Insect Control: Biological and Synthetic Agents - Index of

124 4: Insect Growth- and Development-Disrupting Insecticides
(DDC) and at the same time repress their own
expression.
4.1.1.2. Ecdysone receptors The ecdysone receptor
complex is a heterodimer of two proteins,
ecdysone receptor (EcR) and ultraspiracle (USP),
which is a homolog of the mammalian retinoic
acid receptor (RXR) (Yao et al., 1992, 1995;
Thomas et al., 1993). In several insects, both
EcR and USP exist in several transcriptional and
splice variants, presumably for use in a stage- and
tissue-specific way (review: Riddiford et al., 2001).
Both EcR and USP are members of the steroid receptor
superfamily that have characteristic DNA and
ligand binding domains. Ecdysteroids have been
shown to bind to EcR only when EcR and USP
exist as heterodimers (Yao et al., 1993), although
additional transcriptional factors are required for
ecdysteroid dependent gene regulation (Arbeitman
and Hogness, 2000; Tran et al., 2000). Moreover,
EcR can heterodimerize with RXR to form a functional
ecdysteroid receptor complex in transfected
cells (Yao et al., 1992; Tran et al., 2000). cDNAs
encoding both EcR and USPs from a number of
dipteran (Koelle et al., 1991; Imhof et al., 1993;
Cho et al., 1995; Kapitskaya et al., 1996; Hannan
and Hill, 1997, 2001; Veras et al., 1999), lepidopteran
(Kothapalli et al., 1995; Swevers et al., 1995),
coleopteran (Mouillet et al., 1997; Dhadialla
and Tzertzinis, 1997), homopteran (Zhang et al.,
2003; Dhadialla et al., unpublished data; Ronald
Hill, personal communication), and orthopteran
(Saleh et al., 1998; Hayward et al., 1999, 2003)
insects, tick (Guo et al., 1997) and crab (Chung
et al., 1998) have been cloned. Some of the EcRs
and USPs have been characterized in ligand binding
(Kothapalli et al., 1995; Kapitskaya et al., 1996;
Dhadialla et al., 1998) and cell transfection assays
(Kumar et al., 2002; Toya et al., 2002). In all cases,
the DNA binding domains (DBDs) of EcRs show a
very high degree of homology and identity. However,
homology between the ligand binding domains
(LBDs) of EcRs varies from 70% to 90%, although
all EcRs studied so far bind 20E and other active
ecdysteroids. The DBDs of USPs are also highly
conserved. The USP LBDs, however, show very
interesting evolutionary dichotomy: the LBDs from
the locust, Locusta migratoria, the mealworm
beetle, Tenebrio molitor, the hard tick, Amblyoma
americanum, and the fiddler crab, Uca puglitor,
show about 70% identity with their vertebrate homolog,
but the same sequences from dipteran and
lepidopteran USPs show only about 45% identity
with those from other arthropods and vertebrates
(Guo et al., 1997; Hayward et al., 1999; Riddiford
et al., 2001). The functional significance of
RXR-like LBDs in USPs of primitive arthropods
is not well understood, because EcRs from the
same insects still bind ecdysteroids (Guo et al., 1997;
Chung et al., 1998; Hayward et al., 2003; Dhadialla,
unpublished data for Tenebrio molitor EcR and USP
(TmEcR/TmUSP)).
The crystal structures of USPs from both Heliothis
virescens and Drosophila melanogaster have been
elucidated by two groups (Billas et al., 2001;
Clayton et al., 2001). The crystal structure of USP
is similar to its mammalian homolog RXR, except
that USP structures show a long helix-1 to helix-3
loop and an insert between helices 5 and 6. These
variations seem to lock USP in an inactive conformation
by displacing helix 12 from the agonist conformation.
Both groups found that crystal structures
of the two USPs had large hydrophobic cavities,
which contained phospholipid ligands.
Finally, the crystal structures of Heliothis viresens
EcR/USP (HvEcR/HuUSP) heterodimers liganded
with an ecdysteroid or a nonsteroidal ecdysone
agonist have been determined (Billas et al., 2003;
see Section 4.2.2.2 for more details). The crystal
structure of liganded EcR/USP from the silverleaf
whitefly, Bemesia tabaci, has also been determined
and awaits publication (Ronald Hill, personal
communication).
4.2. Ecdysteroid Agonist Insecticides
4.2.1. Discovery of Ecdysone Agonist Insecticides
and Commercial Products
Although attempts to discover insecticides with an
insect molting hormone activity were made in the
early 1970s (Watkinson and Clarke, 1973), it was
not until a decade later that the first bisacylhydrazine
ecdysone agonist ((2)inFigure 1) was serendipitously
discovered by Hsu (1991) at Rohm and Haas
Company, Springs House, PA, USA. Several years
later, after several chemical iterations of this early
lead, a simpler, unsubstituted, but slightly more
potent analog, RH-5849 ((3) inFigure 1), was discovered
(Aller and Ramsay, 1988). Further work on
the structure and activity of RH-5849, which had
commercial-level broad spectrum activity against
several lepidopteran, coleopteran, and dipteran species
(Wing, 1988; Wing and Aller, 1990), resulted in
more potent and cost-effective bisacylhydrazines with
a high degree of selective pest toxicity (review:
Dhadialla et al., 1998). Of these, three bisacylhydrazine
compounds, all substituted analogs of
RH-5849, coded as RH-5992 (tebufenozide (4);
Figure 1), RH-2485 (methoxyfenozide (5); Figure 1),
and RH-0345 (halofenozide (6); Figure 1) havebeen