Insect Control: Biological and Synthetic Agents - Index of

130 4: Insect Growth- and Development-Disrupting Insecticides
as photoaffinity ecdysteroids by dehydration of the
corresponding 11a-hydroxy ecdysteroids (ajugasterone,
turkesterone, and muristerone A, respectively).
Of the three photoaffinity ecdysteroids, dacryhainansterone
and kaladasterone showed the greatest
potential for use in determining contact amino acid
residues in EcR LBDs. All three dienone ecdysteroids
retained their biological activity in the D. melanogaster
BII cell assay developed by Clement et al.
(1993). However, upon irradiation with UV at
350 nM in the presence of bacterially expressed
DmEcR/DmUSP and CfEcR/CfUSP, both dacryhainansterone
and kaladasterone blocked subsequent
specific binding of tritiated ponasterone A by >70%.
These results clearly showed the potential of using
radiolabeled dienones for further characterization of
their interaction within EcR LBDs.
Photoaffinity analogs of bisacylhydrazines have
also been produced and used to characterize their
binding to EcRs from dipterans and lepidopterans
(Dhadialla et al., unpublished data). A tritiated benzophenone
analog of methoxyfenozide (Figure 5),
coded as RH-131039, displays the above-mentioned
characteristic requirements of a photoaffinity
reagent as well as the specificity to bind to lepidopteran
EcR (CfEcR) with high affinity. Binding to
dipteran EcR (DmEcR) could not be detected. Binding
of either tritiated ponasterone A or tritiated
RH-131039 to CfEcR/CfUSP could be competitively
displaced using excess unlabeled RH-131039,
tebufenozide, ponasterone A, or 20E. These results
indicate that the ecdysteroids and the two nonsteroidal
ecdysone agonists share a common binding
site in the ligand binding pocket of CfEcR/CfUSP
(Figure 6), an observation that has been confirmed
by Billas et al. (2003) in their work on the crystal
structure of liganded HvEcR/HvUSP. However, further
experiments, which can now be done with the
ecdysteroid and bisacylhydrazine photoaffinity compounds,
need to be done to determine the ligand–
receptor contact residues. Similar results were
obtained for AaEcR/AaUSP, but not for DmEcR/
DmUSP, indicating that even within an insect order,
differences in the homology and folding of EcR LBDs
is sufficient to discriminate between affinities of nonsteroidal
ecdysone agonists. The bisacylhydrazine
and ecdysteroid photoaffinity reagents described are
additional useful tools that can be combined with
data from chemical structure–activity relationships
(SAR), analytical (liquid chromatography/mass spectrometery
(LC/MS) and mass laser desorption ionization
(MALDI)), and mutational analysis approaches
to hypothesize and/or define the three-dimensional
space of non-lepidopteran EcR LBDs. However,
the availability of the liganded HvEcR/HvUSP LBD
Figure 5 Structure of the tritiated photoaffinity benzophenone
analog, RH-131039, of methoxyfenozide. The three dots on the
methoxy substitution on the A-phenyl ring indicate tritiation of
the methyl group. The benzophenone group on the B-phenyl
ring can be activated with UV light for it to cross-link to the
nearest amino acid residue.
Figure 6 Fluorograph to show specific binding of tritiated
RH-131039 (PAL; photo affinity ligand) to CDEF domain of Choristoneura
fumiferana EcR (CfEcR) expressed by in vitro transcription
and translation using rabbit reticulocyte system. CfEcR
(CDEF domain) plus CfUSP was incubated with tritiated PAL in
the presence of excess cold PAL, RH-5992, or 20E at the indicated
concentrations under equilibrium binding conditions after
which the reaction mixture was irradiated with UV350 nm for
20 min at 4 C. The reaction mixture was then mixed with SDS-
PAGE sample buffer containing 1 mM DTT, heated for 5 min at
95 C and then subjected to SDS-PAGE. The gel was dried and
fluorographed to get the above image. The numbers on the right
indicate relative migration of molecular weight markers (MWM).
crystal structure provides the needed information for
defining the three-dimensional ligand receptor interaction
for homology modeling of such interactions
with non-lepidopteran EcR LBDs, and for the design
and discovery of new biologically potent ligands as
insecticides for different insects orders.
4.2.3. Mode of Action of Bisacylhydrazines
In the earlier reviews on insecticides with ecdysteroid
and JH activity (Oberlander et al., 1995;
Dhadialla et al., 1998), much of the focus on mode