768 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774
ical at the amino acid level with mEH from rat and human, respectively; and 46 identical with M. sexta
JH-EH Wojtasek and Prestwich, 1996. The T. ni fat body EH also contains the identical amino acid catalytic
triad Glu-403, Asp-226 and His-430 found in M. sexta EH, microsomal EHs from rat Falany et al., 1987,
human Skoda et al., 1988 and rabbit Hassett et al., 1989, and a haloalkane dehalogenase HLD1, Xanthob-
acter autotrophicus Janssen et al., 1989.
The development of inhibitors for mammalian EHs has been somewhat successful. Hammock and co-work-
ers developed chalcone oxides as effective inhibitors for mammalian soluble EH sEH Morisseau et al., 1998;
Borhan et al., 1995; Pinot et al., 1995. Trichloropropene oxide, cyclohexene oxide and glycidyl derivatives have
been shown to be microsomal EH mEH inhibitors Tzeng et al., 1996. Nearly all of the examples of EH
inhibition involve epoxides which typically serve as competitive alternative substrate inhibitors, with the
exception of recent work by Hammock and co-workers Morisseau et al., 1999 that revealed ureas and carbam-
ates could function as inhibitors of murine sEH. The inhibitors of insect EHs are generally not effective and
exhibit considerable variation in activity depending on the insect and substrate used for the assay. Hammock
and co-workers examined several glycidyl ethers and epoxy-alcohol JH analogs as inhibitors, but only the JH
analogs provided significant levels
µ M of inhibition
Casas et al., 1991; Harshman et al., 1991. Typically, mammalian mEH or sEH inhibitors are not as effective
against insect EH. We previously investigated several potential inhibitors
for T. ni JH-EH using crude microsomal enzyme prep- arations Roe et al., 1996; Harris et al., 1999. Inhibitors
designed to mimic a polarized or ionic transition state were only modest competitive inhibitors of T. ni JH-EH
while non-juvenoid long chain aliphatic epoxides were the most potent competitive substrates to JH III. The
most effective inhibitor identified in our earlier studies, methyl 10,11-epoxy-11-methyldodecanoate MEMD,
exhibited an I
50
of 80 µ
M. Mimicry of the length of the JH backbone and of the methyl ester functional group
of JH was found to be important in substrate binding. We now report a more extensive structure activity
relationship SAR study of glycidol-ester and epoxy- ester inhibitors of JH-EH which further examines epox-
ide substitution patterns, and addresses the question of enantioselectivity in epoxide hydration by recombinant
T. ni JH-EH.
2. Materials and methods
2.1. Insects Larvae of the cabbage looper, Trichoplusia ni
Lepidoptera: Noctuidae, were reared on an artificial diet at 27
± 1
° C and a 14 h light: 10 h dark cycle Roe
et al., 1982. Gate I last instars and specifically aged last stadium larvae were selected as previously described
Kallapur et al., 1996. The last stadium in our studies was 4 days in duration with larvae wandering on day 3,
becoming prepupae on day 4 and undergoing ecdysis to pupae by the next day. cDNA library construction from
fat body tissue and library screening as described in Harris et al. 1999 provided plasmid pG6-1 containing
the full-length EH insert, TmEH-1.
2.2. Baculovirus expression Baculovirus expression of the full length epoxide
hydrolase was obtained using the Autographica califor- nica multiple nuclear polyhedrosis virus AcMNPV
vector system. The pG6-1 plasmid was sequentially digested with BamHI Promega and Xho1 Stratagene,
providing a fragment containing the full-length epoxide hydrolase message. This fragment 1908 bp was ligated
using T4 DNA ligase Novagen with the viral transfer vector pBacPak8 pBP8, CLONTECH, Palo Alto, CA,
previously treated with BamHI Promega and Xho1 Stratagene, which provided the recombinant baculo-
virus plasmid pBP8–G6-1. Recombinant baculovirus was obtained by co-transfection of Spodoptera frugip-
erda Sf9 insect cells with pBP8–G6-1, containing the full-length EH, and Bsu361-digested BacPak6 viral
DNA CLONTECH, providing the virus vG6-1. Suc- cessful co-transfection and expression was verified by
Northern blot analysis, using TmEH
237
Roe et al., 1996 as a probe, of EH infected Sf9 cell RNA. All virus
manipulations were according to O’Reilly et al. 1992. 2.3. Assay of EH activity
Sf9 cells were grown to 96 h post-infection with recombinant virus, vG6-1. The cells 3
× 10
7
were pel-
leted by centrifugation at 1000g for 10 min, resuspended in 1.0 ml of sodium phosphate buffer I
= 0.1 M, pH 7.4,
1 mM EDTA and homogenized using a Polytron Brinkmann, Westbury, NY for 30 s at a speed setting
of 4. JH epoxide hydrolase activity of crude cell homo- genates of recombinant EH infected Sf9 cells was
assayed at 30
° C using [
3
H] racemic JH III 12 Cimmol, tritiated at C10, New England Nuclear, Boston, MA
mixed with unlabeled racemic JH III Calbiochem, San Diego, CA Share and Roe, 1988. The final assay con-
centration of JH III was 5 µ
M. The cell homogenates were pre-incubated for 10 min at 30
° C with 0.1
µ M 3-
octylthio-1,1,1-trifluoropropan-2-one OTFP Abdel- Aal and Hammock, 1985 prior to the addition of JH
III to inhibit any esterase activity. JH-EH activity was measured by the partition assay of Share and Roe 1988.
TLC verified the accuracy of the partition assay. The I
50
values were determined using least squares linear
769 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774
regression analysis from at least four different inhibitor concentrations which bracketed the I
50
and fell within the linear range immediate to the I
50
. At each inhibitor concentration, the assays were run in triplicate. Inhibi-
tors were dissolved in ethanol, and the concentration of ethanol due to the addition of inhibitor never exceeded
1 of the reaction volume. Inhibition assays were always compared with ethanol controls.
2.4. Chemical synthesis Chemical reagents were purchased from Aldrich
Chemical Co Milwaukee and purified prior to use by either distillation or chromatography. Alkyllithium
reagents were titrated in toluene using 1,10-phenanthro- line as an indicator. Reactions were typically carried out
under an inert atmosphere of argon using solvents that were distilled immediately prior to use.
1
H,
13
C, and
19
F- NMR spectra were recorded on a 300 MHz spectrometer
in CDCl
3
. Chromatography was performed on silica gel 60, 230–400 mesh ASTM, obtained from EM Science.
Elemental analyses were carried out by Atlantic
Fig. 1. A: Stereospecific synthesis of trisubstituted and trans-disubstituted allylic alcohols, precursors for Sharpless asymmetric epoxidation reac-
tion. B: Stereospecific synthesis of cis-disubstituted allylic alcohol precursors for asymmetric epoxidation.
Microlab Inc. Norcross, GA. High resolution and rou- tine mass spectra were obtained at the Mass Spec-
troscopy Laboratory for Biotechnology at NC State Uni- versity. All new compounds were fully characterized by
spectroscopic methods and combustion or mass spectro- scopic analysis. A description of the chemical synthesis
of the inhibitors is given in Section 3.
3. Results
