U.S. patent application number 09/818769 was filed with the patent office on 2001-10-18 for protease inhibitor assay.
Invention is credited to Bronstein, Irena, Palmer, Michelle, Tillotson, Bonnie, Voyta, John.
Application Number | 20010031478 09/818769 |
Document ID | / |
Family ID | 21902784 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031478 |
Kind Code |
A1 |
Bronstein, Irena ; et
al. |
October 18, 2001 |
Protease inhibitor assay
Abstract
Heterogenous and homogenous assays are provided for the
detection of protease inhibitory activity in a sample or target
compound, taking advantage of the chemiluminescent characteristics
of 1,2-dioxetanes. In the heterogenous assay, a peptide bearing a
cleavage site for the protease of interest is provided with a first
member of a first ligand binding pair at one end, and a first
member of a second ligand binding pair at the other end. The other
member of the first ligand binding pair is attached to a surface,
which binds the peptide, or protease substrate, to the surface. The
peptide substrate is combined with the protease and target compound
or sample. Substrate cleavage, if not inhibited, is allowed to
occur, and any unbound cleaved fragments are removed. An enzyme
complexed with the second member of the second ligand binding pair
is added, and allowed to bind to any of the (uncleaved) first
member of the second ligand binding pair remaining. Unbound complex
is removed, and a 1,2-dioxetane substrate for the enzyme is added.
If any peptide substrate has not been cleaved, the dioxetane will
chemiluminesce, indicating inhibitory activity. In a homogenous
assay, the same substrate bears at one end a fluorescent energy
accepting moiety, and at the other end a 1,2-dioxetane or
precursor. If the substrate is cleaved by the protease, the
dioxetane and the fluorescent moiety are not in close physical
relationship, and no energy transfer occurrs when the dioxetane is
caused to decompose. If cleavage has not occurred, indicating
inhibition, when the dioxetane is caused to decompose, energy is
transferred to the fluorescing entity, which releases light of a
wavelength recognizably distinct from that of the dioxetane.
Inventors: |
Bronstein, Irena; (Newton,
MA) ; Voyta, John; (Sudbury, MA) ; Palmer,
Michelle; (Arlington, MA) ; Tillotson, Bonnie;
(Belmont, MA) |
Correspondence
Address: |
Supervisor, Patent Prosecution Services
PIPER MARBURY RUDNICK & WOLFE LLP
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
21902784 |
Appl. No.: |
09/818769 |
Filed: |
March 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09818769 |
Mar 28, 2001 |
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09035820 |
Mar 6, 1998 |
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6243980 |
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60038940 |
Mar 7, 1997 |
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Current U.S.
Class: |
435/7.5 |
Current CPC
Class: |
C12Q 1/37 20130101; G01N
33/581 20130101; Y10S 436/805 20130101; G01N 33/582 20130101; G01N
2333/81 20130101; Y10S 436/80 20130101 |
Class at
Publication: |
435/7.5 |
International
Class: |
G01N 033/53 |
Claims
1. A method for conducting an assay to determine whether a target
compound exhibits activity as a protease inhibitor, comprising:
combining said protease and said target compound in an environment
further comprising a construct, wherein said environment is such
that said protease cleaves said construct in the absence of
inhibition of said protease, said construct comprising an amino
acid sequence terminating, at a first end, with a first member of a
first ligand binding pair and at a second end with a first member
of a second ligand binding pair, wherein said first member of said
first ligand binding pair binds to a second member of said first
ligand binding pair which second member is bound to a surface,
removing any fragments of said construct cleaved by said protease,
adding a complex of a second member of said second ligand binding
pair complexed with an enzyme and allowing said complex to bind to
any said first member of said second ligand binding pair present,
removing any unbound complex, adding a 1,2-dioxetane which is a
substrate for said enzyme and observing any chemiluminescence
released thereby, wherein emission of said chemiluminescence is
indicative of protease inhibition activity by said target
compound.
2. The method of claim 1, wherein said chemiluminescence observed
is measured, and wherein the amount of chemiluminescence observed
is correlated with the degree of inhibitory activity exhibited by
said compound.
3. The method of claim 1. wherein said assay is a transfer free
single plate endpoint assay.
4. The method of claim 1, wherein said surface is at least a
portion of at least one well of a test plate.
5. The method of claim 1, wherein said second member of said second
ligand binding pair is an antibody.
6. The method of claim 1, wherein said first ligand binding pair is
biotin and a compound which binds to biotin at least as tightly as
avidin.
7. The method of claim 1, wherein said second ligand binding pair
is biotin and a compound which binds to biotin at least as tightly
as avidin.
8. The method of claim 6, wherein said compound which binds to
biotin at least as tightly as avidin is avidin or strepavidin.
9. The method of claim 7, wherein said compound which binds as
tightly as avidin is avidin or strepavidin.
10. The method of claim 1, wherein said first member of said first
ligand binding pair is biotin and said first member of said second
ligand binding pair is fluorescein.
11. The method of claim 1, wherein said second ligand binding pair
is FLAG and an antibody therefor.
12. The method of claim 1, wherein said protease is selected from
the group consisting of a serine protease, a cysteine protease, an
aspartic protease and a metallo proteinase.
13. The method of claim 1, wherein said protease is selected from
the group consisting of HIV-1 protease, caspases, cathhepsins,
hydrolase, L-proteinase, calpain, interleukin converting proteases,
urokinase, trypsin, thrombin, HIV-2 protease, Yapsin I, Yapsin 3,
Plasmepsin I, Plasmepsin II, collagenase, gelatinases, stromelysin,
amino peptidase and elastase.
14. The method of claim 13, wherein said protease is HIV-1
protease.
15. A protease substrate for conducting an assay to determine
protease inhibitory activity in a target compound, said substrate
comprising: a polypeptide, said polypeptide including a cleavage
site specific for said protease, said polypeptide bearing at a
first end a first member of a first ligand binding pair and at
another end a first member of a second ligand binding pair, wherein
said first member of said first ligand binding pair and said first
member of said second ligand binding pair do not bind to each
other.
16. The substrate of claim 15, wherein said first and second ligand
binding pair is comprised, independently, of an antigen and an
antibody therefore, or biotin and a compound which binds as tightly
to biotin as avidin.
17. The substrate of claim 15, wherein said substrate has the
sequence
Fluorescein-Spacer-Ser-Glu-Asu-Tyr-Pro-Ile-Val-Glu-Spacer-Biotin,
or
FLAG-Ser-Nle-Ala-Glu-Phe-Leu-Val-Arg-Ala-Hys-His-Spacer-Biotin.
18. A homogenous assay for detection of protease inhibitory
activity in a target compound, comprising: combining said protease
and said target compound in an environment further comprising a
construct, wherein said environment is such that said protease
cleaves said construct in the absence of inhibition of said
protease, said construct comprising a polypeptide of 2-10 amino
acids, said polypeptide terminating, at a first end, and a moiety
which is a 1,2-dioxetane or precursor thereof which can be
oxygenated in said environment to provide a 1,2-dioxetane moiety
and, at a second end, with an energy accepting fluorescent moiety,
oxygenating said precursor if present to form a 1,2-dioxetane
moiety, causing said 1,2-dioxetane moiety to decompose, and
observing the wavelength of light emitted from said environment,
wherein said wavelength, if the wavelength of said fluorescent
emitter, is indicative of inhibitory activity, and said wavelength,
if the wavelength of said dioxetane, is indicative of absence of
inhibitory activity.
19. A construct for conducting a homogenous assay to determine
protease inhibitory activity in a target compound, comprising: a
polypeptide of 2-10 amino acid residues, wherein said polypeptide
includes a cleavage site specific for said protease, said
polypeptide bearing, at one end, a fluorescent energy accepting
moiety or a chemiluminescece quenching moiety and at another end, a
1,2-dioxetane or 1,2-dioxetane precursor moiety that can be
oxygenated to form a 1,2-dioxetane moiety in the course of said
assay.
20. A kit for conducting an assay to determine whether a protease
inhibitor is present in a sample, comprising in one or more
containers: (a) a peptide which is a substrate for said protease,
wherein said peptide substrate is labeled with (i) a first member
of a first ligand binding pair, and (ii) a first member of a second
ligand binding pair; (b) a chemiluminescent 1,2-dioxetane substrate
containing an enzymatically cleavable group, which substrate is
capable of producing light in the presence of an enzyme which
cleaves said enzymatically cleavable group from said substrate.
21. The kit of claim 20, wherein said protease is HIV-1.
22. The kit of claim 21, wherein said peptide substrate comprises
the amino acid sequence: -Ser-Gln-Asn-Try-Pro-Ile-Val-Gln-.
23. The kit of claim 20, wherein said first member of said second
ligand binding pair is fluorescein.
24. The kit of claim 20, wherein said first member of said first
ligand binding pair is biotin.
25. The kit of claim 20, additionally comprising a polymeric
quaternary onium salt enhancement agent for said 1,2-dioxetane.
26. The kit of claim 25, wherein said enhancement agent comprises
polyvinylbenzyltributyl ammonium chloride.
27. The kit of claim 20, further comprising a enzyme complexed with
a second member of said second ligand binding pair.
28. The kit of claim 27, wherein said enzyme is alkaline
phosphatase.
Description
[0001] This application is a regular National application claiming
priority from Provisional Application, U.S. Application Ser. No.
60/038,940 filed Mar. 7, 1997.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention pertains to the use of chemiluminescent
1,2-dioxetanes in homogenous or heterogeneous assays to detect
proteases inhibitors.
[0004] 2. Discussion of the Background
[0005] The identification of novel therapeutics that block or
inhibit inimical proteases, or proteases that mediate disease
conditions, such as the 11-kd protease encoded by the human
immunodeficiency virus 1(HIV-1) is a key step in slowing the
disease process of AIDS. Retroviral proteases are essential in the
process of viral gag-pol polyproteins of the HIV-1 and HIV-2
viruses. There are a few highly conserved consensus sequences in
retroviral polyproteins, one of which consists of a pentapeptide
(Ser/Thr)-X-X'-(Tyr/Phe)-Pro. Cleavage occurs between the Tyr or
Phe and Pro residues. Blocking activity of these proteases will
interfere with the progression of HIV infection. Although potent
drugs which block HIV protease activity have been found, there is
an ongoing need to find and develop novel inhibitors.
[0006] Current methods utilized in rapid screening of protease
inhibitors are subject to many interferences from a variety of
sources. The most common non-isotopic approach is a fluorescent
assay. In one case, such as in the detection of HIV protease, the
fluorescent substrate is labeled with a fluorescent dansyl group on
one end of a peptide and a quencher on the other. An increase in
fluorescence signal occurs upon cleavage of the protease due to the
fact that the emitter and the quencher are separated as described
in Matayoshi et al. 1990, Science 247: 954-958. Fluorescent
substrates for other proteases can be designed 5 with a terminal
fluorophore which emits a fluorescent signal upon cleavage by the
enzyme.
[0007] Both of the above assay approaches are commonly used as high
throughput assays for screening large chemical, natural product and
combinatorial libraries. These assays tend to have problems related
to autofluorescence of biological components due to the nature of
the molecules which are screened. Many of the compounds and natural
product 10 extracts are colored or fluorescent and are present in
the solution when the assay signal is monitored. This results in an
assay interference which limits the detection sensitivity and the
dynamic range of the assay. This interference can easily be
interpreted as an inhibition of the enzyme, making it difficult to
determine true positive inhibition, thus, requiring extensive
follow-up assays to distinguish true positives from the false
positives.
[0008] U.S. Pat. No. 5,591,591, assigned to Tropix, describes
assays for the detection of proteases wherein a dioxetane compound
bearing a proteolytic enzyme-specific amino acid or peptide, is
added to a sample suspected of containing the protease, and the
amino acid is removed by enzymatic reaction, causing the dioxetane
to decompose and chemiluminescence.
SUMMARY OF THE INVENTION
[0009] An alternative approach to the above homogeneous approaches
is to use homogenous or heterogeneous assays which are not subject
to interference. The present inventors have developed highly
sensitive assays using a chemiluminescent 1,2-dioxetane substrate
for high throughput screening of HIV-1 protease activity. The
present invention provides the advantage of an assay that it is not
subject to interferences from colored or fluorescent compounds, and
therefore is more sensitive and exhibits a greater dynamic range
compared to a direct, fluorescent enzyme assay. This assay, adapted
for the detection of HIV protease inhibitors, utilizes a synthetic
peptide substrate I
(Fam-spacer-Ser-Gln-Asn-Try-Pro-lle-Val-Gln-spacer-(Biotin)-NH2)
whose sequence is derived from the native cleavage site of the Gag
polyprotein. (Fam is used herein to indicate fluorescein). Many
high throughput HIV screening methodologies exist which utilize
large quantities of reagents and involve more laborious
manipulations. The assay of the present invention may be
advantageously formatted as a simple single plate endpoint assay
which is sensitive down to fmoles of captured peptide which is
particularly useful for high throughout screening, although it may
also be presented as a conventional two-step transfer and dilation
capture assay. The assay uses a chemiluminescent 1,2-dioxetane
alkaline phosphatase substrate in an immunoassay format for the
sensitive detection of cleaved peptide. The uncleaved peptide is
recognized by an anti-fluorescein alkaline phosphatase conjugated
antibody. Capture conditions have been optimized to assure a linear
response to cleaved peptide concentration. This response correlates
well to HPLC analysis of cleaved product. The HIV assay has been
validated for acetyl-pepstatin, a known HIV-1 inhibitor. This
robust assay is amenable to automation and can be used to screen
large numbers of compounds in a cost effective 96 well format.
[0010] In an alternative embodiment, a homogenous assay less
sensitive to color or fluorescence interference than prior art
assays is used. In this assay, the same peptide is employed, but
labeled at one terminus with fluorescein or other fluorescing
energy acceptor, and at the other end by a 1,2-dioxetane label
covalently attached to the peptide terminus. After admixture of the
target compound, the dioxetane is caused to decompose by addition
of a chemical (enzymatic or non-enzymatic) or another trigger, such
as application of heat or change in pH. If the peptide has not been
cleaved, the dioxetane is in close physical relationship with the
fluorescent energy acceptor, and upon triggering, energy transfer
assisted fluorescence is observed. The light has a characteristic
color dependent on the fluorescent emitter, such as green for
fluorescein. If the peptide has been cleaved, the chemiluminescent
light of the dioxetane itself, typically a bluer light, is
observed.
DETAILED DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1. Illustration of two labeled HIV-1 substrates I and
II whose sequences are derived from a natural processing site for
aspartyl HIV-1 protease. Incubation of these two HIV-1 peptides
with rec HIV-1 protease results in a specific cleavage between the
Try-Pro bond as reported in Kohl, NE et al.(1988), Proc. Acad. Sci.
USA, 85, 4684 and Billich, SW, et al. (1988), J. Biol. Chem. 263,
17905. These are compared against the 15 prior art substrate as
reported in Mayatoshi et al.
[0012] FIG. 2. HIV-1 Protease Assay. Steps include 1. Addition of
rec HIV-1 protease, acetyl-pepstatin(3) and/or screening compound
to NeutrAvidin coated plates, with a preincubation. 2. Addition of
HIV-1 protease peptide I followed with a 60 min. Incubation at
37.degree. C. 3. Wash out cleaved peptide fragment. 4. Add
anti-fluorescein alkaline phosphatase antibody to detect uncleaved
bound peptide and then 5. Add CSPD/Sapphire II and measure emitted
light in a TR717 luminometer(Tropix).
[0013] FIG. 3. The phosphate group on the CSPD substrate is cleaved
by alkaline phosphatase to generate the unstable anion intermediate
1. Intermediate 1, then undergoes decomposition producing a
long-lived emission at 470 nm.
[0014] FIG. 4. HIV-1 protease peptide I titration on Neutravidin
coated plates. 100 ul of diluted peptide was added to various
densities of coated Neutravidin wells. Plates were washed and
peptide was detected with an anti-fluorescein alkaline phosphatase
conjugate followed with the addition of CSPD/Sapphire II. Light
emission was measured in a TR717 microplate
luminometer(Tropix).
[0015] FIGS. 5(A&B). Comparison of HIV-1 peptide I cleavage by
rec HIV-1 protease by HPLC analysis and capture on Neutravidin
coated plates. 35 nM of rec HIV-1 protease was added to 10 uM HIV
peptide in eppendorf tubes and incubated at 37.degree. C. At
specific time points 0.1% TFA was added to stop the reaction and
tubes placed on ice. Reaction products were analyzed by HPLC.
Injection volumes were 100 ul and absorbency was monitored @490 nm.
The percentage of cleaved product was calculated from the peak
areas. For peptide capture experiments, 100 ul of a 1:50 dilution
was added to Neutravidin (Sug/ml) coated plates and incubated at
ambient temp. for 60 min. after a wash step. captured peptide was
detected with anti-fluorescein conjugated alkaline phosphatase
antibody followed by another wash step and then the addition of
CSPD/Sapphire II. Light emission was measured in a TR717 microplate
luminometer(Tropix).
[0016] FIG. 6. Hydrolysis of EDANS/DABCYL HIV-1 protease Prior Art
Substrate by rec HIV-1 protease. Fluorogenic HIV-1 peptide II was
added to protease buffer pH 4.7 in 96 well plates. Rec HIV-1
protease was added at ambient temperature and immediately read over
a 105 minute time period on a LS50B fluorescence spectrophotometer
(Perkin-Elmer). Excitation was set at 340 nm and emission was set
at 490 nm.
[0017] FIG. 7. Hydrolysis and capture of HIV-1 protease substrate I
on Neutravidin coated plates. Dilutions of rec HIV-1 protease
diluted in protease buffer pH 5.5 were added to wells previously
coated with 0.1 ug/ml Neutravidin. 10 pmoles of protease substrate
was added to start the reaction and the plate was incubated for 1
hour at 37.degree. C. After a wash step, captured peptide was
detected with anti-fluorescein conjugated alkaline phosphatase
antibody followed by another wash step and then the addition of
CSPD/Sapphire II. Light emission was measured in a TR717 microplate
luminometer(Tropix). 100% cleavage of the substrate is not obtained
for substrate 1. The date has been adjusted to reflect this
phenomenon. In actuality, the detected background may be
different.
[0018] FIG. 8. Rec HIV-1 protease inhibition by acetyl-pepstatin in
a HIV-1 capture assay. 2.5 pmoles of rec HIV-1 protease diluted in
protease buffer pH 5.5 was added to Neutravidin (0.1 ug/ml) coated
wells. Dilutions of acetyl-pepstatin in DMSO were added to wells
followed with a preincubation of 5 minutes. 20 pmoles of HIV-1
protease substrate was added and the plate incubated for 1 hour at
37.degree. C. After a wash step captured peptide was detected with
an anti-fluorescein conjugated alkaline phosphatase antibody. After
a second wash step, CSPD/Sapphire II was added and light emission
was measured in a TR717 microplate luminometer(Tropix).
[0019] FIG. 9. Screening compound plate-1001 at 25 uM (5% DMSO) for
rec HIV-1 protease inhibition. 2.5 pmoles of rec HIV-1 protease
diluted in protease buffer pH 5.5 was added to Neutravidin coated
plates. 5 ul from a compound screening plate largely composed of
drug standards was added and preincubated for 5 minutes. 20 pmoles
of HIV-1 protease substrate I was added and the plate was incubated
for 60 minutes at 37.degree. C. After a wash step, captured peptide
was detected with anti-fluorescein conjugated alkaline phosphatase
antibody. After a second wash step, CSPD/Sapphire II was added and
light emission was measured in a TR717 microplate
luminometer(Tropix).
[0020] FIG. 10. HIV FLAG Peptide titration on Neutravidin and
Anti-FLAG MI monoclonal antibody coated plates. 100 ul of diluted
HIV FLAG peptide was added to various densities of pre-coated
Neutravidin or Anti FLAG MI monoclonal antibody coated plates.
Coating was achieved by diluting a given amount of Neutravidin or
Anti-FLAG MI monoclonal antibody and BSA in PBS so as to achieve a
total protein concentration of 5 .mu.g/ml. Plates were incubated
for 1 hour at 37.degree. C. followed by a 3.times. wash with
TBS/0.05% Tween/3 mM CaCl.sub.2. 100 ul of a 1:20,000 dilution of
Avidix-AP was added and incubated for 1 hour at room temp. After 3
washes in TBS/Tween/CaCl.sub.2 buffer and 1 wash with 1.times.
Tris/1 mM MgCl/3 mMCaCl pH 9.8, 100 ul ready to sue CSPD/Sapphire
II was added and wells incubated for 30 min at room temp.
Luminescence was then measured in a TR717 microplate luminometer
(Tropix). The present inventors have discovered novel peptide
capture conditions which permit the assay to be performed as a
single well (plate) assay as opposed to a 2 step transfer and
dilution assay. As shown in FIG. 12, the signals are relatively the
same when done as a one step or two step assay suggesting that
non-specific cleavage of other assay components by the rec HIV-1
protease is nonexistent.
[0021] FIG. 11. Capture of HIV FLAG and HIV FAM Peptides on
Neutravidin (5 ug/ml) coated plates post hydrolysis with rec HIV-1
protease (50 nM). 50 nM rec HIV-1 protease was added to 1 uM HIV
FLAG or HIV FAM peptides in protease buffer (0.1 mM NaAcetate, 1 M
NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/ml BSA, pH 4.7). Biotin at 0.5 mM
and 5 Units of enterokinase were added to control tubes. Reactions
were done for 1 hour at 37.degree. C. in eppendorf tubes. All tubes
were then placed on ice to stop the reaction. An additional 50 nM
rec HIV-1 protease was added to 2 of the reaction tubes and
incubated further for 30 minutes at 37.degree. C. HIV FLAG peptide
containing tubes were diluted 1:100 (10 nM final) and HIV FAM
peptide tubes were diluted 1:100,000 (0.01 nM final) in protease
buffer. 100 ul was added to Neutravidin coated wells (5 ug/ml) and
incubated for 1 hour at room temp. Wells were then washed with
3.times. PBS/Tween/CaCl buffer. 100 ul of 1:5000 dilution of Anti
FITC-AP Fab Fragment was added to HIV FAM peptide containing wells
and 100 ul of a precomplexed Anti-FLAG MI antibody (1:1000) and
Goat anti mouse-AP conjugate (1:10,000) was added to HIV FLAG
peptide containing wells. Plates were incubated for additional hour
at room temp then washed 3.times. in TBS/Tween/CaCl and 1.times.
Tris/MgCl/CaCl pH 9.8. 100 ul ready to use CSPD/Sapphire II was
added to wells and incubated 30 minutes at room temp. Luminescence
was then measured in a TR717 microplate luminometer.
[0022] FIG. 12. Titration of rec HIV-1 Protease I with 100 nM HIV
FLAG Peptide. Diluted rec HIV-1 protease was added to 100 nM HIV
FLAG peptide in protease buffer. Reactions were done either in
eppendorf tubes or directly in wells precoated with Anti-FLAG MI
monoclonal antibody (0.1 ug/ml). Competing FLAG octapeptide at 2.5
uM was added to control tubes and wells. Incubations were done for
1 hour at 37.degree. C., then eppendorf reaction products
transferred to wells, and further incubated for 1 hour at room
temp. Plates were washed 3.times. in PBS/Tween/CaCl buffer followed
with addition of a 1:20,000 dilution of Avidix-AP. Plates were
further incubated for 1 hour at room temp and then washed 3.times.
with PBS/Tween/CaCl buffer and 1.times. with Tris/MgCl/CaCl pH 9.8
buffer. 100 ul CSPD/Sapphire II was added and wells incubated for
30 minutes to room temp. Luminescence was then measured in a TR717
microplate luminometer (Tropix).
[0023] FIG. 13. Inhibition of 12.5 nM rec HIV-1 Protease with
Acetyl-Pepstatin. Assays consisted of reactions with 100 nM HIV
FLAG Peptide in wells coated with Anti-FLAG MI antibody (0.1 ug/ml)
for 1 hour at 37.degree. C. Dilutions of acetyl-pepstatin were
preincubated for 5 minutes with rec HIV-1 enzyme before adding to
HIV FLAG peptide substrate in protease buffer. All additions were
made on a Zymark Rapid Plate. The plate was then washed 4.times.
with TBS/3 mM CaCl.sub.2/0.05% Tween on Tecan 96 plate washer.
100ul of a 1:20,000 dilution of Avidix-AP conjugate was added to
each well and incubated for 1 hour @ room temp. The plate was then
washed 3.times. with above buffer and 1.times. with Tris/MgCl/CaCl
pH 9.8. 100 ul CSPD/Sapphire II was added, incubated for 30 min and
then light emission was measured in a TR717 luminometer (Tropix).
Note: Acetyl-pepstatin is a well known aspartic proteinase
inhibitor and is a reported inhibitor of HIV-1 protease (Pro. Natl.
Sci. U.S.A. 85, 66123, (1988)).
[0024] FIG. 14. Synthetic peptide for homogenous assay. This
peptide, also adapted for HIV-1 protease inhibitor detection, is
derivatized at both ends of the molecule, in fashion similar to
that employed in the heterogenous assay. In this case, however, one
terminus, preferably the carboxy terminus, is labeled with an
energy accepting fluorescent moiety, such as fluorescein, and the
amino terminus is labeled with a 1,2-dioxetane moiety precursor
(14A) which can be photooxygenated in situ to the dioxetane (14B).
The dioxetane may be triggered by other than chemical (enzymatic or
non-enzymatic) means if necessary.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] A peptide substrate is synthesized which contains the
appropriate cleavage site for the target protease. This peptide is
labeled, in a heterogenous assay, with one member of a first ligand
binding pair, such as biotin, on one end and a member of a second
ligand binding pair, such as fluorescein, at the other end. This
peptide is then incubated with the protease and a compound of
interest to be screened for inhibitory activity, in a well or other
solid phase coated with the second binding ligand of said first
ligand binding pair, such as avidin or strepavidin.
[0026] The spacer between the ligand binding pair members and the
peptide of the inventive substrate may be a covalent bond or any
covalent binding moiety which does not interfere with either the
first or second ligand binding pair or substrate cleavage. Among
exemplary sequences are C1-12 alkyls, alkylamino's, carboxylic
acids, or any neutral moiety terminating in coupling
functionalities at either end. The spacer may be provided with
water-solubility enhancing substituents (e.g., carboxy, sulfoxy,
halo, etc.) or, where necessary for enzyme/antibody presentation,
to cyclize the substrate, bridging agents.
[0027] After incubation, the wells are washed, incubated with the
second binding member of said second binding ligand pair conjugated
with an enzyme which is an effective trigger for a 1,2-dioxetane,
such as alkaline phosphatase, washed, incubated with a
1,2-dioxetane substrate such as chlorine substituted phosphate
dioxetane (CSPD) and the signal is measured. Higher signals are
detected in the presence of an inhibitor.
[0028] The 1,2-dioxetanes used as substrates may be any of those
described in any of Tropix' prior patents, including U.S. Pat. Nos.
4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978.614; 5,032,381;
5,112,960, 5,154,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900;
5,336,596; 5,543,295; 5,582,980; 5,605.795; 5,625,007; 5,654,154;
and 5,679,803, which are incorporated by reference herein. The
above mentioned patents disclose 1,2-dioxetanes as chemiluminescent
compounds which can be used as reporter molecules in ultrasensitive
assays that can be conducted quickly without resort to exotic
conditions or elaborate apparatus, for the detection of a variety
of biological molecules. A preferred substrate is CSPD, the
chlorine-substituted counterpart of AMPPD, which is the disodium
salt of 3-(4-methoxy-spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.s-
up.3.7]decan]4-yl)phenyl phosphate.
[0029] The dioxetanes contain a fluorescent chromophore group,
preferably an aryl group such as phenyl or naphthyl and an enzyme
cleavable group, e.g., a phosphate ester, which when cleaved by the
appropriate enzyme, e.g., alkaline phosphatase, forms a negatively
charged substituent (e.g., an oxyanion). This destabilizes the
dioxetane, thereby causing the dioxetane to decompose to form two
carbonyl-containing groups accompanied by the release of light.
[0030] To enhance the chemiluminescent signal, and improve
signal/noise ratio to permit discrimination between background
signals and positive target-responsive signals at very low levels,
a water-soluble enhancement agent may be added to the sample prior
to or concomitant with the introduction of the dioxetane. Specific
enhancement agents which may be used include: quaternary onium
polymeric salts such as polyvinylbenzyltributyl-ammonium chloride
(Sapphire II, Tropix) or any of those disclosed in U.S. Pat. No.
5,336,596 as potential membrane coatings, neutral detergents such
as Tween-20 (Sigma), cationic detergents, such as
cetyltrimethylammonium chloride (CTAB, Sigma) and combinations
thereof.
[0031] Families of proteolytic enzymes which may be targets for
determination of inhibitory agents include
[0032] Cysteine Proteases
[0033] Caspases 1, 2, 3, 6, 7, 8
[0034] Cathepsins (B, H, S and L)
[0035] Hydrolase
[0036] L-proteinases
[0037] Calpain
[0038] Interleukin converting proteases (ICE)
[0039] Serine Proteases
[0040] Urokinase
[0041] Trypsin
[0042] Thrombin
[0043] Cathepsin G
[0044] Aspartic Proteases
[0045] HIV-1 and 2
[0046] Yapsin I and YAP 3
[0047] Plasmepsin I and II
[0048] Cathepsin D and E
[0049] Metalloproteinases
[0050] Collagenase
[0051] Gelatinase A and B
[0052] Stromelysin
[0053] Aminopeptidase
[0054] Elastase
[0055] or any of those mentioned in U.S. Pat. No. 5,591,591. Assay
conditions and applications for specific proteases are also listed
in U.S. Pat. No. 5,591,591. It is possible to use any peptide which
is recognized by the protease of interest. Examples of specific
peptides and proteases are listed in U.S. Pat. No. 5,591,591. In
particular, a peptide is prepared which features a cleavage site
for which the target protease is specific. The resulting peptide is
short, and easily prepared using conventional synthetic
technology.
[0056] A large variety of ligand binding pairs can be used for both
the first and second ligand binding pair employed. Among preferred
labels for either end of the peptide are: Biotin, Fluorescein
(FAM), FLAG, HIS tag (6 histidine amino acid sequence), and Digoxin
(digoxigenin labeled peptide). These are bound by the other member
of the first pair, preferably bound or attached to a solid phase so
as to remain through washing and addition of the other member of
the second pair, added after an opportunity for cleavage to occur,
the other member being complexed with alkaline phosphatase or other
suitable enzyme as a trigger. The principal restriction on the
identity of the binding pairs is that the first binding pair be
distinct from, and not interact with, the second binding pair.
[0057] In addition to alkaline phosphatase, other enzymes which may
be used to cleave the enzymatically cleavable group from the
dioxetane include: acid phosphatases, esterases, decarboxylases,
phospholipase D,.beta.-xylosidase, .beta.-D-frucosidase,
thioglucosidase, .beta.-D-galactosidase, .alpha.-D-galactosidase,
.alpha.-D-glucosidase, .beta.-D-glucosidase, .alpha.-D-mannosidase,
.beta.-D-mannosidase, .beta.-D-fructofuranoside,
.beta.-D-glucosiduronase, and trypsin.
[0058] In addition to being of particular interest as organic
moieties that constitute diagnostic markers, protease enzymes are
also of considerable interest as enzyme labels. Examples of
diagnostic protease markers include cathepsin B (cancer), cathepsin
G (emphysema, rheumatoid arthritis, inflammation), plasminogen
activator (thrombosis, chronic inflammation, cancer) and urokinase
(cancer). Assays for protease detection are therefore needed to
monitor protein stability in various biological and commercial
processes.
[0059] In an alternative embodiment of this assay a homogeneous
chemiluminescent energy transfer assay is provided. In this
approach, one end of the cleavage sequence peptide bears a
1,2-dioxetane or dioxetane precursor. Direct attachment of this
moiety, followed by oxygenation to form the dioxetane, is enabled
in U.S. patent application Ser. No. 08/767,479, allowed and
incorporated herein by reference. The other end of this peptide
bears an energy accepting fluorescent molecule such as fluorescein,
or any of a variety of similar fluorescing moieties, such as those
disclosed in U.S. Pat. Nos. 5,004,565 and 5,208,148 which are
incorporated herein by reference. The peptide is sufficiently short
(no more than about 10 amino acid residues) such that the dioxetane
is in close physical association with the fluorescent label. Upon
triggering of the dioxetane, which can be effected by addition of
an enzyme, or pH alteration, or application of heat or other
triggers, the dioxetane decomposes, emitting energy which excites
the fluorescent moiety which then fluoresces if no cleavage has
occurred (a positive test for protease inhibition). If cleavage has
occurred, the dioxetane and fluorescent moieties are no longer in
close physical relationship, and the light is emitted by the
chemiluminescent dioxetane. The wavelength of the fluorescent
emitter is characteristically shifted markedly from that of the
dioxetane, allowing easy discrimination in a homogenous assay.
[0060] Another approach to a homogeneous assay using an enol lether
or dioxetane label is to use a quenching group in place of the
fluorescent acceptor. In this format, upon triggering of the
dioxetane label, there will be no light from the intact peptide due
to the quenching group. If cleavage of the peptide substrate by a
protease occurs prior to triggering, emission from the dioxetane
group will be observed upon triggering. This quenching process is
subject to the same distance restraints as the energy transfer
process described above. A suitable quenching group for the
substrate shown in FIG. 14 would be a dabcyl group in place of the
fluorescein group. Any non-fluorescent light absorbing dye, which
has an absorption spectra which overlaps the emission spectra of
the donor dioxetane could be used as a quenching group.
[0061] Variations on this approach can be made for many types of
proteases. Direct covalent or hydrophobic attachment of a peptide
substrate to a microwell surface may be required for some
proteases. Alternatively, direct synthesis of the peptide on the
solid phase may also have advantages.
[0062] The heterogenous assay of this invention has been
exemplified, below, by reference to an assay adapted to detect
inhibition of HIV-1 protease. This example is demonstrative only,
and not intended to be limiting.
[0063] Materials and Methods
[0064] Reagents:
[0065] 1. The HIV FLAG peptide was custom synthesized by Genemed
Synthesis, Inc.
[0066] 2. Anti-FLAG MI Monoclonal Antibody, FLAG Octapeptide, and
Enterokinase were purchased from Eastman Kodak Company.
[0067] Neutravidin coated plates: Neutravidin.TM. Biotin Binding
Protein (PIERCE) was diluted to 5 ug/ml or 0.1 ug/ml in BupH.TM.
Carbonate-Bicarbonate buffer (PIERCE), 1 mg/ml BSA (fraction V
Sigma) and 100 ul added to white 96 well plates
(Dynatech-Microlitel) and incubated for 2 hours at 37.degree. C.
The wells were then washed with PBS, 0.1% (v/v) Tween 20 and
blocked with PBS, 0.1% (V/V) Tween 20, 1 mg/ml BSA overnight at 4
degrees.
[0068] Protease assays: Rec HIV-PR1 (affinity purified -BACHEM) was
diluted in 0.1 M NaAcetate, 1M NaCl, 1 mM EDTA, 1 mM DTT, 1 mg/ml
BSA, pH 4.7 or pH 5.5 and added to Neutravidin coated plates. Rec
HIV-1 inhibitor (Acetyl-pepstatin(BACHEM)), compounds from
plate-1001 (Sigma Drug Standards) and/or DMSO was added followed
with a 5 minute preincubation before the addition of HIV-l peptides
I or II, (FAM-spacer-Ser-Gln-Asn-Ty-
r-Pro-lle-Val-Gln-spacer-Biotin (Perkin-Elmer) or
(FLAG-Ser-Nle-Ala-Glu-Ph- e-Leu-Val-Arg-Ala-Lys-His-Spacer-Biotin)
or DABCYL-y-Abu-Ser-Gln-Asn-Tyr-P- ro-lle-Val-Gln-EDANS (BACHEM)
(prior art) respectively. Reactions were done at ambient and/or
37.degree. C. For HPLC analysis, reactions were stopped with 0.1%
TFA and placed on ice before injection. Assays with the BACHEM
fluorogenic substrate were measured directly on a LS50B
fluorescence spectrophotometer (Perkin-Elmer). Neutravidin coated
assay plates with captured biotinylated-fluorescein labeled peptide
I were washed with PBS, 0.1% (V/V) Tween20, 1 mg/ml BSA and
incubated for 60 min. with anti-fluorescein-alkaline phosphatase
Fab fragments (Boehringer Mannheim) at ambient temperature. Plates
were washed again with PBS buffer followed by a lOmM Tris-HCL, 1 mM
MgCl.sub.2 pH 9.8 wash. 100 ul of CSPD/Sapphire II.TM. ready to use
alkaline phosphatase was added and emitted light was measured in a
TR717.RTM. microplate luminometer(Tropix).
[0069] HPLC analysis: Analytical HPLC on the HIV-1 protease
substrate cleavage products was done on a Perkin-Elmer UV/Vis
detector with series 2001c pump, Perkin-Elmer pecosphere 5 C18,
Sum, 4.6 mm.times.15 cm, gradient of water (0.1% TFA) and
CH.sub.3CN (0.1% TFA) from 30% to 60% over 10 minutes at 1 ml/min.
Additions and dilutions were made with a Zymark RapidPlate-96
pipeting station (Zymark Corp.) and washes were done with a Tecan
96PW washer (Tecan).
[0070] The present inventors have successfully developed an
automated screening assay as shown below to search for novel HIV-1
protease inhibitors.
1 AUTOMATION STEPS VOLUME TIME # DESCRIPTION (.mu.L) AUTOMATION
COMPONENT (MIN) 1 Add protease buffer 50 .mu.L Zymark Rapid plate
96-well pipettor 1 min containing 50 nM HIV-1 protease 2 Add test
compound 5 .mu.L Zymark Rapid plate 96-well pipettor 1 min 3 Add
HIV Perkin Elmer 45 .mu.L Zymark Rapid plate 96-well pipettor 1 min
peptide @ 444 nM 4 Incubate (32.degree. C.) 60 min 5 Wash 3 .times.
100 .mu.L Tecan 96PW washer 0.75 min 6 Add Anti-Fluorescein- 100
.mu.L Zymark Rapid plate 96-well pipettor 1 min AP conjugate 7
Incubate (Room Temp.) 30-60 min 8 Wash 5 .times. 100 .mu.L Tecan
96PW washer 1 min 9 Add CSPD + Sapphire 100 .mu.L Zymark Rapid
plate 96-well pipettor 1 min II 10 Read plate Tropix TR717
Luminometer 30 min + 1.4 min
[0071] We have shown that this protease assay can be set up as an
endpoint capture assay with minimal reagent use. An HIV-1 peptide
substrate I concentration of 200 nM and a rec HIV-1 protease
concentration of 25-50 nM have been shown to be optimal working
concentrations with good sensitivity and acceptable signal:noise. A
resonance energy transfer assay reported by Abbott Laboratories,
Matayoshi. E. D. et al., Science 247: 954-958 (1990), utilizes
micromolar amounts of HIV-1 peptide II. This assay is reported as
an assay with a linear detection window of less than 5 minutes,
which we have found to have a poor signal to noise ratio making it
a difficult robotic assay to automate. FIG. 6. This assay gave a
maximum sin ratio of 4, while the inventive assay gives a sin ratio
of greater than 140 when using the FLAG/anti-FLAG ligand binding
pair. By utilizing common laboratory reagents such as biotin and
fluorescein one can eliminate the need to generate sequence
specific monoclonal antibodies as described in the alternative HIV
peptide capture based assay by Fournout, S. et al., Anal. Chem. 69,
1746-1752 (1997). We have shown that DMSO is well tolerated in the
assay. The IC.sub.50 of acetyl-pepstatin in our capture assay was
2-3 uM compared to 0.3 uM reported by Fournout. S. et al.
[0072] This invention has been disclosed in terms of both generic
description and specific example. Variations will occur to those of
ordinary skill in the art, including peptide moiety identities,
specific proteolytic enzymes to be employed, enhancement agents and
enhancement additives, and specific assay formats without the
exercise of inventive faculty. Such variations remain within the
scope of the invention, save for variations excluded by the
recitation of the claims presented below.
* * * * *