Insect Control: Biological and Synthetic Agents - Index of

A4 Addendum: Recent Progress on Mode of
Action of 20-Hydroxyecdysone, Juvenile
Hormone (JH), Non-Steroidal Ecdysone
Agonist and JH Analog Insecticides
T S Dhadialla, Dow AgroSciences LLC,
Indianapolis, IN, USA
ß 2010 Elsevier B.V. All Rights Reserved
A4.1. Recent Progress on Mode of Action of 20-Hydroxyecdysone and Non-Steroidal
Ecdysone Agonist Insecticides 183
The reader is also referred to a more recent publication
on nonsteroidal ecdysone agonist insecticides
(Dhadialla and Ross, 2007). Since the last publication
of this chapter, the crystal structure of the ecdysone
receptor ligand binding domain (LBD) from
the whitefly, Bemesia tabacci (Bt), heterodimeric
ecdysone receptor complex has been published
(Carmichael et al., 2005). Analysis of the amino
acid residues lining the hormone binding pocket
within the EcR subunit indicates that 22 of 25 residues
making hydrogen bonds or nonpolar contacts
with the hormone are highly conserved both in nature
and side-chain rotameric conformation
(Carmichael et al., 2005). The residues that are in
contact with the hormone and that vary between
insects cause remarkably little change in pocket topography.
For example, the effect of replacing
BtEcR Met-272 by the smaller HvEcR Val-384 is
minimized in the region encompassing the hormone
by a change in the rotameric conformation of the
adjacent conserved, non-ligand-interacting residue
BtEcR Leu-308 – HvEcRLeu-420 (see Figure A1).
In fact, regions of the ligand binding pocket surface
in contact with the hormone are remarkably well
conserved, not only in shape, but also in the overall
hydrophobic and polar character.
The same is not true for those parts of the pocket
formed by residues that do not contribute to hormone
interaction. For example, the change in
BtEcR Leu-308 – HvEcRLeu-420 rotameric conformation
described earlier also contributes to
a loosening of residue packing in a region of the
H. virescens pocket adjacent to, but not in contact
with, the steroid side chain (Figure A1). This loosening
is further enhanced by the orientation of HvEcR
Gln-503 toward the surface of the protein, in contrast
to the conformation of the corresponding
BtEcR Met-389, which is directed toward BtEcR
Leu-308 (Figure A1). Calculation of the molecular
surface of the pocket with a 1.2 A ˚ probe reveals an
extension of the H. virescens pocket in this region,
which is absent in the BtEcR protein. It is precisely
here that the bisacylhydrazine inserts itself into the
lepidopteran protein atomic volume, suggesting a
mechanism contributing to differential binding of
the insecticide across taxonomic orders.
In an effort to develop a high-throughput nonradiometric
ligand binding assay for insect EcRs,
Graham et al. (2007) found that fluorescein can be
attached to the end of the ecdysteroid side chain with
little or no effect on binding to the receptor protein.
This led to the development of a recombinant receptor-based
fluorescence polarisation ligand binding
assay, which was readily automated for screening
of a chemical library (Graham et al., 2009). This
high-throughput assay facilitated the discovery of
compounds killing sheep body lice down to
0.5 ppm (Ronald Hill, personal communication).
Hannan et al. (2009) demonstrated that it is possible
to selectively inhibit the synthesis of ecdysone
receptor subunit proteins, using RNAi approaches.
RNAi knock-down of ecdysone receptor synthesis
may find practical applications, for example for
the control of sucking insect pests of agricultural
importance.
Finally, considerable progress has been made in use
of nonsteroidal ecdysone agonist insecticides as
ligands for EcR-based gene switches in plants for
regulated gene expression (Tavva et al., 2006,
2007a, 2007b, 2008). These investigators improved
the sensitivity of the EcR-based gene switch to nanomolar
concentrations of methoxyfenozide compared
to micromolar amounts needed to activate earlier
versions of EcR gene switches. This was achieved by
using gene constructs containing LBD of CfEcR fused
to GAL4 DNA binding domain and VP16 activation
domain fused to LBD of Locusta migratoria retinoid-
X-Receptor (LmRXR) to transform plants. Earlier