Note: Descriptions are shown in the official language in which they were submitted.
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CA 02597131 2007-08-07
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FATTY ACID AMIDE HYDROLASE ASSAY
FIELD OF THE INVENTION
The invention relates to methods for determining the activity of an ammonia-
generating enzyme and to their applications in identifying compounds that
modulate
the enzyme activities.
BACKGROUND OF THE INVENTION
Fatty acid amide hydrolase (FAAH), which may also be referred to as
oleamide hydrolase and anandamide amidohydrolase, is an integral membrane
enzyme. FAAH degrades fatty acid primary amides and ethanolamides, which are
known to serve as endogenous signaling lipids. These include the endogenous
cannabinoid anandamide and the seep-inducing oleamide. (M. P. Patricelli, et
al.,
(1998) Biochemistry 37, 15177-15187; M. Maccarrone, et al., (1998). J. Biol.
Chem.
273, 32332-32339). Although FAAH hydrolyzes a range of fatty acid amides
(FAAs),
FAAH appears to work most effectively on arachidonyl and oleyl substrates (B.
F.
Cravatt, et al., (1996) Nature 384, 83-87; and D. K. Giang, et al., (1997)
Proc. Natl.
Acad. Sci. USA 94, 2238-2242). Inhibitors of FAAH have been demonstrated to
reduce pain, inflammation, and anxiety in animal models.
A number of assays for measuring FAAH activity have been reported. The
majority of these assays utilize radiolabeled-substrates and thin-layer
chromatography, activated charcoal or mass spectrometry to quantitate FAAH
activity. In addition, a spectrophotometric assay using p-nitroanilide as a
substrate
and a fluorescence displacement assay have been reported. However, all of
these
assays have limitations such as low throughput, low sensitivity, or a
requirement for
radioactive material. An assay capable of efficiently, rapidly and accurately
measuring FAAH activity and identifying compounds that inhibit or stimulate
FAAH
and that is also capable of high throughput would be beneficial.
All publications, patents, and patent applications cited herein are hereby
incorporated by reference in their entirety for all purposes.
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SUMMARY OF THE INVENTION
In one aspect, the invention provides a method for measuring the activity of
an
ammonia-generating enzyme. The method involves two coupled reactions. One of
the
reactions is catalyzed by the ammonia-generating enzyme that generates
anunonia as
a product. The other reaction is a reductive amination reaction catalyzed by
glutamate
dehydrogenase that utilizes ammonia as a substrate. To carry out the method, a
reaction mixture is provided that comprises (1) the ammonia-generating enzyme,
(2)
an ammonia-generating substrate for the ammonia-generating enzyme, (3)
glutamate
dehydrogenase, (4) a reductive amination substrate other than ammonia for the
glutamate dehydrogenase, and (5) a reduced form of co-enzyme of glutamate
dehydrogenase. The reaction mixture is incubated under conditions that allow
for
reactions catalyzed by the ammonia-generating enzyme and glutamate
dehydrogenase. The activity of the ammonia-generating enzyme is measured by
measuring the rate of the reductive amination catalyzed by the glutamate
dehydrogenase. In one particular embodiment, the rate of the reductive
amination
catalyzed by the glutamate dehydrogenase is measured by measuring the
consumption
of the reduced form of the co-enzyme.
In another aspect, the invention provides a method for identifying a compound
capable of modulating the activity of an ammonia-generating enzyme. The method
employs the method for measuring the activity of an ammonia-generating enzyme
provided by the present invention. To carry out the method, a test compound is
added
to the reaction mixture and the activity of the ammonia-generating enzyme is
measured. In one embodiment, a method for identifying a compound capable of
modulating the activity of fatty acid amide hydrolase is provided.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation of the effects of altering FAAH
concentration in the presence of a constant concentration of oleamide.
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Figure 2 is a graphical representation demonstrating that the rate of the GDH
coupled FAAH reaction follows Michaelis-Menten kinetics.
Figure 3 is a graphical representation of a GDH-coupled FAAH assay
showing that Compound A inhibits FAAH activity in a dose-responsive manner.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as is commonly understood by one of ordinary skill in the art
to
which this invention belongs. As used herein, the following terms have the
meanings
ascribed to them.
The term "ammonia-generating enzyme" refers to any enzyme that catalyzes a
reaction that yields ammonia as a product. The enzyme may be from any source,
in
any form, and in any purity, may be naturally occurring or recombinant, and
may be a
full-length enzyme or an enzymatically active truncated form thereof.
The term "ammonia-generating substrate" refers to a substrate upon which an
ammonia-generating enzyme can act to produce ammonia as a product.
The term "glutamate dehydrogenase" refers to an enzyme that catalyzes the
reductive amination of a-ketoglutarate to form glutamate. The enzyme may be
from
any source, in any form, and in any purity, may be naturally occurring or
recombinant, and may be a full-length enzyme or an enzymatically active
truncated
form thereof.
The term "reductive amination substrate" of glutamate dehydrogenase refers
to a substrate, other than ammonia, upon which glutamate dehydrogenase can act
in
effecting a reductive amination.
The term "modulate," "modulating," or "modulation" means to change the
activity of an ammonia-generating enzyme in catalyzing the generation of
ammonia.
Such changes include inhibition of the enzyme or activation of the enzyme, and
can
be total or partial.
The term "compound" as used herein refers to any composition of matter,
which can be any synthetic or natural compound or composition, and can be
organic
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and inorganic, including small molecules, peptides, proteins, sugars (mono-
and
polysaccharides), nucleic acids, fatty acids, and the like. The term "test
compound"
refers to any compound or a combination thereof that is being analyzed.
In one aspect, the present invention provides a method for measuring the
activity of an ammonia-generating enzyme by quantitatively detecting the
ammonia
generated by the ammonia-generating enzyme using a coupled reductive amination
reaction catalyzed by glutamate dehydrogenase. The term "coupled" means that
the
reductive amination reaction catalyzed by glutamate dehydrogenase is carried
out
simultaneously and in the same reaction with the reaction catalyzed by the
ammonia-
generating enzyme. The metllod comprises: (a) providing a reaction mixture
comprising a predetermined amount of (1) the ammonia-generating enzyme, (2) an
ammonia-generating substrate for the ammonia-generating enzyme, (3) glutamate
dehydrogenase, (4) a reductive amination substrate other than ammonia for
glutamate
dehydrogenase, and (5) a reduced form of co-enzyme for glutamate
dehydrogenase;
(b) incubating the reaction mixture under conditions that allow for reactions
catalyzed
by the ammonia-generating enzyme and glutamate dehydrogenase; and (c)
measuring
the rate of the reductive amination catalyzed by glutamate dehydrogenase,
wherein
the rate of the reductive amination catalyzed by glutamate dehydrogenase is
indicative of the activity of the ammonia-generating enzyme. This method
provided
by the present invention may be referred to hereinafter as "enzyme activity
assay
method." An example of the reaction scheme utilized in the enzyme activity
assay
method of present invention is depicted below, wherein the ammonia-generating
enzyme is fatty acid amide hydrolase (FAAH) and oleamide is used as the
substrate
for fatty acid amide hydrolase:
o FAAH o
H2N - -~ HO + NH3
oleamide oleic acid
a-{cetoglutarate NADH
GDH
glutamate NAD+
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This method may be used to measure the activity of any ammonia-generating
enzyme by using an appropriate substrate that yields ammonia as a product. One
example of ammonia-generating enzyme is FAAH. Another example of ammonia-
generating enzyme is peptidylarginine deaminase, which catalyzes the
conversion of
the carboxy-terminal Arg residues of various peptides to citrulline residues
with the
generation of ammonia. The optimal amount of the ammonia-generating enzyme
that
may be used in the reaction mixture may vary depending on a number of factors,
such
as the source or specific activity of the enzyme, and incubation conditions,
and can be
readily determined by a person skilled in the art. This method is particularly
useful
for measuring the activity of FAAH. The FAAH may be from any source, may be
naturally occurring or recombinant, and may be a full-length enzyme or
truncated
form thereof. The optimal amount of the FAAH used in the reaction mixture may
vary depending on a number of factors, such as the source or specific activity
of the
enzyme, and the substrate used, and can be readily determined by a person
skilled in
the art. Generally, the concentrations of the FAAH in the reaction mixture are
greater
than about 2 nM, and preferably greater than 10 nM.
The appropriate ammonia-generating substrate for a particular ammonia-
generating enzyme, as well as its optimal amount to be used in the reaction
mixture,
can be readily selected by a person skilled in the art. Suitable ammonia-
generating
substrates for FAAH are fatty acid primary amides. In general, primary amides
as
suitable ammonia-generating substrates of FAAH have the general structure: NH2-
C(O)-R, where R is an alkyl chain that is optionally unsaturated, and may
additionally
be linear or branched as well as substituted or unsubstituted, and could
contain
saturated or unsaturated rings, wherein these rings could be fused or unfused,
and
contain heteroatoms. Where present, the unsaturated bonds of the fatty acid
alkyl
chain may have a cis configuration, such as in cis-9,10-octadecenomaide, cis-
8,9-
octadecenoamide, cis-11,12-octadecendoamide or cis-13, 14-docosenoamide.
Examples of fatty acid primary amides that may be used in the FAAH activity
assay
method of the invention include oleamide, amides of myrtistic acid
(tetradecanoic
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acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid),
caproic acid
(hexanoic acid), caprylic acid (octanoic acid), capric acid (decanoic acid),
lauric acid
(dodecanoic acid), linoleic acid (octadecadienoic acid), linolenic acid
(octadecatrienoic acid), arachidic acid (eicosanoic acid), arachidonic acid
(eicosatetraenoic acid), behenic acid (docosamoic acid), and lignoceric acid
(tetracosanoic acid). Still other fatty acid primary amides, such as the
primary amides
of undecanoic acid, oleic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-
monocaprate, and 1-dodecylazacycloheptan-2-onerespectively. Fatty acid primary
amides are widely available commercially and may be obtained from any source.
The
optimal amount of the fatty acid primary amides in the reaction mixture may
vary
depending on a number of factors, such as the specific fatty acid primary
amide used
or its purity, and can be readily determined by a person skilled in the art.
The amount
of the fatty acid primary amides in the reaction mixture is generally greater
than about
10 M and typically from about 20 to about 500 M. In one embodiment, the
amount
of the fatty acid primary amides in the reaction mixture is about 50 M.
The methods of the invention utilize glutamate dehydrogenase (GDH) in the
GDH-coupled assay. Glutamate dehydrogenase may also be known by other names,
such as glutamic dehydrogenase, glutamic acid dehydrogenase, L-glutamate
dehydrogenase, L-glutamic acid dehydrogenase, NAD(P)-glutamate dehydrogenase,
NAD(P)H-dependent glutamate dehydrogenase, and the like. The reductive
amination
by GDH requires ammonia, a-ketoglutarate and a reduced form of nicotinamide-
adenine dinucleotide (NADH or NADPH) as co-enzyme. The reductive amination
reaction catalyzed by glutamate dehydrogenase is illustrated below:
NH3 + a-ketoglutarate + NADH + H+ ~ glutamate + NAD+ + H20
In this reductive amination reaction, the amount of the ammonia consumed is
in direct proportion to the amount of NADH consumed, or in direct proportion
to the
amount of glutamate produced or NADP produced. GDH that may be used in the
enzyme activity assay method may be from any source, may be naturally
occurring or
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recombinant, and may be a full-length enzyme or truncated forms thereof that
are
enzymatically active. This enzyme is commercially available. (Cat. 49392;
Sigma-
Aldrich Chemicals, St. Louis, MO). The amount of GDH in the reaction mixture
should be sufficiently high in order for allowing for rapid and complete
reaction of
the ammonia that is generated by the ammonia-generating enzyme into glutamate.
The amount of GDH in the reaction mixture is generally not lower than about 1
unit/ml, and is typically about 7 unit/ml or higher.
Any suitable reductive amination substrate of GDH may be used in the
enzyme activity assay method of the present invention. Examples of suitable
reductive amination substrate of GDH include a-ketoglutarate (also known as
"alpha-
ketoglutarate," "2-oxoglutarate," "(x-oxoglutarate," or "2-oxopentanedioate)
and 2-
keto-6-hydroxyhexanoic acid. The amount of the reductive amination substrate
of
GDH in the reaction mixture should be sufficiently high in order for allowing
for
rapid and complete reaction of the ammonia that is generated by the ammonia-
generating enzyme into glutamate. The optimal amount may vary depending on a
number of factors, such as the activity of the ammonia-generating enzyme, the
amount of NADH (NADPH), and the amount or activity of GDH used, and can be
readily determined by a person skilled in the art. Where a-ketoglutarate is
used as the
substrate, its amount in the reaction mixture is generally not lower than 0.1
mM, and
is typically from about 0.3 to about 10 mM. In one embodiment, the
concentration of
a-ketoglutarate in the reaction mixture is about 3 mM.
The reduced form of nicotinamide adenine dinucleotide used in the enzyme
activity assay method of the present invention may be either NADH or NADPH.
The
amount of NADH or NADPH in the reaction mixture should be sufficiently high in
order for allowing for rapid and complete reaction of the ammonia that is
generated
by the ammonia-generating enzyme into glutamate. The optimal amount may vary
depending on a number of factors, such as the activity of the ammonia-
generating
enzyme, the amount of a-ketoglutarate, and the amount or activity of GDH used,
and
can be readily determined by a person skilled in the art. The amount of NADH
or
NADPH in the reaction mixture is generally from about 5 M to about 1000 M,
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preferably from about 50 M to about 300 M, and more preferably from about
100
M to about 200 M.
Other components may be optionally included in the reaction mixture in order
to enhance the method of the invention. For examples, adenosine 5'-diphosphate
(ADP) or guanosin 5'-diphosphate (GDP) may be included in the reaction
niixture to
enhance the activity of GDH. The ADP or GDP in the reaction mixture may be in
any
amount, but is generally greater than 20 M. In one embodiment, ADP is present
in
the reaction mixture at about 2 mM. In addition, a detergent or other
solubilizing
agent may also be included in the reaction mixture to increase the solubility
of an
enzyme, substrate, or any other components in the reaction mixture. Examples
of
suitable detergent or solubilizing agent include Triton X-100 (Sigma-Aldrich
Chemicals, St. Louis, MO) and dimethyl sulfoxide (DMSO).
The reaction mixture is incubated under conditions that allow for the
reactions
catalyzed by the ammonia-generating enzyme and GDH to take place. Typically,
the
reaction mixture is incubated at relatively constant temperatures, usually
between 15
C and 50 C, and more typically at a temperature of between about 20 C and
about
37 C. The reaction mixture is generally maintained at a relatively constant
pH that is
optimal for the reactions, usually between about 4.0 and about 12. In one
embodiment, the reaction mixture is maintained at a pH of from about 7.4 to
about
10.5. The pH of the reaction mixture can be adjusted and maintained using a
suitable
buffer. Examples of suitable buffer include phosphate buffer, a TRIS buffer
(Sigma-
Aldrich Chemicals, St. Louis, MO), and a HEPES buffer (Sigma-Aldrich
Chemicals,
St. Louis, MO). The reaction mixture may be incubated for any duration, from a
few
minutes or shorter to a few hours or longer. An optimal duration may be
determined
based on a number of factors, such as the activity of the ammonia-generating
enzyme
or GDH, the temperature of incubation, the initial amount of the substrates in
the
reaction mixture, and so on, and can be readily determined by a person skilled
in the
art.
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The rate of the reductive amination catalyzed by GDH may be determined by
any suitable method known in the art. For example, the rate of the reductive
amination may be determined by measuring one or more of the following
parameters:
(1) the consumption of the reductive amination substrate, (2) the consumption
of the
reduced form of nicotinamide adenine dinucleotide (NADH or NADPH), (3) the
generation of oxidized nicotinamide adenine dinucleotide (NAD+ or NADP+), and
(4)
the generation of a reductive amination product. One particular method for
determining the rate of the reductive amination catalyzed by GDH is to
spectrophotometrically measure the consumption of the reduced nicotinamide
adenine
dinucleotide (NADH or NADPH) in the reaction mixture. It is known that NADH
and
NADPH each absorbs light strongly at wavelengths between about 290 nm and
about
380 nm, while NAD+, NADP, and other substrates or products of the reactions do
not. Thus, as the NADH or NADPH in the reaction mixture is consumed (i.e.,
oxidized to NAD+, NADP+), the light absorbance of the reaction mixture at the
above
wavelengths decreases. The rate of the consumption of NADH or NADPH is in
directly proportional to the absorbance decrease. As the rate of consumption
of
NADH or NADPH is also directly proportional to the rate of ammonia generation
and, hence, the activity of the ammonia-generating enzyme, the rate of
absorbance
decrease at the above wavelengths is indicative of the activity of ammonia-
generating
enzyme, wherein a faster rate of absorbance decrease indicates a higher
activity of the
ammonia-generating enzyme, and vise versa.
Typically, the light absorbance of the reaction mixture is measured at
wavelengths between about 330 and about 370, and preferably at wavelengths of
about 340 nm. The light absorbance can be measured readily by those skilled in
the
art using conventional spectrophotometric procedures. The measurements of the
parameters of the reductive amination may be taken once at the end of the
incubation
period, at a plurality of time points during the incubation period, or
continuously
during the incubation period. The ammonia-generating enzyme activity can be
quantitated according to methods known in art.
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In another aspect, the present invention provides a method for identifying a
compound capable of modulating the activity of an ammonia-generating enzyme,
wherein the activity of the ammonia-generating enzyme is determined using the
enzyme activity assay method described above. This method may be referred to
hereinafter as "compound screening method" of the invention.
To identify a compound capable of modulating the activity of an ammonia-
generating enzyme, a reaction mixture is incubated in the absence and presence
of a
test compound and the activity of the ammonia-generating enzyme is determined
by
enzyme activity assay method of the invention described above. Specifically,
the
method comprises: (a) providing a reaction mixture comprising a predetermined
amount of (1) an ammonia-generating enzyme, (2) an ammonia-generating
substrate
for the ammonia-generating enzyme, (3) glutamate dehydrogenase, (4) a
reductive
amination substrate other than ammonia for glutamate dehydrogenase, and (5) a
reduced form of co-enzyme for glutamate dehydrogenase; (b) incubating the
reaction
mixture in the absence and presence of a test compound and under conditions
that
allow for reactions catalyzed by the ammonia-generating enzyme and glutamate
dehydrogenase; and (c) measuring the rate of the reductive amination catalyzed
by
glutamate dehydrogenase; wherein a difference in the rate of the reductive
amination
between the presence and absence of the test compound indicates that the test
compound is capable of modulating the activity of the ammonia-generating
enzyme.
A test compound is identified as an inhibitor of the ammonia-generating enzyme
if
the rate of the reductive amination in the presence of the test compound is
lower than
that in the absence of the test compound. Conversely, a test compound is
identified as
an activator of ammonia-generating enzyme if the rate of the reductive
amination in
the presence of the test compound is higher than that in the absence of the
test
compound.
In one embodiment, there is provided a method for identifying a compound
capable of modulating the activity of FAAH, which method comprises: (a)
providing
a reaction mixture comprising a predetermined amount of (1) FAAH, (2) a fatty
acid
primary amide as an ammonia-generating substrate for FAAH, (3) glutamate
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dehydrogenase, (4) oc-ketoglutarate as a reductive amination substrate for
glutamate
dehydrogenase, (5) NADH or NADPH as co-enzyme for glutamate dehydrogenase;
(b) incubating the reaction mixture in the absence and presence of a test
compound
under conditions that allow for reactions catalyzed by FAAH and glutamate
dehydrogenase; and (c) measuring the rate of the reductive amination by
measuring
the consumption of the NADH or NADPH in the reaction mixture, wherein a
consumption of the NADH or NADPH in the presence of the test compound that
differs from that in the absence of the test compound indicates that test
compound is
capable of modulating the activity of FAAH. A test compound will be identified
as an
inhibitor of FAAH if the consumption of the NADH or NADPH in the presence of
the test compound is lower than that in the absence of the test compound.
Conversely,
a test compound will be identified as an activator of FAAH if the consumption
of the
NADH or NADPH in the presence of the test compound is higher than that in the
absence of the test. The FAAH may be from any source, may be naturally
occurring
or recombinant, and may be a full-length enzyme or enzymatically active,
truncated
form thereof. The optimal amount of the FAAH used in the reaction mixture may
vary depending on a number of factors, such as the source or specific activity
of the
enzyme, and the substrate used, and can be readily determined by a person
skilled in
the art. Generally, the concentrations of the FAAH in the reaction mixture are
greater
than about 2 nM, and preferably greater than 10 nM. In one specific
embodiment,
oleamide is used as the fatty acid primary amide, NADH is used as the co-
enzyme of
GDH, and the consumption of NADH is determined by measuring the light
absorbance of the reaction mixture at wavelengths of 340 nm.
The compound screening method of the present invention is useful for rapid
screening of compounds capable of modulating the activity of an ammonia-
generating enzyme, such as FAAH, using automated procedures. Such automated
methods can be readily performed by using commercially available automated
instrumentation and software and known automated observation and detection
procedures. Multi-well formats are particularly attractive for high throughput
and
automated compound screening. Screening methods can be performed, for example,
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using a standard microplate well format. A microplate reader includes any
device that
is able to read a signal from a microplate (e.g., 96 and 384-well plates).
Such a signal
may be detected spectrophotometrically, such as, for instance, reading the
optical
density of the NADH or NADPH absorbance at a wavelength of 340 nm. However,
other detecting means may also be utilized, such as fluorometry (standard or
time-
resolved), luminometry, or photometry in either endpoint or kinetic assays.
Using
such techniques, a wide variety of compounds can be rapidly and efficiently
screened
for their respective effects on an ammonia-generating enzyme, such as FAAH.
Sample handling and detection procedures can be automated using commercially
available instrumentation and software systems for rapid, reproducible
application of
reagents, and automated screening of target compounds. To increase the
throughput
of a compound administration, currently available robotic systems such as the
BioRobot 9600 from Quagen (Quagen, Inc. Valencia, CA), the Zymate fiom Zymark
(Hopkinton, Mass) or the Biomek from Beckman Instruments (Fullerton, CA), most
of which use the multi-well plate format, could be utilized.
EXAMPLES
The following examples relate to assays for measuring the activity of FAAH
and their utility for identifying compounds capable of modulating the activity
of
FAAH. These examples are for illustrative purposes only and are not offered to
limit
the claimed invention. Various modifications or changes in light of these
examples
will be suggested to persons skilled in the art and are to be included within
the spirit
and purview of this application and scope of the claims.
EXAMPLE 1. Preparation of a Truncated Human FAAH
A truncated human FAAH (hFAAH) was prepared by the method described in
this Example, and was used in the studies described in Examples 2-4 below. The
amino acid sequence of this truncated human FAAH is shown in SEQ ID NO: 1,
which comprises amino acids 32-579 of the full length human FAAH. The E. coli
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codon optimized DNA sequence encoding amino acids 32-579 of the full human
FAAH is shown in SEQ ID No: 2.
A. Cloning and Construction of hFAAH Plasmids
Known molecular biological techniques are utilized to carry out the cloning
and construction of constructs described herein. Such techniques are referred
to, for
example, in Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences
Publishing, Inc., New York, NY,1986; Hames et al., Nucleic Acid Hybridization,
IL
Press, 1985; Molecular Cloning, Sambrook et al., Current Protocols in
Molecular
Biology, Eds. Ausubel et al., John Wiley and Sons; CuiTent Protocols in Human
Genetics, Eds. Dracopoli et al., John Wiley and Sons; Current Protocols in
Protein
Science, Eds. John E. Coligan et al., John Wiley and Sons; and Current
Protocols in
Immunology, Eds. John E. Coligan et al., John Wiley and Sons).
pTrcHis A-hFAAH (encoding human (h) FAAH amino acids 30-579)
The following cDNAs were custom-synthesized and subcloned into a pUC 119
vector (Blue Heron Biotechnology (Bothell, WA)). The E. coli codon
optimization
was carried out by using an optimization algorithm (Blue Heron Biotechnology
(Bothell, WA)).
- pUC119-hFAAH (encoding hFAAH, amino acids 1-579): A cDNA containing
the E. coli codon optimized DNA sequence of hFAAH (encoding amino acids 1-
579) with a 5'-Xho I site, 3' stop codon , and 3'-EcoR I site. (SEQ ID NO: 3).
- pUC1 19-hFAAH (encoding hFAAH, amino acids 30-104): A cDNA containing
the E. coli codon optimized DNA sequence of hFAAH (bp from 94 to 319 of SEQ
ID NO: 3) with a 5'-Xho I site (SEQ ID NO: 4).
The pUC119-hFAAH (encoding hFAAH, amino acids 1-579) was digested
with Xho I - EcoR I and the insert was subcloned into a Xho I - EcoR I-
digested
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pTrcHis A vector (Invitrogen, Cat # V360-20) to generate pTrcHis A-hFAAH
(encoding amino acids 1-579). The pTrcHis A-hFAAH vector was digested with Xho
I-Hind III, and the resulting Xho I-Hind III (approximately 4.5 kb) and Hind
III-Hind
III (approximately 1.5 kb) pieces were ligated with the Xho I-Hind III
fragment (225
bp) generated from digesting the pUC119-hFAAH (encoding amino acids 30-104)
construct to generate an NH2-terminally His-tagged pTrcHis A-hFAAH (encoding
amino acids 30-579 of the full length hFAAH).
pET28a-hFAAH (encodinghFAAH, amino acids 32-579)
The human FAAH construct for subcloning into the prokaryotic expression
vector pET28a(+) (Novagen, Catalog # 69864-3) was generated by PCR from the
pTrcHis A-hFAAH (amino acids 30-579) construct using the following primers:
sense primer, 5'-GGAATTCCATATGTCAGGTCGTCGTACCGCACGTG-3' (SEQ
ID NO: 5) ; and antisense primer, 5'-CCGCTCGAGTTATGAGGATTGTTT
TTCCGGAGTCAT-3' (SEQ ID NO: 6). The resulting PCR product was digested
with Nde I-Xho I, and subcloned into a Nde I-Xho I -digested pET28a(+) vector
to
generate an NH2-terminally His-tagged pET28a-hFAAH (encoding amino acids 32-
579). ,
Summary of Human FAAH Constructs:
pTrcHis A-hFAAH encodes hFAAH, amino acids (30-579):
MGGS H n U-I'iiHGMASMTGGQQMGRTLYDDDDKDRWGSELE----
hFAAH
pET28a-hFAAH encodes hFAAH, amino acids (32-579):
MGSSHN SSGLVPRGSHM----hFAAH
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"----" Does not represent amino acid positions. Each indicated hFAAH
position is contiguous with the specifically indicated leading amino acid
sequence comprising HHHHHH.
B. Expression and Purification of Truncated hFAAH
"RT" describes room temperature, which is typically 25 C 3 . The pET28a-
hFAAH construct encoding aniino acids corresponding to amino acids 32-579 of
wild-type human FAAH was transformed into the E. coli BL21-AI strain. 1.2-
liter
cultures of the freshly transformed expressing strains were grown in
SuperBroth
media in the presence of 30 g/m1 kanamycin at 37 C. At OD600 of
approximately
0.12, the cultures were transferred to RT and induced at OD600 of 0.6 - 0.65
with 100
M IPTG and 0.2% L-arabinose for 20 hours at RT. All operations below were at 4
C unless otherwise noted. The cells were then harvested by centrifugation at
5000 x
g. The cell pellets were washed twice by re-suspending in 700 ml of PBS and
collected by centrifugation at 5000 x g. At this point, the cell paste is
optionally
frozen and stored at - 80 C until needed. The cells were re-suspended in 60
ml of
buffer A (20 mM Tris-HC1, pH 8.0/100 mM NaCI/1% Triton X-100) with stirring.
After adding DNase and RNase (1 mg per 25 g E. coli pellet), the cell
suspension was
incubated for 1 hour at RT with mixing intermittently, cooled on ice for 10
min, and
was sonicated with approximately 40-60 ten-second pulses. The resulting lysate
was
centrifuged at 10,000 x g for 35 minutes and the supernatant was loaded at 0.5
- 1
ml/min onto a 5 ml Ni-column (HiTrap chelating HP column from Amersham
Biosciences (Cat # 17-0409-01) was charged with Ni according to the
manufacturer's
instructions) which has been equilibrated with buffer B (20 mM Tris-HCI, pH
8.0/300
mM NaC1/1% Triton X-100). The resulting flow-through was reloaded onto the
column. The column was washed with 100 ml of buffer B and further washed in
sequence with 50 ml of buffer B containing 10, 20, and 50 mM imidazole,
respectively. The elution was performed in sequence with 25 ml of buffer B
containing 100, 200, 400, and 700 mM imidazole, respectively. The majority of
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FAAH was eluted with buffer B containing 100-200 mM imidazole. The eluted
FAAH was dialyzed against buffer B overnight, frozen in liquid N2, and stored
at -80
C.
EXAMPLE 2. FAAH Dose-Response Curve at 100 M Oleamide
To determine whether the assay provides for quantitative detection of FAAH
activity, the assay was carried out with increasing concentrations of FAAH.
The
reactions were carried out in 96-well clear polystyrene plates. The reaction
mixture
(250 l) contained 50 mM Tris-HCl, pH 8.0, 100 M oleamide, 150 M NADH, 3
mM a-ketoglutarate, 2 mM ADP, 6.0 unit/ml GDH, 0.1% Triton X-100, and the
indicated volumes of approximately 150 nM FAAH. The reactions were incubated
at
37 C and the absorbance at 340 nm was collected over a period of 30 min with
readings taken in 10-second intervals using a SpectraMax Microplate
Spectrophotometer (Molecular Devices, Palo Alto, CA) equipped with Softmax
Pro software (Molecular Devices, Palo Alto, CA). As shown in Figure 1, the
rate of
the reaction increases with increasing concentration of FAAH.
EXAMPLE 3. FAAH Initial Velocity Dependence on Oleamide
Concentration
An assay was carried out to determine whether the GDH-coupled FAAH
assay is compatible with enzyme kinetics and to demonstrate that FAAH follows
typical Michaelis-Menten enzyme kinetics. The reactions were carried out in 96-
well
clear polystyrene plates. The reaction mixture (250 l) contained 50 mM Tris-
HC1,
pH 8.0, 150 M NADH, 3 mM a-ketoglutarate, 2 mM ADP, 12 unit/ml GDH, 0.1%
Triton X-100, the indicated concentrations of oleamide, and approximately 10
nM
FAAH. The reactions were incubated at 37 C and the data were collected as
described in Example 1. Oleamide concentrations are plotted on the x axes and
the
initial rates are plotted on the y axes. The data were fit to the Michaelis-
Menten
equation. As shown in Fig. 2, this GDH-coupled FAAH assay allows measuring
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kinetic constants of FAAH. This data also demonstrates that FAAH follows a
typical
Michaelis-Menten kinetics. The Km value, which is oleamide substrate
concentration
at which the reaction rate is half of its maximal value, was determined to be
19.8 M.
EXAMPLE 4. Measurement of FAAH Inhibition by Compound A.
To further demonstrate the utility of the assay, the inhibitory effects of 1-
oxazolo[4,5-b]pyridin-2-yl-5-phenyl-pentan-1-one ((PNAS, (2000) vol. 97, No.
10:p5042), hereinafter, Compound A) on FAAH activity were examined using the
GDH-coupled FAAH assay. The reactions were carried out in 96-well clear
polystyrene plates. The reaction mixture (200 l) contained 50 mM NaPi, pH
7.4, 50
M oleamide, 150 M NADH, 3 mM a-ketoglutarate, 2 mM ADP, 1 mM
ethylenediaminetetraacetic acid (EDTA), 12 unit/ml GDH, 0.1 % Triton X-100 ,
the
concentrations of Compound A indicated on Figure 3, and approximately 10 nM
FAAH. Oleamide (500 M) dissolved in 25% DMSO and 25% EtOH was used as a
stock solution. Compound A stock solutions, dissolved in 50% DMSO, were used.
The final concentrations of DMSO and EtOH were each 7.5%. The reactions were
incubated at 37 C and the data were collected as described in Example 1. The
results
shown in Figure 3 demonstrate that Compound A inhibits FAAH in a dose-
responsive
manner.
SEQUENCE LISTING GUIDE
SEQ ID NO: 1 - Amino acids 32-579 of full length homo sapien FAAH.
SEQ ID NO: 2 - Nucleotide sequence encoding amino acids 32 to 579 of full
length homo
sapien FAAH, optimized for expression in E.coli.
SEQ ID NO: 3 - The DNA sequence of the E. coli codon optimized sequence of
hFAAH
(amino acids 1-579) with a 5'-Xho I site, 3' stop codon, and 3'-EcoR I site.
SEQ ID NO: 4 - The DNA sequence from bp 94 to bp 319 of SEQ ID NO: 3 with a 5'-
Xho I
site.
SEQ ID NO: 5 and 6- Primers.
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