Yehia A.I. Abdel-Aal

New trifluoropropanone sulfides as highly active and selective inhibitors of insect juvenile hormone esterase

Abstract A series of α,α′-bis(2-oxo-3,3,3-trifluoropropylthio)alkanes was synthesized by the alkylation of the appropriate α,α′-alkanedithiol with 3-bromo-1,1,1-trifluoropropan-2-one. The compounds were tested for in vitro inhibition of insect juvenile hormone esterase (JHE), electric eel acetylcholinesterase (AChE), porcine carboxylesterase, and yeast lipase. Inhibition of hemolymph JHE from fifth instar larvae of the cabbage looper, Trichoplusia ni (Hubner) (Lepidotera: Noctuidae), was in the nanomolar range for many compounds. The inhibition of the other three enzymes was only moderate in comparison to JHE. The inhibition of JHE was compared to carbon chain length, differing heteroatoms, and presence of methyl side-chains. The most effective compound was 1,1,1,16,16,16-hexafluoro-4,13-dithiahexadecane-2,15-dione ( I 50 : 8.2 × 10 −10 M ). The kinetics of inhibition were found to be time-dependent against both JHE and AChE enzymes using representative compounds of both high and low inhibitory potential. Second-order inhibition velocity constants were determined and found to be in agreement with the I 50 values.

Developmental Landmarks and the activity of juvenile hormone and juvenile hormone esterase during the last stadium and pupa of Lymantria dispar

Abstract Changes in juvenile hormone and juvenile hormone esterase activity were monitored during the last stadium and pupa of the gypsy moth, Lymantria dispar. Methods were developed to separate the sexes and to stage larvae using morphological and behavioural markers. Juvenile hormone activity determined by the Galleria melonella bioassay was relatively high during the early development of the last larval stadium; the activity was 5 times higher in females (6th instar) than in males (5th instar). Juvenile hormone activity decreased rapidly during the middle of the stadium and further declined shortly before pupation. Low levels of juvenile hormone activity persisted throughout the pupal stage. Three peaks of juvenile hormone esterase activity were observed during the last stadium in both sexes. The major peak, which occurred during the middle of the stadium, coincided with the time of decline in juvenile hormone activity and maximal weight gain. Juvenile hormone esterase activity was generally higher in females than in males. In pupae, one peak of juvenile hormone esterase activity was observed 3 days after pupation in both sexes; activity was again higher in females. Studies on juvenile hormone esterase inhibition using OTFP revealed similar I50 values for all peaks.

Kinetics of binding and hydrolysis of juvenile hormone II in the hemolymph of Trichoplusia ni (Hübner)

Abstract Juvenile hormone II (JH II) was studied for the kinetic behavior in its reaction with JH-esterase and the binding protein from the hemolymph of the last instar larvae of the cabbage looper, Trichoplusia ni . Steady state kinetics revealed 70.6 nM and 65 nmol substrate hydrolyzed min −1 ml, respectively, for the K m and V max for JH hydrolysis and 175 nM and 8.1 μM, respectively, for the equilibrium dissociation constant ( K d ) and the total concentration of the binding sites for the interaction with the binding protein. The rate constant for the dissociation of bound JH II was experimentally evaluated to be 0.095 min −1 . This in turn enabled the second order rate constant of association to be calculated as 5.41 × 10 5 M −1 min −1 . The slow tight binding inhibitor of JH-esterase, 3-octylthio 1,1,1-trifluoropropan-2-one, was applied to the enzyme for the Ackermann-Potter kinetic analysis. The data from this treatment indicated the compound acts as pseudo-irreversible and stoichiometric inhibitor and allowed determination of k cat (31.8 min −1 ) and the enzyme molar equivalency (1.5 μM) in whole hemolymph. The above determination helped compare the interaction of JH II with the two major components that regulated JH titer following biosynthesis. We conclude that degradation of JH in the hemolymph of prewandering last stadium larvae is described by the kinetic dissociation constant of the hemolymph binding protein. A model based on mass action is presented which may help to describe the distribution of JH in the last larval stadium.

JUVENILE HORMONE ESTERASES IN TWO HELIOTHINES: KINETIC, BIOCHEMICAL AND IMMUNOGENIC CHARACTERIZATION

Juvenile hormone hydrolyzing activity in larval hemolymph of the tobacco budworm, Heliothis virescens, and the corn earworm, H. zea, was characterized by several biochemical approaches. 2. Evidence for enzyme differences between species came from using several alkylthiotrifluoropro- panones as selective inhibitors. 3. The enzyme purified by affinity chromatography from H. zea showed two bands on SDS-PAGE and two activity peaks on narrow range (pH 4-6.5) isoelectric focusing (IEF), while purified enzyme from H. virescens showed only one band on SDS-PAGE and one peak of activity on IEF. 4. ELISA tests using polyclonal antisera elicited by the purified enzyme from each species showed the enzyme from 1-1. virescens and H. zea to be antigenically distinct. 5. A kinetic analysis of enzyme from both species showed that the slow tight binding kinetics, often observed with inhibition by transition state analogs, were both compound and enzyme dependent.

Conjugation of chlorodinitrobenzene with reduced glutathione in the absence and presence of glutathione transferase from larvae of the southern armyworm, Spodoptera eridania

Abstract The reaction of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione in the absence and presence of glutathione transferases from larvae of the southern armyworm, Spodoptera eridania , produced the conjugate 2,4-dinitrophenyl S -glutathione with identical uv absorption spectra. The extinction coefficient was 10.63 m M −1 cm −1 at 340 nm (λ max ). Kinetics of spontaneous conjugation in 0.1 M Tris-HCl buffer (pH 8.0) at 30°C indicated that a reversible complex between CDNB and glutathione preceded the formation of the stable conjugate. The kinetic parameters associated with these two steps were 2.9 × 10 −2 M and 0.22 min −1 for the dissociation equilibrium constant ( K d ) and the first order rate constant of conjugation ( k c ), respectively. Glutathione transferase activity was present almost exclusively in the cytosolic fractions of the gut, head capsule, integument, and fat body of the last instar larva of the southern armyworm. Among the above tissue preparations, the head capsule and gut showed the highest activity per unit protein. Hemolymph glutathione transferase activity was approximately double the activity of the head and gut. The transferase activity from the hemolymph and gut exhibited identical K m values for CDNB and glutathione. This, in addition to a high correlation between the activity of the hemolymph and gut in individual larvae, argues that the two activities are due to the same enzyme(s) and are regulated by similar factors.

Quantitative structure-activity relationship of DDT analogs

Abstract A quantitative structure-activity relationship (QSAR) has been carried out for Phormia regina toxicity by a set of 24 p,p′ -disubstituted analogs of DDT. The toxicity data are from the extensive studies of R. L. Metcalf. Preliminary examination of the data indicated that toxicity was parabolically related to molar refractivity, MR, of the ring substituents. This enabled parabolic regression to be evaluated. Multiple regression analysis relating toxicity and the substituent constants MR, Taft steric parameter ( E s ), hydrophobicity (π), and polar effect (σ) showed that MR dominated the regression equations. The implication of these physicochemical properties in the interaction of DDT analogs with the receptor site is discussed.