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NOTE POUR LE TOME / VOLUME NOTE:
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COMPOSITIONS AND METHODS FOR CONTROLLING INSECTS
RELATED TO THE OCTOPAMINE RECEPTOR
FIELD OF THE INVENTION
The present invention relates to compounds, compositions and method for
controlling
insects.
BACKGROUND OF THE INVENTION
Animals have chemosensory and mechanosensory systems that recognize a large
array of
environmental stimuli, generating behavioral responses. Many behavioral
studies have been
conducted to understand the genetics of these systems. The olfactory system
plays a crucial role
in the survival and maintenance of species, particularly in insects.
Biogenic amines serve a neurotransmitter or neuromodulator role in the
olfactory system.
The biogenic amine, octopamine, has a prominent role in insects and other
invertebrates as it is
involved in the regulation of multiple physiological events, for example,
effects on muscular
systems, sensory organs, endocrine tissues as well as learning and behavior,
Octopamine (OA)
occurs in large amounts in the nervous systems of species representing the
phylum Arthropoda,
including the classes Insecta and Crustacea. OA has a broad spectrum of
biological roles in
insects acting as a neurotransmitter, neurohormone and neuromodulator. OA
exerts its effects
through interaction with at least four classes of membrane bound receptors
that belong to the
family of G-protein coupled receptors (GPCRs). All members of GPGRs share the
C01111no11 lllOtlf
of seven transmembrane (TM) domains.
When a GPCR is activated, depending on its type and the protein to which it
binds,
changes in intracellular concentrations of cAMP, Ca'~ or both often take
place. Since changes in
intracellular levels of CAMP or Ca'+ are the most commonly found cellular
responses to biogenic
amine treatments (e.g., serotonin, dopamine, octopamine, etc.), they are used
to functionally
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classify receptor subtypes. As a result of GPCR activation, intracellular CAMP
levels can either
be elevated or reduced. The cellular response strictly relies on the
specificity of interaction
between the receptor and the G protein (See e.~., Gudermann T, I~allcbrenner
F, Schultz G . 1996,
"Diversity and selectivity of receptor-G protein interaction," Annu Rey
Pharmacol Toxicv 1 36:
429-459; and Gudermann T, Schoneberg T, Schultz G. 1997, "Functional and
structural
complexity of signal transduction via G-protein-coupled receptors," Annu Rev
Neurosci 2 0: 399-
427, both of which are incorporated herein by this reference). When the
receptor binds to Gs-type
protein, the activated Gas subunit will interact with adenylyl cyclase (AC) in
the plasma
membrane. This leads to an increase of AC activity and production of CAMP from
ATP.
Several biogenic amine receptors are also known to inhibit AC activity. This
effect is
mediated by interaction of the receptor with inhibitory G protein (Gi).
Interaction of AC wvith
activated Gai subunits most likely competes witlybinding of activated Gas
subunits and t(~ ereby
interferes with AC activation.
Another pathway that is activated by several biogenic amine receptors results
in a rise of
intracellular Ca2+ levels. In such a scenario the amine-activated receptor
binds to G protein's of
the Gq/o family (See e.g., supya, Gudermann et al., 1996 and Gudermann et al.,
1997). TI~ a
activated Gaq/o subunits bind to and stimulate phospholipase C (PLC) activity.
The enzyme
hydrolyzes a membrane-bound substrate, phosphatidylinositol 4,5-bisphosphate
which giv es rise
to two second messengers IP3 and DAG. After binding of IP3 to its receptors,
the calciwn
channel pore is opened and Ca2+ is released into the cytoplasm. Ca''+ ions
play a vital role in the
regulation of many cellular functions by binding to members of large family of
Ca'+-bindimg
proteins and/or directly controlling enzymatic or ion channel activities.
Multiple insect species have been utilized to understand the biological
functions amd
pharmacological characteristics of octopamine receptors. Studies with
Peripdaneta afne~i~ana
(American cockroach) have provided insight into the pharmacology and second
messenger
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signaling of octopamine through octopamine receptors. For example, octopamine
has been found
to activate adenylate cyclase in certain cells in this species. Furthermore,
octopamine has been
found to increase inositol triphosphates in certain cells in this species.
As the octopaminergic system is believed to be unique to invertebrate
physiology, this
pathway has been proposed to offer a target for invertebrate pesticides with
potential for low
vertebrate toxicity, Formamidine-like chemicals have been found to be
octopaminergic agonists
and inhibit the uptake of sodium-sensitive octopamine in cel-tain insects; for
example, the
formamidine pesticides chlordimefonn and demethylchloridimeform were found to
target the
octopamine signaling pathway in cel-tain invertebrates, including
Pe~~iplar7eta an~enicaJZa, To
provide insight into the design of octopamine agonists that could be used as
potential insecticides,
structure function analyses have been performed with 2-(arylimino)oxazolidines
and 2-
(substituted benzylamino)-2-oxazolines in regard to activation of the
octopamine sensitive
adenylate cyclase in certain cells in Peg°iPlaf~eta Aroe~ica~a. More
recently, it has been suggested
that one site of action for the insecticidal activity of plant essential oils
against Per~ilalaneta
anze~~icaf~a is the octopaminergic system and that octopamine receptors may be
targeted by these
compounds, as described in Enan, E., 2001, "Insecticidal activity of essential
oils: octopaminergic
sites of action," Comp. Biochem. Physiol. C Toxicol. Pharmacol. 130, 325-327,
which is
incorporated herein by this reference.
Identifying plant essential oils and combinations thereof, having 111SeCt-
COlltl'Olllllg activity
is particularly desirable given that many such compounds do not produce
unwanted or harmful
affects on humans, other animal species, and certain plants. However,
identifying the most
effective plant essential oils and combinations thereof requires random
selection and use of
tedious screening methods, which, given the vast number of plant essential
oils and possible
combinations thereof, is a substantially impossible task.
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As such, there is a need in the art for an improved method for screening
compounds and
compositions for insect control activity.
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SUMMARY OF THE PRESENT INVENTION
The present invention addresses the above identified problems, and others, by
providing a
screening method for identifying compounds and compositions that are effective
insect control
agents; a screening method for identifying compounds and compositions that are
effective
species-specific insect control agents; compounds and compositions isolated
from the screening
methods; cell lines expressing an octopamine receptor; and isolated nucleic
acid molecule
sequences.
DESCRIPTION OF THE DRAWINGS
Figure lA is an alignment of the nucleic acid sequence and the translated
amino acid
sequence from Pa oar, of SEQ ID NO: 1 and SEQ ID NO: 2;
Figure 1B is the nucleic acid sequence fr0111 Pa oal of SEQ ID NO: 1, with the
seven
putative transmembrane domains (TM) overlined and numbered 1 through 7, the
stop codons (SC)
underlined, and the initiation codon (M) underlined;
Figure 2 is an alignment of the translated amino acid sequences of Pa oar of
SEQ ID NO: 2
and OAMB of SEQ ID NO: 3, with the seven putative transmembrane domains (TM)
overlined
and numbered 1 through 7;
Figure 3A is saturation binding curve of'H-yohimbine to Pa oar, where total
binding is
designated by the squares, nonspecific binding is designated by the triangle,
and specific binding
is designated by the inverted triangle;
Figure 3B is saturation binding curve of 3H-yohimbine to OAMB, where total
binding is
designated by the squares, nonspecific binding is designated by the triangle,
and specific binding
is designated by the inverted triangle;
Figure 4 is a hydropathy profile of Pa oar with the transmembrane domains (TM)
numbered 1 through 7;
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Figure 5 depicts the similarity between octopamine and tyramine receptors -
lrom different
insect species;
Figure 6 is a graph depicting the change of intracellular cAMP levels in HEK-
293 cells
expressing Pa oal in response to treatment with various concentrations of
either octopamine (OA)
or tyramine (TA); and
Figures 7 is a graph depicting the change in intracellular calcium levels in
HEK-293 cells
expressing Pa oal in response to treatment with either 100 nM octopamine (OA)
or 100 nM
tyramine (TA);
Figure 8 is a bar graph depicting the change in intracellular CAMP levels in
HEK-293
expressing Pa oal in response to treatment with 0, 100 nM, or 1 EIM octopamine
(OA) in the
presence and absence of 20 L~M BAPTA/AM, a calcium chelator;
Figure 9 is a bar graph depicting the cAMP response to octopamine through Pa
oai and
OAMB expressed in HEK-293 cells where the cells expressing either receptor are
treated with 10
p,M octopamine and the level of CAMP is determined;
Figures 10A and lOB are graphs depicting the calcium response to octopamine
through Pa
oal and OAMB, respectively, expressed in HEIR-293 cells;
Figure 11 is a depiction of the chemical structures ofp-cymene [methyl( 1-
methylethyl)benzene], eugenol [2-methoxy-4-(2-propenyl)phenol], traps-anethole
[ I -methoxy-4-
(I-propenyl)benzene], cinnamic alcohol [3-phenyl-2-propen-1-of], a-terpineol
[p-menth-1-en-8-
0l], methyl salicylate [2-hydroxybenzoic acid methyl ester], 2-phenylethyl
propionate, and
geraniol [3,7-dimethyl-2,6-octadien-1-of];
Figure 12 is a bar graph depicting the efi°ect of certain plant
essential oils on speci lic
bllldlllg of 3H-yohimbine to Pa oai and OAMB;
Figure 13 is a bar graph depicting the effect of certain plant essential oils
on cAMP levels
in HEK-293 cells expressing either Pa oal or OAMB; and
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Figures 14A-14F are graphs depicting the effect of certain plant essential
oils on
intracellular calcium [Ca2+]; levels in HEK-293 cells either transfected with
Pa oa, or OAMB.
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes: a screening method for identifying compounds
and
compositions that are effective insect control agents; a screening method for
identifying
compounds and compositions that are effective species-specific insect control
agents; compounds
and compositions isolated from the screening methods; transfected cell lines;
alld isolated IluclelC
acid molecule sequences.
The present invention includes: an isolated nucleic acid molecule sequence
which encodes
a protein that binds a biogenic amine, resulting in changes in intracellular
concentrations of
CAMP, Ca'+, or both, having a nucleotide sequence.of SEQ ID NO: l, or a
fragment or derivative
thereof andJor having an amino acid sequence of SEQ ID NO: 2, or a fragment or
derivative
thereof; an isolated nucleic acid molecule of having at least about
30°fo similarity to the nucleotide
sequence of SEQ ID NO: 1, wherein the isolated nucleic acid molecule encodes a
protein,
resulting in changes in intracellular concentrations of cAMP, Ca2+, or both;
an isolated nucleic
acid molecule of having at least about 30% similarity to the nucleotide
sequence of SEQ ID NO:
l, wherein the molecule encodes an octopamine receptor or a protein having an
amino acid
sequence of SEQ ID NO: 2, or a fragment or derivative thereof; and an isolated
nucleic acid
molecule having a nucleotide sequence of SEQ ID NO: 1, or a fragment or
derivative thereof,
wherein the molecule encades a protein designated Pa oal. SEQ ID NO: 1 and SEQ
ID NO: 2 are
shown in alignment in Figure 1A and SEQ ID NO: 1 is also provided in Figure 1
B. Fragments
and derivatives of the sequences shall include transmembrane domains (TM) 3, 5
and 6.
Fragments and derivatives of the sequences may exclude, for example, portions
upstream of TM
1, portions upstream of TM 2, or portions downstream of TM 7.
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The present invention also includes: a strain of cells including a DNA vector
having a
nucleic acid sequence of SEQ ID NO:1; a strain of cells expressing an
octopamine receptor cloned
from an insect species of interest; a strain of cells expressing an octopamine
receptor cloned from
Pe~ipla~reta Anae~~icaha (Pa oar ); a strain of cells expressing a protein
having an amino acid
sequence of SEQ ID NO: 2, or fragments or derivatives thereof, wherein the
fragments or
derivatives thereof bind octopamine; a strain of cells expressing an
octopamine receptor cloned
from Drosoplzila n~elay7ogaste~~ (OAMB); a strain of cells expressing a
protein having an amino
acid sequence of SEQ ID NO: 3, or fragments or derivatives thereof, wherein
the fragments or
derivatives thereof bind octopamine. The transfected cells may be mammalian
cells or insect
cells; for example, they may be African green monkey kidney COS-7 cells (COS-7
cells) or
human embryonic kidney-293 cells (HEIR-293 cells).
The present invention also includes a screening method of using a cell line
expressing an
octopamine receptor to identify compounds and compositions that are effective
insect control
agents. .For example, the octopamine receptor expressed by the cell line may
be Pa oal; or have
an amino acid sequence of SEQ ID NO: 2, or fragments or derivatives thereof,
wherein the
fragments or derivatives thereof bind octopamine.
The present invention also includes a method of using multiple cell lines,
wherein the cell
lines are transfected with octopamine receptors from different insect species
of interest, to identify
compounds and compositions that are effective species-specific insect control
agents. For
example, a cell line expressing Pa oal and a cell line expressing OAMB could
be used to screen
compounds and compositions having insect control activity which is specific to
Peg°i~laneta
Anzef°ica~ra or to DT~os~plzila n~elahogaste~~.
The present invention also includes compounds and compositions having the
ability to
control target insects, which compounds and/or compositions are identified
using the screening
methods of the present invention. These compounds and/or compositions may
include
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compounds that are general regarded as safe (GRAS compounds) meaning that they
do not
produce unwanted or harmful affects on humans and other non-target animal
species and that they
are exempt from the Environmental Protection Agency's (EPA) pesticide
registration
requirements. The compounds and/or compositions of the present invention
include certain plant
essential oils identified using the screening methods of the present
invention.
The CO111pOLl1ldS alld CO111pOS1tIO11S Of the pl'e5e11t I11Ve11t1011 COlltr0l
111S2CtS by targeting aI7
octopamine receptor, resulting in a disruptive change in the intracellular
levels of cAMP, Ca2+ or
both. For purposes of simplicity, the term insect has been and shall be used
through out this
application; however, it should be understood that the term insect refers, not
only to insects, but
also to arachnids, larvae, and like invertebrates. Also for purposes of this
application, the term
"insect control" shall refer to repelling or killing an insect.
The present invention is fu l-ther illustrated by the following specific but
non-limiting
examples.
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EXAMPLE 1
PREPARATION OF STABLYTRANSFECTED COS-7 CELL LINES and HEK-293
CELL LINES WITH OCTOPAMINE RECEPTOR
A. ISOLATION OF A cDNA ENCODING A G-PROTEIN-COiJPLED RECEPTOR
FROM PERIPLANETA AMERICANA
G protein-coupled receptors from insects and a tick that are demonstrated to
be
octopamine receptors or have significant DNA similarity to known octopamine
receptors are
aligned using the program DNAStar (Ma). The following degenerate
oligonucleotides are
designed based on this alignment: Transmembrane (TM) VI oligonucleotide
5'TACAAGCTTTG(C, T~TGG(C, T) (G, T) (A, C, G, T)CC(A, C, G,T)TTCTT3' (SEQ ID
NO:
4), and TM VII oligonucleotide 5'CATGCGGCCGCTTT(A, C, G, T) (A, CAA, C) (A,
G)TA(A,
C, G T)CC(A, C)AGCCA3' (SEQ ID NO: 5), The underlined sequence corresponds to
the TM
regions.
The TM VI oligonucleotide contains a Hinc~lll site and the TM VII
oligonucleotide
contains a Notl site flanking the TM sequences. Total RNA from the heads of
mixed sex adult
American cocla~oaches that have the antennae excised is prepared by
ultracentrifugation through
cesium chloride, as described in Chirgwin et al., 18 Biochemistry 5294-5299
(1979), and is
reverse transcribed into cDNA using random hexamers and murine leukemia virus
reverse
transcriptase (Applied Biosystems, Foster City, CA), The polymerase chain
reaction (PCR) is
performed on this cDNA using AmpliTaq polymerase (Applied Biosystems) and the
TM VI and
VII oligonucleotides at final concentrations of about 5 pM, The reaction
conditions are about
95 ~C, about 5 min for about one cycle; about 95 UC, about 45 s, about 40 ~C,
about 2 min, about
72 ~C, about 30 s for about three cycles; about 95 ~C, about 45 s, about 55
~C, about 2 171111; about
72 ~C, about 30 s for about 37 cycles; and about 72 ~C, about 10111111 for
about one cycle,
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Products are digested with Hi~rdlll and Notl and ligated into pBI~-RSV
(Stratagene, La
Jolla, CA). Inserts are sequenced and compared to 1C110W11 genes by
Seal'Chlllg the NCBI database
with the Blast program.
To obtain the corresponding cDNA for an approximately 101 nucleotide fragment
with the
highest similarity to octopamine receptors from other species, 5' and 3' rapid
amplification of
cDNA ends (RACE) are performed using the SMART RACE cDNA amplification system
(Clontech, Palo Alto, CA), Poly(A) RNA is prepared from total RNA isolated
from the head of
Pei~ipla~eta amerieana using an oligo-dT column as per the manufact~.irer's
protocol (A111e1'S11a111
Biosciences, Piscataway, NJ). The poly(A) RNA is used as template in the RACE
reverse
transcription reaction for production of 5' and 3' RACE cDNA as per the
manufacturer's
instructions. The gene specific oligonucleotides used for the RACE PCR are 5'
RACE
oligonucleotide 5'CAGTAGCCCAGCCAGAAGAGGACGGAGAAG3' (SEQ ID NO: 6), and 3'
RACE oligonucleotide 5'GCTGGCTGCCGTTC'TTCACCATGTACCTGG3' (SEQ ID NC): 7), 5'
RACE and 3' RACE polymerase chain reactions are each about 50 y1 and consist
of about 2.5 Etl
of the respective cDNA reaction, about 0.2 ~.M of the gene specific
oligonucleotide and the
additional RACE components including Advantage 2 polymerase as per the
manufacturer
(Clontech). The cycling conditions for the 5' RACE are about 95 ~C, about 1
min for about one
cycle; about 94 ~C, about 20 s, about 72 ~C, about 3 min for about five
cycles; about 94 ~C, about
s, about 70 ~C, about 10 s, about 72 ~C, about 3 111111 for about five cycles;
about 94 ~C, about
20 20 s, about 68 ~C, about 10 s, about 72 ~C, about 3 niin for about 32
cycles; and about 72 ~C, about
10 min for about one cycle.
An approximately 1.9 lcb product is gel purified and further, amplified using
the same
oligonucleotides, Advantage 2 polymerase and cycling parameters of about 95
~C, about 3 min for
about one cycle; about 94 ~C, about 20 s, about 68 ~C, about 10 s? about 72
nC, about 3 min for
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about 35 cycles; and about 72 ~C, about 10 min for about one cycle. To
facilitate T/A ligation, the
product is A-tailed by precipitating with ethanol, resuspending in 1 x PCR
Buffer II (Applied
Biosystems), 2 mM MgCla, 1 mM dATP and 0.05 U AmpliTaq per p l and mcubatlng
at about
72 ~C for about 15 min. The PCR product is ligated into pBI~-RSV (Stratagene)
that has been
digested with S~~aI and T-tailed using dTTP and AmpliTaq. The insert is
sequenced on both
strands by automated fluorescent DNA sequencing (Vanderbilt Cancer Center).
The cycling conditions for the 3' RACE reaction are about 95 ~C, about 1 m in
for about
one cycle; about 94 ~C, about 5 s, about 72 ~C, about 3 min for about five
cycles; about 94 ~C,
about 5 s, about 70 ~C, about 10 s, about 72 ~C, about 3 min for about five
cycles; about 94 ~C,
about 5 s, about 68 ~C, about 10 s, about 72 ~C, about 3 min for about 32
cycles; and about 72 ~C,
about 10 min for about one cycle. The product of this reaction is A-tailed,
subcloned and
sequenced as for the 5' R ACE product.
B. GENERATION OF THE OPEN REALIING FRAME FOR OCTOPAMINE
RECEPTOR (Pa onl)
Oligonucleotides used to amplify the open reading frame are a sense
oligonucleotide 5'
CAGGAATTCATGAGGGACGGGGTTATGAACGCTAG 3' (SEQ ID NO: 8), and an antisense
oligonucleotide 5' GCTTCTAGATCACCTGGAGTCCGATCCATCGTTG 3' (SCQ ID NO: 9)
Sequences corresponding to the open reading frame are underlined, The sense
oligonucleotide
contains an EeoR1 restriction site and the antisense oligonucleotide an.~'baI
restriction site, These
oligonucleotides are used in a polymerase chain reaction that included the
5'RACE cDNA as
template and VENT polymerase (New England Biolabs, Beverly, MA),
The product is subcloned into the plasmid pAcS.l/VS-His (Invitrogen Life
Technologies,
Carlsbad, CA) at the EcoRI and Xl~czl restriction sites and sequenced. This
plasmid is designated
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pAc-Pa oar, For mammalian cell expression, a Kozalc sequence is inserted using
a sense
oligonucleotide 5'ACAGAATTCGCCACCATGAGGGACGGGGTTATGAACGCTAG 3' (SEQ
ID NO: 10) and an internal antisense oligonucleotide that contains an Xlrol
site 5'
TTGAGGGCGCTCGAGGACGTC 3' (SEQ ID NO: 11), The sense oligonucleotide contains
an
EcoRl site, These oligonucleotides are used in a polymerase chain reaction
that includes pAc-Pa
oar as template and VENT polymerase_ The product is inserted at EcoRI and
~Yhol sites into pAc-
Pa oal, in which the corresponding EcoRl and .~hol fragment have been removed,
The product is
sequenced, The entire open reading frame is then transferred into pCDNA3
(Invitrogen Life
Technologies, Carlsbad, CA) at Ec~RI and Apal restriction sites, and this
plasmid is designated
pCDNA3-Pa oa,.
C. AMPLIFICATION AND SUBCLONING OF OAMB, AN OCTOPAMINE
RECEPTOR FROM THE FRUIT FLY, .UROSOPIIILA MELANOGASTER
The Drosophtla nxela~rogastef° head cDNA phage library GH is obtained
through the
Berkeley Drosophila Genome Project (www.fruittl .~), Phage DNA is purified
from this library
using a liquid culture lysate as described in Lech, Current Protocols in
Molecular Bioloay, John
Wiley & Sons, Inc., pp. 1 (2001), Oligonucleotides designed to amplify the
open reading frame of
D~°osoplzila Jraelayzogastef~ OAMB C011S15t of the sense
oligonucleotide 5'
CAGGAATTGGCCACCATGAATGAAACAGAGTGCGAGGATCTC 3' (SEQ ID NO: 12) and
the antisense oligonucleotide 5' AATGCGGCCGCTCAGCTGAAGTCCACGCCCTCG 3' (SEQ
ID NO: 13), Sequences corresponding to the open,, reading frame are
underlined. A I~ozalc
sequence is included in the sense oligonucleotide In addition, the 5'
oligonucleotide includes an
EcoRl restriction site and the 3' oligonucleotide a Notl site,
For amplification by the polymerase chain reaction, about 200 ng of the GH
library DNA
is used as template with about 0.5 EiM of each oligonucleotide and VENT DNA
polymerase (New
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G
England Biolabs). Cycling conditions are about 95 C, about 5 min for about one
cycle; about
95 ~C, about 30 s and about 70 ~C, about 1.5 min for about 40 cycles; and
about 70 ~C, about 10
min for about one cycle. The product is digested with EcoRI and Notl, ligated
into pCDNA3 and
sequenced on both strands by automated fluorescent DNA sequencing (Vanderbilt
Cancer
Center).
D. ISOLATION OF cDNA ENCODING OCTOPAMINE RECEPTOR (Pa oal)
A polymerase chain reaction with degenerate oligonucleotides corresponding to
regions of
TM VI and TM VII of previously identified octopamine receptors is used to
isolate an
approximately 101 nucleotide fi~agment of cDNA from the head of Per~ipdaneta
aYne~~ieavra. This
eDNA fragment is used to design gene specific oligonucleotides to amplify the
frill-length eDNA
of the corresponding gene by RACE, This method generates overlapping S' and 3'
segments that
include the original cDNA fi~agment from TM VI to TM VII indicating these
segments originate
from the same cDNA, The eDNA includes an approximately 1887 nucleotide open
reading frame
and 5' and 3' untranslated regions (Genbanl: accession number is AY333178),
The predicted
initiation codon is preceded by an in-frame stop codon, indicating that the 5'
end of the open
reading frame is included in the cDNA and that the encoded protein will be
full length. This
cDNA and encoded protein are designated Pa oar.
The open reading frame encodes a protein of approximately 628 amino acids with
a
predicted molecular mass of about 68,642 Da. Hydropathy analysis by the method
described in
I~yte et al., J. Mol. Biol. 157, 105-132 (1982), with a window of about nine
amino acids indicates
about seven potential transmembrane spanning damains, In addition, a protein
BLAST search
fords similarity of Pa oat to the rhodopsin family of 7 transmembrane G
protein-coupled receptors
contained within the conserved domain database,
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The BLAST analysis also indicates Pa oar, is most s imilar to other biogenic
amine
receptors, As melitioned above, all members of GPCT~s share the common motif
of seven
transmembrane (TM) domains. Of these seven domains, TM 3, 5 and 6 comprise the
binding
sites. Compared to proteins with defined functions, Pa oar is most closely
related to OAMB, an
octopamine receptor from the fruit fly 17~~osophila ~rzelafzogaster~ and to
Lym- oar, an octopamine
receptor from the pond snail Lyn~f~aea stagrralis), Sequence similarity is
also detected with
vertebrate alA adrenergic receptors and invertebrate tyramine receptors,
Protein alignment
indicates Pa oal is about 51% identical to OAMB, 37% identical to Lym oar, and
about 27%
identical to both the insect tyramine receptors Tyr-Loc from Locusta
migi~atoi~ia and Tyr-Dro
from D~osoplzila n~elarlogaste~°, Sequence conservation between Pa oar,
OAMB and Lym oai, is
greatest within the TM domains, as shown in Figure 2. The regions of lowest
similarity among
these three proteins are in the amino terminus extending into TM l,
extracellular loop 2 (between
TM IV and V), intracellular loop 3 (between TM V and VI) and the carboxyl
termini following
TM VII.
E. CELL CULTITRE AND TRANSFECTION OF CELLS
Cell culture reagents may be obtained from Sigma-Aldrich (St Louis, MO), or as
otherwise indicated, African green monkey kidney COS-7 cells and human
embryonic kidney
(HEK)-293 cells are obtained from American 'Type Culture Collection (Manassas,
VA). COS-7
cells are grown in Dulbecco's modified Eagle's medium (about 4.5 g glucose/I)
and about 10%
fetal bovine serum, HETC-293 cells are grown in Dulbecco's modified Eagle's
medium (about 1 g
glucose/1), about 5% fetal bovine serum and about 5% newborn calf serum, Both
types of media
are supplemented with about 100 U penicillin G/ml, about 100 pg
streptomycin/ml and about 0.25
pg amphotericin B/ml) except during Lipofectamine 2000 transfections.
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Lipofectamine 2000 and Opti-MEM I media may be obtained from Invitrogen Life
Technologies (Carlsbad, CA), COS-7 cells are transiently transfected using
Lipofectamine 2000.
Cells are plated at about 1.5 x 10G cells per dish (about 55 cm2) in about 10
llll gl'OWth 11'ledlLllll
without antibiotics the day before transfection, For each dish, about 30 p1
Lipofectamine 2000 in
about 1 ml Opti-MEM I medium is mixed with about 12 pg plaslnid DNA in about 1
ml Opti-
MEM I medium and added to the cells after an approximately 20 min incubation
at 1'00111
temperature. The cells are harvested for membrane preparation 24 h following
transfection.
For stable transfections of HEK-293 cells, about 1 x 10~' cells in about 2.5
ml growth
media without antibiotics are plated into dishes (about 10 cm') the day before
transfection, For
transfection, about 10 p1 Lipofectamine 2000 is added to about 250 p1 Opti-MEM
I medium. This
is mixed with about 4 pg plasmid DNA in about 250 p1 OptiMEM I medium. After
an
approximately 20 min incubation at room temperature, the approximately 500 EII
of solution is
added to cells in a single dish, Cells are split about 24 h after transfection
into growth media
containing about 0.8 mg 6418 sulfate/ml (Mediatech II1C., Heradon, VA), Clonal
lines are
selected and assayed for receptor expression with whole cell binding by
incubating about 500,000
cells in about 1 ml phosphate buffered saline (PBS; 137 mM NaCI, 2.7 mM KC 1,
10 mM
Na2HP0~, 1.4 mM ICI-IZP04 (pH 7.4)) with about 2 nM 3H-yohilnbine for about 30
171111 at about
27 ~C, Cells are pelleted by centrifugation, washed with PBS, and then
transferred to scintillation
vials. Nonspecific binding is determined by including about 50 yM phentolamine
in the binding
reaction.
F. EFFICACY OF CELLS LINES TRANSFECTED WITH OCTOPAMINE
RECEPTORS FOR SCREENING COMPOUNDS AND COMPOSITIONS FOR
OCTOPAMINE RECEPTOR INTERACTION
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0
All steps are performed at about 4 C or on ice, Cells are harvested in growth
media by
scraping from the dishes and then rinsing dishes with PBS, The cells are
centrifuged at about
I OOOg for abOLlt 3 111111, washed with PBS and centrifilged again, The cells
are suspended in ice
cold hypotonic buffer (10 mM Tris-CI (pH 7.4)), incubated on ice for about 10
min, and lysed
using a glass Bounce homogenizer and tight glass pestle (I~ontes Glass Co.,
Vineland, NJ) with
about 10 strokes, Nuclei are pelleted by centrifugation at about 600g for
about 5 111111, The
supernatant is decanted and centrifuged at about 30,OOOg for about 30 min to
pellet a crude
membrane fraction, The pellet is suspended, in binding buffer (50 mM Tris-C1,
5 mM MgCl2 (pH
7.4)), Protein concentration is determined by the Bradford assay (Bio-Rad
Laboratories, Hercules,
I 0 CA), Membranes are frozen on dry ice and stored at about -75 ~C in
aliquots.
Antagonists and biogenic amines are obtained from Sigma-Aldrich (St, Louis,
MO).
Octopamine is the mixed isomeric form DL-octopamine, 3H-yohitnbine is obtained
from Perkin
Elmer Life Sciences (Boston, MA), Radioligand binding is performed with about
7.5-IS yg
membrane protein inabout250 y1 binding buffer for about 30 min at about 27 ~C
while shaking at
about 100 rpm, Reactions are terminated by addition of abaut 3 ml ice cold
binding buffer and
filtered over GF/C filters (Whatman International, Maidstone, England) that
have been soaked for
about 30 min in about 0.3% polyethylenimine (Sigma-Aldrich), Filters are
rinsed again with about
3 m1 binding buffer, For the determination of ICS; and B",a~, a range of 3H-
yohilnbine is used from
about 0.5 to 50 nM, and about 50 ~,M phentolamine is used as a competitor to
determine
nonspecific binding, To determine K;, of different ligands, about 2 nM 3H-
yohimbine is used with
a concentration range of competitor that gives from 0% to 100% competition,
Binding data is
analyzed by nonlinear regression using the software GraphPad Prism (San Diego,
CA),
For pharmacological binding experiments, Pa oa;, is expressed in COS-7 cells
by transient
transfection. Membrane fractions are analyzed to determine total, nonspecific
and specific
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binding of 3H-yohimbine, as shown in Figure 3A. The Kd and B",~X for specific
binding are
determined to be about 28.4 nM and about 11.8 pmol/mg protein, respectively.
Membrane
fractions from COS-7 cells transiently transfected with empty pCDNA3 do not
demonstt ate
specific binding. The high affinity binding of 3H-yohimbine by Pa oa; indicate
that this is a
suitable ligand to be used for competition binding experiments.
The octopamine receptor OAM:B from DJ°osophila n~elaroogastef°
is ampliFed by the
polymerase chain reaction. Saturation binding analysis with 3H-yohimbine is
performed with
OAMB expressed in GOS-7 cells, as shown in Figure 3B. The I~~ and B",ax are
determined to be
about 43.0 nM and about 8.04 plllol/111~, respectively.
Competitive binding with various biogenie amines is utilized to determine the
affinities for
potential natural ligands of Pa oal. Referring now to Table A, below, DL-
Octopamine has the
lowest I~; (about 13.3 p,M) for Pa oat followed by tyramine (about 31.0 p.M).
The decreasing
order of affinity for the biogenic amines is octopamine > tyramine > dopamine
> serotonin. The
binding affinities for octopamine and tyramine are determined for this
receptor. The K; (mean ~
standard deviation) of octopamine and tyramine for OAMB are about 8.20 ~2.60yM
and about
33.8 ~ 7.93 wM, respectively. These values are similar to those obtained for
Pa oa;. The affinity
of octopamine is about 2.3-fold higher than tyramine for Pa oa;, and for OAMB,
the affi pity of
octopamine is about 4.1-fold higher than tyramine, indicating that octopamine
is the likely
endogenous ligand for Pa oa;.
Liga.nd I~; (yM)
Bioge~ic As~tihe
Octopamine 13.3 ~ 2.4
Tyramine 31.0 ~ 1.9
Dopamine 56.6 ~ 8.0
Serotonin 77.4 ~ 11.6
AYl tagOl2lSl
Chlorpromazine ' 0.012 ~ 0.003
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Phentolamine 0.023 ~ 0.009
Mianserin 0.048 ~ 0.013
Metoclopramine 4.76 ~ 1.32
Table A
In addition to using the affinity of octopamine receptors for specific
antagonists as a
method for classifying these receptors, antagonists may be used to analyze the
effects of
octopamine on adenylate cyclase activity in the brain, ventral nerve cord and
hen iocytes of
Penipla~reta arne~ieana. A pharmacological profile is developed for Pa oal
using these
antagonists. With reference to Table A, in order of decreasing affinity, the
profile of the
antagonists is chlorpromazine > phentolamine > mianserin > metoclopramide.
EXAMPLE 2
STRUCTURAL FEATURES OF CLONED AMERICAN COCKROACH OCTOPAMINE
RECEPTOR (Pa oa,)
The Pa oar cDNA of 2268 by which includes an 1887 nucleotide open reading
frame and
5' and 3' untranslated regions is set forth in Figures 1A, 1B and SEQ ID NO:
1. With reference
to Figure 1B, the predicted initiation codon (M) is preceded by an in-frame
stop codon (SC). This
indicates that the 5' end of the open reading frame is included in the cDNA
and that the encoded
protein would be full length.
With reference to Figure 4, hydropathy analysis by the method of Kyte and
Doolittle with
a window of 9 amino acids indicates that this sequence shares the common motif
of 7 potential
transmembrane scanning domains. See I~yte and Doolittle, 1982, J. Mol. Biol.
157, I05-132. A
phylogenic comparison of invertebrate biogenic amine receptor sequences
reveals that both
OAMB and Pa oa, sequences share ~ 45% similarity, which is illustrated in
Figure 5. Pa oar
clusters with octopamine and tyramine receptors from different insect species.
Similarity between
1p
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these receptors is analyzed using BLAST search aiid calculated based on
protein alignment using
DNASTAR software program. Pa oal is used as a reference for comparisons with
other receptors.
With reference to Figure 2, protein alignment indicates sequence conservation
between Pa
oal and OAMB is greatest within the transmembrane domains (TMs). The regions
of lowest
similarity among these two proteins are in the amino terminus extending into
TM1, extracellular
loop2 between TM4 and TMS, intracellular loop between T-'MS and TM6 and the
carboxy termini
following TM7.
EXAMPLE 3
EFFECTS OF TREATMENT WITH OCTOPAMINE ON CELLS EXPRESSING THE
OCTOPAMINE RECEPTOR (Pa oal~
A. EFFECT OF TREATMENT ON [CAMP]
Twenty-four hours before cell treatment, about 300, 000 I-IEK-293 cells are
plated in about
1 ml media with about 0.8 mg G418/ml into multi-well dishes (e.g., 12-well,
4.5 cm'). For cell
treatment, the media is aspirated and about 1 ml PBS with about 300 pM IBMX
and the test
reagent is added. Cells are incubated at about 37 ~C for about 20 min, and the
PBS is then
aspirated. Cells are incubated with about 70% ethanol for about 1 h at about -
20 ~C. The cellular
debris is centrifiiged and then the supernatant is removed and lyophilized to
dryness. The amount
of CAMP in the extract is determined by using a cAMP binding protein fro111
the ~H-cAMP
Biotralc assay system (Amersham Biosciences) as per the manufacturer's
instructions. To test the
effects of calcium chelation on cAMP levels, the cells are incubated with
about 20 y 1 V 1
BAPTA/AM (Calbiochem Biochemicals, La Jolla, CA) for about 10 min before the
addition of
the test reagents.
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Octopamine has been demonstrated to increase levels of the second messenger
cAMP in
brain, thoracic ganglion and hemocytes from Peoiplaa2eta arne~~ica~a. To
deterlllllle WhlCh SeCOlld
messenger signaling pathways octopamine could affect through the Pa oal
receptor, HEIR-293
cells are stably transfected with pCDNA3-Pa oat or pCDNA3 without an insert as
a control In the
control HEIR-293 cells, neither DL-octopamine nor tyramine at concentrations
up to about 100
pM has significant effects on CAMP levels,
A clone transfected with pCDNA3-Pa oal having a high specific binding t0 3H-
yohl111b111e
is selected for second messenger analysis, BOth octopamme alld tyramine are
able t0 increase the
levels of cAMP in these cells in a dose dependent manner, as shown in Figure
6, The >JCSOS for
the octopamine and tyramine mediated increases in cAMP are about 1.62 and
97.7pM,
respectively (p < 0.05). Octopamine is more potent than tyramine in the cAMP
response as a
statistically significant increase in CAMP over the basal level (about 0.48
pmol CAMP) is first
detected with about 10 11M octopamine (about 1.2 pmol CAMP) (p < 0.05). The
cAMP
concentration with about 10 nM tyramine is about 0.50 pmol CAMP, and therefore
not statistically
significant from the basal level (p > 0.05), A concentration of about 1 EIM
tyramine results in an
increase in CAMP to about 1.2 pmol. In addition, about 100 IIM octopamine
leads to an
approximately 911-fold increase in cAMP compared to an approximately 215-fold
increase for
about 100 p,M tyramine. Since these assays are performed in the presence of
the
phosphodiesterase inhibitor IBMX, the increases in CAMP is determined to be
through activation
of adenylate cyclase. As such, it appears that the Pa oal receptor is an
octopamine receptor, the
Pa oal receptor may be targeted to effect a disruptive change in intracellular
levels of cAMP,
controlled targeting of the receptor allows for insect control, and the cell
lines stably expressing
the Pa oal receptor may be used to screen compounds and compositions for
insect control activity.
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B. EFFECT OF TREATMENT ON cAlVn' AND (Ca2+]
To determine cAMP levels in cells, about 24-hovers before cell treatment,
300,000 HEK-
293 cells are plated in 1 mL media with 0.8 mg G418/n~L lllt0 111L11t1-dlSheS
(4.5 cm2). For cell
treatment, the media is aspirated and 1 mL PBS with 3~0 p.M IBMX and the test
reagent is added.
Cells are incubated at 37°C for 20 min, and the PBS is then aspirated.
Cells are incubated with .
70°e° ethanol for 1 hour at-20°C. The cellular debris is
centrifuged and then the supernatant is
removed and lyophilized to dryness. The amount of c~MP in the extract is
determined by using a
cAMP binding protein from the 3H-cAMP Biotrak assay system (Amersham
Biosciences,
Piscataway, NJ) as per the manufactzlrer's instructions.
To determine Ca2+ levels in the cells, HEK-293 cells are washed once with
Hank's
balanced salt solution (137 mM NaCI, 5.4 mM ICCI, 0.3 mM NaZHP04, 0.4 mM
ItH~P04, 4.2
mM NaHC03, 1 mM CaCl2, 1 mM MgSO4, and 5.6 mM glucose (pH 7.4)) (HBSS). Cells
are
collected by scraping and are suspended at about 750,000 cells/ml in HBSS with
about 5 p.M
Fura-2 AM (Sigma-Aldrich), Cells are incubated at abo ut 37 ~C for about 1 h
in the dark,
centrifuged, suspended in HBSS at about 750,000 cellsJml and used for calcium
measurements A
spectrofluol°emeter with Felix software from Photon Te chnology
International (Lawrencevi Ile,
NJ) is used for the fluorescence measurements and data- collection,
Octopamine has been demonstrated to modulate; intracellular calcium levels in
cultured
hemocytes of Malacosonza disstria. Also, in hemocytes from Peg~ipla~eta
annericafna, octopamine
lead to an increase in inositol triphosphate which likely will lead to
increases in calcium in these
cells as well, The ability of both octopamine and tyran W na to modulate
calcium levels in the 1-IEK-
293 clone expressing Pa oat is determined, Neither about 10 yM octopamine nor
about 10 pM
tyramine modulates intracellular calcium levels in collt~'ol HEIR-293 cells
transfected with
pCDNA3 lacking an insert, However, when about 100 r1M octopamine is added to
the Pa oa,
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expressing HEIR-293 cells, a rapid increase in intracellular calcium is
detected, as shown in Figure
7. In these same cells, about 100 nM tyramine does not modulate intracellular
calcium levels, as
shown in Figure 7,
Testing of these amines at additional concentrations indicates that the lowest
concentration
of octopamine that increases intracellular calcium levels is about 10 nM.
Tyramine is found to
increase intracellular calcium when a concentration of about 1 p.M or higher
is tested, These
increases in intracellular calcium by about 10 nM octopamine and about 1 pM
tyramine are to a
similar level, both of which is lower than the increase in calcium mediated by
about 100 nIVI
octopamine, This result is similar to that obtained with the cAMP assay in
that an approxim ately
100-fold increase in tyramine concentration compared to about 10 nM octopamine
is required to
give a similar level of response,
As such, it appears that the Pa oar receptor.is an octopamine receptor, the Pa
oar rec eptor
may be targeted to effect a disruptive change in intracellular levels of Cap+,
controlled targeting of
the receptor allows for insect control, and the cell lines stably expressing
the Pa oar receptoa- may
be used to screen compounds and compositions for insect control activity.
Octopamine is found to increase both cAMP and calcium in HEK-293 cells
expressing Pa
oar and the calcium increase is detected immediately upon octopamine addition.
As such, tL-~e
possibility that calcimil is leading to a secondary increase in cAMP levels in
the cells expressing
Pa oar is tested. The intracellular calcium chelator BAPTA/AM is used,
BAPTA/AM at abut 20
pM is found to inhibit the increase in free intracellular calcium when about 1
yM octopami 3~e is
added to the Pa oar-expressing cells, Octopamine-mediated changes in cAMP
levels are cotmpared
in the absence and presence of about 20 p.M BAPTA/AM, cAMP levels following
treatment with
either about 100 nM or about 1 ~,M octopamine, as well as basal cAMP levels,
are not four d to be
statistically different, whether in the absence or presence of about 20 ~M
BAPTA/AM, as shown
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in Figure 8. This indicates that the increase in CAMP by octopamine results
from direct coupling
of Pa oar to a G protein that leads to activation of adenylate cyclase,
malting the expression of Pa
oa; in HEK-293 cells a good model for adenylate cyclase-modulated insect
control through this
receptor and the cell lines stably expressing the Pa oat receptor useful for
screening compounds
and compositions for insect control activity.
EXAMPLE 4
RECEPTOR BINDING AND CHANGES IN cAMP AND INTRACELLTJLAR CAZ+ IN
RESPONSE TO OCTOPAMINE TREATMENT
For radioligand binding studies, the binding of 3H-yohimbine to membranes
isolated from
COS-7 cells expressing Pa oa; and octopamine receptor'(OAMB) from
17r°osoplaila ~raela~rogaste~°
Are performed. See Bischof and Enan, 2004, Insect Biochem. Mol. Biol. 34, pp.
511-521, which
is incorporated herein by this reference. The data shown in Table B
demonstrates that the affinity
of Pa oa; to the radioligand is abaut 1.5 fold higher than OAMB. Radioligand
binding using 3H-
yohimbine is performed on membranes expressing either either Pa oar or OAMB.
For the
determination of ICd and B",~, a range of 3H-yohimvine is used from 0.5 to 50
n M, and 50 yM
phentolamine is used as a competitor to determine nonspecific binding. To
determine IC; of
octopamine, 4 nM 3H-yohimbine is used with a concentration range of octopam
ine that gives from
0 to 100% competition.
OAR Species I~d B",a, Ki
(nM) (pmole receptor/mg (yM)
protein)
OAR species 28.4 11.80 13.30
OAMB 43.0 8.04 8.20
Table B
With reference to Figure 9, OA (10 pM) increases the level of cAMP in HEIR-293
cells
permanently expressing either OAMB or Pa oar. With reference to Figures 10A
and l OB, OA (10
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l.~M) increases the level [Ca'+]; in HEK-293 cells permanently expressing
either OAMB or Pa oa;,
where HEK-293 cells expressing either receptor are incubated for 30s before
the addition of 10
1.~M octopamine (OA). The arrow in the figures indicates addition of the
amine. The fluorescence
ratio determined from excitation with 340 and 380 nm is plotted to indicate
changes in [Ca''+];
levels. These increases are mediated through the OAR as judged by the
insignificant changes in
cAMP level and [Ca2~]; in cells transfected with an empty vector then treated
with 10 yM OA
(data not shown).
EXAMPLE 5
L 0 EFFECTS OF TREAMENT WITH PLANT ESSENTIAL OILS ON CELLS
EXPRESSING THE OCTOPAMINE RECEPTOR
In this example, membranes isolated from COS-7 cells expressing the receptor
are used
for receptor binding studies and HEK-293 cells are.used for CAMP and [Ca'+];
studies. Plant
15 essential oils, including: ~-cymene [methyl(I-methylethyl)benzene], eugenol
[2-methoxy-4-(2-
propenyl)phenol], traps-anethole [I-methoxy-4-(1-propenyl)benzene], cinnamic
alcohol [3-
phenyl-2-propen-1-of], a,-terpineol [p-menth-I-en-8-of], methyl salicylate [2-
hydroxybenzoic acid
methyl ester], 2-phenylethyl propionate, and geraniol [3,7-dimethyl-2,6-
octadien-1-of], are
obtained from Gity Chemical (West Haven, CT) and tested for insect control
activity. The
20 chemical structures of these compounds are set forth in Figure 11.
A. RECEPTOR BINDING ACTIVITY
The binding activity of 3H-yohimbine to membranes expressing Pa oa; or OAMB is
performed in the presence and absence of three structurally related plant
essential oil
25 monoterpenoids, which are selected based on their insecticidal activity,
the absence or presence
and location of the hydroxyl group and a spacing group within the molecule.
Membrane protein
(10 yg) expressing Pa oa; is incubated with 4 nM 3H-yohimbine in the presence
and absence of 50
25.
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p,M of the test chemical. The specific activity is calculated as the
difference between counts in
the presence and absence of test chemical. Specific binding is calculated by
determining
nonspecific binding with 50 yM tested plant essential oils and subtracting
nonspecific binding
fr 0111 total binding.
With reference to Figure 12, depicting specific binding of 3H-yohimbine to Pa
oai and
OAMB, while eugenol and cinnamic alcohol decrease the binding of 3H-yohimbine
to membranes
expressing either Pa oar or OAMB as compared to the corresponding control,
trans-anethole
decreases the 3H-yohimbine binding activity to only Pa oal. It is also found
that eugenol and
trans-anethole are more potent inhibitors against Pa oar than OAMB, while
cinnamic alcohol is
more potent against OAMB than Pa oal. The data suggested insect species
differences in receptor
binding in response to monoterpenoids.
B. EFFECTS OF TREATMENT ON [CAMP]
Figure 13 depicts the effect of certain plant essential oils on cAMP levels in
HEIL-293
cells expressing either Pa oai or OAMB. HEIR-293 cells stably expressing
either receptor are
treated with 300 ECM IBMX and the effect oftested plant essential oils (50
p.M) on basal CAMP
levels is measured.
Eugenol (50 ~~M) significantly decreases the cAMP level (24%) in cells
expressing Pa oar
but slightly decreased cAMP level in cells expressing OAMB. A 22% increase in
cAMP level in
cells expressing OAMB is fOUlld in response to treatment with (SO yM) traps-
anethole. Cinnamic
alcohol (50 p.M) induces slight increase in CAMP level in both cell models.
C. EFFECT OF TREATMENT ON INTRACELLULAR CALCIUM
MOBILIZATION
To address whether changes in [Ca2+]; in octopamine receptor-expressing cells
in response
to 25 yM of tested plant essential oils is mediated specifically through the
receptor, cells
2 (~
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transfected with an empty plasmid (pCDNA3) are treated with either test
chemicals or solvent
only and changes in [Ca2+]; are monitored. In cells transfected with an empty
plaslnid, none of the
test chemicals induce remarkable changes in [Cap+]; levels as compared with
cells treated with the
solvent (data not shown).
On the other hand, changes in [Ca2+]; level in cells expressing either OAMB or
Pa oa; in
response to test chemicals is remarkably high. Figures 14A-14F, depict the
effect of cinnamic
alcohol (Figures 14A and 14B), eugenol (Figures 14C and 14D), and t-anethole
(Figures 14E and
14F) on intracellular calcium [Ca2+]; levels in HEK-293 cells either
transfected with Pa oar or
OAMB. HEK-293 cells are incubated for 30s before the addition of 25 pM tested
agents. The
arrow in the figures indicates addition of tested agents. The fluorescence
ratio detel'nlllled fl'Olll
excitation with 340 mn and 380 nm is plotted to indicate transient increase in
[Ca2+]; levels.
Generally, changes in [CaZ~'~]; in cells expressing OAMB is more pronounced
than changes
in cells expressing Pa oal. Based on increased [Ca~''+]; level in cells
expressing OAMI3, cinnamic
alcohol is the most potent agent tested in this example, followed by eugenol
and traps-anethole.
In cells expressing Pa oal, eugenol is the most potent agent tested in this
example, followed by
cinnamic alcohol then traps-anethole. The data suggest that elevation pattern
of [Caz-F]; levels is
chemical-dependent. While application of octopamine induces an immediate but
transient peak (~
sec) in [Ca2+]; level, as shown in Figure 9, the peaked [Ca2+]; level is
slower in onset and has a
longer recovery time (more than 3 min) in response to treatment with tested
plant essential oils.
20 In cells expressing OAMB, the increase in [GaZ+]; level in response t0
C11111a1111C alCOl101 is
slower than the other two chemicals. In Pa oa;-expressing cells, the increase
in [Ca'+]; in
response to traps-anethole is slower than eugenol and cinnamic alcohol. Thus,
the efficacy of
coupling of both cloned octopamine receptors to different second messenger
signaling varies with
the chemical used.
D. SUMMARY OF THE EFFECTS OF TREATMENT WITH CERTAIN PLANT
ESSENTIAL OILS
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The present example studies the molecular interaction of plant essential oils
with
octopamine receptors fi~om different insect species. Based on the
characteristic features of
octopamine receptors from American cocla~oach and fruit fly, the example
characterizes certain
molecular basis for insect species differences in response to plant essential
oils. AIthOLIgII trans-
anethole does not have a significant effect on bmdmg t0 OAMB while eugenol and
cinnamic
alcohol do (Figvlre 12), only traps-anethole increases cAMP level (Figure 13)
and [Ca''~'~]; (Figures
14A-14F) through OAMB. These findings suggest that, in the case of traps-
anethole, ionic
interaction between the tested agent and the receptor is not critical for the
activation of signaling
down stream to OAMB.
On the other hand, while both eugenol and cinnamic alcohol decrease the
binding activity
to Pa oa; (Figure 12), only eugenol decreases cAMP levels through this
receptor (Figure 13).
However, these two chemicals increase [Ca'+]; through Pa oa; and OAMB (Figures
14A-14F).
The data demonstrates that activation of Pa oal by traps-anethole and cinnamic
alcohol is not
primarily coupled to cyclic nucleotide system. It appears that it is coupled
to IP3-system, which
activates the release of Ca2+ ions from internal stores. Activation of Pa oa;
by eugenol is Found to
be coupled to both adenylate cyclase/cAMP and IP3/Ca2+ signaling cascades.
Therefore, the
current changes in cellular responses suggest that tested plant essential oils
differing by only a
single hydroxyl group or methoxy group in their chemical structure are capable
of differentially
coupling each octopamine receptor to different second messenger systems. The
data also suggest
that, activation of single GPCR such as Pa oal or OAMB, may potentially couple
to multiple
second messenger systems. Thus, a single receptor may have a different
pharmacological profile
depending on which second messenger system is activated. The variability of
the transmembrane
regions and N-termini of Pa oal and OAMB might determine the selectivity of
tested
monoterpenoids. In addition, conservation of cel-tain transmembrane motifs and
the variability of
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the intracellular loops might enable Pa oar and OAMB to discriminate among the
various G-
protein subtypes upon treatment with tested monoterpenoids.
Protein alignment indicate that the regions of lowest similarity among these
two proteins
ar a in the amino terminus extending into TM1, extracellular loop2 between TM4
and TMS,
intracellular loap between TM5 and TM6 and the carboxy termini following TM7
(Figure 2). On
the other hand, protein alignment indicates sequence conservation between Pa
oai and OAMB is
greatest within the transmembrane domains (TMs).
EXAMPLE 6
TOXICITY TESTING AGAINST CERTAIN INSECT SPECIES
Toxicity bioassay against the wild type Drosophila nzelan~gaster fly and
American
cockroach is performed to address insect species 5peciticity in response to
certain plant essential
oils and to determine whether the cellular changes in Pa oar and OAMB cell
models in response
to treatment with tested essential oils correlate with their insecticidal
activity.
Dnosophila n-aelaaogaste~ wild type strain is purchased from Carolina
Bialogical Supply
Company (Burlington, NC). Flies carrying the inactive (iav) mutation that
exhibit low locomotor
activity and poor mating success, both of which are associated with a
deficiency in octopamine
synthesis are obtained from Bloomington Drosopl7ila Stocl: Center (flybase ID
FBaI 0005570,
stoclc# BL-6029 iav).
Plant essential oils, including: p-cymene [methyl(1-methylethyl)benzene],
eugenol [2-
methoxy-4-(2-propenyl)phenol], traps-anethole ~l-methoxy-4-(1-
propenyl)benzene], cinnamic
alcohol [3-phenyl-2-proper-1-of], a,-terpineol [p-month-1- en-8-of], methyl
salicylate [2-
hydroxybenzoic acid methyl ester], 2-phenylethyl propionate, and geraniol [3,7-
dimethyl-2,6-
octadien-1-of], are obtained from City Chemical (West Haven, CT) and tested
for insect control
activity. The chemical structures of these compounds are set forth in Figure
11.
2~~
CA 02563886 2006-10-13
WO 2005/092016 PCT/US2005/009223
Acetonic solutions of plant essential oils are prepared and different
concentrations of each,
that give from 10% - 100% mortality, are applied by topical application.
Controls are treated with
the same volume (0.5 yl/insect) of acetone. Replicates, with 5 insects per
replicate, are used for
each concentration. The mortality is calculated 24 hours after treatment. Data
are subjected to
probit analysis to determine LDSO value for each compound. See Finney, 1971,
Probit Analysis,
3'd Ed., Cambridge University Press, London, pg. 333.
To determine whether the octopamine/octopamine receptor (OA/OAR) system is
involved
in the toxicity of tested plant essential oils, octopamine synthesis mutant
(iav) DT~o.s~oplaila
mela~ogaste~~ strain is topically treated with a dose eduivalent to the
determined LDSO for wild
type strain. For this study, the LDSO values of eight monoterpenoid plant
essential oils (p-cymene,
eugenol, traps-anethole, cinnamic alcohol, a-terpineol, methyl salicylate,
phenylethyl propionate,
and geraniol) are determined against wild type as described above and being
used to treat the
octopamine mutant (iav) fruit fly. Controls are treated with the same volume
(0.5 E~l/fly) of
acetone. The mol-tality is calculated 24 hour after treatment. Multiple
replicates and 5 flies per
replicate are used for the bioassay of each chemical. Data are subjected to
probit analysis to
determine LDso value for each chemical. See Finney, 1971.
To determine insect species differences in response to plant essential oil
1110170terpel'lOldS, the
toxicity of certain monoterpenoids is determined against fruit fly and
American cockroach. Based
on the calculated LDSO values, shown 111 Table C, cinnamic alcohol is the most
toxic chemical
tested in the example (LDSO = 1.65 ~.g/fly) against wild type fruit fly
strain, followed by eugenol
(LDso = 1.90 p.g/fly), and traps-anethole (LDSO = 6.00 l.lg/fly). Cugenol is
about 2-Fold and about
27-fold more toxic against American cockroach than cinnamic alcohol and traps-
anethole,
respectively.
Plant essential oil ~ D. >'r2ela~ogastey~ ~ P. Anaericarza
CA 02563886 2006-10-13
WO 2005/092016 PCT/US2005/009223
Cirlnamic alcohol 1.G5 98
Eugenol 1.90 47
Traps-anethole 6.00 1300
Table C
To determine whether the OA/OAR system mediates the toxicity of certain plant
essential
oil monoterpenoids, fruit flle5 CaTlylllg the dCIV IllLItat10115, which are
highly susceptible to the
octopalnine analoguep-cresol, are used in parallel with wild type fruit fly
strain in the taxicity
bioassay test. The toxicity Of CII111a1111C alCOh0l, eugenol, traps-anethole
and 2-phenyethyl
propionate is remarlcably increased when they are topically applied to the
ierv strain, as shown in
Table D.
Wild/type %Mortality at
LDSO of wild/type
Chemical name LDso valuesDrosophila melczsaogaster strain
(l.~g/ily) Wild/type ierv
cinnamic alcohol 1.G5 30.0% 80.0!
eugenol 1.90 53.3% 80.0%
traps-anethole 6.00 40.0% 100.0%
methyl salicylate 7.50 40.0% 4G.G%
geraniol 10.50 G0.0% G0.0%
a-terpineol 13.00 46.G% G0.0%
2-phenylethyl propionate14.50 53.3% 80.0%
p-cymene 25.00 40.0% 40.0%
Table D
31
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However, mutation of the octopamine synthesis does not affect the toxicity ofp-
cymene,
methyl salicylate, and geraniol. Therefore, the current data suggests a
correlation between agents
111d11Clllg Celhllar Changes 111 CIOllal cells expressing octopamine receptors
and their insecticidal
activity. The data also suggests that the insecticidal activity of cinnamic
alcohol, eugenol, trans-
anethole and 2-phenyethyl propionate is mediated through the
octopamine/octopamine receptor
system. From these data it can be concluded that the increase in the
insecticidal activity of these
chemicals results from the deficiency of octopamine synthesis in iav mutants
because low
octopamine levels may be unable to compete against the toxic effect of these
chemicals.
As mentioned above, the toxicity data demonstrates significant differences
between the
toxicity of the tested chemicals against each insect (Table C). The toxicity
data also demonstrates
differences between the two insects in response to each chemical. The toxicity
data against wild
type and octopamine mutant (iav) fruit fly suggests that the toxicity of
cinnamic alcohol, eugenol
and traps-anethol is mediated through octopamine/octopamine receptors system.
Among certain
other plant essential oils tested against both strains of fruit fly only the
toxicity of 2-phenylethyl
propionate is mediated through octopamine receptors. Collectively the data
suggest a correlation
between cellular changes and toxicity of certain plant essential oils.
In the present example, chemical-structure relationships of plant essential
oil monoterpenoids
against wild type fruit fly suggest certain structural features required for
chemical-receptor
interaction. Among these features are the presence and location of a hydroxyl
group, and a
spacing group such as lnethoxy group. The rank order of toxicity demonstrates
that cyclic
alcohols and phenolic compounds are more toxic than other monoterpenoids such
as acyclic
alcohols and esters. The efficacy of each compound is found to be determined
by the presence
and location of the spacing group on the benzene ring. For example, although
the phenolic
derivative, eugenol, and propenyl benzene, traps-anethol, contain the same
spacing group (-
32.
CA 02563886 2006-10-13
WO 2005/092016 PCT/US2005/009223
OCH3) on position 2 and l, respectively, eugenol is 3-fold more toxic against
wild type flies than
traps-anethole (Figure 11 and Table D).
In summary, the similarities and differences between both Pa oar and OAMB
sequences are
determining features in the toxicity differences of certain plant essential
oil monoterpenoids.
Additionally, it appears that the octopamine receptor mediates the
insecticidal properties of
cinnamic alcohol, eugenol, traps-anethole and 2-phenylethyl propionate and, in
part, the toxicity
of a-terpineol against Droso~hila fzzelanogaste~° fly. Furthermore, it
appears that the presence of
an electronegative group SLICK as hydroxyl group, and different spacing
groups, may be required
for the insecticidal activity of plant essential oils through octopamine
receptor.
'~ ~' a'
It will be apparent to those skilled in the art that various modifications and
variations can
be made in the present ITlVelltloll Wlthollt departing from the scope or
spirit of the invention. It is
intended that the Specification and Example be considered as exemplary only,
and not intended to
limit the scope and spirit of tile invention. The references and publications
cited herein are
incorporated herein by this reference.
Unless otherwise indicated, all numbers expressing quantities of ingredients,
properties
such as reaction conditions, and so forth used in the Specification, Examples,
and Claims are to be
understood as being modified in all instances by the term "about."
Accordingly, unless indicated
to the contrary, the numerical parameters set forth in the Specification,
Example, and Claims are
approximations that may vary depending upon the desired properties sought to
be determined by
the present invention.
33
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