Biotechnology for Crop Protection - ACS Publications - American

Chapter 15

Inhibition of Juvenile Hormone Esterase by Transition-State Analogs A Tool for Enzyme Molecular Biology Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 12, 2018 at 12:03:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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András Székács , Bruce D. Hammock , Yehia Α. I. Abdel-Aal , Matthew Philpott , and György Matolcsy 1

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Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616 Plant Protection Institute of the Hungarian Academy of Sciences, Budapest, Hungary 2

A summary of Transition State Theory is presented, as it applies to the design of trifluoromethyl ketone esterase inhibitors. Possible mechanisms for the inhibition of the enzyme are discussed. A new series of compounds, α,α'-alkanebisthiotrifluoro-propanones was synthesized and showed excellent in vitro and moderate in vivo inhibition of the insect juvenile hormone esterase from the fifth instar larvae of Trichoplusia ni (cabbage looper). The potency of the above series was also screened for its ability to inhibit other esterases of toxicological and pharmacological significance. Trifluoroketones are discussed as an example of the importance of chemistry in biotechnology approaches. I t i s g e n e r a l l y r e c o g n i z e d t h a t t h e w i d e s p r e a d use o f i n s e c t i c i d e s has two s e r i o u s s i d e - e f f e c t s : e v o l u t i o n o f i n s e c t i c i d e r e s i s t a n c e i n i n s e c t s and danger t o e n v i r o n m e n t a l and human s a f e t y . The c o n t i n u e d use o f i n s e c t i c i d e s r e q u i r e s s t r i c t e r a g r i c u l t u r a l p r a c t i c e s as w e l l as t h e improvement o f t h e c h e m i c a l s t h e m s e l v e s . E q u a l l y as i m p o r t a n t , however, new s t r a t e g i e s f o r development o f i n s e c t i c i d a l agents a r e needed ( 1 - 3 ) . The e l u c i d a t i o n o f enzyme-substrate i n t e r a c t i o n s has e s t a b l i s h e d new paradigms l e a d i n g t o t h e d i s c o v e r y o f b i o l o g i c a l l y a c t i v e compounds. One such paradigm i s t h e " T r a n s i t i o n S t a t e Theory" a s i t a p p l i e s t o t h e mechanism o f e n z y m a t i c r e a c t i o n s . Based on t h i s t h e o r y , s e r i e s o f t r a n s i t i o n s t a t e a n a l o g i n h i b i t o r s known as t r i f l u o r o m e t h y l ketones have been s y n t h e s i z e d i n o u r l a b o r a t o r y . Our t a r g e t has been an i n s e c t enzyme o f d e v e l o p m e n t a l a n d r e p r o d u c t i o n a l i m p o r t a n c e , j u v e n i l e hormone e s t e r a s e (4-12). The development o f those e x t r e m e l y p o t e n t i n h i b i t o r s s e r v e d s e v e r a l aims: i n a d d i t i o n t o p r o v i d i n g " t r a d i t i o n a l " i n h i b i t o r s t h a t c a n be u s e d t o b l o c k t h e enzyme and s t u d y i t s b i o c h e m i c a l a n d p h y s i o l o g i c a l consequences, t h e new group o f compounds l e d t o p o w e r f u l l i g a n d s f o r t h e h i g h y i e l d a f f i n i t y chromatography 0097-6156/88/0379-0215$06.00/0 « 1988 American Chemical Society

Hedin et al.; Biotechnology for Crop Protection ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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p u r i f i c a t i o n o f t h i s low abundance enzyme (13-15). The pure enzyme, j u v e n i l e hormone e s t e r a s e (JHE) i s a v e r y s u i t a b l e c a n d i d a t e f o r s e v e r a l b i o t e c h n o l o g i c a l approaches from t h e use o f the enzyme as a probe f o r e n d o c r i n e r e g u l a t i o n , t o t h e more d e t a i l e d r e s e a r c h o f t h e m o l e c u l a r b i o l o g y and m o l e c u l a r g e n e t i c s o f t h e enzyme, i t s e l f . As d i s c u s s e d above t h e b i o l o g i c a l r o l e o f JHE i n l e p i d o p t e r o u s l a r v a e was e s t a b l i s h e d u s i n g h i g h l y s p e c i f i c e s t e r a s e i n h i b i t o r s . A p p l i c a t i o n o f t h e s e compounds l e d t o g i a n t l a r v a e w h i c h were l o c k e d i n t h e f e e d i n g s t a g e . The u s e o f t r i f l u o r o k e t o n e a f f i n i t y columns l e d t o t h e p u r i f i c a t i o n o f l a r g e amounts o f t h e enzyme. T h i s a v a i l a b i l i t y f i r s t l e d t o a doser e s p o n s e , l i n e a r w i t h t h e q u a n t i t y o f t h e enzyme a p p l i e d , o b s e r v e d f o r t h e d i r e c t i n j e c t i o n o f t h e enzyme i n t o s e v e r a l i n s e c t s p e c i e s , showing t h a t i n j e c t e d JHE has a c l e a r a n t i - J H e f f e c t . S i n c e such an e f f e c t i s d e s i r a b l e i n a g r i c u l t u r e , i t becomes i m p o r t a n t t o c l o n e t h e enzyme. Here a g a i n t h e a f f i n i t y p r o c e d u r e from c h e m i c a l approaches p r o v i d e d t h e p r o t e i n n e c e s s a r y f o r development o f m o l e c u l a r p r o b e s . The i n j e c t i o n o f p u r i f i e d enzyme i n r a b b i t s r e s u l t e d i n JHE s p e c i f i c a n t i b o d i e s needed f o r s c r e e n i n g an e x p r e s s i o n l i b r a r y as w e l l as f o r f u r t h e r immunoassay s t u d i e s . The a f f i n i t y p u r i f i e d p r o t e i n a l s o allowed c l a s s i c a l aminoacid s c r e e n i n g t o be done w h i c h l e d t o t h e s y n t h e s i s o f o l i g o n u c l e o t i d e probes f o r c o n f i r m i n g p o s i t i v e s from t h e e x p r e s s i o n l i b r a r y . H o p e f u l l y t h e c l o n e d message c a n be i n s e r t e d i n t o b a c u l o v i r u s v e c t o r s w h i c h l e a d t o p r e c o c i o u s p r o d u c t i o n o f t h e enzyme. T h i s , i n t u r n , s h o u l d r e s u l t i n a n t i - j u v e n i l e hormone e f f e c t s such as c e s s a t i o n o f f e e d i n g and d e v e l o p m e n t a l a b n o r m a l i t i e s . These r e s u l t s and f u t u r e a p p l i c a t i o n s o f t r i f l u o r o m e t h y l k e t o n e s show t h e importance o f t h e " t r a d i t i o n a l " c h e m i c a l o p t i m i z a t i o n o f compounds i n v a r i o u s b i o t e c h n o l o g i c a l approaches. I n t h i s p r e s e n t a t i o n t h e a u t h o r s w i s h t o g i v e a summary o f t h e l e a d i n g r e s e a r c h paradigm, T r a n s i t i o n S t a t e Theory r e s u l t i n g i n t r i f l u o r o m e t h y l k e t o n e s , a group o f h i g h l y e f f e c t i v e i n h i b i t o r s o f JHE. The same c o n c e p t s c a n be a p p l i e d t o a v a r i e t y o f p o l a r i z e d c a r b o n y l s , carbamates, phosphates and phosphonates as i n h i b i t o r s o f e s t e r a s e s and p r o t e a s e s . Clear targets i n the i n s e c t i c i d e f i e l d w i l l be enzymes i n v o l v e d i n i n s e c t i c i d e m e t a b o l i s m and neurohormone p r o c e s s i n g enzymes. J u v e n i l e Hormone

Esterase

Our l a b o r a t o r y i s c o n c e r n e d w i t h t a r g e t i n g p o t e n t i a l i n s e c t i c i d e s t h a t d i s r u p t normal development and metamorphosis i n i n s e c t s . J u v e n i l e hormones ( J H s ) , a c t i n g i n c o n c e r t w i t h t h e s t e r o i d hormone ecdysone, a r e b e l i e v e d t o c o n t r o l t h e t i m i n g o f t h e l a r v a l - l a r v a l m o l t s , l a r v a l - p u p a l and p u p a l - a d u l t t r a n s f o r m a t i o n s o f t h e i n s e c t s . I t has been demonstrated t h a t t h e e v e n t s l e a d i n g t o p u p a t i o n a r e i n i t i a t e d by r e d u c t i o n o f t h e J H t i t e r i n t h e hemolymph. I n a d d i t i o n t o a c e s s a t i o n o f b i o s y n t h e s i s , t h i s r e d u c t i o n i n JH t i t e r i s c o n t r o l l e d by d e g r a d a t i v e m e t a b o l i s m (16,17). H y d r o l y s i s o f t h e e p o x i d e and e s t e r f u n c t i o n a l i t i e s p r e s e n t i n a c t i v e J H a r e two r o u t e s o f d e g r a d a t i o n and subsequent i n a c t i v a t i o n o f J H ( 1 8 ) . The p r i m a r y r o u t e o f JH m e t a b o l i s m i n t h e hemolymph o f l a s t s t a d i u m l e p i d o p t e r o u s l a r v a e i s e s t e r h y d r o l y s i s , and i t i s c a t a l y z e d by the enzyme j u v e n i l e hormone e s t e r a s e (JHE). JHE has been shown t o


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play a c r u c i a l r o l e i n i n i t i a t i n g pupation i n lepidopterous insects (19); s e l e c t i v e i n h i b i t i o n o f t h i s enzyme p r e v e n t s JH h y d r o l y s i s and causes a d e l a y i n the o n s e t o f p u p a t i o n ( 2 0 ) . T r a n s i t i o n S t a t e Theory and JH E s t e r

Hydrolysis

I n e v e r y c h e m i c a l r e a c t i o n the r e a c t a n t s are i n e q u i l i b r i u m w i t h an u n s t a b l e a c t i v a t e d complex, the t r a n s i t i o n s t a t e complex (TS), w h i c h decomposes t o g i v e the p r o d u c t . I n h i s p i o n e e r i n g work, L i n u s P a u l i n g p o i n t e d out t h a t f o r p r o m o t i n g a r e a c t i o n w i t h o u t i n f l u e n c i n g i t s e q u i l i b r i u m c o n s t a n t , the enzyme s h o u l d have much h i g h e r a f f i n i t y f o r the t r a n s i t i o n s t a t e o f a r e a c t i o n t h a n f o r e i t h e r the s u b s t r a t e o r the p r o d u c t ( s ) , t h e r e b y p u s h i n g a r e a c t i o n i n the d e s i r e d d i r e c t i o n by c o n t i n u o u s l y removing i t s t r a n s i t i o n s t a t e (21). Based on t h i s i d e a , e x t r e m e l y p o t e n t i n h i b i t o r s can be d e v e l o p e d f o r a g i v e n e n z y m a t i c r e a c t i o n i f one can s y n t h e s i z e " t r a n s i t i o n s t a t e mimics" (TSM): s t a b l e c h e m i c a l compounds r e s e m b l i n g the t r a n s i t i o n s t a t e ( 2 2 ) . Because t r a n s i t i o n s t a t e s may have l i f e t i m e s o f o n l y s e v e r a l nanoseconds, i n most c a s e s , i t i s i m p o s s i b l e t o observe them d i r e c t l y . However, t h e r e are numerous l i n e s o f e v i d e n c e f o r the e x i s t e n c e o f a t e t r a h e d r a l - l i k e t r a n s i t i o n s t a t e f o r non-enzymatic e s t e r h y d r o l y s i s : a) s u b s t i t u t i o n a t a c a r b o n y l group (as i s the case o f the h y d r o l y s i s o f e s t e r s ) most o f t e n p r o c e e d s by a t e t r a h e d r a l mechanism, a s e c o n d - o r d e r a d d i t i o n - e l i m i n a t i o n ( f o r a r e v i e w o f t h i s mechanism, see ( 2 3 ) ) ; b) the k i n e t i c s are pseudof i r s t o r d e r e i t h e r i n the s u b s t r a t e o r i n the n u c l e o p h i l e , as p r e d i c t e d by the mechanism; c) f o r the 0 l a b e l e d e s t e r s , the 0 i s o t o p e i s d e t e c t a b l e i n b o t h p r o d u c t s ( i n a "normal" S^2 r e a c t i o n a l l the 0 i s o t o p e s s h o u l d remain i n the a c i d f u n c t i o n a l i t y ) ( 2 4 ) ; d) i n a few c a s e s t e t r a h e d r a l i n t e r m e d i a t e s have been i s o l a t e d o r detected s p e c t r a l l y (25). E s t e r h y d r o l y s i s o f the JHs i s shown i n E q u a t i o n 1. We have no e x a c t thermochemical d a t a measured f o r t h i s r e a c t i o n . I n g e n e r a l , the h y d r o l y s i s o f u n s a t u r a t e d l o n g c h a i n a l i p h a t i c a c i d s i s t h e r m o d y n a m i c a l l y n e u t r a l under s t a n d a r d c o n d i t i o n s (26,27) ( i . e . the h e a t o f h y d r o l y s i s f o r c i s - and t r a n s - o l e i c a c i d i s -1.7 and +0.8 k J / m o l , r e s p e c t i v e l y ) , w h i c h shows t h a t t h e s e r e a c t i o n s are g e n e r a l l y not f a v o r e d b a s e d s o l e l y on thermodynamic c o n s i d e r a t i o n s . I n aqueous s o l u t i o n e s t e r h y d r o l y s i s i s d r i v e n by the h i g h c o n c e n t r a t i o n o f water. The j u v e n i l e hormones w i l l be more r e s i s t a n t t o a c i d or base c a t a l y s e d h y d r o l y s i s because i n t h e s e compounds c o n j u g a t i o n w i t h the α,/3-unsaturation g r e a t l y s t a b i l i z e s the c a r b o n y l f u n c t i o n a l i t y ( 1 9 ) . T h e r e f o r e , the n o n c o n j u g a t e d TS s h o u l d be d i f f i c u l t t o form, and t h i s s i t u a t i o n w i l l be r e f l e c t e d by a h i g h a c t i v a t i o n energy f o r the u n c a t a l y z e d h y d r o l y s i s o f JH. The e s t e r h y d r o l y s i s c a t a l y z e d by h y d r o l a s e enzymes (E) proceeds a c c o r d i n g t o E q u a t i o n 2 and the f r e e energy diagram i s shown i n F i g u r e 1. A f t e r the p r e l i m i n a r y n o n c o v a l e n t b i n d i n g s t e p ( [ E S ] ) the enzyme becomes complementary i n s t r u c t u r e t o the s u b s t r a t e ( o r i t s TS) and forms a c o v a l e n t adduct w i t h i t (ES). R e l e a s e o f the f i r s t p r o d u c t (MeOH) r e s u l t s i n the acyl-enzyme (EX), w h i c h h y d r o l y s e s t h r o u g h a c o v a l e n t enzyme-product adduct (EP) t o the a p p r o p r i a t e c a r b o x y l i c a c i d and the f r e e enzyme. I t has been shown 1 8

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t h a t the r a t e l i m i t i n g s t e p i n t h e r e a c t i o n i s the p r o d u c t r e l e a s e from the acyl-enzyme ( 2 8 ) . Any compound t h a t i s r e c o g n i z e d by the enzyme as a TS w i l l b i n d t o i t c o m p e t i t i v e l y w i t h the s u b s t r a t e . I t i s i m p o r t a n t t o n o t e , however, t h a t the mechanism o f enzymatic r e a c t i o n i s n o t s i m p l y the r e a c t i o n o f the enzyme w i t h the t r a n s i t i o n s t a t e o f the u n c a t a l y z e d r e a c t i o n ; i t can be seen by the c a r t o o n i n F i g u r e 1, t h a t the enzymatic h y d r o l y s i s o f e s t e r s proceeds t h r o u g h s e v e r a l i n t e r m e d i a t e s and t r a n s i t i o n s t a t e s ; a p u t a t i v e TSM compound might resemble any o r s e v e r a l o f t h e s e s t a t e s . Thus, t h e c o n c e p t o f a " t r a n s i t i o n s t a t e mimic" i s somewhat o f a misnomer. T r i f l u o r o m e t h y l Ketones as T r a n s i t i o n S t a t e M i m i c s An exemplary a p p l i c a t i o n o f T r a n s i t i o n S t a t e Theory i n d e v e l o p i n g h i g h l y a c t i v e i n h i b i t o r s f o r e s t e r a s e enzymes i s the c a s e o f t r i f l u o r o m e t h y l k e t o n e s ( 2 9 ) . R e p l a c i n g the a l k o x y group o f the c a r b o x y l i c e s t e r s by a t r i f l u o r o m e t h y l group r e s u l t s i n h i g h l y p o l a r i z e d ketones which are s e n s i t i v e to n u c l e o p h i l i c a t t a c k . Subsequently, i n t h e p r e s e n c e o f t r a c e amounts o f w a t e r , the k e t o form w i l l be h y d r a t e d and be i n e q u i l i b r i u m w i t h the c o r r e s p o n d i n g geminal d i o l . The g e m i n a l d i o l i s t e t r a h e d r a l i n geometry and i n t h e o r y resembles the t r a n s i t i o n s t a t e o f the u n c a t a l y z e d h y d r o l y s i s o f e s t e r s (TSX). Thus, a c c o r d i n g t o the o r i g i n a l P a u l i n g - t h e o r y , t r i f l u o r o m e t h y l k e t o n e s s h o u l d b i n d s t r o n g l y t o e s t e r a s e enzymes. I n t h i s r e a c t i o n t h e y form h e m i k e t a l s w i t h the s e r i n e p r e s e n t a t the a c t i v e s i t e o f the enzyme; two p o s s i b l e r e a c t i o n mechanisms a r e enzyme a d d i t i o n t o the c a r b o n y l (30) o r c o n d e n s a t i o n w i t h the g e m i n a l d i o l (31) (enhanced by the h y d r o p h o b i c a l i p h a t i c c h a i n i n the m o l e c u l e ) ( E q u a t i o n 3 ) . The most l i k e l y c h e m i c a l r e a c t i o n i s a d d i t i o n t o the c a r b o n y l as shown i n the top p o r t i o n o f E q u a t i o n 3; however, i n aqueous s o l u t i o n the m a j o r i t y o f the compound e x i s t s as the g e m i n a l d i o l . I f t h i s s i t u a t i o n p r e d o m i n a t e s , e i t h e r the e q u i l i b r i u m between the d i o l and c a r b o n y l form must be f a s t on the time s c a l e o f our a b i l i t y t o measure enzyme r e a c t i o n s , o r the Ieo's are i n f a c t f a r lower t h a n we have r e p o r t e d . A l t e r n a t i v e l y , the h y d r a t e d c a r b o n y l c o u l d r e a c t d i r e c t l y w i t h t h e enzyme. T h i s c o u l d happen i n two ways. I n one case the TSM c o u l d be h e l d n e a r the c a t a l y t i c s i t e due t o i n t e r a c t i o n w i t h the R group. The r e l a t i v e abundance o f the c a r b o n y l m i g h t t h e n be f a v o r e d i n a nonaqueous m i c r o e n v i r o n m e n t . T h i s p r o c e s s c o u l d even be a c c e l e r a t e d c a t a l y t i c a l l y . I f there i s enzyme i n v o l v e m e n t i n p r o d u c t i o n o f the c a r b o n y l from the g e m i n a l d i o l , t h e n t h e s e TSMs c o u l d be c o n s i d e r e d " s u i c i d e " s u b s t r a t e s . A n o t h e r a l t e r n a t i v e e x p l a n a t i o n i s a "normal" S^2 type r e a c t i o n between a s e r i n e a n i o n and a p r o t o n a t e d g e m i n a l d i o l w i t h w a t e r as the l e a v i n g group. These a l t e r n a t i v e pathways a r e n o t m u t u a l l y e x c l u s i v e , b u t a d d i t i o n a l work on the k i n e t i c s and s t r u c t u r a l b i o c h e m i s t r y o f the i n t e r a c t i o n w i l l be needed t o i n d i c a t e the predominant pathway. The r e a c t i o n r e s u l t s i n an adduct w i t h the enzyme (ETSM) w i t h no e s t e r C-OR bond p r e s e n t i n the m o l e c u l e t o be c l e a v e d . I t i s c l e a r from E q u a t i o n 3 t h a t the h e m i k e t a l may o n l y r e a c t by the r e l e a s e o f the t r i f l u o r o m e t h y l ketone by the enzyme through the t r a n s p r o t o n a t i o n t r a n s i t i o n s t a t e , t h e r e b y a c t i n g as a r e v e r s i b l e


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TSX + Ε

ETSM reaction coordhate

F i g u r e 1. Schematic p r e s e n t a t i o n o f t h e f r e e energy changes i n non-enzymatic (ETX) and e n z y m a t i c r e a c t i o n s (EX) and i n t h e r e a c t i o n o f a h y p o t h e t i c a l t r a n s i t i o n s t a t e mimic (TSM) w i t h t h e enzyme.


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Η»

Θ

Ο

Enz-OH

ΓΟ 2) , and s e r i n e e s t e r a s e s , such as a c e t y l c h o l i n e s t e r a s e (13,14), c a r b o x y l e s t e r a s e s ( 1 0 ) , JHE and o t h e r e s t e r a s e s w i t h v a r y i n g s e l e c t i v i t y . I n a s e r i e s o f some j u v e n o i d - l i k e t r i f l u o r o m e t h y l k e t o n e s and compounds o f the s t r u c t u r e A, l , l , l - t r i f l u o r o - 2 - t e t r a d e c a n o n e (TFT) was f o u n d t o be h i g h l y a c t i v e and s e l e c t i v e a g a i n s t JHE ( I s o * l x l 0 " M ) as compared t o α-naphthyl a c e t a t e e s t e r a s e (α-NaE) o r t r y p s i n ( 4 - 6 ) . 7

CH (CH ) -C(0)CF 3

2

n

3

A CH (CH ) -SCH C(0)CF 3

2

n

2

3

Β

I n t r o d u c i n g a s u l f u r atom β t o the c a r b o n y l s i g n i f i c a n t l y i n c r e a s e d the a c t i v i t y o f the r e s u l t i n g compounds (B) on JHE and some b u t n o t a l l o t h e r e s t e r a s e s , p o s s i b l y by b i o i s o s t e r i c a l l y m i m i c k i n g (35) the α,β double bond o f the n a t u r a l JHs. Based on t h i s f i n d i n g , a s e r i e s o f a l i p h a t i c , a r o m a t i c (Z»â) and t e r p e n o i d (9) d e r i v a t i v e s were s y n t h e s i z e d . The most a c t i v e compound o f these s e r i e s was 3 - o c t y l t h i o - l , 1 , 1 - t r i f l u o r o - 2 - p r o p a n o n e (OTFP). I n a r e c e n t r e p o r t , by making a s l i g h t m o d i f i c a t i o n i n t h e s t r u c t u r e o f the p a r e n t compound, two a d d i t i o n a l compounds w i t h s l i g h t l y h i g h e r a c t i v i t y have been s y n t h e s i z e d ( 3 6 ) . TFT and the a l i p h a t i c t r i f l u o r o m e t h y l k e t o n e s appeared t o be c l a s s i c a l c o m p e t i t i v e i n h i b i t o r s , w h i l e many t r i f l u o r o p r o p a n o n e s u l f i d e s were found t o be r e v e r s i b l e b u t s l o w and t i g h t b i n d i n g i n h i b i t o r s o f JHE (2,11,12). The o u t s t a n d i n g a c t i v i t y o f some members o f the t h i o t r i f l u o r o propanones p r o v e d u s e f u l as l i g a n d s and e l u t i n g a g e n t s f o r the a f f i n i t y p u r i f i c a t i o n o f JHE (13-15) . T h i s new method made p o s s i b l e s p e c i f i c p u r i f i c a t i o n o f JHE w i t h u n u s u a l l y h i g h y i e l d s , making p o s s i b l e new r e s e a r c h i n the a r e a s o f immunochemistry and m o l e c u l a r b i o l o g y f o r t h i s h i g h l y a c t i v e b u t low abundant e s t e r a s e . I n our l a b o r a t o r y , we f e e l t h a t the s c i e n t i f i c and p r a c t i c a l use o f these i n h i b i t o r s , by no means, has r e a c h e d a peak. An o b v i o u s need i s t o t r y d i f f e r e n t l i g a n d s f o r a f f i n i t y e l u t i o n . Therefore, a new c l a s s o f t r a n s i t i o n s t a t e a n a l o g s was s y n t h e s i z e d and t e s t e d f o r t h e i r i n h i b i t o r y p o t e n t i a l a g a i n s t JHE, c h o l i n e s t e r a s e and malathionase.


222


Synthesis Compounds o f the g e n e r a l s t r u c t u r e C and D were s y n t h e s i z e d c h a r a c t e r i z e d (See T a b l e I )

CF C(0)CH2S-(CH2) -SCH C(0)CF3 3

n

I - X

CF C(0)CH S-R-SCH C(0)CF 3

R:

C

2

n: 3 - 12

and

2

2

-(CH ) -0-(CH ) -(CH ) -S-(CH ) -ÇH(CH ) CH -ÇH(CH ) CH 2

2

2

2

2

2

2

2

2

2

2

3

D

3

XI XII XIII

3

XIV

3

The compounds were s y n t h e s i z e d a c c o r d i n g t o a p r e v i o u s l y p u b l i s h e d p r o c e d u r e (7) w i t h the m o d i f i c a t i o n t h a t t r i e t h y l a m i n e was u s e d t o a c c e l e r a t e the r e a c t i o n by n e u t r a l i z i n g the HBr. Et N ---> C F C ( 0 ) C H S - R - S C H C ( 0 ) C F 3

HS-R-SH + 2 B r C H C ( 0 ) C F 2

3

3

2

2

3

The s t a r t i n g mercaptans were e i t h e r p u r c h a s e d from A l d r i c h C h e m i c a l s o r s y n t h e s i z e d from the a p p r o p r i a t e α,α'-dihaloalkane v i a the t h i o u r e a method ( 3 7 ) . The y i e l d s were between 59% and 98% f o r compounds I I I and V, r e s p e c t i v e l y , and i n g e n e r a l h i g h e r t h a n 85% depending on the speed o f the a d d i t i o n o f t r i e t h y l a m i n e . (Chemical d e t a i l s w i l l be d i s c u s s e d e l s e w h e r e : Székâcs, Α.; Hammock, B.D.; A b d e l - A a l , Y.A.I.; P h i l p o t t , M.; M a t o l c s y , G.: P e s t . Biochem. P h y s i o l . . submitted). Enzyme Assays The i n h i b i t i o n r a t e f o r a l l i n h i b i t o r a s s a y s was measured from the i n i t i a l v e l o c i t i e s w i t h i n the l i n e a r t i m e - a c t i v i t y r e l a t i o n s h i p s o f the c o n t r o l and i n h i b i t e d samples. The s t a n d a r d d e v i a t i o n s were c a l c u l a t e d f o r the Isο v a l u e s o f the compounds a g a i n s t the t h r e e enzymes u s e d and the maximal v a l u e s were used t o compare the s t a t i s t i c a l v a r i a b i l i t y w i t h i n each a s s a y t e s t . JHE: For the d e t e r m i n a t i o n o f JHE i n h i b i t i o n by the t i t l e compounds, the r a d i o m e t r i c p a r t i t i o n method (3B) was used. Hemolymph JHE from Day 2 o f the f i f t h i n s t a r l a r v a e o f Γ. ni was u s e d ( L D ) , d i l u t e d 1:500 w i t h 0.08M phosphate b u f f e r (pH-7.4 w i t h 0.1% p h e n y l t h i o u r e a t o i n h i b i t t y r o s i n a s e s ) . The main r e a s o n f o r c h o o s i n g t h i s i n s e c t was t h a t a g r e a t d e a l o f e f f o r t has been p u t i n t o the c h a r a c t e r i z a t i o n o f l a r v a l c a r b o x y l e s t e r a s e s and JHE i n Γ. ni (39,40). I n L s D l a r v a e , the JHE t i t e r i s n e a r i t s maximum (19). CIO H l a b e l e d JH I I I (New E n g l a n d N u c l e a r ) and u n l a b e l e d JH I I I (Calbiochem) were used as s u b s t r a t e s o l u b i l i z e d i n a b s . e t h a n o l . 5

2

2

3


15.

SZEKACS ET AK

223


A c e t y l c h o l i n e s t e r a s e (AChE): The l y o p h i l i z e d enzyme from e l e c t r i c e e l (Sigma) was d i s s o l v e d i n 0.05M phosphate b u f f e r (pH-7.4) a t a c o n c e n t r a t i o n o f 20 /ig/mL. A c e t y l t h i o c h o l i n e i o d i d e was u s e d as s u b s t r a t e a t a f i n a l c o n c e n t r a t i o n o f 5x10" M i n buffer. 5 , 5 ' - D i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) a t a f i n a l c o n c e n t r a t i o n o f 3 . 8 x l 0 " M was used t o m o n i t o r t h e r e l e a s e d t h i o c h o l i n e a c c o r d i n g t o a p u b l i s h e d p r o c e d u r e (41) w i t h s l i g h t m o d i f i c a t i o n . Acetone was used as a s o l v e n t f o r t h e i n h i b i t o r s . M a l a t h i o n a s e (ME): F o r measuring t h e i n h i b i t i o n o f m a l a t h i o n e s t e r a s e a c t i v i t y , g e n e r a l c a r b o x y l e s t e r a s e from p o r c i n e l i v e r (Sigma) was used a t a f i n a l c o n c e n t r a t i o n o f 16 /xg p r o t e i n / m L i n 0.1M T r i s HC1 b u f f e r (pH-7.5). The p r o c e d u r e i n v o l v e s an i n d i r e c t d e t e r m i n a t i o n o f t h e m a l a t h i o n a s e a c t i v i t y by c o u p l i n g t h e h y d r o l y s i s o f m a l a t h i o n t o t h e r e d u c t i o n o f a t e t r a z o l i u m dye ( 4 2 ) . An a c e t o n e s o l u t i o n o f m a l a t h i o n was u s e d as s u b s t r a t e t o a f i n a l c o n c e n t r a t i o n o f 3x10" M. 4

2

4

Table I .

compound number I II III τ

ι

VI

b

VIII IX

D

5

x

b

XI XII XHIb XIV b

I n h i b i t i o n o f E s t e r a s e s by a , a ' - a l k a n e b i s thiotrifluoropropanones

number of carbons

JHE

3 4 5 6 7 8 9 10 11 12 2-0-2 2-S-2 3' 4'

9., 8 7 x l 0 " 3.,06x10" 7,,00x10" 1.,50x10" 1,,68x10" 8.. 1 7 x 1 0 " 2.,67x10' 4.,54x10" 1..33x10" 8..15x10" 2..16x10' 4..94x10" 4,.68x10" 8,.20x10"

M o l a r Iso

Value*

ME

AChE 7

9

9

8

8

9

9

8

9

8

9

7

9

10

1.14x10" 8.44x10' 4.30x10" 2.98x10" 2.98x10" 7.78xl0" 1.34x10" 4.35xl0" 5.25xl0" 1.03x10' 6.01x10" 7.12x10" 3.47x10" 8.12x10"

[M]

5

6

6

6

6

e

5

e

e

5

6

6

6

7

3.63xl0" 3.79xl0* 2.38x10* 9.02x10' 4.89x10' 4.62xl0" 5.00x10" 1.66x10" 1.03x10" 3.02x10' 8.58x10' 3.53x10" 4.56x10" 5.22x10"

e

6

6

7

8

6

4

5

3

3

6

6

6

6

JH I I I was used as a s u b s t r a t e f o r JHE from Γ. n i , a c e t y l t h i o c h o l i n e i o d i d e f o r e l e c t r i c e e l AChE and m a l a t h i o n f o r c a r b o x y l e s t e r a s e from p o r c i n e l i v e r . The d i t h i o l was p r e p a r e d from a l k y l - d i b r o m i d e .

Summarized i n T a b l e I a r e t h e Iso v a l u e s f o r α,α'-alkanebist h i o t r i f l u o r o k e t o n e s a g a i n s t JHE, AChE and m a l a t h i o n a s e . A l l the t e s t e d compounds showed much h i g h e r i n h i b i t o r y p o t e n c y a g a i n s t JHE t h a n a g a i n s t t h e o t h e r two enzymes. The c o m p a r a t i v e p o t e n c y i n f a v o r o f JHE adds t o the e v i d e n c e t h a t t h e s e t r i f l u o r o m e t h y l k e t o n e s a r e t r a n s i t i o n s t a t e mimics (TSMs) o f t h e enzyme n a t u r a l s u b s t r a t e s ( J H s ) . However, t h e s l o p e s o f t h e i n h i b i t i o n c u r v e s f o r a l m o s t a l l t h e compounds were lower w i t h JHE t h a n w i t h t h e o t h e r


224


two enzymes. P o s s i b l y t h i s i s due t o t h e f a c t t h a t JHE from Γ. ni c o n t a i n s m u l t i p l e J H c a t a l y t i c s i t e s (43-45), as i t has been shown f o r t h e t o b a c c o hornworm (Manduca sexta)(13). E x c e p t f o r compounds I I I and I V , t h e i n h i b i t i o n c u r v e s f o r m a l a t h i o n a s e i n h i b i t i o n h a d the h i g h e s t s l o p e s . The maximal v a l u e s o f t h e p e r c e n t a g e s t a n d a r d e r r o r o f p l s o were 0.77%, 0.18% and 0.66% o f t h e mean f o r t h e JHE, AChE and m a l a t h i o n a s e a s s a y s , r e s p e c t i v e l y . The q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s o f t h e t e s t e d compounds a r e beyond t h e scope o f t h i s paper. However, t h e e m p i r i c a l s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s b a s e d on t h e number o f the c a r b o n atoms i n t h e a l k y l c h a i n i s d i f f e r e n t f o r each o f t h e esterases assayed. I t i s w o r t h n o t i n g t h a t i n t h e JHE a s s a y t h e t r e n d o f t h e d a t a showed s i m i l a r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p t o t h e 3 - a l k y l t h i o 1 , 1 , l - t r i f l u o r o - 2 - p r o p a n o n e s (Β), w h i c h adds some a d d i t i o n a l e v i d e n c e f o r t h e r a t i o n a l d e s i g n o f t h e s e TSMs o f J H s . I n t h i s a s s a y , however, a d e f i n i t e peak o c c u r s a t c a r b o n number e i g h t , f o r the compound 1,1,1,16,16,16-hexafluoro-4,13-dithia-hexadecane-2,15dione ( I s o : 8 . 2 x l 0 " M ) , which suggests t h a t a f t e r reaching t h i s p a r t i c u l a r s i z e , t h e compound might be a b l e t o i n t e r a c t w i t h t h e enzyme(s) i n two c a t a l y t i c s i t e s ( o r h i g h l y h y d r o p h i l i c p a r t s ) a t the same time. I n c o n t r a s t , c h o l i n e s t e r a s e d i d n o t seem t o r e s p o n d i n a systematic f a s h i o n to the s t r u c t u r a l v a r i a t i o n . Malathionase showed i n t e r m e d i a t e response w i t h a g e n e r a l l y d e c r e a s i n g p o t e n c y as a f u n c t i o n o f t h e number o f c a r b o n atoms i n t h e m o l e c u l e . l o

In Vivo

Assays

U s i n g t h e i r r e v e r s i b l e i n h i b i t o r EPPAT, Sparks and Hammock (4) d e m o n s t r a t e d t h a t p u p a t i o n c o u l d be d e l a y e d i n Γ. ni presumably by i n h i b i t i n g JHE and t h e r e b y m a i n t a i n i n g an a b n o r m a l l y h i g h J H and low p r o t h o r a c i c o t r o p i c hormone (PTTH) l e v e l . TFT ( l a c k i n g t h e t h i o e t h e r m o i e t y ) f a i l e d t o cause t h i s e f f e c t w h i l e 0TFP was e f f e c t i v e i n d e l a y i n g p u p a t i o n when r e p e a t e d doses o f 0.1-0.2 μτηοΐ were a p p l i e d t o p i c a l l y on day 1 and 2 o f t h e f i f t h l a r v a l i n s t a r (L5D1 and L D ) ( 7 ) . The two most e f f e c t i v e in v i t r o i n h i b i t o r s o f JHE i n t h e new s e r i e s , V I and I I , were s c r e e n e d in vivo f o r t h e i r a b i l i t y t o d e l a y p u p a t i o n i n Trichoplusia n i , r e l a t i v e t o t h e a b i l i t y o f OTFP, a r e s p o n s e shown t o be c o n c u r r e n t w i t h t h e s e l e c t i v e i n h i b i t i o n o f 5

2

JHE (2,2).

The compounds (2 /iL o f 1x10"*M EtOH s o l u t i o n ) were t o p i c a l l y a p p l i e d t o T. ni on L D i and L D a t 4, 12 and 17 h r s AL0 ( a f t e r l i g h t s on) and t h e time o f p u p a t i o n was r e c o r d e d . I n the c o n t r o l group, l a r v a e t r e a t e d w i t h 2 mL EtOH, 90-100% o f l a r v a e h a d pupated by L5D5 9 AM. A t f i n a l doses o f 0.2 /xmol, V I was as e f f e c t i v e as OTFP i n d e l a y i n g p u p a t i o n r e l a t i v e t o t h e e t h a n o l c o n t r o l s , w i t h o n l y 45-75% o f t h e l a r v a e p u p a t i n g by L 5 D 5 . A t t h i s c o n c e n t r a t i o n , t r e a t m e n t w i t h χΐ d i d n o t d e l a y p u p a t i o n s i g n i f i c a n t l y , 75-100% p u p a t e d by LsDs. s

5

2


15. SZEKACS ET AL.


225

Conclusion A l t h o u g h t r i f l u o r o m e t h y l k e t o n e s a c t i n g on JHE w i l l p r o b a b l y not r e s u l t i n e f f e c t i v e i n s e c t i c i d e s (delaying pupation i s generally not a d e s i r e d b i o l o g i c a l e f f e c t i n a g r i c u l t u r e ) , continued research on them has l e d t o s e v e r a l p r a c t i c a l r e s u l t s as w e l l as t o new t h e o r e t i c a l considerations. Knowledge g l e a n e d b y r e s e a r c h w i t h t r i f l u o r o m e t h y l k e t o n e s w i l l l e a d t o a more complete u n d e r s t a n d i n g o f the r o l e o f JHE i n i n s e c t development. W i t h t h i s u n d e r s t a n d i n g o f the mechanisms r e g u l a t i n g normal development i n i n s e c t s , new avenues may be opened f o r t a r g e t i n g new and s p e c i f i c ways o f i n s e c t c o n t r o l . T h i s work w i t h j u v e n i l e hormone e s t e r a s e p r o v i d e s a n i c e example o f c h e m i s t r y , b i o c h e m i s t r y and m o l e c u l a r b i o l o g y as complementary t e c h n o l o g i e s ( 1 9 ) . These p o t e n t i n h i b i t o r s were f i r s t u s e d t o demonstrate the e s s e n t i a l r o l e o f JHE i n i n s e c t development. A knowledge o f the c h e m i s t r y and b i o c h e m i s t r y o f the i n t e r a c t i o n o f t h e s e t r a n s i t i o n s t a t e mimics w i t h JHE l e d t o t h e development o f a f f i n i t y p u r i f i c a t i o n systems f o r t h i s low abundance enzyme ( 1 3 ) . L a r g e amounts o f the pure p r o t e i n a l l o w e d more d e t a i l e d k i n e t i c s t u d i e s as w e l l as the development o f a n t i b o d i e s and n u c l e i c a c i d probes u s e d t o c l o n e the enzyme. I t c o u l d w e l l be t h a t the r e s u l t i n g c l o n e s can be e n g i n e e r e d i n t o b a c u l o v i r u s e s o r other vectors for insect c o n t r o l . I n a d d i t i o n , s t u d i e s from t h i s and o t h e r l a b o r a t o r i e s i l l u s t r a t e t h a t b y m i m i c k i n g h y p o t h e t i c a l t r a n s i t i o n s t a t e s one can d e v e l o p e x c e p t i o n a l l y p o t e n t enzyme i n h i b i t o r s . These compounds w i l l advance our u n d e r s t a n d i n g o f c a t a l y t i c mechanisms, s e r v e as probes f o r u n r a v e l i n g the r o l e s o f p a r t i c u l a r enzymes i n c a t a l y t i c p r o c e s s e s , and a l l o w t h e i r r a p i d p u r i f i c a t i o n . They a r e a l s o p r o m i s i n g c o m m e r c i a l l y as p e s t i c i d e s , p e s t i c i d e s y n e r g i s t s , as w e l l as p h a r m a c e u t i c a l s . From such work i t seems c l e a r t h a t b o t h c h e m i c a l and b i o t e c h n o l o g i c a l approaches w i l l be u s e d a l o n e and i n c o m b i n a t i o n i n the development o f b i o l o g i c a l l y a c t i v e m a t e r i a l s . Acknowledgments The a u t h o r s w i s h t o e x p r e s s t h e i r s i n c e r e a p p r e c i a t i o n t o E r i c D i e t z e f o r the v a l u a b l e c o n s u l t a t i o n s and t o K a t h l e e n Dooley f o r h e r c a r e f u l work i n t y p i n g the m a n u s c r i p t . T h i s work was s u p p o r t e d by g r a n t s ES02710-07, DCB-8518697, and 85-CRCR-1-1715 from NIEHS, NSF, and USDA, r e s p e c t i v e l y . A.S. was s u p p o r t e d b y a f e l l o w s h i p from F u l b r i g h t Program #33917 ( I n s t i t u t e o f I n t e r n a t i o n a l E d u c a t i o n ) . B.D.H. i s a Burroughs Wellcome S c h o l a r i n T o x i c o l o g y .

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