Probing Bioactive Mechanisms - American Chemical Society

Chapter 19

Analysis of Binding at 4-Aminobutyric Acid Receptor Sites by Structure—Activity Relationships 1

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 30, 2016 | http://pubs.acs.org Publication Date: November 14, 1989 | doi: 10.1021/bk-1989-0413.ch019

Philip S. Magee and James W. King

2

1BIOSAR Research Project, Vallejo, CA 94591 and School of Medicine, University of California, San Francisco, CA 94143 U.S. Army Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, Aberdeen, M D 21010-5423 2

Series of GABA-ergic compounds were analyzed through the expressed binding response (IC50) in the brain, spinal cord and uptake systems of man and various animals. The technique used was multiple regression of the pIC50 values against the variations in substructural features (1.0/0.0). Nearly a l l substructural factors made negative contributions relative to the basic GABA structure. Spacing is c r i t i c a l and binding occurs in a sterically restricted lipophilic cleft between the amino and carboxyl sites. The cleft exhibits chiral selection and the most probable binding mechanism is ion-paring. There a r e two c l a s s e s o f n e u r a l r e c e p t o r s f o r b i n d i n g o f t h e i n h i b i t o r y n e u r o t r a n s m i t t e r , 4 - a m i n o b u t y r i c a c i d (GABA). Of p r i m a r y i n t e r e s t t o t h i s study i s t h e GABA^ r e c e p t o r which p o p u l a t e s b o t h p r e - and p o s t - s y n a p t i c n e u r a l gaps i n t h e CNS. Of l e s s e r i n t e r e s t i s t h e p r e - s y n a p t i c GABAg r e c e p t o r and v a r i o u s n o n - n e u r a l r e c e p t o r sites. These s i t e s a r e r e a d i l y c l a s s i f i e d by b l o c k i n g r e s p o n s e s t o b i c u c u l l i n e (I) o r p i c r o t o x i n i n ( 2 ) . The p o s t - s y n a p t i c GABA^ r e c e p t o r i s a membrane embedded complex m e d i a t i n g t h e i n f l u x and e f f l u x of c h l o r i d e i o n and p o s s e s s i n g a l l o s t e r i c b i n d i n g s i t e s f o r b e n z o d i a z e p i n e s , p i c r o t o x i n i n and some a v e r m e c t i n s ( 3 ) . R e c i p r o c a l a l l o s t e r i c m o d u l a t i o n s among t h e f o u r c l a s s e s o f r e c e p t o r s i t e s a r e e a s i l y observed i n v i t r o . The r e c e p t o r has been shown t o be a g l y c o p r o t e i n , l i k e most membrane p r o t e i n s ( 4 ) . Two and p o s s i b l y t h r e e GABA r e c e p t o r s i t e s on t h e complex c a n be i d e n t i f i e d k i n e t i c a l l y by use o f S c a t c h a r d p l o t s ( 5 - 9 ) . Krogsgaard-Larsen presents e v i d e n c e f o r t h r e e b i n d i n g s i t e s (6) which he terms Low, Medium and H i g h ( 3 ) . A f u r t h e r c o m p l i c a t i o n i s t h e p r e s e n c e o f an endogeneous p r o t e i n i n h i b i t o r b i n d i n g t o t h e h i g h a f f i n i t y s i t e s (10,11). These s i t e s c a n be exposed f o r b i n d i n g s t u d i e s t h r o u g h a complex washing p r o t o c o l (9) u s i n g t h e n o n - i o n i c d e t e r g e n t , t r i t o n X-100 ( 1 2 ) . Johnson and co-workers have shown e x t r a o r d i n a r y i n c r e a s e s i n GABA b i n d i n g t o r a t b r a i n p r e p a r a t i o n s by T r i t o n X-100 e x t r a c t i o n ( 1 3 ) . 0097-6156789/0413-028im00/0 © 1989 American Chemical Society

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


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MECHANISMS

A v a r i e t y o f GABA r e l a t e d c h e m i c a l s b i n d t o t h e GABA^ s i t e s t o p r o duce b o t h a g o n i s t and a n t a g o n i s t r e s p o n s e s (3,14,15). B i c u c u l l i n e ( 1 ) , p i c r o t o x i n i n ( 2 ) , i s o - T H I P and iso-THAZ appear t o be s p e c i f i c GABA r e c e p t o r a n t a g o n i s t s (3,14). Muscimol, THPI, i s o g u v a c i n e and p i p e r i d i n e - 4 - s u l f o n i c a c i d (P4S) a r e s p e c i f i c GABA a g o n i s t s w h i l e muscimol, THPO, g u v a c i n e and n i p e c o t i c a c i d a r e s p e c i f i c GABA uptake i n h i b i t o r s (15,17). Muscimol, d e r i v e d from the mushroom Amanita m u s c a r i a , i s q u i t e t o x i c , t e n times more p o t e n t than GABA as an ago n i s t , and v e r y s p e c i f i c i n b i n d i n g o n l y t o h i g h a f f i n i t y s i t e s (5,18). DeFeudis and co-workers have r e p o r t e d a p p r o x i m a t e l y t w i c e as many b i n d i n g s i t e s f o r muscimol as f o r GABA ( 1 9 ) . Of t h e known s p e c i f i c a g o n i s t s , THPI i s t h e o n l y one c a p a b l e o f p e n e t r a t i n g t h e b l o o d - b r a i n b a r r i e r t o e x e r t a n a l g e s i c and a n t i c o n v u l s a n t e f f e c t s ( r e f e r e n c e s c i t e d i n 6). C l i n i c a l r e s p o n s e s o f GABA a g o n i s t s and a n t a g o n i s t s have been r e v i e w e d ( 2 0 ) . Of r e l a t e d i n t e r e s t i s the r e p o r t t h a t b o t h a n e s t h e t i c and c o n v u l s a n t b a r b i t u a t e s enhance GABA b i n d i n g i n a dose dependent manner, presumably an a l l o s t e r i c e f f e c t ( 1 3 ) . S t e r e o c h e m i s t r y i s c l e a r l y i n v o l v e d i n the b i n d i n g o f GABA and GABA-ergic compounds. Andrews and J o h n s t o n p o s t u l a t e t h a t GABA b i n d s to GABA r e c e p t o r s i n an extended c o n f o r m a t i o n and t o GABAg r e c e p t o r s i n a f o l d e d conformation (21). These c o n s i d e r a t i o n s l e d B l o c k and K i n g t o a d e t a i l e d c o n f o r m a t i o n a l s t u d y o f GABA, muscimol and n i p e c o t i c a c i d based on X-ray c r y s t a l d a t a ( 2 2 ) . Differences i n binding a f f i n i t i e s o f s t e r e o i s o m e r i c GABA-ergic compounds range from s m a l l to l a r g e (14,17). A

A s t a t i s t i c a l a p p r o a c h t o mapping the GABA r e c e p t o r s i t e s i s presented i n t h i s study. We attempt t o a n a l y z e t h e c o n t r i b u t i o n s o f key s u b s t r u c t u r e s t o the measured b i n d i n g a f f i n i t i e s o f GABA-ergic compound s e r i e s . O l s e n has n o t e d v a r i a t i o n s i n t h e r a n k o r d e r p o t e n cy o f GABA a n a l o g s between systems ( 2 3 ) . By use o f m u l t i p l e r e g r e s s i o n a n a l y s i s , we a r e a t t e m p t i n g t o q u a n t i f y these o b s e r v a t i o n s i n terms o f s u b - s t r u c t u r e c o n t r i b u t i o n s ( F i g u r e 1 ) . 1

R e s e a r c h Method. Most s t u d i e s i n v o l v e c a r e f u l l y measured I C 5 0 s f o r a s u b s t a n t i a l number o f GABA-ergic compounds on membrane o r c e l l u l a r preparations. Most o f t h e p r e p a r a t i o n s a r e t r e a t e d by complex p r o t o c o l s i n v o l v i n g e x t r a c t i o n w i t h T r i t o n X-100 ( 9 ) . I n many e x p e r i —-

n

ments, t h e GABA r e c e p t o r s i t e s a r e f i r s t s a t u r a t e d w i t h H-GABA, 3 3 3 H-muscimol, H-diazepam o r H-P4S f o l l o w e d by measurement o f t h e c o n c e n t r a t i o n o f GABA-ergic compound r e q u i r e d t o d i s p l a c e 50% o f t h e bound t r a c e r ( I C 5 0 ) . C o r r e c t i o n s f o r n o n - s p e c i f i c b i n d i n g a r e made to r e f i n e t h e v a l u e s . These numbers a r e c o n v e r t e d t o p I C 5 0 s ( l o g 1/IC50) f o r r e g r e s s i o n a g a i n s t the p r e s e n c e o r absence o f s p e c i f i c substructures. I n d i c a t o r v a r i a b l e s ( I • 1.0/0.0) a r e used t o code the p r e s e n c e or absence o f a key s u b s t r u c t u r e . R e g r e s s i o n o f r e a l numbers (pIC50 s) a g a i n s t a m a t r i x o f i n d i c a t o r v a r i a b l e s i s a v a l i d p r o c e dure f o r l a r g e s e t s , as i n t h e F r e e - W i l s o n method. However, many o f the s e t s i n t h i s s t u d y a r e s m a l l (n * 7-10) and i t i s p r o b a b l e t h a t s t a t i s t i c a l measures f o r these s e t s a r e o n l y a p p r o x i m a t e . The o v e r a l l c o n s i s t e n c y o f s u b s t r u c t u r e dependence i n b o t h s m a l l and l a r g e r s e t s i s c o n s i d e r e d t o v a l i d a t e these measures i n a s e m i - q u a n t i t a t i v e sense. f

f


19.

M A G E E & KING

Binding at 4-Aminobutyrk Acid Receptor Sites283


O

MUSCIMOL

GUVACINE

NIPECOTIC ACID

ISOGUVACINE

PIPERIDINE-4-SULFON1C ACID

THPO

Figure 1. G A B A agonists and antagonists.


284

PROBING BIOACTIVE MECHANISMS


There a r e major v a r i a t i o n s i n c h e m i c a l s t r u c t u r e among the s e t s , making g e n e r a l c l a s s i f i c a t i o n o f s u b - s t r u c t u r e s d i f f i c u l t . We have attempted t o use i n d i c a t o r v a r i a b l e s t h a t d e s c r i b e comparable changes i n the same b i n d i n g r e g i o n o f d i f f e r e n t s e t s a l t h o u g h the compounds d e s c r i b e d may not be i d e n t i c a l . The f o l l o w i n g v a r i a b l e s a r e t y p i c a l . IHETS « 1.0 IHETO « 1.0 IRNG « 1.0 IDB « 1.0 IME » 1.0 I20H, I30H 1.0 IS03 1.0 INSUB « 1.0 ILNG, ISL, ISHT » 1.0 INH2 1.0 IR = ING

for for for for for for for for for for

isothiazole ring isoxazole ring aliphatic ring closure c o n j u g a t e d double bond or equivalent r i g i d i t y b r a n c h i n g m e t h y l group 2- or 3-OH groups SO3H r e p l a c e m e n t o f COOH a l k y l - s u b s t i t u t e d amino l o n g and s h o r t s p a c i n g (2- o r 4 - C H s ) a d d i t i o n a l NH group f

2

£

1.0 f o r R - c o n f i g u r a t i o n i n c h i r a l 1.0 f o r u n u s u a l or "bad" f e a t u r e s

38

analogs

A r e a s o f study a r e d i v i d e d i n t o 1. B r a i n S t u d i e s , 2. S p i n a l Cord S t u d i e s , 3. Non-Competitive B i n d i n g S t u d i e s , 4. C e l l u l a r Uptake Studies. Brain

Studies

Human. D i s p l a c e m e n t o f H-GABA from 7 d i f f e r e n t r e g i o n s o f human b r a i n t i s s u e by GABA and 8 o t h e r GABA-ergic compounds p r o v i d e s a p o w e r f u l o v e r v i e w o f the GABA b i n d i n g s i t e ( 2 4 ) . Despite s e v e r a l f o l d v a r i a t i o n s i n the ICSO's f o r these r e g i o n s , a l l p I C 5 0 s a r e c o l i n e a r ( r - 0.938-0.983). The S u b s t a n t i a N i g r a c o r r e l a t e s lowest (r - 0.938-0.966), w h i l e the r i c h e s t GABA b r a i n r e g i o n , C e r e b e l l a r C o r t e x , c o r r e l a t e s h i g h e s t w i t h the o t h e r s ( r » 0.946-0.983). More o v e r , a l l p r o v i d e e q u i v a l e n t SAR e q u a t i o n s c o n t a i n i n g the same f a c tors with s i m i l a r loadings. The f o l l o w i n g e x p r e s s i o n f o r b i n d i n g a t the Caudate Nucleus i s t y p i c a l o f the 9 e q u a t i o n s . f

pIC50(CN) - -1.35 T « 6.18 n 9

IRNG - 0.74 2.28 r - 0.985

IHETS + 0.88 4.02 s - 0.251

IDB

- 2.93 IME 10.77 F « 32.56

+

7.76

In a r e l a t e d s t u d y c o n t a i n i n g h y d r o x y l a t e d and s u l f o n i c a c i d a n a l o g s , we a g a i n f i n d e x c e l l e n t a d d i t i v i t y o f s u b s t r u c t u r a l f e a t u r e s (25). Data a r e membranes. pIC50 =• 0.813 T « 2.15 n - 11

f o r i n h i b i t i o n o f ^H-GABA b i n d i n g to human c e r e b e l l a r

IRNG - 1.20 13OH - 0.770 IS03 - 3.16 ISHT - 4.25 INSUB + 2.60 2.16 6.43 10.52 r - 0.981 s - 0.522 F - 25.98

7.78

Other s t u d i e s on human c e r e b e l l a r membranes d e a l w i t h c l o s e l y r e l a t e d a n a l o g s d i f f e r i n g m o s t l y by m e t h y l s u b s t i t u t i o n i n the 2,3and 4 - p o s i t i o n o f GABA (26,27). The SAR e q u a t i o n s show n e a r l y p e r f e c t a d d i t i v i t y o f the p o s i t i o n a l m e t h y l e f f e c t s , a l l w i t h s t r o n g n e g a t i v e c o e f f i c i e n t s (-1.55 to - 2 . 5 1 ) .


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M A G E E & KING

285

Binding at 4-Aminobutyric Acid Receptor Sites

Mouse and Rat. D i s p l a c e m e n t 3H-GABA from a mouse b r a i n membrane p r e p a r a t i o n shows a d d i t i v e r e s p o n s e s o f s u b s t r u c t u r e s s i m i l a r to those o b s e r v e d i n the human b r a i n ( 2 8 ) .


pICSO « -1.94 T « 6.18 n - 14

ISL - 0.98 IS03 - 1.05 IRNG - 2.71 INH2 - 1.67 I0H + 2.12 2.71 6.58 2.70 r - 0.962 s - 0.536 F 19.70

7.07

B i n d i n g s t u d i e s i n r a t b r a i n p r e p a r a t i o n s i n v o l v e a broad diver s i t y o f compounds and r e v e a l a v a r i e t y o f i n t e r e s t i n g e f f e c t s . In h i b i t i o n o f %-GABA b i n d i n g by O l s e n and co-workers shows the c r i t i c a l i t y o f the 4-carbon s p a c i n g o f GABA as w e l l as s e n s i t i v i t y to m e t h y l s u b s t i t u t i o n alpha to the amino group ( 2 9 ) . pIC50 - -2.37 T 7.16 n - 14

ISL - 1.05 I4ME + 3.49 r * 0.909

6.72

s - 0.481

F - 26.32

Another study c o n f i r m s the s p a c i n g c r i t i c a l i t y and d i f f e r e n t n e g a t i v e c o n t r i b u t i o n s o f R- and S - c o n f i g u r e d i n the 4 - p o s i t i o n ( 3 0 ) . D i f f e r e n t n e g a t i v e e f f e c t s f o r — groups a r e a l s o a p p a r e n t . Data a r e f o r d i s p l a c e m e n t o f rat b r a i n receptor s i t e s .

shows m e t h y l groups 2- and 3-0H 3 H-GABA i n

pIC50 « -1.67 I4ME(R) - 2.14 I4ME(S) - 1.53 ILNG - 1.69 I20H - 0.84 BOH + T « 3.40 3.75 3.25 3.13 2.10 n = 13 r - 0.890 S « 0.576 F = 5.33

6.61

Bovine. A s i n g l e s t u d y o f b o v i n e c o r t e x membranes by K r o g s g a a r d L a r s e n and co-workers p r o v i d e s some comparison w i t h human, mouse and r a t p r e p a r a t i o n s ( 3 1 ) . The s e l e c t i o n o f compounds does not r e v e a l much d e t a i l , but r e a f f i r m s the importance o f o p t i m a l s p a c i n g . Data a r e f o r c o m p e t i t i v e d i s p l a c e m e n t o f H-GABA and H-P4S. The p I C 5 0 s a r e c o l i n e a r ( r * 0.963). 3

pIC50(GABA) « 0.67 T 2.68 n - 13

IHETO - 1.98 ISL + 6.90 7.92 r - 0.931 s - 0.377

pIC50(P4S) 0.43 IDB - 1.99 ISL + T « 1.16 4.46 (not s i g n i f i c a n t ) n - 14 S p i n a l Cord

3

r - 0.820

f

F - 32.73

6.88

s - 0.680

F -

11.26

Studies

Cat. K r o g s g a a r d - L a r s e n and co-workers p r e s e n t a d a t a s e t composed p r i n c i p a l l y o f h e t e r o c y c l i c and c y c l i c GABA-ergic a n a l o g s ( 3 1 ) . Data are IC50*s f o r b i n d i n g to r e c e p t o r s i t e s i n c a t s p i n a l c o r d . Nega t i v e e f f e c t s f o r the i s o t h i a z o l e and a l i c y c l i c r i n g s a r e c l e a r l y defined.



286 pIC50 « T n -

-1.35 2.44 14

IHETS - 1.21 3.00 r - 0.867

IRNG - 2.67 ING + 2.01 4.81 s » 0.710 F 10.08


Rat. E q u i m o l a r p o t e n c i e s f o r d e p o l a r i z a t i o n o f d o r s a l ( D R ) and v e n t r a l (VR) r o o t f i b e r s o f the r a t s p i n a l c o r d have been measured f o r a v a r i e d s e t o f GABA-ergic compounds ( 3 2 ) . There a r e too many f e a t u r e s i n the d a t a s e t ( 6 ) f o r h i g h c o r r e l a t i o n o f non-IC50 d a t a , but the dominant f e a t u r e s a r e e a s i l y e x t r a c t e d . LogVR and LogDR a r e c o l i n e a r (r « 0.891). LogDR T « n -

-0.72 2.76 12

IRNG - 1.27 I30H + 0.306 2.70 r - 0.748 s « 0.429

LogVR « T -

-0.73 2.70

IRNG - 1.15 I30H + 0.260 2.35

12

r - 0.724

n -

Non-Competitive B i n d i n g

s * 0.447

F -

5.72

F -

4.95

Studies

B i n d i n g a t the GABA complex r e c e p t o r s i t e has

an a l l o s t e r i c

effect

o J

f

on the b i n d i n g o f H-diazepam a t i t s r e c e p t o r s i t e . EC50 s of diaze pam b i n d i n g have been measured f o r a v a r i e d s e t o f c y c l i c and a c y c l i c GABA-ergic compounds i n r a t f o r e b r a i n membranes ( 3 3 ) . The c o r r e l a t i o n shows n e g a t i v e c o n t r i b u t i o n s by m e t h y l groups a l p h a to amino and e s p e c i a l l y those i n the R - c o n f i g u r a t i o n . These two e f f e c t s a r e c o n founded i n the a n a l y s i s i n t h a t R-Me groups a r e c o u n t e d t w i c e . The o t h e r major e f f e c t i s the v e r y weak r e s p o n s e of c y c l i c v s . a c y c l i c s t r u c t u r e s (IRNG). pEC50 -2.54 IRNG - 1.73 IME - 1.60 IME(R) + 5.48 T = 6.97 3.94 3.65 n 11 r - 0.952 s - 0.506 F « 22.75 In a n o t h e r s t u d y o f n i n e o p t i c a l isomers o f s u b s t i t u t e d GABA, c r o t o n a t e and muscimol a n a l o g s , I C 5 0 s were measured f o r c o m p e t i t i v e b i n d i n g a g a i n s t H-GABA, H-THPI and H-P4S ( 3 4 ) . These were compared w i t h the n o n - c o m p e t i t i v e ( s t i m u l a t e d ) EC50 b i n d i n g o f H-diazepam. A l l p I C 5 0 s and the pEC50 were c o l i n e a r ( r - 0.905-0.993), d e s p i t e two o r d e r s o f magnitude i n the c o n c e n t r a t i o n r e s p o n s e . Correlations a r e weak ( r • 0.571-0.696) f o r t h i s s e t (n * 9 ) , but the major nega t i v e f a c t o r i s the double-bond r i g i d i t y o f the c r o t o n a t e s and m u s c i mols ( c o e f f i c i e n t s » -0.77 to -1.46). 1

3

3

3

3

f

B r a i n and

C e l l u l a r Uptake

Studies

E x t r a s y n a p t i c r e c e p t o r s i n the s u p p o r t i v e g l i a l c e l l s o f the CNS a r e r e s p o n s i b l e f o r the uptake ( s y n a p t i c gap c l e a r a n c e ) and t r a n s p o r t o f GABA t o t e r m i n a t e the n e u r o t r a n s m i s s i o n . These r e c e p t o r s i t e s d i f f e r markedly from the GABA^ s i t e s i n SAR response to GABA a n a l o g s . Other uptake s i t e s w i t h i n the n e u r o n a l s t r u c t u r e appear to d i f f e r from those i n the g l i a l c e l l s a c c o r d i n g t o c o m p a r a t i v e s t u d i e s by Schousboe


19.

MAGEE

287 Binding at 4-Aminobutyric Acid Receptor Sites

& KING

and co-workers ( 3 5 ) . Data a r e f o r a c y c l i c and c y c l i c GABA a n a l o g s w i t h uptake measured i n c u l t u r e d a s t r o c y t e s and mouse b r a i n m i n i slices. The p I C 5 0 s show o n l y moderate c o l i n e a r i t y ( r • 0.780), though t h e r e g r e s s i o n e q u a t i o n s show s i m i l a r f a c t o r s . f


pIC50(mouse b r a i n ) T n pIC50(astrocytes) « T « n -

-1.10 I4ME + 0.649 IR - 0.585 IRNG + 4.16 4.18 2.18 2.22 12 r - 0.856 s - 0.416 F - 7.31 -1.21 I4ME + 0.743 IR + 3.98 2.84 1.53(weak) 12 r - 0.699 s - 0.700

F - 4.29(weak)

R i n g s t r u c t u r e s and 4-methyl groups a r e n e g a t i v e f a c t o r s w h i l e the R - c o n f i g u r a t i o n i s f a v o r e d f o r b o t h c y c l i c and a c y c l i c a n a l o g s . The c o r r e l a t i o n s a r e weak t o modest but have t h e m e r i t o f b e i n g t h e o n l y a n a l y z a b l e s e t s known t o u s . S t r u c t u r a l c o m p o s i t i o n i n f i v e o t h e r d a t a s e t s was such t h a t no c l e a r d e d u c t i o n s were p o s s i b l e (28, 36-39). Summary o f S u b s t r u c t u r a l B i n d i n g E f f e c t s 3

B r a i n and S p i n a l Cord (CNS). In the b i n d i n g o f H-GABA t o human c e r e b e l l a r membranes, r i n g - c l o s e d a n a l o g s show enhanced p I C 5 0 s ( c o e f f i c i e n t « 0.813). T h i s i s an e x c e p t i o n as a l l o t h e r c o n t r i b u tions are negative. The p r e s e n c e o f a c o n j u g a t e d double bond i s p o s i t i v e i n t h e Caudate N u c l e u s f o r H-GABA b i n d i n g (IDB - 0.88) as i s the o x a z o l i n e h e t e r o - r i n g i n b o v i n e c o r t e x membranes (IHET = 0.67). These s m a l l p o s i t i v e e f f e c t s a r e opposed by g e n e r a l l y l a r g e n e g a t i v e binding e f f e c t s i n a l l other f a c t o r s . f

3

IRNG IHETS IME, I4ME INSUB I20H, I30H INH2 IS03 ISHT, ILNG, I S L Non-Competitive IRNG IME, IME(R) -

-0.72 -0.74 -1.05 -4.25 -0.84 -2.71 -0.77 -1.53

t o -1.35(4) t o -1.35(2) t o -2.93(4)

(#) o f c o e f f i c i e n t s

t o -1.69(6) t o -0.93(2) t o -3.16(7)

Binding -2.54 -1.60 t o -1.73(2)

C e l l u l a r Uptake IRNG I4ME IR »

-0.585 -1.10 t o -1.21(2) 0.649 - 0.743(2) -

(R-configuration i s favored)

Conclusions. The f a c t o r s o u t l i n e d i n the Summary o f S u b s t r u c t u r a l E f f e c t s l e a d t o a number o f b i n d i n g s i t e c o n c l u s i o n s .


288


The r e c e p t o r s i t e i s c l e a r l y asymmetric. C h i r a l i t y i s important, b o t h by s i m p l e i n s p e c t i o n o f c a s e s and s u p p o r t e d by d i s c r e t e subs t r u c t u r a l c o n t r i b u t i o n s . The R - c o n f i g u r a t i o n f o r b o t h c y c l i c and a c y c l i c a n a l o g s i s f a v o r e d a t uptake s i t e s . I f we assume the amino and c a r b o x y l groups t o b i n d s p e c i f i c a l l y , then the c a v i t y between these s i t e s i s most l i k e l y a f l e x i b l e , narrow gap. Congeners are p e r m i t t e d to b i n d ( f l e x i b i l i t y ) , but a l l s u b s t i t u e n t s from the 2- t o 4 - p o s i t i o n reduce the b i n d i n g energy ( s t e r i c o b s t r u c t i o n ) . T h i s i s t r u e even f o r s m a l l H-bonding groups l i k e NH and OH. There a r e no H-bonding s i t e s i n the gap, hence these groups e x e r t a s i m p l e s t e r i c e f f e c t i n the same sense as a CH3~group. The v a l l e y between the amino and c a r b o x y l b i n d i n g s i t e s i s c l e a r l y h y d r o p h o b i c . It i s pro b a b l e t h a t the b i n d i n g o f muscimol i s s p e c i a l and o c c u r s w i t h the r i n g i n a v e r t i c a l , r a t h e r than f l a t o r i e n t a t i o n . The r e l a t i v e weak ness o f t h i o m u s c i m o l b i n d i n g s u g g e s t s a d i f f e r e n c e i n a c i d i t y r a t h e r than a s t e r i c e f f e c t f o r a m a r g i n a l l y l a r g e r r i n g .


2

The f a c t t h a t s u l f o n i c a n a l o g s b i n d w i t h r e s p e c t a b l e a f f i n i t y s u g g e s t s two f e a t u r e s about the b i n d i n g mechanism. F i r s t , t h e r e a r e s t e r i c r e q u i r e m e n t s f o r the a c i d group ( 1 - p o s i t i o n ) s i m i l a r t o those f o r the 2- to 4 - p o s i t i o n s . The b i n d i n g s i t e f o r the a c i d group a l s o l i e s i n a r e s t r i c t e d c a v i t y . Second, z w i t t e r i o n i c b i n d i n g i s assured f o r a l l a n a l o g s as the s u l f o n i c d e r i v a t i v e s a r e 100.00% z w i t t e r i o n i c w i t h no measurable e q u i l i b r i a to n e u t r a l s p e c i e s . T h i s would suggest t h a t b i n d i n g i s by i o n - p a r i n g w i t h no a c t i v a t i o n energy, much l i k e acetylcholine. D i f f u s i o n - c o n t r o l l e d i o n - p a i r i n g may be the o n l y p r o c e s s f a s t enough f o r a n e u r a l r e s p o n s e . Another f a c t o r r e l a t i n g to the b i n d i n g mechanism i s the aminoc a r b o x y l a t e s p a c i n g , a f a c t o r t h a t i s s t r o n g l y n e g a t i v e whenever the s p a c i n g i s too s h o r t [(CH2)2l or too l o n g [ ( ^ 2 ) 4 ] . I t i s important to n o t e t h a t b i n d i n g i s not p r e v e n t e d i n these c a s e s , but o n l y weak ened. T h i s f a c t , and the many c o l i n e a r i t i e s o b s e r v e d , s u p p o r t s an i o n - p a i r i n g mechanism t h a t depends o n l y on a d i s t a n c e f u n c t i o n . Ion p a i r s a r e i n t a c t out to 5 A i n n o n - p o l a r s o l v e n t s such as benzene. I t i s r e a s o n a b l e then to assume t h a t such p a i r s a r e i n t a c t a t 2 % d i s t a n c e s on a l i p o p h i l i c enzyme s u r f a c e . T h i s degree o f l a t i t u d e would e x p l a i n the p o s i t i v e but weaker b i n d i n g o f a l l p o o r l y spaced analogs. There i s n o t h i n g i n t h i s study t h a t p r o v i d e s a l e a d to h i g h e r GABA-ergic a c t i v i t y as v i r t u a l l y a l l s u b s t r u c t u r a l f a c t o r s a r e nega t i v e i n b i n d i n g energy. The message may s i m p l y be t h a t t h e r e i s no point i n searching f o r b e t t e r analogs. R a t h e r , the d i r e c t i o n o f r e s e a r c h s h o u l d be toward d e g r a d a b l e pro-GABA or -muscimol a n a l o g s t h a t e f f i c i e n t l y l o a d the drug i n t o the CNS. Acknowledgment. T h i s study was s u p p o r t e d by the C h e m i c a l Systems L a b o r a t o r y (Aberdeen P r o v i n g Ground, MD) under C o n t r a c t No. DAAD0586-M-Q973. We w i s h to thank P r o f e s s o r John H. B l o c k o f Oregon S t a t e U n i v e r s i t y f o r h i s e x c e l l e n t G A B A - r e l a t e d l i t e r a t u r e s e a r c h under a subcontract.


19.

MAGEE & KING

Binding at 4-Aminobutyrk Acid Receptor S i t e 289

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MECHANISMS

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