Potent, Rationally Designed Inhibitors of Squalene Synthase

Chapter 7

Potent, Rationally Designed Inhibitors of Squalene Synthase 1

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Scott A. Biller , Michael J. Sofia , Jeffrey W. Abt , Barbara DeLange, John K. Dickson, Jr., Cornelia Forster , Eric M . Gordon, Thomas Harrity, David R. Magnin, Joseph Marretta , Lois C. Rich, and Carl P. Ciosek, Jr. 3

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Downloaded by CORNELL UNIV on June 7, 2017 | http://pubs.acs.org Publication Date: July 2, 1992 | doi: 10.1021/bk-1992-0497.ch007

Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543

The first class of potent inhibitors of squalene synthase is described. The lead inhibitor, ether 10, was discovered as a result of rational design on the basis of a proposed mechanism for the enzymatic reaction. The ether oxygen was positioned to hydrogen bond with a putative active site acid catalyst and was found to make a substantial contribution to the inhibitor-enzyme binding energy. The structural requirements for potent inhibition are discussed.

W i t h the d i s c o v e r y o f the m e v i n i c a c i d f a m i l y of H M G - C o A r e d u c t a s e i n h i b i t o r s , the i n h i b i t i o n of de novo c h o l e s t e r o l b i o s y n t h e s i s h a s b e e n v a l i d a t e d as a m e t h o d for the t r e a t m e n t o f h y p e r c h o l e s t e r o l e m i a i n h u m a n s (1-3). T h r e e m e m b e r s o f this f a m i l y , l o v a s t a t i n , p r a v a s t a t i n a n d s i m v a s t a t i n , are c u r r e n t l y i n c l i n i c a l use. I n a d d i t i o n , a l a r g e n u m b e r o f s y n t h e t i c a n d s e m i s y n t h e t i c a n a l o g u e s h a v e b e e n r e p o r t e d a n d are i n v a r i o u s stages o f d e v e l o p m e n t (4-8). A l t h o u g h H M G - C o A r e d u c t a s e i n h i b i t o r s are safe a n d h i g h l y e f f i c a c i o u s d r u g s (2, 3), the s t u d y o f a l t e r n a t i v e targets i n the c h o l e s t e r o l b i o s y n t h e t i c p a t h w a y s h o u l d p r o v i d e a d d i t i o n a l i n s i g h t i n t o the m o d u l a t i o n of l i p i d h o m e o s t a s i s .

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Corresponding authors Current address: Lilly Research Labs, Indianapolis, IN 46285 Current address: SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406 Current address: University of Illinois, Biotechnology Center B62-1, Urbana, IL 61801

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0097-6156/92/0497-O065S06.00/0 © 1992 American Chemical Society

Nes et al.; Regulation of Isopentenoid Metabolism ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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REGULATION OF ISOPENTENOID METABOLISM


W e p r o p o s e d (9-11) that the i n h i b i t i o n o f s q u a l e n e s y n t h a s e m a y h a v e a d v a n t a g e s o v e r i n t e r v e n t i o n e a r l i e r i n the c h o l e s t e r o l b i o s y n t h e t i c p a t h w a y ( F i g u r e 1). S q u a l e n e s y n t h a s e (12-14) c a t a l y z e s the r e d u c t i v e d i m e r i z a t i o n o f f a r n e s y l d i p h o s p h a t e (1, F P P ) to s q u a l e n e (2). T h i s e n z y m e o c c u p i e s a strategic site at the f i n a l b r a n c h p o i n t i n the p a t h w a y . S e l e c t i v e i n h i b i t i o n of s q u a l e n e s y n t h a s e s h o u l d not d i r e c t l y s u p p r e s s the p r o d u c t i o n o f the n o n - s t e r o l i s o p r e n e m e t a b o l i t e s i n c l u d i n g u b i q u i n o n e , d o l i c h o l , isopentenyl f - R N A and isoprenylated proteins. This chapter d e s c r i b e s o u r p r o g r e s s t o w a r d s the d e s i g n , s y n t h e s i s a n d e v a l u a t i o n o f s q u a l e n e s y n t h a s e i n h i b i t o r s (9-11, 15). O u r u l t i m a t e g o a l is the d i s c o v e r y o f a s u p e r i o r agent for treatment o f h y p e r c h o l e s t e r o l e m i a .

Acetyl CoA

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Cholesterol

a = HMG-CoA Reductase b = Squalene Synthase

Mevalonic Acid

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TOp

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F P P (1)

Isoprenyl Proteins

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F i g u r e 1. I s o p r e n e B i o s y n t h e s i s P a t h w a y : a n a b b r e v i a t e d r e p r e s e n t a t i o n of the b i o s y n t h e t i c routes to c h o l e s t e r o l a n d the n o n - s t e r o l i s o p r e n e s .

E n z y m o l o g y . S q u a l e n e s y n t h a s e is a m i c r o s o m a l p r o t e i n a n d i s t y p i c a l l y s t u d i e d as a c r u d e m i c r o s o m a l p r e p a r a t i o n f r o m l i v e r o r yeast (12-14). W e h a v e d e v e l o p e d a n assay for s q u a l e n e s y n t h a s e a c t i v i t y w h i c h u t i l i z e s the 100,000 χ g m i c r o s o m a l p e l l e t d e r i v e d f r o m rat l i v e r h o m o g e n a t e as a s o u r c e o f e n z y m e a n d gas c h r o m a t o g r a p h y to q u a n t i t a t e s q u a l e n e synthesis f r o m u n l a b e l e d F P P s u b s t r a t e (9, 10). T h e p r o d u c t i o n o f s q u a l e n e is m o r e c o m m o n l y m o n i t o r e d b y the i n c o r p o r a t i o n o f r a d i o l a b e l f r o m H - o r C F P P (12, 14). D i v a l e n t m e t a l i o n ( M g or M n ) a n d r e d u c e d p y r i d i n e n u c l e o t i d e ( N A D H o r N A D P H ) are r e q u i r e d for the e n z y m a t i c r e a c t i o n . 3

+ 2

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+ 2

T h e p u r i f i c a t i o n o f s q u a l e n e s y n t h a s e i n s o l u b l e f o r m has b e e n a c h a l l e n g e . R e c e n t l y , R i l l i n g a n d c o w o r k e r s (16) s u c c e e d e d i n p u r i f y i n g s o l u b i l i z e d s q u a l e n e s y n t h a s e f r o m y e a s t to h o m o g e n e i t y . N o n i o n i c d e t e r g e n t s , m e t h a n o l a n d s u c r o s e w e r e f o u n d to e n h a n c e the s t a b i l i t y o f the s o l u b l e e n z y m e . T w o r e s e a r c h g r o u p s r e p o r t e d the c l o n i n g a n d e x p r e s s i o n o f the s q u a l e n e s y n t h a s e g e n e f r o m the yeast Saccharomyces cerevisiae (17, 18). T h e gene codes for a p r o t e i n o f 444 a m i n o a c i d s ( M = 51.7 k D ) . T h e C - t e r m i n a l h y d r o p h o b i c d o m a i n w a s i d e n t i f i e d as a l i k e l y m e m b r a n e s p a n n i n g r e g i o n w h i c h s e r v e s to a n c h o r the p r o t e i n i n the e n d o p l a s m i c r e t i c u l u m . S q u a l e n e s y n t h a s e a c t i v i t y is c o o r d i n a t e l y r e g u l a t e d (19) b y i n t r a c e l l u l a r s t e r o l c o n c e n t r a t i o n s (20-22), i n a m a n n e r s i m i l a r to H M G - C o A reductase. r



7. BILLER ET AL.

Inhibitors of Squalene Synthase

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R e a c t i o n M e c h a n i s m . T h e s q u a l e n e s y n t h a s e r e a c t i o n is a t w o s t e p t r a n s f o r m a t i o n w h i c h p r o c e e d s t h r o u g h the i n t e r m e d i a t e c y c l o p r o p a n e , p r e s q u a l e n e d i p h o s p h a t e (3, S c h e m e 1) (23). T h e s o l u b l e , p u r i f i e d e n z y m e (16) a n d the r e c o m b i n a n t e n z y m e (17, 18) f r o m y e a s t p r o d u c e b o t h p r e s q u a l e n e d i p h o s p h a t e a n d s q u a l e n e , i n d i c a t i n g that the t w o t r a n s f o r m a t i o n s are c a t a l y z e d b y a s i n g l e p r o t e i n . I n the first step (12, 13), o n e e q u i v a l e n t o f F P P (the p r e n y l d o n o r ) u n d e r g o e s a loss o f the pro-Sh y d r o g e n a n d i n o r g a n i c d i p h o s p h a t e (PPi) f r o m C - l , c o n c o m i t a n t w i t h the i n s e r t i o n o f C - l i n t o the C - 2 / C - 3 a l k e n e o f the s e c o n d e q u i v a l e n t o f F P P (the p r e n y l a c c e p t o r ) to f o r m 3. I n the s u b s e q u e n t s t e p (12, 13, 24), presqualene diphosphate undergoes a reductive rearrangement i n w h i c h a s e c o n d m o l e c u l e o f P P i is released a n d N A D ( P ) H is c o n s u m e d to f o r m the s y m m e t r i c a l d i m e r , squalene. T h e d o n o r a n d acceptor b i n d i n g sites e x h i b i t i n d e p e n d e n t s e l e c t i v i t y for the acceptance o f m o d i f i e d F P P a n a l o g u e s as substrates, i n d i c a t i n g that the t w o sites are d i s t i n c t (25, 26).

It has b e e n p r o p o s e d that the i n i t i a l process i n v o l v e d i n b o t h steps o f the s q u a l e n e s y n t h a s e r e a c t i o n is h e t e r o l y s i s o f the C - O P P i b o n d to f o r m a n i o n p a i r ( S c h e m e 2) (12, 24, 27). I n the case o f s t e p 1, a l l y l c a t i o n 4 is p r o d u c e d , w h e r e a s i n the case o f step 2, the c y c l o p r o p y l c a r b i n y l c a t i o n 5 is f o r m e d . T h i s p r o p o s a l is s u p p o r t e d b y c h e m i c a l m o d e l s t u d i e s for step 2 as w e l l as e l e g a n t i n h i b i t i o n s t u d i e s i n v o l v i n g m i m i c s o f i o n p a i r 5 (12, 24, 27). S e v e r a l l i n e s o f e v i d e n c e i n d i c a t e that the c l o s e l y r e l a t e d p r e n y l transferase r e a c t i o n c a t a l y z e d b y F P P s y n t h a s e p r o c e e d s via a l l y l i o n p a i r s a n a l o g o u s to 4 (27-31). I n a d d i t i o n , h e t e r o l y s i s o f d i p h o s p h a t e esters to f o r m s t a b i l i z e d c a r b o n i u m i o n s is a d o c u m e n t e d aspect o f the i n t r i n s i c c h e m i s t r y o f t h i s f u n c t i o n a l g r o u p (32). T h e s t u d i e s d e s c r i b e d i n t h i s c h a p t e r center o n the i n i t i a l process o f i o n p a i r f o r m a t i o n . S p a c e d o e s n o t


68


allow for discussion of the fascinating mechanistic transformations subsequent to the initial step and the reader is referred to several key reviews (12-14, 24, 27).

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D i p h o s p h a t e Surrogates In the d e s i g n o f i n h i b i t o r s o f s q u a l e n e s y n t h a s e b a s e d o n the s t r u c t u r e o f s u b s t r a t e 1, as w e l l as r e l a t e d t r a n s i t i o n states a n d i n t e r m e d i a t e s , stable s u r r o g a t e s for the d i p h o s p h a t e m o i e t y h a v e to b e d e v i s e d . A l t h o u g h the d i p h o s p h a t e is c r u c i a l for b i n d i n g a n d c a t a l y s i s , i t is u n s u i t a b l e as a n i n h i b i t o r c o m p o n e n t d u e to its c h e m i c a l ( h i g h a n d l o w p H ) (32) a n d biological (phosphatases) lability. W e have evaluated a n u m b e r of p o t e n t i a l d i p h o s p h a t e s u r r o g a t e s (9-11), a n d c o n c e n t r a t e h e r e o n the ( p h o s p h i n y l m e t h y l ) p h o s p h o n a t e ( P M P ) m o i e t y (9, 15). P a r e n t P M P 6a is s i m p l y the r e s u l t o f r e p l a c i n g b o t h the r e a c t i v e a l l y l i c a n d a n h y d r i d e o x y g e n s o f F P P w i t h C H 2 (9). T h i s m o d i f i c a t i o n affords a stable m o l e c u l e w h i c h is u n a b l e to u n d e r g o the i n i t i a l step o f the s q u a l e n e s y n t h a s e r e a c t i o n . A priori, it w a s d i f f i c u l t to p r e d i c t w h e t h e r e i t h e r o f these t w o o x y g e n s w a s c r u c i a l for i n t e r a c t i o n w i t h the e n z y m e . U p o n e v a l u a t i o n i n the rat l i v e r m i c r o s o m a l s q u a l e n e s y n t h a s e a s s a y , 6a p r o v e d to b e a n effective i n h i b i t o r of the e n z y m e , I50 = 31.5 μ Μ ( T a b l e 1). L i n e w e a v e r - B u r k a n a l y s i s i n d i c a t e s that 6a is c o m p e t i t i v e w i t h respect to F P P w i t h K j = 10 μ Μ ( F i g u r e 2A). C o m p a r i s o n w i t h the M i c h a e l i s c o n s t a n t for F P P u n d e r o u r assay c o n d i t i o n s ( a p p a r e n t K = 12.7 μ Μ ) reveals that 6a a n d F P P b i n d to the e n z y m e w i t h s i m i l a r a f f i n i t y . W e i n f e r f r o m t h i s t h a t n e i t h e r the a n h y d r i d e n o r a l l y l i c o x y g e n s o f F P P are critical for substrate b i n d i n g . m


7. BILLERETAL.

69



R

T a b l e 1. I n h i b i t i o n o f S q u a l e n e S y n t h a s e b y C o m p o u n d s 6, 7 a n d 8* Cpd Cpd ΐ5ο (μΜ) 6a 7a 31.5 6b 12.2 7b 6c 8a -40 % @ 600 *For assay conditions, see references 9 and 10.

I 0 (μΜ) 29.9 15.5 42 5

T h e r e s u l t s of o u r i n i t i a l s t u d y o n P M P i n h i b i t o r s r e l a t e d to 6a are p r e s e n t e d i n T a b l e 1. 1 , 3 - D i e n y l P M P 6 b e x h i b i t e d i m p r o v e d a c t i v i t y r e l a t i v e to 6a, s u g g e s t i n g that a v i n y l g r o u p m a y be a s u p e r i o r substitute for the a l l y l i c C - O b o n d . T h e d i f l u o r o - P M P s u r r o g a t e of 7a a n d 7b w a s i n t e n d e d to m o r e c l o s e l y m i m i c the d i p h o s p h a t e of F P P w i t h respect to p K v a l u e s (33) , t h e r e b y f a v o r i n g the f u l l y i o n i z e d f o r m at p h y s i o l o g i c a l p H . T h e e q u i v a l e n t i n h i b i t o r y p o t e n c i e s o f the c o r r e s p o n d i n g P C H 2 P (6a, 6 b ) a n d P C F 2 P ( 7 a , 7 b ) a n a l o g u e s i n d i c a t e that b o t h 6 a n d 7 c a n a c h i e v e the p r o t o n a t i o n state n e c e s s a r y for b i n d i n g to the e n z y m e . T h e g e r a n y l d e r i v a t i v e 6c is at least 20-fold less potent t h a n its f a r n e s y l c o u n t e r p a r t 6 b . T h i s is c o n s i s t e n t w i t h r e p o r t s that g e r a n y l d i p h o s p h a t e is n o t a substrate (34) a n d is a p o o r i n h i b i t o r (35) for s q u a l e n e synthase. I n h i b i t o r 8a (36), a n a

1/(Π>ΡμΜ)

1/ΕΡΡμΜΐ

F i g u r e 2. L i n e w e a v e r - B u r k a n a l y s i s o f the i n h i b i t i o n o f rat l i v e r m i c r o s o m a l s q u a l e n e synthase b y 6a ( F i g u r e 2 A ) a n d 10 ( F i g u r e 2 B ) .


70


a n a l o g u e w h i c h retains the a n h y d r i d e l i n k a g e , possesses a c t i v i t y c o m p a r a b l e to 6 a , p r o v i d i n g further e v i d e n c e that the a n h y d r i d e o x y g e n is n o t e s s e n t i a l . I n r e l a t e d w o r k (37), P o u l t e r , M c C l a r d a n d c o w o r k e r s d e m o n s t r a t e that the P M P a n a l o g u e s o f i s o p e n t e n y l - a n d d i m e t h y l a l l y l d i p h o s p h a t e are i n h i b i t o r s o f the p r e n y l transferase, F P P s y n t h a s e . R a t i o n a l D e s i g n of Potent P M P Inhibitors


R a t i o n a l e . P M P i n h i b i t o r s 6a,b a n d 7a,b b i n d to s q u a l e n e s y n t h a s e w i t h a n a f f i n i t y w h i c h is c o m p a r a b l e to that o f the substrate F P P a n d t h u s r e p r e s e n t a s t a r t i n g p o i n t for f u r t h e r s t u d i e s . A s a b a s i s for r a t i o n a l d e s i g n , w e f o c u s e d o n a h y p o t h e t i c a l m o d e l for F P P b i n d i n g to the d o n o r site o f the e n z y m e a n d s u b s e q u e n t i o n - p a i r f o r m a t i o n ( F i g u r e 3). W e e n v i s i o n that

F i g u r e 3. H y p o t h e t i c a l m o d e l of the d o n o r site o f s q u a l e n e s y n t h a s e , b o u n d to: (A) s u b s t r a t e F P P , (B) the a l l y l c a t i o n - d i p h o s p h a t e i o n p a i r 4 a n d ( C ) e t h e r i n h i b i t o r 10. T h e a c t i v e s i t e f u n c t i o n A - H r e p r e s e n t s the p u t a t i v e a c i d catalyst a n d X r e p r e s e n t s c a t i o n i c s i d e chain(s) w h i c h i o n p a i r w i t h the p h o s p h a t e g r o u p s . +



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BILLER ET AL.

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F P P i s b o u n d to the d o n o r site o f s q u a l e n e s y n t h a s e via h y d r o p h o b i c i n t e r a c t i o n s w i t h the i s o p r e n e s u b u n i t a n d i o n i c i n t e r a c t i o n s w i t h the highly charged diphosphate moiety (Figure 3A). W e propose two m e c h a n i s m s b y w h i c h the e n z y m e m i g h t c a t a l y z e the g e n e r a t i o n o f i o n p a i r 4 f r o m e n z y m e b o u n d 1. F i r s t , w e e x p e c t that the e n z y m e interacts m o r e s t r o n g l y w i t h the t w o fragments f o r m e d i n the c o n v e r s i o n o f 1 to 4 as the d i s t a n c e b e t w e e n t h e m a p p r o a c h e s the transition-state s e p a r a t i o n . T h e e n z y m e m i g h t a c c o m p l i s h this b y p o s i t i o n i n g the i s o p r e n e a n d d i p h o s p h a t e b i n d i n g p o c k e t s s u c h that o p t i m a l b i n d i n g e n e r g i e s are a c h i e v e d at the t r a n s i t i o n - s t a t e d i s t a n c e . I n this m a n n e r , the a c t i v e site s e r v e s to stretch the C - O P P j b o n d . S e c o n d , w e p r o p o s e that s q u a l e n e s y n t h a s e u t i l i z e s an a c t i v e site a c i d catalyst to p r o m o t e i o n - p a i r f o r m a t i o n ( A - H i n F i g u r e 3). T h i s a c i d is e x p e c t e d to interact w e a k l y w i t h substrate 1 d u e to the p o o r b a s i c i t y of the ester o x y g e n , b u t m u c h m o r e s t r o n g l y as the b a s i c p h o s p h a t e a n i o n o f 4 is f o r m e d . I d e n t i c a l a r g u m e n t s c a n be a p p l i e d to the f o r m a t i o n o f i o n - p a i r 5 f r o m 3. F o l l o w i n g the a b o v e a n a l y s i s , w e a t t e m p t e d to create m o r e p o t e n t P M P i n h i b i t o r s b y : (1) i n c r e a s i n g the s e p a r a t i o n b e t w e e n the i s o p r e n y l s u b u n i t a n d the P M P s u r r o g a t e i n o r d e r to a p p r o x i m a t e t h e i o n p a i r s e p a r a t i o n a n d / o r (2) i n c o r p o r a t i n g f u n c t i o n s i n the l i n k i n g c h a i n to i n t e r a c t w i t h the p u t a t i v e a c t i v e site a c i d . T h e f o r m e r m a y be d i f f i c u l t to a c c o m p l i s h , s i n c e w e are r e s t r i c t e d to n o r m a l c o v a l e n t b o n d l e n g t h s , a l i m i t a t i o n n o t s h a r e d b y t r a n s i t i o n states. R e s u l t s . L i n k e r h o m o l o g 6 d (9), a n a t t e m p t to a p p r o x i m a t e the t r a n s i t i o n state s e p a r a t i o n b e t w e e n the f r a g m e n t s , is a c t u a l l y a s o m e w h a t w e a k e r i n h i b i t o r of the e n z y m e than 6a ( T a b l e 2). I n c o r p o r a t i o n o f a p h o s p h o n a t e ester o x y g e n a t o m i n the l i n k i n g c h a i n to interact w i t h the p u t a t i v e a c t i v e site a c i d ( e x a m p l e 9) results i n a 4 - f o l d increase i n a c t i v i t y r e l a t i v e to 6 d . T h i s m o d e s t i m p r o v e m e n t is n o t s u r p r i s i n g , s i n c e a n ester o x y g e n , l i k e that of F P P itself, is expected to f o r m a r e l a t i v e l y w e a k h y d r o g e n b o n d w i t h T a b l e 2. L i n k e r M o d i f i c a t i o n s o f the P M P I n h i b i t o r s * v

Ο

Ο _

o- oCpd

Linker X

ΐ5θ(μΜ)

Cpd

Linker X

I50 ( μ Μ ) 0.15

31.5

12

-CH OCH -

6d

-CH CH -

67.0

13

-CH CH OCH -

79.3

9

-CH 0

16.0

14

-NH +CH -

16.0

10

-OCH -

0.05

15

-SCH -

122

6a

-CH 2

2

2

2

2

2

2

2

2

2

2

2

2

11 -OCH CH 2.3 *For assay conditions, sec references 9 and 10. 2

2




72

the a c i d m o i e t y . W h e n a m o r e b a s i c e t h e r o x y g e n w a s i n s e r t e d i n the l i n k i n g c h a i n (example 10), a p r o f o u n d increase i n i n h i b i t o r y p o t e n c y r e s u l t e d (15). E t h e r 10 (I50 = 0.05 μ Μ ) is 1 3 4 0 - f o l d m o r e p o t e n t t h a n its c a r b o n isostere 6 d as a n i n h i b i t o r o f s q u a l e n e s y n t h a s e ! I n h i b i t o r 10 w a s d e m o n s t r a t e d to be c o m p e t i t i v e w i t h respect to F P P , K j = 0.037 μ Μ ( F i g u r e 2B). F u r t h e r m o d i f i c a t i o n s o f the l i n k i n g r e g i o n (15) w e r e a i m e d at r e v e a l i n g s t r u c t u r a l features that are i m p o r t a n t for i n h i b i t i o n ( T a b l e 2). T h e p o s i t i o n of the ether o x y g e n r e l a t i v e to the p h o s p h i n i c a c i d is c r i t i c a l . I n s e r t i o n o f a n a d d i t i o n a l C H 2 b e t w e e n the e t h e r a n d p h o s p h i n i c a c i d results i n a 4 6 - f o l d loss i n a c t i v i t y ( e x a m p l e 11), w h e r e a s h o m o l o g a t i o n o n the o t h e r s i d e o f the ether ( e x a m p l e 12) leads to just a 3 - f o l d loss. F u r t h e r h o m o l o g a t i o n o f 12 to 13, h o w e v e r , is h i g h l y d e l e t e r i o u s . C o n s i s t e n t w i t h the r e q u i r e m e n t f o r a h y d r o g e n b o n d a c c e p t o r is t h e u n i m p r e s s i v e i n h i b i t o r y p o t e n c i e s o f a m m o n i u m i o n 14, w h i c h is a n Η - b o n d d o n o r , a n d thioether 15, w h i c h is a p o o r Η - b o n d acceptor. Enzyme-Inhibitor B i n d i n g Energy It w o u l d be u s e f u l for future w o r k i n the area o f m o l e c u l a r r e c o g n i t i o n to a r r i v e at a q u a n t i t a t i v e a c c o u n t i n g o f the c o n t r i b u t i o n m a d e b y the e t h e r o x y g e n to e n z y m e - i n h i b i t o r i n t e r a c t i o n s . In o r d e r to a c c o m p l i s h t h i s , w e c o m p a r e ether 10 to its c a r b o n isostere 6 d ( F i g u r e 4). T h e difference b e t w e e n the e n z y m e b i n d i n g energies of 10 a n d 6 d i n a q u e o u s s o l u t i o n ( A A G t e r ) is c a l c u l a t e d to b e 4.3 k c a l / m o l u t i l i z i n g t h e e x p r e s s i o n : A A G t e r = - R T l n [ l 5 o ( 1 0 ) / l 5 o ( 6 d ) ] . T h e d i f f e r e n t i a l b i n d i n g e n e r g y i n the a b s e n c e o f s o l v e n t ( A A G ) (38) is a m o r e m e a n i n g f u l m e a s u r e o f the d i f f e r e n c e b e t w e e n the t w o i n h i b i t o r s , s i n c e , b y f a c t o r i n g o u t s o l v a t i o n , A A G is s o l e l y a f u n c t i o n o f the i n t e r a c t i o n s w i t h i n the E-I c o m p l e x . U s i n g the t h e r m o d y n a m i c c y c l e i n F i g u r e 4 as s u g g e s t e d b y the w o r k of Bartlett (38-41), the e x p r e s s i o n i n e q 1 for A A G t r is d e r i v e d . S o l v i n g for AAGgas affords e q 2, s i n c e A A G 1 V - E is z e r o . T o f u r t h e r s i m p l i f y t h i s e x p r e s s i o n (38), w e m a k e the a s s u m p t i o n that the t e r m AAGSO1V-E-I is a l s o z e r o , s i n c e i t is l i k e l y that w a t e r is e x c l u d e d f r o m the b i n d i n g site o f b o t h E - I c o m p l e x e s . If a n y t h i n g , t h i s a p p r o x i m a t i o n s h o u l d l e a d to a n underestimation of A A G , s i n c e the s o l v a t i o n e n e r g y o f E - 6 d h a s the p o t e n t i a l to be l a r g e r t h a n that of E-10 i n the e v e n t that w a t e r is accessible to the a c t i v e site f u n c t i o n a l i t y (eg. the p u t a t i v e a c i d c a t a l y s t ) w h i c h i s satisfied b y the ether i n the case o f E - 1 0 . T h u s , A A G is a p p r o x i m a t e d as the s u m o f A A G r (4.3 k c a l / m o l ) a n d A A G 1 V - I , w h e r e A A G I - I i s t h e difference b e t w e e n the s o l v a t i o n energies o f 10 a n d 6 d . w a

w a

g a s

g a s

w a

e

S 0

g a s

g a s

w a t e

S 0

S O

V

A c c o r d i n g to the e m p i r i c a l c o r r e l a t i o n s o f H i n e (42), a n ether o x y g e n c o n t r i b u t e s 4.2 k c a l / m o l m o r e t h a n a C H 2 to the s o l v a t i o n e n e r g y . T h u s , AAG for 10 r e l a t i v e to 6 d is e s t i m a t e d to b e 8.5 k c a l / m o l . I n w a t e r , a p p r o x i m a t e l y h a l f o f this e n e r g y is e x p e n d e d f o r the d e s o l v a t i o n o f the e t h e r f u n c t i o n . A s s u m i n g that 10 a n d 6 d b i n d to s q u a l e n e s y n t h a s e i n the s a m e m a n n e r , w e s p e c u l a t e that t h i s l a r g e d i f f e r e n t i a l b i n d i n g e n e r g y is d u e to h y d r o g e n b o n d i n g o f the ether o x y g e n to a n active-site a c i d catalyst g a s


7. BILLER ET AL.


73

as i l l u s t r a t e d i n F i g u r e 3 - C . T h e ether o x y g e n i s o f i n t e r m e d i a t e b a s i c i t y r e l a t i v e to the w e a k l y b a s i c p h o s p h a t e ester o x y g e n i n F i g u r e 3 - A a n d the s t r o n g l y b a s i c p h o s p h a t e a n i o n i n F i g u r e 3 - B , a n d c a n b e c o n s i d e r e d to b e a m i m i c o f a species a l o n g the r e a c t i o n c o o r d i n a t e f r o m A to B . L a r g e A A G h a v e b e e n o b s e r v e d p r e v i o u s l y for i s o s t e r i c f u n c t i o n a l g r o u p r e p l a c e m e n t s i n i n h i b i t o r s o f t h e r m o l y s i n (38, 39) a n d n u c l e o s i d e d e a m i n a s e s (40, 41).

g a s

A G water

F + τ ^water ^ water

E - I water

1

- A G solv-I

- A G solv-E


E AAG*

= A A G

g

a

s

-AAG

+

s o l v

A G gas

*gas _

E

A G solv-E-I

- A A G solv-I + A A G

A A G

A A G

E - I gas

g a s

+

AAG

AAG

gas = water + s o l v - I " solv-E-I = 4.3 + 4 . 2 + 0 = 8.5 k c a l / m o l

s o l v

e(

( l

. . E

2

I

(eq D

)

F i g u r e 4. T h e r m o d y n a m i c c y c l e is u t i l i z e d to estimate the d i f f e r e n t i a l b i n d i n g e n e r g y o f 10 r e l a t i v e to 6 d i n the absence o f s o l v e n t ( A A G

g a s

).

A n a l t e r n a t i v e e x p l a n a t i o n for the l a r g e d i f f e r e n t i a l b i n d i n g e n e r g y is a n i n d i r e c t effect o f the ether o n the i n t e r a c t i o n o f t h e P M P s u r r o g a t e w i t h t h e e n z y m e . If a n y t h i n g , the e l e c t r o n w i t h d r a w i n g c a p a b i l i t i e s o f the e t h e r w o u l d b e e x p e c t e d to decrease the b i n d i n g e n e r g y o f t h e adjacent p h o s p h i n i c a c i d g r o u p (38, 43). A g a i n , t h i s w o u l d l e a d t o a n u n d e r e s t i m a t i o n o f the c o n t r i b u t i o n m a d e b y the ether. Further Structure-Activity Studies M o d i f i c a t i o n s o f the P M P D i p h o s p h a t e Surrogate. Studies were undertaken to d e t e r m i n e w h e t h e r the c o m p l e t e t r i a c i d P M P s u r r o g a t e w a s e s s e n t i a l for potent i n h i b i t i o n . T h e strong interaction offered b y the ether does not c o m p e n s a t e f o r t h e loss o f either p h o s p h o r u s m o i e t y , as e v i d e n c e d b y the p o o r a c t i v i t y e x h i b i t e d b y 16 a n d 17 ( T a b l e 3). F u r t h e r a t t e m p t s t o r e d u c e the t r i a c i d P M P s u r r o g a t e o f 10 to a d i a c i d r e s u l t e d i n c o n s i d e r a b l e loss o f p o t e n c y as e v i d e n c e d b y e x a m p l e s 18 - 23. I n the case o f p h o s p h i n y l acetate

24, Iso = 102 μ Μ

25, X = C H , 1 2

5 0

= 31.8 u M

26, X = 0 , 1 ^ = 8.7 μ Μ


74

REGULATION OF ISOPENTENOID METABOLISM T a b l e 3. M o d i f i c a t i o n s o f the P M P D i p h o s p h a t e S u r r o g a t e *

Cpd

10

X

C

0

0

II

II

00


16

ξ-Ρ-000

e

0

0

II

II

ξ— P s ^ P - O M e '

19

C

1

Cpd

0.05

20

C

1

II

0 0
II

H

\—P^P-OCH 0 -

94

3

75

*For assay condirions, see references 9 and 10. 2 1 , the ether a n a l o g u e is 8-fold m o r e p o t e n t t h a n the c o r r e s p o n d i n g ester l i n k e d c o m p o u n d 24; h o w e v e r , this is a m i n o r i m p r o v e m e n t c o m p a r e d to the s a m e m o d i f i c a t i o n i n the P M P series (9 vs 10). P h o s p h i n y l f o r m a t e 22 is a c t u a l l y less p o t e n t t h a n isosteres 2 5 a n d 26(11), s u g g e s t i n g that the p h o s p h i n y l formates b i n d e i t h e r to a different site o n the e n z y m e (eg, the acceptor site), o r to the same site b u t i n a different m a n n e r . M o d i f i c a t i o n o f the I s o p r e n e S u b u n i t . T h e results o f o u r s t u d i e s o n the m o d i f i c a t i o n o f the f a r n e s y l s u b u n i t o f 10 are p r e s e n t e d i n T a b l e 4. B e g i n n i n g w i t h the t e r m i n a l i s o p r e n e u n i t , r e d u c t i o n o f the o l e f i n ( e x a m p l e 27) a n d further r e m o v a l o f a C H 3 g r o u p ( e x a m p l e 28) b o t h l e d to > 10-fold loss i n p o t e n c y r e l a t i v e to 10. T h e s e s a m e m o d i f i c a t i o n s to F P P itself h a v e b e e n r e p o r t e d to afford g o o d substrates for s q u a l e n e s y n t h a s e (26, 34). D e l e t i o n o f the entire t e r m i n a l i s o p r e n e u n i t ( g e r a n y l a n a l o g u e 29) effects a n a l m o s t c o m p l e t e l o s s o f i n h i b i t o r y a c t i v i t y . T h i s is c o n s i s t e n t w i t h o u r p r i o r o b s e r v a t i o n w i t h g e r a n y l P M P 6c (vide supra), a n d c o n f i r m s the r e q u i r e m e n t for a c o m p l e t e f a r n e s y l c h a i n - l e n g t h for s t r o n g e n z y m e inhibitor binding. T h e e n z y m e is m o r e tolerant of s t r u c t u r a l v a r i a t i o n s i n the i s o p r e n e u n i t p r o x i m a l to the ether. R e m o v a l o f the v i n y l i c m e t h y l g r o u p ( e x a m p l e


7. BILLER ET AL.

IS


30) , h o m o l o g a t i o n to a n e t h y l g r o u p ( e x a m p l e 33) a n d r e p l a c e m e n t w i t h c h l o r i d e ( e x a m p l e 34) a l l r e s u l t i n m i n i m a l loss o f p o t e n c y . W h e n the first t w o m o d i f i c a t i o n s are m a d e to F P P itself, the r e s u l t i n g a l l y l i c d i p h o s p h a t e s a r e r e p o r t e d to b e e x t r e m e l y p o o r s u b s t r a t e s f o r the e n z y m e (25, 26). S u r p r i s i n g l y , r e p l a c i n g the p r o x i m a l (E)-olefin w i t h a n a c e t y l e n e ( e x a m p l e 31) o r a ( Z ) - o l e f i n ( e x a m p l e 32) l e a d s to c o m p l e t e r e t e n t i o n o f i n h i b i t o r y a c t i v i t y . M o l e c u l a r m o d e l i n g studies suggest that b o t h 31 a n d 32 c a n attain c o n f o r m a t i o n s that o v e r l a p w i t h 10 at b o t h the t e r m i n a l i s o p r e n e u n i t a n d the e t h e r - P M P p o r t i o n s of the m o l e c u l e , w h i l e b r i d g i n g the t w o t e r m i n i via d i f f e r e n t r o u t e s . T h e s e r e s u l t s i n d i c a t e that t h e r e is a n e l e m e n t o f s t r u c t u r a l f l e x i b i l i t y i n the i n t e r i o r r e g i o n o f the i n h i b i t o r .


T a b l e 4. M o d i f i c a t i o n s o f the I s o p r e n y l S u b u n i t * Ο II

ο II

.P.

ο- ο Cpd

R

οR

ΙδοίμΜ)

Ι50 (μΜ)

Cpd

10

0.05

33

0.078

27

0.53

34

0.107

28

0.59

35

0.155

29

>300

36

0.315

30

0.17

37

0.218

31

0.066

38

0.412

32

0.047

39

0.178

*For assay conditions, see references 9 and 10.

S u m m a r y o f S A R S t u d i e s . T h e results of o u r S A R studies o n P M P i n h i b i t o r s o f s q u a l e n e s y n t h a s e are s u m m a r i z e d i n F i g u r e 5. T h e r e q u i r e m e n t s for p o t e n t e n z y m e i n h i b i t i o n are: (1) a n intact, t r i a c i d P M P s u r r o g a t e , (2) a n ether o x y g e n adjacent to the P M P s u r r o g a t e , a n d (3) a f a r n e s y l - l e n g t h i s o p r e n e s u b u n i t . A c e r t a i n degree o f s t r u c t u r a l f l e x i b i l i t y is a l l o w e d i n the i s o p r e n e s u b u n i t that is p r o x i m a l to the ether.


76


Diphosphate Surrogate (PMP)


-triacid

required-

Required Terminal Isoprene F i g u r e 5. A n a t o m y o f a S q u a l e n e S y n t h a s e I n h i b i t o r F u r t h e r B i o l o g i c a l E v a l u a t i o n o f 10 The i n h i b i t i o n of squalene synthase b y inhibitor 10 i n h u m a n liver a n d yeast m i c r o s o m e s w a s c o m p a r e d to that i n rat l i v e r m i c r o s o m e s u n d e r o u r s t a n d a r d assay c o n d i t i o n s (9, 10) ( T a b l e 5). A l t h o u g h the results f r o m b o t h m a m m a l i a n sources w e r e n e a r l y i d e n t i c a l , 1 0 is m u c h less effective against the yeast e n z y m e . T h e a p p a r e n t K v a l u e s for F P P i n the rat (12.7 μ Μ ) a n d yeast (25 μ Μ ) m i c r o s o m a l systems are s i m i l a r . U p o n e x p r e s s i n g the r e l a t i v e p o t e n c y for the t w o e n z y m e p r e p a r a t i o n s as ( K m / K i ) t / ( K m / K i ) y a s t / d e m o n s t r a t e d to be 16-fold m o r e active against the rat vs the yeast e n z y m e . T h i s m a y be a n i n d i c a t i o n that the a c t i v e sites o f the yeast a n d m a m m a l i a n e n z y m e s are s i g n i f i c a n t l y d i f f e r e n t . I n a d d i t i o n , 10 i n h i b i t s c h o l e s t e r o l b i o s y n t h e s i s f r o m C - a c e t a t e i n w h o l e , f r e s h l y - i s o l a t e d h e p a t o c y t e s (I50 = 2.7 μ Μ ) . T h e p a r e n t P M P a n a l o g u e 6a is i n a c t i v e i n the w h o l e cell assay at u p to 100 μ Μ (11). m

1 0

r a

e

14

T a b l e 5. Effect of 10 o n S q u a l e n e B i o s y n t h e s i s i n M i c r o s o m e s a n d o n Cholesterol Biosynthesis i n Rat Hepatocytes Assay

Ki 150 ( μ Μ ) 0.037 0.05 0.038 — 1.18 2.6 Yeast M i c r o s o m e s ' 2.7 W h o l e Rat H e p a t o c y t e s ^ — (a) For standard assay conditions, see ref s 9 and 10. (b) From frozen human liver. (c) Recombinant, overexpressed enzyme (17). (d) For assay, see ref. 11. 3

Rat L i v e r M i c r o s o m e s H u m a n Liver Microsomes ' 3

3

1 5

0


i s

7.

BILLER ET AL.

77


S y n t h e s i s o f I n h i b i t o r 10


T h e s y n t h e t i c r o u t e to e t h e r - P M P 10 ( S c h e m e 3) is g e n e r a l a n d w a s u t i l i z e d to p r e p a r e m o s t o f the i n h i b i t o r s d e s c r i b e d h e r e i n . T w o m e t h o d s for the p r e p a r a t i o n o f a l k o x y p h o s p h o n a t e 41 f r o m f a r n e s o l 40 w e r e e v a l u a t e d . R h o d i u m - c a t a l y z e d c a r b e n e i n s e r t i o n (44) o f d i a z o m e t h y l p h o s p h o n a t e 42 (45) p r o v i d e d d i m e t h y l ester 4 1 a i n m o d e s t y i e l d . T h e r e a c t i o n o f the l i t h i u m a l k o x i d e d e r i v e d f r o m f a r n e s o l w i t h triflate 43 (46) a f f o r d e d the c o r r e s p o n d i n g d i i s o p r o p y l ester 4 1 b i n 92 % y i e l d . T r i f l a t e 43 is p r e p a r e d f r o m d i i s o p r o p y l p h o s p h i t e i n t w o steps ( p a r a f o r m a l d e h y d e , E t 3 N , 100 ° C (47); t r i f l u o r o m e t h a n e s u l f o n i c a n h y d r i d e , i - P r 2 N E t , ether, -78 to 0 ° C , 52 % o v e r a l l ) . E i t h e r 41a o r 4 1 b c a n be u s e d to c o m p l e t e the s y n t h e s i s f o l l o w i n g o u r p r e v i o u s l y r e p o r t e d m e t h o d o l o g y f o r the p r e p a r a t i o n o f P M P i n h i b i t o r s (48). I n the case of 4 1 b , c o n v e r s i o n to a c i d c h l o r i d e 4 4 w a s Scheme 3

42

43

(a) 0.2 equiv Rh (OAc) , 2 cquiv 42, PhH, RT, 36 %. (b) BuLi, THF, -78 °C; 43, 0 °C, 92 %. (c) KOH, H 0 , i-PrOH, 100 °C; TMSNEt C H C 1 , oxalyl chloride, cat DMF, C H C 1 . (d) LiCH PO(OMe) , THF, -78 °C, 79% overall from 41b. (e) TMSBr, C H C 1 2,4,6-collidine; KOH, H Q , 93 %. 2

2

2

2

2/

2

2

2

2/

2

2

2

2


78


followed by coupling with the l i t h i u m anion of dimethyl m e t h y l p h o s p h o n a t e t o p r o v i d e P M P t r i e s t e r 4 5 (79 % y i e l d f r o m 4 1 b ) . B r o m o t r i m e t h y l s i l a n e (49) p r o m o t e d d e e s t e r i f i c a t i o n g a v e i n h i b i t o r 10 i n 93 % y i e l d .


Conclusion In s u m m a r y , o u r s t u d i e s h a v e l e d to the first f a m i l y o f p o t e n t i n h i b i t o r s o f s q u a l e n e s y n t h a s e . I n h i b i t o r 10 w a s d i s c o v e r e d as t h e r e s u l t o f r a t i o n a l d e s i g n o n the basis o f a p r o p o s e d m e c h a n i s m for the e n z y m a t i c catalysis o f s q u a l e n e b i o s y n t h e s i s . T h i s s t u d y r e v e a l e d a s u r p r i s i n g l y large c o n t r i b u t i o n o f a n e t h e r o x y g e n to the i n h i b i t o r - e n z y m e b i n d i n g e n e r g y . W e s p e c u l a t e that this o x y g e n is e n g a g e d i n a h y d r o g e n b o n d i n g i n t e r a c t i o n w i t h a k e y a c t i v e site a c i d c a t a l y s t . F u r t h e r s t r u c t u r e - a c t i v i t y s t u d i e s r e v e a l that a t r i a c i d d i p h o s p h a t e s u r r o g a t e a n d a f a r n e s y l - l e n g t h i s o p r e n y l s u b u n i t are r e q u i r e d for o p t i m a l i n h i b i t o r y a c t i v i t y , a n d that a d e g r e e o f s t r u c t u r a l f l e x i b i l i t y is t o l e r a t e d i n the i s o p r e n y l p o r t i o n o f the i n h i b i t o r . A c k n o w l e d g e m e n t s . T h e a u t h o r s w o u l d l i k e to t h a n k D r s . P h i l i p M . S h e r , W i l l i a m H . K o s t e r a n d R i c h a r d E . G r e g g for t h e i r h e l p f u l c o m m e n t s o n this m a n u s c r i p t , D r . V a l S. G o o d f e l l o w for assistance w i t h t h e m o l e c u l a r m o d e l i n g s t u d i e s , a n d D r . Jeffrey M . H o e g o f t h e N a t i o n a l I n s t i t u t e s o f H e a l t h f o r a s a m p l e o f h u m a n l i v e r tissue.

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