. (pre IL-1.beta.) (10-50 .mu.g/ml prepared as described
in Black et al. I) incubated with 10 .mu.l of IL-1.beta. pro
polypeptide or another substance suspected of having IL-1.beta. pro
biological activity. The incubation proceeds for approximately one
hour at approximately 37.degree. C. and is terminated by the
addition of 15 .mu.l of 2.times.SDS sample buffer followed by
boiling for five minutes. The boiled sample is electrophoresed on a
SDES-polyacrylamide gel and placed onto a Western blot using an
IL-1.beta. C-terminal-specific monoclonal antibody, such as 16F5
described in Black et al. I.
[0039] FIG. 1 also shows a nucleotide sequence encoding a 404 amino
acid sequence having IL-1.beta. pro biological activity. The
present invention further comprises an isolated DNA sequence
encoding IL-1.beta. pro or a derivative, analog or allelic variant
thereof displaying biological activity to proteolytically cleave a
human precursor IL-1.beta. 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
FIG. 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 FIG. 1 sequence from nucleotide 1 to nucleotide 1232 and encode
a polypeptide displaying biological activity to proteolytically
cleave a human precursor IL-1.beta. 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-1.beta. pro polypeptide encoded by any of the foregoing DNA
inserts and sequences.
[0040] Inventive DNA sequences that detectably hybridize to the
FIG. 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.degree. C. in a 6.times.SSC solution
followed by washing at about 68.degree. C. in a 0.6.times.SSC
solution.
[0041] Antisense oligonucleotides can be synthesized (by
conventional phosphodiester techniques such as by Synthecell,
Rockville, Md.) that are complementary to unique regions of at
least 18 bases at the initiation codon (TACCGGCTGTTCCAGGAC, Seq.
I.D. No. 4) or (TACCTATTCTGGGCTCGA, Seq. I.D. No. 5) complementary
to bases 18-36 and 168 to 196, respectively in FIG. 1, at the
N-terminus of mature IL-1.beta. pro (TTGGTCGATACGGGTGT, Seq. I.D.
No. 6) complementary to bases 374 to 392 in FIG. 1, at the
approximate C terminus after protease cleavage (CACCACACCAAATTTCTA,
Seq. I.D. No. 7) complementary to bases 890 to 908 in FIG. 1, or at
a region immediately 5' to the termination codon
(ATGGAGAAGGGTCCTGTA, Seq. I.D. No. 8) complementary to bases 1205
to 1229 in FIG. 1.
[0042] The primary amino acid structure of IL-1.beta. 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 amino acid sequence mutants or derivatives. Covalent
derivatives of IL-1.beta. pro are prepared by linking particular
functional groups-to IL-1.beta. pro amino-acid side chains or at
the N-terminus or C-terminus of the IL-1.beta. pro polypeptide.
[0043] Other derivatives of IL-1.beta. pro within the scope of this
invention include covalent or aggregative conjugates of IL-1.beta.
pro or its fragments with other proteins or polypeptides, such as
by synthesis in recombinant culture as N-terminal or C-terminal
fusions. For example, the conjugated polypeptide may be a signal
(or leader) polypeptide sequence at the N-terminal region of
IL-1.beta. pro polypeptide which co-translationally or
post-translationally directs transfer of the IL-1.beta. pro
polypeptide from its site of synthesis to a site inside or outside
of the cell membrane or wall (e.g., the yeast .alpha.-factor
leader). IL-1.beta. pro polypeptide fusions can comprise
polypeptides added to facilitate purification and identification of
IL-1.beta. pro (e.g., poly-His). Further, the amino acid sequence
of IL-1.beta. pro can be linked to the peptide
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (Hopp et al. BioTechnology 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 cells.
[0044] The present invention further includes IL-1.beta. pro
polypeptides with or without associated native-pattern
glycosylation. IL-1P, pro expressed in yeast or mammalian
expression systems (e.g., COS-7 cells) may be similar or
significantly different in molecular weight and glycosylation
pattern than the native human IL-1.beta. pro polypeptide. This
depends upon the choice of expression system. Expression of
IL-1.beta. pro polypeptides in bacterial expression systems, such
as E. coli, provides non-glycosylated molecules.
[0045] Functional mutant analogs of human IL-1.beta. pro can be
synthesized, for example, with inactivated N-glycosylation sites by
oligonucleotide synthesis and ligation or by site specific
mutagenesis techniques. The IL-1.beta. 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-.PHI.-.OMEGA. where .PHI. is any amino acid except Pro and
.OMEGA. is Ser or Thr. In this sequence, carbohydrate residues are
covalently attached at the Asn side chain.
[0046] IL-1.beta. pro analogs or derivatives may also be obtained
by mutations of the IL-1.beta. pro DNA sequence. An IL-1.beta. pro
mutant derivative, as referred to herein, is a polypeptide
substantially homologous to IL-1.beta. pro but which has an amino
acid sequence different from native IL-1.beta. pro because of a
deletion, insertion or substitution.
[0047] IL-1.beta. 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.
[0048] Bioequivalent analogs of IL-1.beta. pro polypeptides
(defined as polypeptides having IL-1.beta. pro biological activity)
can 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.
For example, 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.
[0049] Generally, substitutions are made conservatively by
substituting and amino acid having physiochemical characteristics
resembling those of the replaced residue. Further substitutions may
be outside of the "core" sequence needed for IL-1.beta. pro
biological activity. Subunits of IL-1.beta. pro may be constructed
by deleting terminal or internal residues or sequences. The
resulting polypeptide should have IL-1.beta. pro biological
activity as defined herein.
[0050] The terms "IL-1.beta. pro", "human IL-1.beta. protease"
include, but are not limited to, analogs or subunits of IL-1.beta.
pro which are substantially similar to human IL-1 pro and/or which
exhibit the substrate-specific proteolytic biological activity
associated with IL-1.beta. pro as described herein.
[0051] The term "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. However, the net effect is the same
protease biological activity characteristic of the reference human
IL-1.beta. pro polypeptide. For example, a derivative can have a
truncated sequence comprising a "core region" or a sequence of
amino acids necessary for the specific protease biological activity
characteristic of IL-1.beta. pro. Substantially similar IL-1.beta.
pro derivatives will be greater than about 30% similar to the
corresponding sequence of human IL-1.beta. pro and have IL-1.beta.
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.
More preferably, the derivative polypeptides will have greater than
80% amino acid sequence homology to human IL-1.beta. pro
polypeptide.
[0052] 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.
[0053] "Biologically active", as used herein, refers to IL-1.beta.
pro biological activity to cleave a particular amino acid sequence
at the peptide bond between an Asp residue and an Ala or Gly
residue.
[0054] "Recombinant", as used herein, 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. As a product, "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) will
be free of glycan. Polypeptides expressed in yeast may have a
glycosylation pattern different from that expressed in mammalian
cells.
[0055] The IL-1.beta. pro protease has a highly restricted
substrate specificity. Human precursor IL-1.beta. polypeptide has
an amino acid sequence His-Asp-Ala-Pro for residues 115-118. Human
IL-1.beta. pro cleaves this sequence between residues 116.and 117
(Asp-Ala) to form human mature IL-1.beta. polypeptide. Changing
Asp-116 to Ala in a human precursor IL-1.beta. polypeptide by
site-directed mutagenesis prevented cleavage of the mutant
IL-1.beta. polypeptide derivative.
[0056] Isolated human IL-1.beta. pro was able to cleave at its
specific substrate site even when the tertiary structure of the
substrate precursor IL-1.beta. polypeptide was altered by
denaturing the substrate polypeptide in boiling water. Precursor
human IL-1.beta. was denatured by boiling a solution of precursor
IL-1.beta. for fifteen minutes. Denaturation had little effect on
the ability of human IL-1.beta. pro to be able to cleave human
precursor IL-1.beta. into mature IL-1.beta.. Thus, the tertiary
structure of the substrate polypeptide does not significantly
contribute to the reaction with the enzyme IL-1.beta. pro.
[0057] IL-1.beta. pro biological activity was determined by a
protease assay. As the IL-1.beta. pro enzyme is salt sensitive,
samples having a salt concentration greater than 50 mM were
initially desalted. Samples can be desalted, for example, by
applying 100 .mu.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 minutes at
1876.times.g. The assay was conducted by incubating a mixture of
five .mu.l (30 ng) of purified human IL-1.beta. precursor and 10
.mu.l of the sample to be tested for IL-1.beta. protease biological
activity for 60 minutes at 37.degree. C. A control sample was
similarly incubated to check for endogenous IL-1. The control
sample mixture contained 5 .mu.l of 10 mM Tris-HCl, pH 8.1 and 5 mM
dithiothreitol instead of IL-1.beta. precursor. The control sample
incubations were terminated by addition of SDS (sodium dodecyl
sulfate) in sample buffer followed by five minutes of boiling.
[0058] All incubated assay mixtures were electrophoresed on 0.75
mm-thick SDS, 14% polyacrylamide slab gels, using a discontinuous
system, such as the one described in Laemmli, Nature, 277:680
(1970). Western blots were performed following electrophoresis by
transferring the proteins onto nitrocellulose (Sartorius) and
probing using an 20 .mu.g/ml solution of purified IL-1.beta.
COOH-terminal-specific monoclonal antibody (i.e., 16F5). The blot
was developed using Horseradish Peroxidase Color Developing Reagent
(Bio Rad). One hundred ng of purified mature IL-1.beta. was used as
a control 17,500 dalton marker on the Wester blot.
[0059] Human IL-1.beta. pro enzyme was obtained and purified from
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
10.sup.8/ml. The suspended cells were frozen and thawed three times
and the lysates stored at -80.degree. C. until further use. Prior
to purification, the lysates were thawed and then centrifuged for
20 minutes at 47,800.times.g at 4.degree. C. The supernatant was
taken for further purification. The freeze-thawing procedure
repeated four times released over 50% of the IL-1.beta. pro
activity into the supernatant. Additional freeze-thaws did not
increase the yield of soluble material.
[0060] The human IL-1.beta. pro polypeptide was purified in a
six-step process described below. All the chromatography steps were
performed at 4.degree. 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.
[0061] 1. Approximately 500-600 ml of lysate supernatant was
diluted 1:2 in 10 mM Tris-HCl and 5 mM dithiothreitol, pH 8.1
("buffer A") to reduce ionic strength of the lysate to <20 mM.
pH was adjusted to 8.1. The diluted lysate supernatant was applied
to a DEAE-Sephacel column (20.times.4.4 cm, Pharmacia Fine
Chemicals), equilibrated with buffer A. The flow rate was 120
ml/hour. 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-1.beta. pro activity and stored for
further purification. The IL-1.beta. 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-1.beta., which eluted
between 0.06 and 0.11 M NaCl, and endogenous precursor IL-1.beta.
which eluted between 0.12 and 0.18 M NaCl.
[0062] 2. The pooled active fractions from the DEAE column were
diluted in 50 mM potassium phosphate buffer, 5 mM dithiothreitol,
pH 7.0 ("buffer B"). A 14.times.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/hour. 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.
Fractions were collected as 10 ml volumes, analyzed for IL-1.beta.
pro activity, and stored for further purification. IL-1.beta. 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-1.beta. eluted between 0.05 and 0.08 M potassium phosphate.
[0063] 3. A 20.times.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 allow IL-1.beta.
pro to bind to the column. Diluted fractions were applied to the
Blue Agarose column at a 30 ml/hour rate. The column was washed
with three column volumes of buffer A. The proteins were eluted
with five column volumes of a linear gradient ranging from 0.1 to 1
M NaCl in buffer A. Ten ml fractions were collected, analyzed for
IL-1.beta. pro activity and stored for further purification.
IL-1.beta. 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.
[0064] 4. A 95.times.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-1.beta. 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-1.beta. 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 starting protein
contaminants have been separated from IL-1.beta. pro. However, PAGE
(polyacrylamide gel electrophoresis) analysis of the fractions
still revealed several protein bands that did not correlate with
IL-1.beta. pro biological activity.
[0065] 5. Fractions from the Sephadex column which contained
protease activity were pooled and the pool was concentrated on
pretreated Centriprep 10 Concentrators to a 500 .mu.l volume. Since
protein concentration of the Sephadex pool was low (<30
.mu.g/ml), pretreatment of the centripreps with bovine serum
albumin reduced loss of IL-1.beta. pro activity during
concentration. Extensive washing of the treated centripreps prior
to use prevented contamination of samples with albumin.
Pretreatment was accomplished by centrifuging 15 ml of 1% bovine
serum albumin (BSA) in centripreps for 30 minutes, decanting the
remaining solution, and washing with 10 mM Tris-HCl. A Mono P5/20
FPLC chromatofocusing 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
.mu.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/hour flow rate. One ml fractions were collected and analyzed for
pH and biological protease activity. This chromatofocusing step
increased the purity of IL-1.beta. pro a further 100 fold and
allowed for the visualization of a single protein band that
correlated with IL-1.beta. pro biological activity. IL-1.beta. 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 .mu.l to 50 .mu.l.
[0066] 6. The fractions were subjected to electrophoresis on a
polyacrylamide gel (PAGE), followed by electroblotting onto
polyvinyl difluoride membrane paper (PVDF, Millipore
Immobilin-P.RTM.) at 300 mA for 30 minutes. The PVDF membrane was
stained with Coomassie Blue. There were five major bands with
molecular weights of approximately 45,000, 43,000, 36,000, 22,000
and 18,000 daltons. The 22,000 dalton band correlated with
IL-1.beta. pro activity and was sequenced.
[0067] The N-terminal sequence of the 22,000 dalton band yielded an
amino acid sequence described herein. A mature human IL-1.beta. 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. In the first stage PCR procedure; fully degenerate
PCR primers were designed and made from the N-terminal amino acid
sequence. The degenerate primers were used to amplify IL-1.beta.
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-1.beta. pro DNA (nucleotides
1-17) and to contain an Eco RI restriction site. Primer #1 had the
sequence: TABLE-US-00001 (Seq. I.D. No. 9)
5'-GTCGAATTCAA(T/C)CCNGCNATGCCNAC-3'.
[0068] Primer #2 was designed to cross-hybridize to IL-1.beta. pro
DNA (complementary to nucleotides 31-47 and contain an Xba I
restriction site. Primer #2 had the sequence: TABLE-US-00002 (Seq.
I.D. No. 10) 5'-GTCTCTAGAAG(T/C)TTNAC(A/G)TTNCC(T/C)TC-3'.
[0069] PCR amplification was performed with thermus acuatius
polymerase (Perkin-Elmer Cetus) in 100 .mu.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-1.beta. pro as
determined by purification and N-terminal sequence analysis.
[0070] The second stage of the PCR procedure made Primer #3
composed of nucleotides 1-17 (FIG. 1) and a Not I restriction site
and Primer #4 containing 20 T residues and a Not I restriction
site. Primers #3 and #4 were added to the THP-1 CDNA library
described above and PCR amplified for 6 cycles at 94.degree. C. for
1 minute, 50.degree. C. for 1 minute and 72.degree. C. for 1
minute. Southern analysis of the PCR amplified clone using a 17
base oligonucleotide probe (complementary to nucleotides 16-32 in
FIG. 1) found a band at approximately 1000 bp that was also found
to posses IL-1.beta. pro biological activity. The 1000 bp DNA was
gel purified, subjected to a similar second round of PCR and
subcloned into pGem-5 for sequencing. The nucleotide sequence of
this clone is shown in FIG. 1.
[0071] In the third stage of PCR cloning, full length IL-1.beta.
pro clones were isolated from a cDNA library prepared from
peripheral blood neutrophils. We found that neutrophils expressed
IL-1.beta. pro mRNA. We isolated two clones (p48 and p214) with
IL-1.beta. pro specific inserts of 1367 and 1360 base pairs,
respectively. The DNA sequence shown in FIG. 1 is a composite of
all the IL-1.beta. pro clones. The amino acids encoded by all of
the IL-1.beta. pro clones we found were identical.
[0072] IL-1.beta. 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.
[0073] IL-1.beta. pro is a cytoplasmic enzyme. As the purified
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.
[0074] The DNA sequence shown in FIG. 1 was expressed in a
mammalian cell (e.g., COS-7 cells). For mammalian cell expression,
synthetic oligonucleotide primers were made to amplify the entire
coding domain of IL-1.beta. pro. The 5' primer TABLE-US-00003 (Seq.
I.D. No. 11) (5'-ATATCGGTACCGCCTCCAGCATGCCTCCGGCAATGCCCA
CATC-3')
contained an Asp 718 restriction site and an initiator Met residue
fused to the N-terminus of the enzyme (nucleotides 1-20).
[0075] The 3' primer TABLE-US-00004 (Seq. I.D. No. 12)
(5'-CTGCTAGATCTGCCCGCAGACATTCATACAG-3')
[0076] contains a Bgl II restriction site and is complementary to
nucleotides 883-902 of FIG. 1. The PCR generated fragment was
ligated into pDC303 mammalian vector, as described in Mosley et
al., Cell, 59:335-348 (1989).
[0077] Human IL-1.beta. pro is preferably produced by recombinant
DNA techniques. A recombinant DNA expression system inserts a clone
encoding human IL-1.beta. pro polypeptide or a derivative thereof
with biological activity into an expression vector. The expression
vector is inserted into a host cell. The host cell's protein
syntheses machinery synthesizes the recombinant human IL-1.beta.
pro polypeptide.
[0078] Suitable host cells for expression of mammalian IL-1.beta.
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 subtilis, 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. Cell-free translation
systems could also be employed to produce mammalian IL-1.beta. 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, N.Y., (1985).
[0079] When an IL-1.beta. pro polypeptide or derivative thereof is
expressed in a yeast host cell, the nucleotide sequence (e.g.,
structural gene) that codes on expression for an IL-1.beta. pro
polypeptide or derivative thereof may include a leader sequence.
The leader sequence enables improved extracellular secretion of
translated polypeptide by a yeast host cell.
[0080] Alternatively, in a prokaryotic host cell, such as E. coli,
the IL-1.beta. 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-1.beta. pro
polypeptide or derivative thereof. Moreover, prokaryotic host cells
may be used for expression and disulfide processing.
[0081] The recombinant expression vectors carrying the recombinant
IL-1.beta. 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. Examples of 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) cells.
[0082] Transformed host cells are cells which have been transformed
or transfected with IL-1.beta. pro or a derivative thereof
structural gene nucleotide sequences. Expressed IL-1.beta. 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.
[0083] 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 (ATTC
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-1.beta. pro structural gene
sequence. Other commercially vectors include, for example, pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega
Biotec, Madison, Wis., USA).
[0084] Promoter sequences are commonly used for recombinant
prokaryotic host cell expression vectors. Common promotor sequences
include .beta.-lactamase (penicillinase), lactose promoter system
(Chang et al., Nature, 275:615, 1978; and Goeddel et al., Nature,
281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,
Nucl. Acids Res,. 8:4057, 1980; and EPA 36,776) and tac promoter
(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic
host cell expression system employs a phage .lamda. P.sub.L
promoter and a cI875ts thermolabile repressor sequence. Plasmid
vectors available from the American Type Culture Collection which
incorporate derivatives of the .lamda. P.sub.L promoter include
plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and
pPLc28 (resident in E. coli RR1 (ATCC 53082)).
[0085] Human IL-1.beta. pro polypeptides and derivative
polypeptides may be expressed in yeast host cells, 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 .mu. yeast plasmid, an autonomously replicating sequence (ARS), a
promoter region, sequences for polyadenylation, and sequences for
transcription termination. Preferably, 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., J.
Biol. Chem., 255:2073, (1980)] or other glycolytic enzymes [Hess,
et al., J. Adv. Enzyme Reg., 7:149, (1968); and Holland et al.,
Biochem. 17:4900, (1978)], 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. Other
suitable vectors and promoters for use in yeast expression are
further described in Hitzeman, EP-A-73,657.
[0086] Yeast vectors can be assembled, for example, using DNA
sequences from pBR322 for selection and replication in E. coli
(Amp.sup.r 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 .alpha.-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 .alpha.-factor leader sequence
directs secretion of heterologous polypeptides. The .alpha.-factor
leader sequence is often inserted between the promoter sequence and
the structural gene sequence. [See, e.g., Kurjan et al., Cell,
30:933, (1982); and Bitter et al., Proc. Natl. Acad. Sci. USA,
81:5330, (1984).] 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.
[0087] 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.sup.+ transformants in a selective medium, wherein
the selective medium consists of 0.67% yeast nitrogen base, 0.5%
casamino acids, 2% glucose, 10 .mu.g/ml adenine and 20 .mu.g/ml
uracil.
[0088] Yeast host cells transformed by vectors containing ADH2
promoter sequence may be grown for inducing expression in a "rich"
medium. An example of a rich medium is one consisting of 1% yeast
extract, 2% peptone, and 1% glucose supplemented with 80 .mu.g/ml
adenine and 80 .mu.g/ml muacil. Derepression of the ADH2 promoter
occurs when glucose is exhausted from the medium.
[0089] Mammalian or insect host cell culture systems could also be
employed to express recombinant IL-1.beta. pro polypeptide or
derivatives thereof. Examples of suitable mammalian host cell lines
include the COS-7 lines of monkey kidney cells [Gluzman, Cell,
23:175, (1981)], L cells, C127 cells, 3T3 cells, 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.
[0090] Transcriptional and translational control sequences in
mammalian host cell expression vectors may be provided by viral
sources. For example, commonly used 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 SV40 viral genome, for example, SV40
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 III site toward the BglI
site located in the SV40 viral origin of replication site is
included.
[0091] Further, mammalian genomic IL-1.beta. 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)].
[0092] Purified human IL-1.beta. pro polypeptides or derivatives
thereof are prepared by culturing transformed host cells under
culture conditions necessary to express IL-1.beta. pro polypeptides
or derivatives thereof. The expressed polypeptides are purified
from culture media or cell extracts. For example, supernatants from
cultured transformed host cells can secrete recombinant IL-1.beta.
pro polypeptide into culture media. The IL-1.beta. 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. For example, a suitable purification matrix is
an IL-1.beta. pro inhibitor or an antibody molecule specific for an
IL-1.beta. pro polypeptide or derivative thereof and bound to a
suitable support. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendent
diethylaminoethyl (DEAE) groups. The matrices can be acrylamide,
agarose, dextran, cellulose or other types commonly employed in
protein purification. Alternatively, a cation exchange step can be
employed. Suitable cation exchangers include various insoluble
matrices comprising sulfopropyl or carboxymethyl groups.
Sulfopropyl groups are preferred.
[0093] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify an IL-1.beta. pro polypeptide
composition. Some or all of the foregoing purification steps, in
various combinations, can also be employed to provide a homogeneous
recombinant protein. Alternatively, some or all of the steps used
in the purification procedure described herein can also be
employed.
[0094] Recombinant polypeptide produced in bacterial culture is
usually isolated by initial disruption of the host cells,
extraction from cell pellets of an insoluble polypeptide, or from
the supernatant of a soluble polypeptide, followed by one or more
concentration, salting-out, ion exchange or size exclusion
chromatography steps. Finally, 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-thaw cycling, sonication,
mechanical disruption, or use of cell lysing agents.
[0095] Transformed yeast host cells generally express IL-1.beta.
pro polypeptide as a secreted polypeptide. This simplifies
purification. Secreted recombinant polypeptide from a yeast host
cell fermentation can be purified by methods analogous to those
disclosed by Urdal et al., [J. Chromatog., 296:171, (1984)]. Urdal
et al., describe two sequential, reversed-phase HPLC steps for
purification of recombinant human IL-2 on a preparative HPLC
column.
[0096] II. Interleukin 18 Protease Inhibitors
[0097] The inhibitors 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
preIL-I.beta..
[0098] The amino acid sequence of preIL-1.beta. is presented in
Seq. I.D. No. 3 and in FIG. 2 taken from March, C. J. et al.,
Nature (London), 315(6021):641-647 (1985). Mature IL-I.beta. is
represented by the C-terminal 153 amino acid residues of preIL-1.
Thus, the N-terminal of IL-1.beta. is the Ala residue at position
117 of Seq. I.D. No. 3.
[0099] Amino acid residues can be expressed by the full name (e.g.,
alanine) or by the three letter designation (e.g., Ala). This
application will use the three letter designation.
[0100] The naturally occurring 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.
[0101] As used herein, the phrase "corresponds to" means that a
particular sequence of an inhibitor compound may differ 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.
[0102] Examples of substitutions that do not materially alter
inhibitory activity-are -replacement-of the Ala at position 112 of
Seq. I.D. No. 3 with Ser or Gly; replacement of the Tyr at position
113 of Seq. I.D. No. 3 with Phe; replacement of the Val at position
114 of Seq. I.D. No. 3 with Leu, Ile or Met and replacement of the
His at position 115 of Seg. I.D. No. 3 with Phe, Pro, a positively
charged amino acid such as Lys, Arg, His or Tyr, or the use of D
isomers.
[0103] The C-terminal amino acid residue of the inhibitor compounds
of the present invention is aspartic acid (Asp). Asp has a side
chain of the formula CH.sub.2--COOH. Preferably, the Asp side chain
carboxyl group is protected to facilitate synthesis of the
compounds of the present invention.
[0104] 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.
[0105] In a preferred embodiment, the Asp side chain protecting
moiety is connected to the Asp side chain via an ester linkage,
which linkage is subject to cleavage by naturally occurring
intracellular esterase enzymes. In this way, 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-1.beta. pro is predominantly
located.
[0106] As used herein, the phrase "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 present
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. Pat. Nos. 4,652,552 and 4,636,492.
[0107] As used herein, the phrase "electronegative leaving group"
refers to chemical groups susceptible to nucleophilic attack by an
amino acid residue in the enzyme active site, thus modifying
IL-1.beta. pro such that IL-1.beta. pro cannot interact with and
cleave preIL-1.beta..
[0108] The compounds of the present invention inhibit the catalytic
activity of IL-1.beta. pro in a reversible or an irreversible
fashion. As used herein, "irreversible" means the formation of a
covalent bond between the enzyme and the inhibitor.
[0109] The reversibility of IL-1.beta. pro activity is a function
of the electronegative leaving group. When the electronegative
leaving group is a diazoalkyl ketone, the inhibition of IL-1.beta.
pro is irreversible and the compound is an irreversible inhibitor.
When the electronegative leaving group is an aldehyde, the
inhibition of IL-1.beta. pro is reversible and the compound is a
reversible inhibitor.
[0110] The compounds of the present invention have the formula:
Z-Q.sub.2-Asp-Q.sub.1 where Z is an N-terminal blocking group;
[0111] Q.sub.2 is 0 to about 4 amino acids such that the sequence
Q.sub.2-Asp corresponds to at least a portion of the sequence
Ala-Tyr-Val-His-Asp, residues 112 to 116 of Seq. I.D. No. 3; and
[0112] Q.sub.1 is an electronegative leaving group.
[0113] In a preferred embodiment, Z is C.sub.1-C.sub.6 alkyl,
benzyl, acetyl, C.sub.1-C.sub.6 alkoxycarbonyl, benzyloxycarbonyl
or C.sub.1-C.sub.6 alkyl carbonyl. As used herein, "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. In a more preferred embodiment, Z is
t-butoxycarbonyl (t-Boc), acetyl or benzyloxycarbonyl (Cbz).
[0114] Q.sub.2 is preferably 1 amino acid. Preferably Q.sub.2 is
His, Phe, Pro, or Tyr. Most preferably, Q.sub.2 is His or Phe.
[0115] Q.sub.1 is preferably an aldehyde, a diazoalkyl ketone or a
haloalkyl ketone. As used herein in reference to electronegative
leaving groups, "alkyl" refers to linear or branched chain radicals
having l to 3 carbon atoms, which may be optionally substituted as
herein defined. Representative alkyl groups include methyl, ethyl,
propyl and the like. More preferably, Q.sub.1 is an aldehyde or
fluoromethyl (CH.sub.2F) ketone.
[0116] The 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. Biophys., 162:56 (1974) ; U.S. Pat. No.
4,582,821; U.S. Pat. No. 4,644,055; Kettner, C. A. et al., Arch.
Biochem. Biophys., 165:739 (1974); Dakin, H. D. and West, R., J.
Biol. Chem., 78:91 (1928); Rasnick, D., Anal. Biochem., 149:461
(1985).
[0117] Compounds-having a fluoromethyl electronegative leaving
group are preferably synthesized by the Rasnick procedure.
[0118] 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 HCl, HBr or HI to produce the
desired N-blocked, C-terminal haloalkyl ketone peptide or amino
acid.
[0119] 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.degree. C. for about two hours to allow for CO.sub.2
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. The N-blocked, fluoroalkyl ketone peptide is then eluted
from the gel and purified.
[0120] 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).
Alternatively, 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).
[0121] III. Methods of Treatment and Pharmaceutical
Compositions
[0122] The present invention provides methods of using therapeutic
compositions comprising an effective amount of IL-1.beta. pro
polypeptides and derivatives thereof in a suitable diluent and
carrier. For therapeutic use, purified IL-1.beta. pro or a
biologically active derivative thereof is administered to a
patient, preferably a human, for treatment in a manner appropriate
to the indication. Thus, for example, IL-1.beta. pro compositions
administered to suppress autoimmunity can be given by bolus
injection, continuous infusion, sustained release from implants, or
other suitable technique. Typically, an IL-1.beta. pro therapeutic
agent will be administered in the form of a pharmaceutical
composition comprising purified polypeptide in conjunction with
physiologically acceptable carriers, excipients or diluents. Such
carriers will be nontoxic to patients at the dosages and
concentrations employed. Ordinarily, the preparation of such
compositions entails combining IL-1.beta. 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 stabilizers and
excipients. Neutral buffered saline or saline mixed with
conspecific serum albumin are exemplary appropriate diluents.
[0123] The inhibitor compounds of the present invention are useful
in inhibiting the physiological actions of interleukin 1 by
preventing formation of biologically active IL-1.beta.. The
inhibition of IL-1.beta. pro results in a decrease in active
IL-1.beta. levels and a concomitant increase in preIL-1.beta.,
which compound is biologically inactive.
[0124] 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.
[0125] Mammals needing treatment for an inflammatory disorder or
prevention of an autoimmune condition are administered effective
amounts of the inhibitor compounds of this invention either alone
or in the form of a pharmaceutical composition.
[0126] The pharmaceutical compositions of the present invention
include one or more of the compounds of this invention formulated
into compositions together with one or more non-toxic
physiologically acceptable carriers, adjuvants or vehicles which
are collectively referred to herein as carriers, for parenetral
injection, for oral administration or solid or liquid form, for
rectal or topical administration, and the like.
[0127] The compositions can be administered to humans and animals
either orally, rectally, parenterally (intravenously,
intramuscularly or subcutaneously) intracisternally,
intravaginally, intraperitoneally, locally (powders, ointments or
drops), or as a buccal or nasal spray.
[0128] Compositions suitable for parenteral injection may comprise
physiologically acceptable sterile aqueous or nonaqueous solutions,
dispersions, suspension or emulsions and sterile powders for
reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers, diluents,
solvents or vehicles include water, ethanol, polyols
(propyleneglycol, polyethyleneglycol, glycerol, and the like),
suitable mixtures thereof, vegetable oils (such as olive oil) 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.
[0129] These compositions may also contain 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.
[0130] If desired, and for more effective distribution, the
compounds can be incorporated into slow release or targeted
delivery systems such as polymer matrices, liposomes, 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.
[0131] Solid dosage forms for oral administration include capsules,
tablets, pills, powders and granules. In such solid dosage forms,
the active compound is admixed with at least one inert customary
excipient (or carrier) such as sodium citrate of dicalcium
phosphate or (a) fillers or extenders, as for example, starches,
lactose, sucrose, glucose, mannitol and silicic acid, (b) binders,
as for example, carboxymethylcellulose, alignates, gelatin,
polyvinylpyrrolidone, sucrose and acadia, (c) humectants, as for
example, glycerol, (d) disintegrating agents, as for example,
agar-agar, calcium carbonate, potato or tapioca starch, alginic
acid, certain complex silicates 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, (h)
adsorbents, as for example, kaolin and bentonite, and (i)
lubricants, as for example, talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate or
mixtures thereof. In the case of capsules, tablets and pills, the
dosage forms may also comprise buffering agents.
[0132] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethyleneglycols, and the like.
[0133] Solid dosage forms such as tablets, dragees, capsules, pills
and granules can be prepared with coatings and shells, such as
enteric coatings and others well known in the 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.
[0134] The active compounds can also be in micro-encapsulated form,
if appropriate, with one or more of the above-mentioned
excipients.
[0135] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compounds, the liquid
dosage forms may contain inert diluents commonly used in the art,
such as water or other solvents, solubilizing agents and
emulsifiers, as for example, ethyl alcohol, isopropyl alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in
particular, cottonseed oil, groundnut oil, corn germ oil, olive
oil, caster oil and sesame oil, glycerol, tetrahydrofurfuryl
alcohol, polyethyleneglycols and fatty acid esters of sorbitan or
mixtures of these substances, and the like.
[0136] Besides such inert diluents, the composition can also
include adjuvants, such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring and perfuming agents.
[0137] Suspensions, in addition to the active compounds, may
contain suspending agents, as for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, or mixtures of these substances, and the
like.
[0138] 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.
[0139] Dosage forms for topical administration of a compound of
this invention include ointments, powders, sprays and inhalants.
The active component is admixed under sterile conditions with a
physiologically acceptable carrier and any needed preservatives,
buffers or propellants as may be required. Ophthalmic formulations,
eye ointments, powders and solutions are also contemplated as being
within the scope of this invention.
[0140] The compounds of the present invention can also be
administered in the form of liposomes. As is known in the art,
liposomes are generally derived from phospholipids or other lipid
substances. Liposomes are formed by mono- or multi-lamellar
hydrated liquid crystals that are dispersed in an aqueous medium.
Any non-toxic, physiologically acceptable and metabolizable lipid
capable of forming liposomes can be used. The present compositions
in liposome form can contain, in addition to the IL-1.beta. pro
inhibiting compounds of the present invention, stabilizers,
preservatives, excipients, and the like. The preferred lipids are
the phospholipids and the phosphatidyl cholines (lecithins), both
natural and synthetic.
[0141] Methods for form lipsomes are known in the art. See, for
example, Methods in Cell Biology, Ed. by Prescott, Volume XIV,
Academic Press, New York, N.Y. p. 33 et seq., (1976).
[0142] Actual dosage levels of active ingredient in the
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.
[0143] The total daily dose of the inhibitor 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 per
kilogram of body weight. Dosage unit compositions may contain such
amounts of such submultiples thereof as may be used to make up the
daily dose. It will be understood, however, that the specific dose
level for any particular patient will 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.
[0144] The following examples are for the purposes of illustration
and not by way of limitation.
EXAMPLES
Example 1
Substrate Specificity of IL-1.beta. pro
[0145] This example illustrates the range of substrate specificity
of purified human IL-1.beta. 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-1.beta.
(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-1.beta. 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.degree. C., 1 hr) in the presence of anisole as scavenger
(HF:anisole 9:1). After evaporation of HF, the substrate peptide
resin mixtures were washed with diethyl ether and extracted with
15% (w/v) acetic acid, lyophilized and purified on reverse phase
high performance liquid chromatography (RP-HPLC) on a Vydac C18,
2.2 cm.times.25 cm column. Trifluoroacetic acid (0.1%) in water was
solvent A and 0.1% trifluoroacetic acid in acetonitrile was solvent
B for the mobile phases.
[0147] 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
.sup.252Cf 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.
[0148] 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.
[0149] Purified human IL-1.beta. pro enzyme (10 .mu.l ), peptide
substrate in water (10 .mu.l), and 10 mM Tris buffer, pH 8.0
containing 25% v/v glycerol (10 .mu.l ) were mixed and the mixtures
were incubated at 37.degree. C. for four hours. The reaction was
quenched with by adding 1 M glycine/HCl buffer pH 2.0 (10 .mu.).
The samples were then analyzed using RP-HPLC with a Vydac C18
column (0.46 cm.times.25 cm) and eluting with a linear gradient
from 100% solvent A to 70% solvent A/30% solvent B over 30 minutes
at a flow rate of 1 ml/min. The effluent was monitored at 280 nm
absorbing product. A comparison of peak area of product peptide to
that of total peak area of substrate and product yielded the extent
of peptide cleavage, because the area under the combined substrate
and product peaks was constant and independent of the amount of
cleavage by the IL-1.beta. pro enzyme. Identities of peptide
product peaks were confirmed by amino acid analysis and by mass
spectrometry.
[0150] Table 1 shows the relative reactivities of a series of eight
peptide substrates that were subject to digestion by purified
IL-1.beta. pro enzyme. TABLE-US-00005 TABLE 1 Reactivity Relative
Peptide Sequence To Peptide 1 1 Ala-Tyr-Val-His- 1.00
Asp-Ala-Pro-Val-Arg-Ser (Seq. I.D. No. 13) 2 Ala-Tyr-Val-His-
<0.01 Asn-Ala-Pro-Val-Arg-Ser (Seq. I.D. No. 14) 3
Ala-Tyr-Val-His- <0.05 Glu-Ala-Pro-Val-Arg-Ser (Seq. I.D. No.
15) 4 Ala-Tyr-Val-His- <0.01 (D-Asp)-Ala-Pro-Val-Arg-Ser (Seq.
I.D. No. 16) 5 Ala-Tyr-Val-His- 3.40 Asp-Gly-Pro-Val-Arg-Ser (Seq.
I.D. No. 17) 6 Ala-Tyr-Val-His- <0.05 Asp-Val-Pro-Val-Arg-Ser
(Seq. I.D. No. 18) 7 Ala-Tyr-Val-Phe- 0.50 Asp-Ala-Pro-Val-Arg-Ser
(Seq. I.D. No. 19) 8 Ala-Tyr-Val-His- 0.47 Asp-Ala-Ala-Val-Arg-Ser
(Seq. I.D. No. 20)
[0151] The cleavage site can be described with the corresponding
human precursor IL-1.beta. amino acid residues as follows: P2 P1
P1' P2' His-Asp-Ala-Pro Changing the L-aspartic acid residue of
peptide 1 to either asparagine (peptide 2), glutamic acid (peptide
3) or D-aspartate (peptide 4) has a profound effect on the ability
of IL-1.beta. pro to cleave the substrate. These data establish the
requirement of an L-aspartate residue in the P1 position for this
enzyme to be able to cleave a substrate.
[0152] Peptides 5 and 6 represent changes in the P1' position of
the human precursor IL-1.beta. cleavage site. Replacing alanine
with glycine (peptide 5) results in a substrate that is 3.4 times
more reactive than peptide 1. However, changing the same residue to
a valine (peptide 6) effectively prevents proteolytic cleavage. The
fact that with a glycine residue in P1' the peptide is cleaved more
readily suggests that the alanine residue in human precursor
IL-1.beta. polypeptide is not critical for substrate binding, while
the result with a valine residue in the P1' position indicates low
steric tolerance at the P1' position. Thus, it seems unlikely that
the IL-1.beta. pro enzyme or derivatives thereof will cleave
anywhere other than between Asp-Gly and Asp-Ala residues.
[0153] Peptides 7 and 8 represent changes to the P2 and P2' sites,
respectively. Changing the proline of peptide 1 to an alanine
yielded a substrate which was still cleaved by human IL-1.beta. pro
but only half as efficiently as the peptide with human IL-1.beta.
native sequence. A similar result was obtained when the histidine
of peptide 1 was replaced with a phenylalanine. These data suggest
that human IL-1.beta. pro enzyme tolerates conservative
replacements of both residues and that the P2 and P2' positions are
not as vital for activity as the amino acids at the P1 and P1'
positions.
Example 2
Effect of Substrate Length
[0154] This example illustrates the effect of substrate peptide
length on the ability of human IL-1.beta. 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-1.beta. pro cleavage site of human
precursor IL-1.beta.. The results are shown in Table 2 below:
TABLE-US-00006 TABLE 2+HX,1/32 !? ? Reactivity? ? !?? Relative?
!Peptide? Sequence? To Peptide 1 1 Ala-Tyr-Val-His- 1.00
Asp-Ala-Pro-Val-Arg-Ser- (Seq. I.D. No. 13) 9 Glu-Ala-Tyr-Val- 0.74
His-Asp-Ala-Pro- (Seq. I.D. No. 21) 10 Tyr-Val-His-Asp- 2.40
Ala-Pro-Val-Arg- (Seq. I.D. No. 22) 11 Val-His-Asp-Ala- Not cleaved
Pro-Val- (Seq. I.D. No. 23) 12 His-Asp-Ala-Pro- Not cleaved (Seq.
I.D. No. 24)
The eight amino acid peptide
(Ac-Tyr-Val-His-Asp-Ala-Pro-Val-Arg-NH.sub.2) is cleaved most
efficiently while the four and six amino acid peptides are not
cleaved. Thus, IL-1.beta. pro has a minimum number of amino acid
residues necessary for substrate peptide cleavage.
Example 3
Synthesis of IL-1.beta. Protease Inhibitors
[0155] A. Synthesis of Boc-Asp-CH.sub.2F.
[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.times.50 ml), saturated NaHCO.sub.3
(2.times.50 ml), and saturated NaCl (2.times.50 ml), followed by
drying over anhydrous MgSO.sub.4. The solvent was then removed by
evaporation under reduced pressure. The resulting oil was applied
to a 2.5.times.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-CH.sub.2F, Boc-Tyr-Asp-CHF and
Boc-Phe-Asp-CH.sub.2F.
[0159] Boc-Asp-CH.sub.2F 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.degree. C. Cold
ether may then be added to the mixture. The ether is evaporated and
toluene added to co-evaporate residual TFA. The deprotected peptide
(H-Asp-CH.sub.2F) 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 coupling reagent. Bodanszky, supra.
[0160] C. Synthesis of Ac-His-Asp-CH.sub.2F, Ac-Pro-Asp-CH.sub.2F,
Ac-Tyr-Asp-CH.sub.2F and Ac-Phe-Asp-CH.sub.2F.
[0161] The Boc protecting groups may be removed from the compounds
made in accordance with the method of Example 3B using
trifluoroacetic acid as described above. Each deprotected compound
may then be acetylated with acetic anhydride and diisopropylamine
(DIAE) according to standard techniques. Stuart et al., supra.
[0162] D. Synthesis of Cbz-His-Asp-CH.sub.2F,
Cbz-Pro-Asp-CH.sub.2F, Cbz-Tyr-Asp-CH.sub.2F and
Cbz-Phe-Asp-CH.sub.2F.
[0163] The Boc protecting group may be removed from
Boc-Asp-CH.sub.2F prepared according to the method of Example 3A
using TFA as described above. Benzyloxycarbonyl-protected amino
acids (i.e., Cbz-His-OH, Cbz-Phe-OH, Cbz-Tyr-OH, Cbz-Pro-OH)
available from commercial sources (Bachem, Philadelphia, Pa.) can
then be coupled to the deprotected Asp using a symmetric anhydride
coupling procedure. Bodanszky, supra.
Example 4
Inhibition of IL-1.beta. pro Activity
[0164] Boc-Asp-CH.sub.2F was tested for its ability to inhibit
IL-1.beta. pro catalyzed degradation of preIL-I.beta. using an in
vitro assay method. Black, et al., J. Biol. Chem., 263(19): 9437
(1988). The results of this study are shown in FIG. 2.
Boc-Asp-CH.sub.2F was prepared in accordance with the method of
example of 1A.
[0165] A. Production of preIL-I.beta.
[0166] Precursor preIL-1.beta. polypeptide was obtained from E.
coli using standard recombinant DNA techniques. Black, supra.
Recombinant preIL-1.beta. was expressed in E. coli under the
control of the phage .lamda. P.sub.L promoter and cI857.sup.ts
thermolabile repressor. Using standard recombinant DNA techniques,
pLNIL-1.beta.F was constructed by Iigating the following DNA
segments: (I) 6160 base pairs of Nco I/Hind III-digested
pLNIL-1.beta. (12) containing the vector (conferring ampicillin
resistance), codons 134-269 and 3' noncoding regions of
HuIL-1.beta.; (2) complementary synthetic oligonucleotides encoding
residues 1-6 of IL-1.beta. and Nco I and Sst I complementary ends;
and (3) a 380-base pair Sst I/Hind III restriction fragment from
plasmid IL-1.beta.-6(1) encoding residues 7-133. The ligation
mixture was transformed into the tetracycline-resistant host
RRI:pRK248cI.sup.ts (12) and correctly assembled plasmids were
identified by restriction analysis of DNA isolated from
transformants resistant to both ampicillin and tetracycline.
Transformants containing pLNIL-1.beta.F were tested for the
production of preIL-1.beta. by SDS-PAGE analysis of cultures grown
in super induction medium to A.sub.600 of 0.5 and derepressed for
1-20 h by elevation of temperature from 30.degree. to 42.degree. C.
A protein of about 31,000 daltons was apparent in samples from
pLNIL-I.beta.F containing cultures but not in control cultures
lacking the IL-1.beta. coding region. Immuno-dot blot analysis with
an anti-IL-1.beta. monoclonal antibody (MAb) and purified
recombinant mature IL-1.beta. as a standard indicated that the
cultures contained approximately 2.5-5.0 .parallel.g/ml of
preIL-1.beta..
[0167] B. Extraction of preIL-1.beta. from E. coli
[0168] Cell pellets from 2.5 liters of transferred E. coli culture
were resuspended in 20 ml of 30 mM Tris-HCl buffer (pH 9.5)
containing 5 mM ethylenediaminetetraacetic acid (EDTA), 500
.mu.g/ml of lysozyme, and 1 mM phenylmethanesulfonyl (PMSF). The
cell suspensions were homogenized using a Polytron homogenizer
(Brinkmann Instruments), rapidly frozen in a Dry Ice/methanol bath,
and then thawed. Next, 200 ml of 30 mM Tris-HCl buffer (pH 8.0)
containing 150 mM NaCl and 1 mM PMSF was added to the suspensions,
which were then homogenized until a uniform homogenate was
obtained. The suspensions were incubated for 30 minutes at
4.degree. C., then centrifuged at 4.degree. C. for 60 minutes at
3800.times.5 g. The supernatant fractions were carefully decanted
and filtered to remove any particular matter. The pellets were
re-extracted in 200 ml of 30mM Tris-HCl buffer (pH 8.0), containing
150 mM NaCl, 8 M urea, and 1 mM PMSF. Since both the Tris and the
urea extracts contained substantial amounts of the preIL-1.beta.,
both were purified as described below.
[0169] C. Purification of preIL-I.beta.
[0170] All chromatographic procedures were carried out at 4.degree.
C. All fractions were assayed for protein concentration, and
conductivity was measured where appropriate. After each
chromatographic step, fractions were analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis SDS-PAGE (with a 10-20%
gradient of polyacrylamide), followed by silver staining, and by
Western blot using a MAb generated against purified mature
IL-1.beta..
[0171] The extracts were diluted 1:4 in H.sub.2O, the pH was
adjusted to 8.1, and the material was loaded at 100 ml/h onto a
25.times.2.5 cm Q-Sepharose column. For the Tris extract, the
column was equilibrated in 10 mM Tris-HCl (pH 8.1). For the urea
extract, the column was equilibrated in 10 mM Tris-HCl (pH 8.1), 2
M urea. The columns were washed with 8 column volumes of 10 mM
Tris-HCl (pH 8.1), and the bound proteins eluted with a linear
gradient (three column volumes) ranging from 0 to 1.5 M NaCl in 10
mM Tris-HCl (pH 8.1). Fractions of 7.5 ml were collected and stored
at 4.degree. C. until the next step of the purification.
[0172] The Q-Sepharose fractions containing the preIL-I.beta. (as
determined by Western blot analysis) were pooled and diluted 1:10
in 10 mM Tris-HCl (pH 8.1). The column was washed with 4 column
volumes of the starting buffer.
[0173] The Tris-HCl solution was applied to a 20.times.5 cm column
of phenyl-Sepharose CL-4B that had been equilibrated in 10 mM Tris
HCl buffer (pH 8.1) containing 0.2 M (NH.sub.4).sub.2SO.sub.4. The
column was washed with 3 column volumes of the starting buffer and
then material was eluted initially with 4 column volumes of a
decreasing linear gradient of (NH.sub.4).sub.2SO.sub.4, generated
with 0.2 and 0 M solutions in 10 mM Tris-HCl buffer (pH 8.1).
Finally, the material was eluted with 2 column volumes of 10 mM
Tris-HCl (pH 8.1). Fractions containing partially purified
preIL-1.beta. were pooled, dialyzed against PBS, and stored at
-70.degree. C. until use.
[0174] D. Proteolytic Treatment of preIL-I.beta.
[0175] 5 .mu.l of preIL-1.beta. (about 50 .mu.g/ml in PBS) was
mixed with 10 .mu.l of purified IL-1.beta. pro (15-75 .mu.g/ml in
PBS) and incubated at 37.degree. C. for 30 minutes. The incubation
was terminated by placing the samples on Dry Ice or by the addition
of SDS sample buffer. PMSF 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-Vac
concentrator and dissolved in SDS sample buffer.
[0176] E. Western Blot Analysis of Proteolytic Products
[0177] SDS-PAGE was carried out with 12% polyacrylamide gels. 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 minutes at room
temperature. We used a MAb specific for mature IL-1.beta. to probe
the blot. MAb was added to a concentration of 9 .mu.g/mI, and the
incubation was continued for 30 minutes. 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 .mu.l diluted
into 10 ml of Tris-buffered saline).
[0178] The data show that Boc-Asp-Ch.sub.2F completely inhibits the
generation of mature IL-I.beta. from preIL-1.beta. at a
concentration of 5 .mu.M and partially inhibits generation of
mature IL-1.beta. at a concentration of 1 .mu.M.
Example 5
Biological activity of IL-1.beta. pro When Transfected into COS-7
Cells
[0179] We inserted a cDNA corresponding to amino acids 120 to 404
into a mammalian 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-1.beta.. After two days, cells were radiolabeled with .sup.35S
and IL-1.beta. specific proteins were immunoprecipitated from cell
lysates. The immunoprecipitates were analyzed by SDS-PAGE and
autoradiography. We found that transfected COS-7 cells can process
precursor IL-1.beta. to mature IL-1.beta. only if the cells were
co-transfected with a plasmid encoding IL-1.beta. pro. Cells
co-transfected with a control plasmid or cells mock transfected did
not show any processing of precursor IL-1.beta.. Thus, IL-1.beta.
pro, lacking the N-terminal 119 amino acids enables cells to
process precursor IL-1.beta. to the mature form of this protein.
Sequence CWU 1
1
26 1 1373 DNA Homo sapiens CDS (18)..(1229) 1 aaaaggagag aaaagcc
atg gcc gac aag gtc ctg aag gag aag aga aag 50 Met Ala Asp Lys Val
Leu Lys Glu Lys Arg Lys 1 5 10 ctg ttt atc cgt tcc atg ggt gaa ggt
aca ata aat ggc tta ctg gat 98 Leu Phe Ile Arg Ser Met Gly Glu Gly
Thr Ile Asn Gly Leu Leu Asp 15 20 25 gaa tta tta cag aca agg gtg
ctg aac aag gaa gag atg gag aaa gta 146 Glu Leu Leu Gln Thr Arg Val
Leu Asn Lys Glu Glu Met Glu Lys Val 30 35 40 aaa cgt gaa aat gct
aca gtt atg gat aag acc cga gct ttg att gac 194 Lys Arg Glu Asn Ala
Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp 45 50 55 tcc gtt att
ccg aaa ggg gca cag gca tgc caa att tgc atc aca tac 242 Ser Val Ile
Pro Lys Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr 60 65 70 75 att
tgt gaa gaa gac agt tac ctg gca ggg acg ctg gga ctc tca gca 290 Ile
Cys Glu Glu Asp Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala 80 85
90 gat caa aca tct gga aat tac ctt aat atg caa gac tct caa gga gta
338 Asp Gln Thr Ser Gly Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val
95 100 105 ctt tct tcc ttt cca gct cct cag gca gtg cag gac aac cca
gct atg 386 Leu Ser Ser Phe Pro Ala Pro Gln Ala Val Gln Asp Asn Pro
Ala Met 110 115 120 ccc aca tcc tca ggc tca gaa ggg aat gtc aag ctt
tgc tcc cta gaa 434 Pro Thr Ser Ser Gly Ser Glu Gly Asn Val Lys Leu
Cys Ser Leu Glu 125 130 135 gaa gct caa agg ata tgg aaa caa aag tcg
gca gag att tat cca ata 482 Glu Ala Gln Arg Ile Trp Lys Gln Lys Ser
Ala Glu Ile Tyr Pro Ile 140 145 150 155 atg gac aag tca agc cgc aca
cgt ctt gct ctc att atc tgc aat gaa 530 Met Asp Lys Ser Ser Arg Thr
Arg Leu Ala Leu Ile Ile Cys Asn Glu 160 165 170 gaa ttt gac agt att
cct aga aga act gga gct gag gtt gac atc aca 578 Glu Phe Asp Ser Ile
Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr 175 180 185 ggc atg aca
atg ctg cta caa aat ctg ggg tac agc gta gat gtg aaa 626 Gly Met Thr
Met Leu Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys 190 195 200 aaa
aat ctc act gct tcg gac atg act aca gag ctg gag gca ttt gca 674 Lys
Asn Leu Thr Ala Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala 205 210
215 cac cgc cca gag cac aag acc tct gac agc acg ttc ctg gtg ttc atg
722 His Arg Pro Glu His Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met
220 225 230 235 tct cat ggt att cgg gaa ggc att tgt ggg aag aaa cac
tct gag caa 770 Ser His Gly Ile Arg Glu Gly Ile Cys Gly Lys Lys His
Ser Glu Gln 240 245 250 gtc cca gat ata cta caa ctc aat gca atc ttt
aac atg ttg aat acc 818 Val Pro Asp Ile Leu Gln Leu Asn Ala Ile Phe
Asn Met Leu Asn Thr 255 260 265 aag aac tgc cca agt ttg aag gac aaa
ccg aag gtg atc atc atc cag 866 Lys Asn Cys Pro Ser Leu Lys Asp Lys
Pro Lys Val Ile Ile Ile Gln 270 275 280 gcc tgc cgt ggt gac agc cct
ggt gtg gtg tgg ttt aaa gat tca gta 914 Ala Cys Arg Gly Asp Ser Pro
Gly Val Val Trp Phe Lys Asp Ser Val 285 290 295 gga gtt tct gga aac
cta tct tta cca act aca gaa gag ttt gag gat 962 Gly Val Ser Gly Asn
Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp 300 305 310 315 gat gct
att aag aaa gcc cac ata gag aag gat ttt atc gct ttc tgc 1010 Asp
Ala Ile Lys Lys Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys 320 325
330 tct tcc aca cca gat aat gtt tct tgg aga cat ccc aca atg ggc tct
1058 Ser Ser Thr Pro Asp Asn Val Ser Trp Arg His Pro Thr Met Gly
Ser 335 340 345 gtt ttt att gga aga ctc att gaa cat atg caa gaa tat
gcc tgt tcc 1106 Val Phe Ile Gly Arg Leu Ile Glu His Met Gln Glu
Tyr Ala Cys Ser 350 355 360 tgt gat gtg gag gaa att ttc cgc aag gtt
cga ttt tca ttt gag cag 1154 Cys Asp Val Glu Glu Ile Phe Arg Lys
Val Arg Phe Ser Phe Glu Gln 365 370 375 cca gat ggt aga gcg cag atg
ccc acc act gaa aga gtg act ttg aca 1202 Pro Asp Gly Arg Ala Gln
Met Pro Thr Thr Glu Arg Val Thr Leu Thr 380 385 390 395 aga tgt ttc
tac ctc ttc cca gga cat taaaataagg aaactgtatg 1249 Arg Cys Phe Tyr
Leu Phe Pro Gly His 400 aatgtctgcg ggcaggaagt gaagagatcg ttctgtaaaa
ggtttttgga attatgtctg 1309 ctgaataata aacttttttt gaaataataa
atctggtaga aaaatgaaaa aaaaaaaaaa 1369 aaaa 1373 2 404 PRT Homo
sapiens 2 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile
Arg Ser 1 5 10 15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu
Leu Leu Gln Thr 20 25 30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys
Val Lys Arg Glu Asn Ala 35 40 45 Thr Val Met Asp Lys Thr Arg Ala
Leu Ile Asp Ser Val Ile Pro Lys 50 55 60 Gly Ala Gln Ala Cys Gln
Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp 65 70 75 80 Ser Tyr Leu Ala
Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly 85 90 95 Asn Tyr
Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro 100 105 110
Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly 115
120 125 Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg
Ile 130 135 140 Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp
Lys Ser Ser 145 150 155 160 Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn
Glu Glu Phe Asp Ser Ile 165 170 175 Pro Arg Arg Thr Gly Ala Glu Val
Asp Ile Thr Gly Met Thr Met Leu 180 185 190 Leu Gln Asn Leu Gly Tyr
Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 Ser Asp Met Thr
Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 Lys Thr
Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235
240 Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu
245 250 255 Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys
Pro Ser 260 265 270 Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala
Cys Arg Gly Asp 275 280 285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser
Val Gly Val Ser Gly Asn 290 295 300 Leu Ser Leu Pro Thr Thr Glu Glu
Phe Glu Asp Asp Ala Ile Lys Lys 305 310 315 320 Ala His Ile Glu Lys
Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp 325 330 335 Asn Val Ser
Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg 340 345 350 Leu
Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu 355 360
365 Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala
370 375 380 Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe
Tyr Leu 385 390 395 400 Phe Pro Gly His 3 269 PRT Homo sapiens 3
Met Ala Glu Val Pro Glu Leu Ala Ser Glu Met Met Ala Tyr Tyr Ser 1 5
10 15 Gly Asn Glu Asp Asp Leu Phe Phe Glu Ala Asp Gly Pro Lys Gln
Met 20 25 30 Lys Cys Ser Phe Gln Asp Leu Asp Leu Cys Pro Leu Asp
Gly Gly Ile 35 40 45 Gln Leu Arg Ile Ser Asp His His Tyr Ser Lys
Gly Phe Arg Gln Ala 50 55 60 Ala Ser Val Val Val Ala Met Asp Lys
Leu Arg Lys Met Leu Val Pro 65 70 75 80 Cys Pro Gln Thr Phe Gln Gln
Asn Asp Leu Ser Thr Phe Phe Pro Phe 85 90 95 Ile Phe Glu Glu Glu
Pro Ile Phe Phe Asp Thr Trp Asp Asn Glu Ala 100 105 110 Tyr Val His
Asp Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp 115 120 125 Ser
Gln Gln Lys Ser Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala 130 135
140 Leu His Leu Gln Gly Gln Asp Met Glu Gln Gln Val Val Phe Ser Met
145 150 155 160 Ser Phe Val Gln Gly Glu Glu Ser Asn Asp Lys Ile Pro
Val Ala Leu 165 170 175 Gly Leu Lys Glu Lys Asn Leu Tyr Leu Ser Cys
Val Leu Lys Asp Asp 180 185 190 Lys Pro Thr Leu Gln Leu Glu Ser Val
Asp Pro Lys Asn Tyr Pro Lys 195 200 205 Lys Lys Met Glu Lys Arg Phe
Val Phe Asn Lys Ile Glu Ile Asn Asn 210 215 220 Lys Leu Glu Phe Glu
Ser Ala Gln Phe Pro Asn Trp Tyr Ile Ser Thr 225 230 235 240 Ser Gln
Ala Glu Asn Met Pro Val Phe Leu Gly Gly Thr Lys Gly Gly 245 250 255
Gln Asp Ile Thr Asp Phe Thr Met Gln Phe Val Ser Ser 260 265 4 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 4 taccggctgt tccaggac 18 5 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 tacctattct gggctcga 18 6 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 6 ttggtcgata cgggtgt 17 7 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 7 caccacacca aatttcta 18 8 18 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 8 atggagaagg gtcctgta 18 9 26 DNA Artificial
Sequence Description of Artificial Sequence Synthetic primer 9
gtcgaattca ayccngcnat gccnac 26 10 26 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 10 gtctctagaa
gyttnacrtt nccytc 26 11 43 DNA Artificial Sequence Description of
Artificial Sequence Synthetic primer 11 atatcggtac cgcctccagc
atgcctccgg caatgcccac atc 43 12 31 DNA Artificial Sequence
Description of Artificial Sequence Synthetic primer 12 ctgctagatc
tgcccgcaga cattcataca g 31 13 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 13 Ala Tyr Val
His Asp Ala Pro Val Arg Ser 1 5 10 14 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 14 Ala Tyr Val
His Asn Ala Pro Val Arg Ser 1 5 10 15 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 15 Ala Tyr Val
His Glu Ala Pro Val Arg Ser 1 5 10 16 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 16 Ala Tyr Val
His Asp Ala Pro Val Arg Ser 1 5 10 17 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 17 Ala Tyr Val
His Asp Gly Pro Val Arg Ser 1 5 10 18 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 18 Ala Tyr Val
His Asp Val Pro Val Arg Ser 1 5 10 19 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 19 Ala Tyr Val
Phe Asp Ala Pro Val Arg Ser 1 5 10 20 10 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 20 Ala Tyr Val
His Asp Ala Ala Val Arg Ser 1 5 10 21 8 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 21 Glu Ala Tyr
Val His Asp Ala Pro 1 5 22 8 PRT Artificial Sequence Description of
Artificial Sequence Synthetic peptide 22 Tyr Val His Asp Ala Pro
Val Arg 1 5 23 6 PRT Artificial Sequence Description of Artificial
Sequence Synthetic peptide 23 Val His Asp Ala Pro Val 1 5 24 4 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
peptide 24 His Asp Ala Pro 1 25 8 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 25 Asp Tyr Lys
Asp Asp Asp Asp Lys 1 5 26 1659 DNA Homo sapiens 26 aaaaggagag
aaaagcctaa aagagagtgg gtagatggcc gacaaggtcc tgaaggagaa 60
gagaaagctg tttatccgtt ccatgggtga aggtacaata aatggcttaa ggtagaaggt
120 gaaggaaata ctggatgaat tattacagac aagggtgctg aacaaggaag
agatggagaa 180 agtaaaacgt gaaaatgcta cagtttatag aaaagaagaa
cgcttatgga taagacccga 240 gctttgattg actccgttat tccgaaaggg
gcacaggcat gccaaatttg catcacatac 300 cggataagtg aaagtgataa
tttgtgaaga agacagttac ctggcaggga cgctgggact 360 ctcagcagat
caaacatctg gaaattacct taattgagga aagaaagaaa attatgcaag 420
actctcaagg agtactttct tcctttccag ctcctcaggc agtgcaggac aacccagcta
480 tgcccacagg gaacggaaga gtgaatcctc aggctcagaa gggaatgtca
agctttgctc 540 cctagaagaa gctcaaagga tatggaaaca aaagtcggca
gttaagtaga acaggagaga 600 tttatccaat aatggacaag tcaagccgca
cacgtcttgc tctcattatc tgcaatgaag 660 aatttgacag tagagtgaag
aatgtttgag taattcctag aagaactgga gctgaggttg 720 acatcacagg
catgacaatg ctgctacaaa atctggggta cagcgtaaaa taaatttgga 780
aaaagggatg tgaaaaaaaa tctcactgct tcggacatga ctacagagct ggaggcattt
840 gcacaccgcc cagagcacaa gtatatgagg gcggacctct gacagcacgt
tcctggtgtt 900 catgtctcat ggtattcggg aaggcatttg tgggaagaaa
cactctgagg aagaaaatat 960 acacaagtcc cagatatact acaactcaat
gcaatcttta acatgttgaa taccaagaac 1020 tgcccaagtt tgaaggacag
aacaggagaa taagaaaccg aaggtgatca tcatccaggc 1080 ctgccgtggt
gacagccctg gtgtggtgtg gtttaaagat tcagtaggaa gattgggaaa 1140
aaaggtttct ggaaacctat ctttaccaac tacagaagag tttgaggatg atgctattaa
1200 gaaagcccac atagagaaga aactaaatag ttgagatttt atcgctttct
gctcttccac 1260 accagataat gtttcttgga gacatcccac aatgggctct
gtttttattg aggtggtaac 1320 caaggagaag ggaagactca ttgaacatat
gcaagaatat gcctgttcct gtgatgtgga 1380 ggaaattttc cgcaaggttc
gatttggaga gaagtttgag attagcttca tttgagcagc 1440 cagatggtag
agcgcagatg cccaccactg aaagagtgac tttgacaaga tgtttctacc 1500
tcgttcccag gacattaaaa taaggaaact gtatgaatgt ctgcgggcag gaagtgaaga
1560 gatcgttctg taaaaggttt ttggaattat gtctgctgaa taataaactt
tttttgaaat 1620 aataaatctg gtagaaaaat gaaaaaaaaa aaaaaaaaa 1659
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