R

Figure 14 Chemical structures of newecdysone agonists reported in literature: (1) 3,5-di-t-butyl-4-hydroxy-N-isobutyl-benzamide (DTBHIB) from Sumitomo; (2) 8-O-acetylhrapagide from Merck Research Laboratories, Westpoint, PA, USA and (3) tetrahydroquino-line from FMC Corporation, Princeton, NJ, USA. In (3) R = halide.

(DTBHIB; Figure 14) and Elbrecht et al. (1996) at Merck Research Laboratories, Westpoint, PA, USA, reported the isolation of an iridoid glycoside, 8-O-acetylharpagide (Figure 14), from Ajuga reptans. Both these compounds were reported to induce 20E-like morphological changes in Drosophila Kc cells, as well as competitively displace tritiated ponasterone A from Drosophila ecdysteroid receptors with potencies similar to that of RH-5849, the unsubstituted bisacylhydrazine. However, the insec-ticidal activity of these compounds was not described. Attempts to replicate the results of Mikitani (1996) using DTBHIB and analogs failed to demonstrate that these compounds were competitive inhibitors of tritiated ponasterone A binding to DmEcR/DmUSP produced by in vitro transcription and translation (Dhadialla, unpublished observations). On the other hand, Dinan et al. (2001) demonstrated that the results obtained by Elbrecht etal. (1996) were due to co-purification of ecdyster-oids in their 8-O-acetylharpagide preparation. When used as a highly purified preparation, Dinan et al. (2001) found that 8-O-acetylharpagide was not active as an agonist or an antagonist in D. melanogaster BII cell bioassay, and neither did it compete with tritiated ponasterone A for binding to the lepidopteran ecdysteroid receptor complex from C. fumiferana.

Finally, scientists at FMC discovered a new tetrahydroquinoline (THQ) class of compounds (Figure 14) that competitively displaced tritiated ponasterone A from both dipteran (D. melanogaster) and lepidopteran (H. virescens) EcR and USP heterodimers (unpublished data). Interestingly, the most active analogs of this class of compounds bound the DmEcR/DmUSP with much higher affinity than the HvEcR/HvUSP. This is the reverse of what was observed with bisacylhydrazines. When tested for ligand binding to EcR/USP proteins from

L. migratoria, B. argentifoli, and T. molitor, to which bisacylhydrazines show no measurable affinity, members of the THQ were found to bind with measurable affinity (mM range; Dhadialla and colleagues, unpublished results). Further work on this chemistry was continued at Rohm and Haas Company, Spring House, PA, USA, and its subsidiary, RheoGene LLC, Malvern, PA, USA, which resulted in the synthesis of a number of analogs that were active in transactivating reporter genes fused to different insect EcRs and heterodimeric partners (L. migratoria RXR, and human RXR) in mouse NIH3T3 cells (Michelotti et al., 2003).

The discovery of THQs with ligand binding activities to various EcRs (Michelotti et al., 2003), some of which do not interact with bisacylhydrazines, and the interpretation of X-ray crystal structure results of liganded HvEcR/HvUSP (Billas et al., 2003) provides good evidence for the potential to discover new chemistries with ecdysone agonist activities. It should also be possible to design chemistries that specifically interact with EcR/USPs from a particular insect order.

6.3.2.9. Noninsecticide Applications of Nonsteroidal Ecdysone Agonists; Gene Switches in Animal and Plant Systems

A number of researchers started to explore the utilization of the ecdysone receptor as an inducible gene switch due to the knowledge that neither mammals nor plants have ecdysone receptors, and the discovery of bisacylhydrazine as true ecdysone agonists with reduced risk ecotoxicology and mammalian profiles. Gene switches are inducible gene regulation systems that can be used to control the expression of transgenes in cells, plants, or animals. There is a recognized need for tightly regulated eukaryotic molecular gene switch applications, such as for gene therapy, and in understanding the role played by specific proteins in signaling pathways, cell differentiation, and development (Allgood and Eastman, 1997). Other applications include large-scale production of proteins in cells, cell-based high-throughput screening assays, and regulation of traits in both plants and animals. Unlike all other previous and current insecticides, commercial nonsteroidal ecdysone agonists are the first class of insect toxic chemistry that has led to such a high level of interest and utility outside the insecticide arena. The following is a brief overview of the use of bisacylhydrazine insecticides and EcRs as gene switches in mammalian and plant systems.

6.3.2.9.1. Gene switch in mammalian systems

Christopherson et al. (1992) demonstrated that DmEcR could function as an ecdysteroid-dependent transcription factor in human 293 cells cotrans-fected with an RSV-based expression vector that encoded DmEcR and a reporter gene, which contained four copies of Drosophila Hsp27 linked to a MTV promoter-CAT construct. They demonstrated that the activity of DmEcR could not be activated by any of the mammalian steroid hormones tested. Even amongst the ecdysteroids, the phytoecdysteroid muristerone A was the best trans-activator of the reporter gene, while 20E was not as effective. These authors further demonstrated that chimeric receptors containing the LBD of DmEcR and the DBD of glucocorticoid receptor could also function when cotransfected with plasmids containing glucocorticoid receptor response elements fused to a reporter gene. Subsequently, No et al. (1996) demonstrated that an EcR-based gene switch consisting of DmEcR, Homo sapiens RXR, and appropriate response elements fused to a reporter gene in mammalian cells or transgenic mice could be transactivated with micromolar levels of murister-one A. Muristerone was shown to maintain its activity when injected into mice and was not found to be toxic, teratogenic, or inactivated by serum binding proteins. Moreover, overexpression of modified DmEcR and RXR did not appear to to be toxic, at least in transfected cells. This study, like an earlier one (Yao et al., 1995), demonstrated that HsRXR could substitute for its insect homolog and heterodimer partner for EcR, USP. However, in all these studies, the range of activating ligands was limited to ecdysteroids and, in particular, to muris-terone A. In addition, RXR, which is present in almost all mammalian cells, was found not to be as good a partner for EcR as USP. Hence, very high endogenous levels were necessary for stimulation with muristerone A to occur.

With the availability of EcR and USPs from other insects, especially lepidopteran insects, and understanding the high affinity of nonsteroidal ecdysone agonists like tebufenozide and methoxyfenozide for lepidopteran EcRs/USPs, Suhr et al. (1998) demonstrated that the B. mori EcR (BmEcR), unlike DmEcR, could be transactivated to very high levels in mammalian cells with tebufenozide without adding an exogenous heterodimer partner like RXR. The endogenous levels of RXR were enough to provide the high transactivation levels. The work by Suhr et al. (1998) further demonstrated that while the D domain (hinge region) of EcR was necessary for high-affinity heterodimerization with USP, both D and E (LBD) domains were necessary for high-affinity interaction with RXR. By creating chimeric EcR using parts of E-domains from BmEcR and DmEcR, these investigators showed that a region in the middle of the E-domain (amino acids 402 to 508) in BmEcR constituted the region conferring high specificity for tebufenozide. Subsequent research by others has mainly focused on using EcRs and USPs from other insects as well as using different promoters for expression of the transfected genes (Kumar et al., 2002; Dhadialla et al., 2002; Palli and Kapitskaya, 2002; Palli et al., 2002, 2003). Based on EcR homology modeling studies and site-directed mutagenesis of selected amino acid residues, Kumar et al. (2002) found that a CfEcR mutant, which had an A110P mutation in the LBD, was responsive only to nonsteroidal ecdysone agonists, including methoxyfenozide, but not to any of the ecdysteroids tested. This demonstrated that the affinity and functional specificity of an EcR for a ligand can be altered, thus offering the possibility of using multiplexed EcR-based gene switches that could regulate different traits with different ligands. Palli et al. (2003) developed a two-hybrid EcR based gene switch, which consisted of constructs containing DEF domains of CfEcR fused to the GAL4 DNA binding domain, and CfUSP or Mus musculus retinoid X receptor (MsRXR) EF domains fused to the VP16 activation domain. These constructs were tested in mammalian cells for their ability to drive a luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. This combination gave very low level basal activation of the reporter gene in the absence of the induc-er. In the presence of the inducer, there was a rapid increase in the expression of the luciferase reporter gene, reaching levels as high as 8942-fold greater than basal level by 48 h. Withdrawal of the ligand resulted in 50% and 80% reduction of the reporter gene by 12 h and 24 h, respectively.

These studies clearly demonstrate the potential of utilizing EcR based gene switches that can be activated by ecdysteroids and nonsteroidal ecdysone agonists like tebufenozide, methoxyfenozide, and others. The fact that both tebufenozide and methox-yfenozide are registered as commercial insecticides, and have proven reduced risk mammalian and ecotoxicology profiles, makes them very attractive as inducers of the EcR based gene switches. The work of Kumar et al. (2002) clearly demonstrates the potential of mutating EcR to change its ligand specificity, thus opening additional possibilities of extending the use of the EcR gene switch in a multiplexed manner.

6.3.2.9.2. Gene switch for trait regulation in plants The reader is referred to very good recent reviews on this topic that not only describe the EcR-based chemically inducible gene regulation systems, but also other systems that have utility in plants (Jepson etal., 1998; Zuo and Chua, 2000; Padidam, 2003). This section is restricted to descriptions of EcR-based gene switch systems.

In the mid-1990s, a number of agricultural companies initiated research to exploit the use of the EcR-based gene switch and nonsteroidal ecdysone agonists like tebufenozide as chemical inducers for regulation of traits (for example, fertility, flowering, etc.) in plants. Initial work was done using DmEcR and DmUSP as components of the gene switch (Goff et al., 1996). In this case, the researchers used chimeric polypeptides (GAL4 DBD fused to LBD of DmEcR and VP16 activation domain fused to DmUSP) to activate the luciferase reporter gene fused to GAL4 response element in maize cells. In the presence of 10 mM tebufenozide, about 20- to 50-fold activation of luciferase expression was obtained. Subsequently, a number of researchers developed variants of EcR gene switches using different chimeric combinations of heterologous DBD, LBDs from different lepidopteran EcRs, and transactivation domains that could interact with appropriate response elements to transactivate a reporter gene in a ligand-dependent manner (Martinez et al., 1999a; Unger et al., 2002; Padidam et al., 2003). For example, Jepson et al. (1996) and Martinez et al. (1999b) used chimeric H. virescens EcR (HvEcR) composed of glucocorti-coid receptor transactivation and DBD fused to LBD of HvEcR and GUS reporter gene fused to glucocorticoid receptor response element for trans-fection of maize and tobacco protoplast. In both cases, weak transactivation of the GUS reporter gene was obtained with tebufenozide and murister-one A, though the response with the latter was much lower than with tebufenozide. However, 10 mM and higher concentrations of the two ligands were required to transactivate the reporter genes.

Padidam et al. (2003) used a chimeric C. fumifer-ana EcR (CfEcR) composed of a CfEcR LBD, GAL4 and LexA DBDs, and VP16 activation domains that could be activated with methoxyfenozide in a dose-dependent manner from a GAL4- or LexA-response element to express a reporter gene. These researchers used Arabidopsis and tobacco plants for transformation with the gene switch components and obtained several transgenic plants that had little or no basal level of expression in the absence of meth-oxyfenozide. In the presence of methoxyfenozide, reporter expression was several fold higher than in the absence of methoxyfenozide. The above studies provided ample evidence of the utility of EcR gene switch, especially those that utilize EcR from a lepi-dopteran species, and tebufenozide and methoxyfe-nozide as chemical inducers for trait regulation in plants.

Demonstration of the utility of EcR-based gene switch for trait regulation in maize was demonstrated by Unger et al. (2002). A mutation in maize (MS45), which results in male-sterile phenotype, could be reversed by complementation to gain fertility using methoxyfenozide-dependent chimeric receptor gene switch to express the wild-type MS45 protein in tapetum and anthers. These researchers used the EcR LBD from, O. nubialis to generate a chimeric receptor. The chimeric receptor was introduced into MS45 maize with the MS45 gene fused to the GAL4 response element, which in the absence of methox-yfenozide were male sterile. However, application of methoxyfenozide to plants containing either a constitutive promoter or anther specific promoter resulted in the restoration of fertility to MS45 plants grown in either the greenhouse or the field.

It is interesting to note that in all the above studies, except those by Goff et al. (1996), reporter transactivation response to tebufenozide, methoxy-fenozide, or muristerone A via the EcR gene switch was obtained without the requirement of an exogenous heterodimeric partner (USP or RXR), suggesting that there may be other factor(s) in plants that can substitute for USP as a partner for EcR, or that EcR can function as a homodimer. However, as far as is known, there is no evidence of EcR binding an ecdysteroid or nonsteroid ligand in the absence of USP or RXR. Irrespective, these studies provide ample demonstration of the utility of EcR-based gene switch, which can be regulated with an ecdysteroid or a nonsteroidal ecdysone agonist. The use of nonsteroidal ecdysone agonists, like any of the commercialized products, is attractive because their reduced risk for the environment and mammals, birds, aquatic animals, and beneficial arthropods has been found to be acceptable. The specificity and the high affinity with which the bisa-cylhydrazines bind ecdysteroid receptors also offers the opportunity to utilize different ligands and ecdy-sone receptors to regulate more than one trait in a plant or an animal system.

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