Receptor Mechanisms and Biochemical Rationales - Advances in

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B. B E L L E A U

1

Departments of Biochemistry and Chemistry, Faculties of Medicine and Science, University of Ottawa, Ottawa, Ontario, Canada

The following function

biochemical

are analyzed:

(b) kinetic

approaches,

biophysical linergic

and

adrenergic

systems,

relationships

regard

to

binding

do not modulate The cholinergic

receptor

be

provide

reduce

free

and

parameters

receptors

regulatory include

unit of

times of interact

entially

or quasi-symmetrical

or

accessory agonists,

sites for drugs.

these sites would

symmetrical

receptors.

analogous

cause of their topography, with

antagonists in

structurally

binding

conclu with

the diffusion

additional

or and

uninformative

and the anionic

would

allosteric

relationships

Agonists

Neurotransmitter

sites which

and which

specificities.

the

to cho

based on linear

to be

the same physical

acetylcholinesterase homologous.

are shown

from

as applied

The following

functions

and

approaches,

(d) receptor

in drugs.

additivity

energy

structure

approaches

standpoints

to structure—action

quasi-symmetry

sions are drawn:

control

(c) molecular

and biochemical

control sites in relation molecular

aspects of receptor

(a) extra-thermodynamic

Be

prefer molecules.

Τ T n t i l a b o u t 10 years ago ( I , 2 ) , n e u r o t r a n s m i t t e r - a n d d r u g - r e c e p t o r ^

interactions h a d b e e n t r e a t e d p h e n o m e n o l o g i c a l l y rather t h a n i n

terms of m o d e r n s t r u c t u r a l c h e m i s t r y .

Since t h e n , progress i n r e c e p t o r

c h e m i s t r y has b e e n too m o d e s t to a l l o w clear-cut p r e d i c t i o n s of i m m e d i a t e t a n g i b l e v a l u e i n the field o f d r u g d e s i g n . Nonetheless, hopes are h i g h p r e s e n t l y that t h e r a t i o n a l d e s i g n of n e w t h e r a p e u t i c agents m a y n o l o n g e r be a d r e a m — a n i m p r e s s i o n w h i c h c a n m a r k e d l y affect f u t u r e e x p e r i m e n t a l approaches to d r u g d i s c o v e r y . T h e s e hopes or expectations a r e b a s e d l a r g e l y o n extrapolations f r o m a v a r i e t y of e x t r a - t h e r m o d y n a m i c 1

Present address: Department of Chemistry, McGill University, Montreal, Canada. 141

In Drug Discovery; Bloom, Barry, et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1971.

142

DRUG DISCOVERY

r e l a t i o n s h i p s b e t w e e n substituent effects i n s m a l l m o l e c u l e s ( o r l i g a n d s ) a n d b i n d i n g o n m o d e l systems.

H o w e v e r , the s t r u c t u r a l c h e m i s t r y of the

receptors of e x c i t a b l e m e m b r a n e s is as y e t p o o r l y u n d e r s t o o d , a n d e v e n less is k n o w n a b o u t the m e c h a n i s m s u n d e r l y i n g t h e i r response to l i g a n d s . A t first, t h e n , the q u e s t i o n n a t u r a l l y arises as to w h a t q u a l i t a t i v e r e l e v a n c e h a v e studies w i t h m o d e l systems to the structure a n d r e g u l a t i o n of m e m b r a n e receptors? A l l e x p l a n a t i o n s of the effects of s t r u c t u r a l or substituent

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1971 | doi: 10.1021/ba-1971-0108.ch006

changes o n b i n d i n g o r o n c h e m i c a l reactions are a l w a y s b a s e d o n some k i n d of c o m p a r i s o n w i t h m o d e l s , w h i c h m a y be s i m p l e or c o m p l e x .

In

the latter case, m a n y of the c o m p l e x i t i e s are, so to speak, a v e r a g e d o u t i n the c o m p a r i s o n s , thus a l l o w i n g f o r the g e n e r a t i o n of s i m p l e m e c h a n i s t i c p i c t u r e s . T h i s is the s i t u a t i o n , f o r instance, w h e n the h y d r o p h o b i c b o n d i n g t h e o r y ( 3 ) is u s e d to e x p l a i n the i n h e r e n t l y c o m p l e x k i n e t i c responses of e n z y m e s to o r g a n i c l i g a n d s (4,

5).

O n the other h a n d , the use

of

s i m p l e m o d e l s , s u c h as those f o r substituent effects o n h y d r o p h o b i c transfer forces, m a y o f t e n r e q u i r e m o r e c o m p l e x a n d m o r e e m p i r i c a l e x p l a n a tions because s e v e r a l a d d i t i o n a l s i m p l e m o d e l s ( f o r steric or e l e c t r o n i c effects, f o r e x a m p l e ) m u s t b e c o n s i d e r e d s i m u l t a n e o u s l y to a c h i e v e c o r r e lations w i t h effects o n a r e a l system (6).

I n a n y event, w h e n s t r u c t u r a l

or substituent effects are e x p l a i n e d i n terms of the same effects i n a m o d e l system, the parameters w h i c h are c o m p a r e d are a l w a y s of a t h e r m o d y n a m i c n a t u r e — u s u a l l y free energies i n the case of ligand—protein or d r u g r e c e p t o r i n t e r a c t i o n s — a n d so c a n n o t t e l l us a n y t h i n g a b o u t r e a c t i o n paths, as is w e l l k n o w n .

O n l y o c c a s i o n a l l y h a v e enthalpies a n d entropies b e e n

u s e d i n s u c h c o m p a r i s o n s (7, 8),

a n u n f o r t u n a t e o m i s s i o n since t h e y c a n

h e l p n a r r o w the c h o i c e of p o s s i b l e i n t e r p r e t a t i o n s .

It is clear, t h e n , that

the q u a l i t y of the answers w h i c h studies w i t h m o d e l systems c a n s u p p l y d e p e n d s c r i t i c a l l y o n the nature of the questions w h i c h are a s k e d of the model.

If n o n - s p e c i f i c questions are a s k e d , the p a r a m e t e r of s p e c i f i c i t y ,

w h i c h characterizes r e a l r e c e p t o r systems ( a n d a l l l i f e processes, f o r that m a t t e r ) c a n h a r d l y a p p e a r i n the answers, thus f o r c i n g the c o n c l u s i o n that specific d r u g s m a y not yet be d e s i g n e d r a t i o n a l l y t h r o u g h e x t r a p o l a tions f r o m m o d e l s w h i c h i n c o r p o r a t e o n l y w h a t is c o m m o n to a l l systems.

Extrothermodynamic Approaches O f t e n , s i m p l e a d d i t i v i t y r e l a t i o n s h i p s b e t w e e n one t h e r m o d y n a m i c p r o p e r t y or the other a n d some s u i t a b l e substituent p r o p e r t i e s are f o u n d (6),

b u t these r e l a t i o n s h i p s are not p a r t of t h e r m o d y n a m i c s p e r se

they are e x t r a - t h e r m o d y n a m i c ) .

(i.e.,

It is i n t e r e s t i n g that m a n y l i n e a r free

e n e r g y r e l a t i o n s h i p s b e t w e e n affinity f o r m o d e l systems a n d c e r t a i n s u b stituent p r o p e r t i e s c o u l d b e f o u n d (6).

H e n c e , the f o r m a l i n t e r a c t i o n

m e c h a n i s m s d e d u c e d f r o m s u c h m o d e l studies a p p e a r to h a v e a n exact


6.

BELLEAU

Receptor

143

Mechanisms

c o u n t e r p a r t i n r e a l systems of v e r y c o m m o n o c c u r r e n c e .

Accordingly, the

m e a s u r e d a d d i t i v e effects of l i g a n d - s u b s t i t u e n t s i n r e a l c o m p l e x systems m u s t h a v e some f a i r l y c o m m o n p h y s i c a l d i m e n s i o n s w h i c h , at first a p p r o x i m a t i o n , are i d e n t i c a l to those c o n t r o l l i n g t h e b e h a v i o r of t h e s i m p l e m o d e l systems.

P a r t processes o f s u c h w i d e o c c u r r e n c e

can only be

d r i v e n b y c o m p o n e n t forces w h i c h are c o m m o n to m a n y systems. C l e a r l y , o n l y e x t e r n a l forces ( s u c h as solute—solvent i n t e r a c t i o n s ) a p p l y so i n d i s -


c r i m i n a t e l y , w i t h t h e consequence that t h e l i g a n d - s u b s t i t u e n t s w i l l h a p p e n to p e r t u r b some p r o p e r t y w h i c h is c o m m o n to a l l t h e i r m a c r o m o l e c u lar partners

[ s u c h as p a r a l l e l changes i n t h e surface free e n e r g y o r t h e

v o l u m e s o f t h e systems

(see

below)].

I n other

words, non-specific

p e r t u r b a t i o n s w i l l b e i n d u c e d , as is t h e case i n g e n e r a l enzyme—receptor i n h i b i t i o n , b y h o m o l o g o u s series of substrate analogs

(9, JO). L i g a n d -

i n d u c e d losses of a specific r e c e p t o r o r e n z y m e p r o p e r t y ( s u c h as c a t a l y t i c a c t i v i t y ) c a n result f r o m m a n y k i n d s of u n p r o d u c t i v e p e r t u r b a t i o n s of t h e a c t i v e sites, so that p o t e n c y i n p r e v e n t i n g a n effector

molecule

f r o m i n i t i a t i n g a specific c o n f o r m a t i o n a l c h a n g e has o n l y t h e d i m e n s i o n of a r e l a t i v e free e n e r g y c h a n g e , w h i c h m a y b e subject t o m o d u l a t i o n s i m p l y b y c h a n g i n g t h e substituent i n t e r a c t i o n w i t h t h e m e d i u m . I t f o l l o w s that

the observation

of a d d i t i v i t y f u n c t i o n s b a s e d

o n l y o n AG

changes cannot serve to i d e n t i f y t h e b i n d i n g r e a c t i o n paths, w h i c h m u s t d i s t i n g u i s h o n e r e c e p t o r system f r o m another, a n d e v e n t h o u g h e x t r a p o lations of t h e f u n c t i o n s m a y serve to i m p r o v e o v e r - a l l s t r e n g t h o f b i n d i n g , n o n - s p e c i f i c interactions w i l l t e n d to d o m i n a t e t h e p i c t u r e . I n fact, i t is d e v i a t i o n s f r o m a d d i t i v i t y f u n c t i o n s w h i c h are i n d i c a t i v e of s p e c i f i c i t y effects, a measure of w h i c h is sometimes g i v e n b y t h e n u m b e r o f s e m i e m p i r i c a l parameters w h i c h m u s t b e c o n s i d e r e d s i m u l t a n e o u s l y t o a c h i e v e correlations w i t h r e a l systems ( 6 )

T h e b a s i c q u e s t i o n as to w h e t h e r t h e

r e s p e c t i v e effects of activators ( n e u r o e f f e c t o r s )

a n d inhibitors

(antago-

nists) measure t h e same t h e r m o d y n a m i c p r o p e r t y d i d n o t e v e n attention u n t i l r e c e n t l y ( 1 0 , 11; see below).

a l l o w i n g a n i n s i g h t i n t o t h e m o l e c u l a r m e c h a n i s m s of r e c e p t o r to

neuroeff ectors

a r e therefore

needed

attract

Other more suitable models badly

(see

below).

responses Before

e l a b o r a t i n g o n this subject i t w o u l d b e advantageous to d e s c r i b e b r i e f l y some recent advances i n k n o w l e d g e of t h e k i n e t i c b e h a v i o r o f t h e e x c i t a b l e m e m b r a n e receptors as i t bears o n t h e p r o b l e m of i n t e r a c t i o n specificities.

Kinetic Approaches L i t t l e a t t e n t i o n n e e d b e d e v o t e d here to t h e c l a s s i c a l k i n e t i c analyses of d r u g - r e c e p t o r interactions dose—response curves.

as b a s e d o n t h e g e n e r a l a p p e a r a n c e

of

T h i s a p p r o a c h to r e c e p t o r m e c h a n i s m s has b e e n

d o c u m e n t e d f u l l y , a n d n o s i g n i f i c a n t progress has b e e n m a d e since w h i c h


144

DRUG DISCOVERY

c a n h e l p a c h i e v e a n i m p r o v e d u n d e r s t a n d i n g i n s t r u c t u r a l terms.

This

a p p r o a c h has s e r v e d essentially to s h o w that the response c u r v e of c e r t a i n m e m b r a n e s to g r a d e d increases i n l i g a n d concentrations o f t e n obeys the L a n g m u i r i s o t h e r m (12)

a n d that s t r u c t u r a l l y r e l a t e d d r u g s c a n i n d u c e

different m a x i m a l responses ( a p h e n o m e n o n at the o r i g i n of the c o n c e p t of " i n t r i n s i c a c t i v i t y " ) (12). (13)

H o w e v e r , it has b e e n n o t e d i n r e c e n t years

that d e p e n d i n g o n the t y p e of e x c i t a b l e

membrane, the

dose-


response curves c a n b e s l i g h t l y S - s h a p e d , a n d sometimes e x t r e m e l y s h a r p (as i n " a l l - o r - n o t h i n g r e s p o n s e s " ) .

T h e s e observations suggest a p r e v i -

o u s l y u n s u s p e c t e d p r o p e r t y of receptors a n d associated m o l e c u l e s .

By

e x t r a p o l a t i n g f r o m the k n o w n b e h a v i o r of r e g u l a t o r y e n z y m e s (14,

15),

a m o d e l was proposed where receptor protomers w o u l d display cooperat i v e responses t y p i c a l of h i g h l y o r d e r e d structures (16).

B a s e d o n the

a s s u m p t i o n that the r e c e p t o r c a n exist i n o n l y t w o c o n f o r m a t i o n a l states, a t h e o r e t i c a l treatment, u s i n g the m o l e c u l a r

field

approximation

(17)

of the c o o p e r a t i v e c o n f o r m a t i o n a l t r a n s i t i o n , a l l o w e d the c o n s t r u c t i o n of dose—response curves c l o s e l y a p p r o x i m a t i n g c e r t a i n r e a l situations. A s i n t h e case of the t h e o r e t i c a l t r e a t m e n t of r e g u l a t o r y e n z y m e b e h a v i o r

(15),

the b a s i c a s s u m p t i o n w a s m a d e that the r e v e r s i b l e c o n f o r m a t i o n a l changes of the p r o t o m e r pre-exist l i g a n d b i n d i n g .

H o w e v e r , recent e x p e r i m e n t a l

e v i d e n c e has a c c u m u l a t e d w h i c h casts serious d o u b t o n the v a l i d i t y of this free e q u i l i b r i u m m o d e l (18,

19).

It n o w seems m o r e e v i d e n t that

phase-transitions i n o r d e r e d structures r e q u i r e n u c l e a t i o n , a step w h i c h was also i n c o r p o r a t e d i n a recent t r e a t m e n t of o s c i l l a t i n g p h a s e transitions as r e l a t e d to c o o p e r a t i v e effects i n steady-state branes (20).

t r a n s p o r t across m e m -

N e v e r t h e l e s s , c o o p e r a t i v i t y of r e c e p t o r units is a n i m p o r t a n t

factor i n propagating m e m b r a n e depolarizations a n d nerve impulses and offers a r a t i o n a l basis f o r a l l - o r - n o t h i n g processes i n g e n e r a l . T h e r e a l i t y of c o o p e r a t i v e p h e n o m e n a at the r e c e p t o r l e v e l of m e m b r a n e

lattices

denies the existence of s i m p l e a d d i t i v i t y f u n c t i o n s b e t w e e n l i g a n d affinities a n d substituent p r o p e r t i e s a n d f u r t h e r suggests that those l i g a n d s w h i c h m a y a n t a g o n i z e receptors b y c o o p e r a t i v e m e c h a n i s m s

(assuming

that m o r e t h a n t w o r e c e p t o r c o n f o r m a t i o n s c a n d i s p l a y c o o p e r a t i v i t y ) s h o u l d be s o u g h t a c t i v e l y . T h i s aspect of d r u g r e s e a r c h is e m p h a s i z e d below.

Molecular Approaches Biophysical Aspects.

T h e n a t u r e of the m o l e c u l a r responses of re-

ceptors to activators ( or nucleators ) a n d i n h i b i t o r s ( or antagonists ) m u s t b e u n d e r s t o o d i n m o l e c u l a r terms i f 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 are to b e r a t i o n a l i z e d . P h e n o m e n o l o g i c a l l y , r e c e p t o r responses are r e v e a l e d w h e n the n a t u r e of the m e d i u m i n contact w i t h excitable m e m b r a n e s is


6.

BELLEAU

Receptor

145

Mechanisms

v a r i e d b y a d d i n g salts, acids, o r v a r i o u s o r g a n i c l i g a n d s , thus l e a d i n g to a s u d d e n c h a n g e i n t h e p h y s i c o c h e m i c a l p r o p e r t i e s o f the m e m b r a n e s . s h a r p t r a n s i t i o n c a n best b e a s c r i b e d to c o n f o r m a t i o n a l changes m a y b e c o o p e r a t i v e ; see above)

This

(which

i n some s p e c i a l b i o p o l y m e r s , f r o m a

p h y s i c a l state w h i c h n o r m a l l y braces a s t r u c t u r e d m a t r i x o f l i p r o t e i n s to o n e w h i c h spurs a cascade o f b i o p h y s i c a l events u l t i m a t e l y p r o d u c i n g d e p o l a r i z a t i o n of the m e m b r a n e ,


relaxation

of muscle

fibers.

a n d i n s e v e r a l cases c o n t r a c t i o n or

T h e receptors

f o r the n e u r o t r a n s m i t t e r s

a c e t y l c h o l i n e ( A C h ) a n d the c a t e c h o l a m i n e h o r m o n e s a r e t h e best k n o w n examples o f s u c h specific b i o p o l y m e r s . T h e y are g e n e r a l l y b e l i e v e d to b e proteins, a n d t h e phase

t r a n s i t i o n f r o m t h e " b r a c i n g " state Β to the

" s p u r r i n g " state S c a n b e n u c l e a t e d b y A C h a n d r e l a t e d q u a t e r n a r y salts or b y n o r e p i n e p h r i n e ( N E ) a n d some of its analogs.

B y analogy w i t h

the crystallites a n d other h i g h l y o r d e r e d p o l y m e r s , the r e v e r s i b l e phase t r a n s i t i o n B ^± S c a n g i v e rise to s i m p l e s i g m o i d s , w h o s e steepness o f slope d e p e n d s o n t h e degree o f c o o p e r a t i v i t y b e t w e e n t h e receptors

(see

above ). T h e p r i n c i p l e s at w o r k m a y b e i l l u s t r a t e d b y t h e c l a s s i c a l m o d e l of t h e h e l i x - c o i l t r a n s i t i o n i n p o l y p e p t i d e s a n d p r o t e i n s (21).

However,

f o r t h e case at h a n d , it is best to i m a g i n e that the r e c e p t o r t r a n s i t i o n involves a change

f r o m o n e o r d e r e d state to another

structured one,

rather than a r a n d o m coil w h i c h w o u l d h a r d l y allow for ready reversi bility.

O b v i o u s l y , nucleators o f t h e t r a n s i t i o n m a y b i n d o n b o t h t h e Β

a n d S states, a n d r e v e r s a l of t h e t r a n s i t i o n ( S —» Β ) m a y r e q u i r e n u c l e a t i o n b y other effectors receptors.

w h i c h m a y b e released

i n t h e S state o f t h e

W e n o w c o m e to t h e case w h e r e l a r g e g r o u p s o f l i g a n d s o r

drugs combine w i t h the receptor

w h i l e b e i n g u n a b l e to n u c l e a t e t h e

t r a n s i t i o n f r o m t h e Β to t h e S state.

M a n y of these l i g a n d s or d r u g s

p r e v e n t t h e a c t i o n of nucleators b y a process w h i c h often f o l l o w s the l a w of c o m p e t i t i v e k i n e t i c s (12),

so that the o b s e r v e d t h e r m o d y n a m i c p r o p

erty m e a s u r e d b y these m u t u a l e x c l u s i o n effects has t h e d i m e n s i o n o f a r e l a t i v e A G c h a n g e ( i n t h e absence o f c o o p e r a t i v i t y ) , w h i c h b e i n g s u b stituent sensitive m a y a l l o w t h e generation of e x t r a - t h e r m o d y n a m i c r e l a tionships (see

above).

H o w e v e r , the t r u l y i m p o r t a n t q u e s t i o n for t h e f u t u r e i s : w h a t t h e r m o d y n a m i c p r o p e r t y is m e a s u r e d b y t h e o b s e r v e d effects of n u c l e a t o r s o r agonists?

I n other w o r d s , d o nucleators a n d antagonists m o d u l a t e t h e

same p h y s i c a l v a r i a b l e s ? f o l l o w i n g reason.

A n e g a t i v e a n s w e r appears most l i k e l y f o r t h e

T h e seasoned

m e d i c i n a l c h e m i s t has f r e q u e n t l y o b

s e r v e d that a n increase i n t h e size o r h y d r o p h o b i c b u l k o f agonist s u b stituents w i l l often t r a n s f o r m t h e m i n t o antagonists

(10, 12), thus s u g

gesting s t r o n g l y that a n excess o f a p o l a r b u l k a l l o w s t h e p a r t i c i p a t i o n o f p h y s i c a l v a r i a b l e s w h i c h are n o t n o r m a l l y d i s t u r b e d b y the nucleators themselves.

A c c o r d i n g l y , agonists a n d antagonists

must produce


their

146

DRUG DISCOVERY

effects b y w a y of different p h y s i c a l i n t e r a c t i o n m e c h a n i s m s .

Hence, if

the m e a s u r e d t h e r m o d y n a m i c p r o p e r t y f o r antagonist effects is a r e l a t i v e AG change, this m a y n o t b e true f o r nucleators.

I n other w o r d s , agonist

potencies cannot b e a s i m p l e f u n c t i o n of affinity f o r t h e receptors, as is frequently implied

(12).

R a t h e r , a s s u m i n g that Β a n d S states a t t a i n

e q u i l i b r i u m , r e l a t i v e n u c l e a t o r potencies s h o u l d b e a d i r e c t f u n c t i o n o f the p o s i t i o n o f t h e c o n f o r m a t i o n a l e q u i l i b r i u m , so that p o t e n c y


h a v e the d i m e n s i o n of a c o n f o r m a t i o n a l free e n e r g y c h a n g e .

would

I t is clear,

t h e n , that agonist affinities f o r receptors or m o d e l systems [ s u c h as a c e t y l cholinesterase

(-22)] h a v e n o b e a r i n g o n t h e degree o f c o n f o r m a t i o n a l

shift i n d u c e d b y nucleators.

Much

confusion w o u l d be eliminated if

these considerations w e r e p r o p e r l y w e i g h t e d . I n contrast to t h e i n h i b i t e d p h y s i c a l state o f the receptor f o r w h i c h m a n y r e l a t i v e l y c h a o t i c , u n p r o d u c t i v e c o n f o r m a t i o n s are possible, i t seems p r o b a b l e that t h e s p u r r e d p h y s i c a l state s h o u l d possess e x c l u s i v e characteristics u n i q u e l y a l l o w i n g f o r c o o p e r a t i v e b e h a v i o r (16).

I d e a l l y , then, nucleators s h o u l d a l l m o d u

late q u a l i t a t i v e l y s i m i l a r p h y s i c a l v a r i a b l e s since o t h e r w i s e m o r e t h a n one s p u r r e d state w o u l d h a v e to b e p o s t u l a t e d .

O n e k e y factor

which

m a y c o n t r i b u t e i m p o r t a n t l y to the m a g n i f i c a t i o n of t h e u n i t a r y m o l e c u l a r response to nucelators is c o o p e r a t i v i t y b e t w e e n r e c e p t o r p r o m o t e r s above),

(see

a n d i f w e assume that besides t h e Β a n d S states, a d d i t i o n a l b u t

u n p r o d u c t i v e states c a n also d i s p l a y c o o p e r a t i v i t y , h i g h l y specific a n d potent t h e r a p e u t i c agents w o u l d b e c o m e accessible.

T h e r e a l i t y o f this

p o s s i b i l i t y appears to b e c o n f i r m e d , at least i n p r i n c i p l e , b y t h e m a n n e r i n w h i c h t h e c o l i c i n class o f a n t i b i o t i c s p r o d u c e t h e i r effects (16,

23).

O t h e r m o d e l s c a n b e c o n s t r u c t e d w h i c h l e a d to s i m i l a r conclusions regarding the divergence of interaction mechanisms antagonists.

f o r agonists a n d

It b e c o m e s o f c o n s i d e r a b l e interest, t h e n , t o d i s c o v e r w h i c h

measurable physical parameter(s)

m a y h a v e d i r e c t r e l e v a n c e to n u c l e a t o r

potencies at the r e c e p t o r l e v e l . A t m e m b r a n e surfaces

macromolecular

structures s u c h as receptors o c c u r at a n aqueous i n t e r f a c e . H e n c e , s o l u t e solvent interactions i n b o t h the i n i t i a l a n d final states o f t h e b i n d i n g partners w i l l c o n t r i b u t e to t h e r e l a t i v e t h e r m o d y n a m i c s t a b i l i t y o f t h e addition complex.

F o r a t i g h t l y p a c k e d g l o b u l a r l i p o p r o t e i n , s u c h as is

p o s t u l a t e d f o r t h e receptor, t h e u p t a k e o f a l i g a n d w i l l necessarily p r o duce a v o l u m e change w h i c h m a y be positive or negative.

T h e volume

c h a n g e w i l l result i n a p a r a l l e l c h a n g e i n t h e h y d r o p h o b i c o r h y d r o p h i l i c area o f t h e r e c e p t o r " m i c e l l e " w h i c h is e x p o s e d to t h e solvent a n d a d j a c e n t macromolecules.

T h e receptor-water

i n t e r f a c i a l tension w i l l

therefore

be c h a n g e d b y t h e l i g a n d , a n d it f o l l o w s that t h e surface f r e e e n e r g y o f the r e c e p t o r

protein must always be changed b y the ligands.

l i g a n d - i n d u c e d v o l u m e changes conformational perturbations.

These

m a y as w e l l b e l o o s e l y r e f e r r e d to as

F o r c o n v e n i e n c e , w e c a n easily c o n c e i v e


Library American Chemical Society 6.

BELLEAU

Receptor

147

Mechanisms

that antagonists or i n h i b i t o r s penetrate the surface of the r e c e p t o r m i c e l l e , c a u s i n g a v o l u m e increase, w h e r e a s nucleators or agonists m a y cause, b y way

of d e s o l v a t i o n , a v o l u m e c o n t r a c t i o n w h i c h m u s t h a v e a m i n i m u m

v a l u e i n o r d e r that the d e s i r e d m a c r o s c o p i c effects m a y b e

observed.

T h i s m o d e l also p r e d i c t s that different p h y s i c a l v a r i a b l e s w o u l d c o n t r o l the s t a b i l i t y of the " c o n t r a c t e d " ( o r s p u r r e d ) a n d the " e x p a n d e d " ( u n productive)

states

respectively.

N o w , the

change

from

the

initial


" b r a c e d " state to a u n i q u e l y s p u r r e d state m u s t o c c u r b y w a y of

a

s t e r e o t y p e d b o n d - b r e a k i n g a n d b o n d - m a k i n g process i n w h i c h t h e a q u e ous i n t e r f a c e m u s t p a r t i c i p a t e a c t i v e l y . T h i s suggests that the e n t h a l p i e s a n d entropies of b i n d i n g s h o u l d b e m a r k e d l y sensitive to s t r u c t u r a l effects i n the nucleators b u t n o t the free-energies since the p o s t u l a t e d u n i q u e n e s s of the p h y s i c a l m e c h a n i s m u n d e r l y i n g the v o l u m e c o n t r a c t i o n s w i l l l e a d to c o m p e n s a t e d

fluctuations

i n the enthalpies a n d entropies of b i n d i n g .

These predictions have recently been experimentally verified t h r o u g h extensive t h e r m o d y n a m i c studies of q u a t e r n a r y salt b i n d i n g o n t h e e n z y m e acetylcholinesterase

(AChE)

as a m o d e l ( 7 ) .

Of

considerable

interest w a s the a d d i t i o n a l finding of a c o r r e l a t i o n b e t w e e n t h e enthalpies of salt b i n d i n g ( b u t n o t the free e n e r g i e s ) a n d the degree of c o n f o r m a t i o n a l response of the e n z y m e (11).

It w a s also n o t e d t h a t p h y s i c a l

changes at the aqueous i n t e r f a c e a p p e a r e d to d o m i n a t e the p i c t u r e , a n d e v i d e n c e was a d d u c e d that i n d e e d different p h y s i c a l v a r i a b l e s m a y b e m o d u l a t e d b y r e c e p t o r nucleators a n d antagonists q u i r e d b y t h e o r y (10).

r e s p e c t i v e l y , as re-

A s i t turns out, agonist potencies a p p e a r to b e a

f u n c t i o n of m o d u l a t i o n i n t e n s i t y ( o r degree of c o n f o r m a t i o n a l s t a b i l i z a t i o n of the S-state), w h e r e a s antagonist potencies are s i m p l e f u n c t i o n s of the t h e r m o d y n a m i c s t a b i l i t y of the u n p r o d u c t i v e a d d i t i o n P a r a l l e l studies w i t h

intact membrane

complexes.

preparations w i l l u n d o u b t e d l y

prove fruitful. L o o k i n g a h e a d , i t w o u l d seem that d r u g s c a p a b l e of i n d u c i n g rec e p t o r rearrangements

b y cooperative mechanisms w o u l d have m u c h

greater v a l u e i n t h e r a p e u t i c s .

T o a c h i e v e this, i n c r e a s e d a t t e n t i o n m a y

h a v e to b e p a i d to p r o t e i n t o p o g r a p h y i f d r u g structures are to b e d e s i g n e d w h i c h are c o m p a t i b l e w i t h l o w e n e r g y r e c e p t o r

conformations

d i s p l a y i n g c o o p e r a t i v i t y . I n this c o n n e c t i o n , it is not i n c o n c e i v a b l e t h a t one of the reasons w h y so m a n y h i g h l y a c t i v e d r u g s are f o u n d a m o n g the n a t u r a l p r o d u c t s ( s u c h as the a l k a l o i d s , f o r instance ) m a y b e r e l a t e d to the

f a c t that

as the

e n d p r o d u c t s of p r o t e i n actions

themselves,

they are necessarily e n d o w e d w i t h " b u i l t - i n k n o w l e d g e " a b o u t p r o t e i n t o p o g r a p h y . It m a y b e w o r t h w h i l e , t h e n , to use the a c c u m u l a t e d k n o w l edge a b o u t the t h r e e - d i m e n s i o n a l structures of p r o t e i n s as a s t a r t i n g p o i n t for d r u g d e s i g n .


148

DRUG DISCOVERY

B i o c h e m i c a l A s p e c t s . A l t h o u g h serious efforts h a v e b e e n m a d e over the past f e w years to isolate a n d c h a r a c t e r i z e t h e n e u r o t r a n s m i t t e r r e c e p tors b y t h e m e t h o d o f affinity l a b e l l i n g (24, 25),

t h e results h a v e thus f a r

been generally disappointing a n d ambiguous. T h e isolation of a muscar o n e b i n d i n g p r o t e i n f r a c t i o n f r o m e l e c t r o p l a x tissue a n d t h o u g h t to b e d i s t i n c t f r o m acetylcholinesterase

( A C h E ) has b e e n r e p o r t e d (26),

but

it is too e a r l y to assess t h e p o s s i b l e significance of these observations.


N e v e r t h e l e s s , s i g n i f i c a n t advances i n t h e in situ s t r u c t u r a l c h e m i s t r y o f b o t h t h e c h o l i n e r g i c a n d a d r e n e r g i c systems h a v e b e e n m a d e i n r e c e n t years, a n d t h e observations m a y suggest n e w avenues t o d r u g research. CHOLINERGIC SYSTEMS: DIRECT AND M O D E L OBSERVATIONS. S i n c e the b a c k g r o u n d of this subject has r e c e n t l y b e e n c o v e r e d e l s e w h e r e o n l y some of t h e i m p o r t a n t n e w h i g h l i g h t s are d i s c u s s e d here.

(27), These

c o n c e r n t h e A C h r e c e p t o r of e l e c t r o p l a x tissue, of t h e m o u s e d i a p h r a g m a n d the f r o g t o e m u s c l e , e s p e c i a l l y i n r e l a t i o n to A C h E

as a m o d e l

receptor. N e w approaches

to t h e c h e m i s t r y of the e e l e l e c t r o p l a x

h a v e b e e n d e s i g n e d b y K a r l i n (24, 28).

receptor

I t w a s s h o w n that d i t h i o t h r e i t o l

( D T T ) r e d u c e s a d i s u l f i d e b o n d at or n e a r the A C h - r e c e p t o r l e v e l , a n d one of t h e l i b e r a t e d thiols c a n b e i r r e v e r s i b l y a l k y l a t e d b y a p p r o p r i a t e t h i o l reagents c a r r y i n g a q u a t e r n a r y m o i e t y . T h e p o s s i b i l i t y is therefore a v a i l a b l e of a c h i e v i n g selective affinity l a b e l l i n g of t h e D D T - g e n e r a t e d t h i o l g r o u p , a n d p r e l i m i n a r y e x p e r i m e n t s a l o n g this l i n e h a v e

already

b e e n c a r r i e d o u t (-29). O f c o n s i d e r a b l e interest w a s the o b s e r v a t i o n that the D T T - t r e a t e d e l e c t r o p l a x is a c t i v a t e d b y h e x a m e t h o n i u m i n s t e a d o f being blocked. T h e r e d u c e d receptor can be reconstituted functionally b y o x i d a t i o n w i t h E U m a n ' s reagent.

A p p a r e n t l y , these t h i o l reagents w e r e

w i t h o u t effect o n s o l u b i l i z e d A C h E (see below, h o w e v e r ) . It thus appears p o s s i b l e that n u c l e a t i o n of phase transitions b y agonists m a y n o r m a l l y b e t r a n s m i t t e d f r o m the r e c e p t o r

to t h e other m e m b r a n e

components b y

w a y of d i s u l f i d e b o n d s a c t i n g as p u l l e r s . T h i s suggests that n e w u s e f u l d r u g s m a y b e d e s i g n e d w h i c h w o u l d b e e n d o w e d w i t h specific i n t r i n s i c p o w e r to affect d i s u l f i d e l i n k s o r to e n g a g e i n d i s u l f i d e i n t e r c h a n g e tions at the r e c e p t o r l e v e l of e x c i t a b l e m e m b r a n e s .

reac-

T h i s area of m e d i c i n a l

c h e m i s t r y has h a r d l y b e e n e x p l o r e d , except i n t h e field o f r a d i o p r o t e c t i v e agents w h o s e m e c h a n i s m s of a c t i o n are s t i l l p o o r l y u n d e r s t o o d . I n r e c e n t years, m u c h attention has b e e n p a i d to t h e r o l e o f c a l c i u m i n r e c e p t o r p h e n o m e n a , besides its b e t t e r k n o w n i n v o l v e m e n t i n e n z y m e a c t i v a t i o n a n d c o n t r a c t i o n o f m u s c l e fibers. E a r l i e r observations b y C s i l l i k and Savay

(30)

a n d N a k a m u r a et al.

co-workers

(32)

to s t u d y c a l c i u m release at t h e m o t o r e n d plates o f

(31)

l e d Tazieff-Depierre and

m o u s e d i a p h r a g m i n r e l a t i o n to r e c e p t o r a c t i v a t i o n a n d i n h i b i t i o n .

Of

c o n s i d e r a b l e interest w a s the o b s e r v a t i o n that w h e r e a s m e m b r a n e d e -


6.

BELLEAU

polarizers

Receptor

(carbachol,

149

Mechanisms succinyldicholine, etc) liberate

c a l c i u m o n the

m o t o r e n d plates, n o n - d e p o l a r i z e r s s u c h as d - t u b o c u r a r i n e f a i l to d o so. It a p p e a r e d that t h e greater t h e d e p o l a r i z i n g p o t e n c y o f t h e effector, t h e l a r g e r w a s t h e a m o u n t o f c a l c i u m l i b e r a t e d . T h i s suggests a n e w a p p r o a c h to t h e e v a l u a t i o n of r e l a t i v e potencies t h r o u g h d i r e c t c h e m i c a l m e t h o d s rather t h a n c l a s s i c a l p h a r m a c o l o g i c a l p r o c e d u r e s .

It w a s also s h o w n t h a t

c a l c i u m l i b e r a t i o n leads to a c t i v a t i o n of v i c i n a l m e m b r a n e - b o u n d A C h E ,


whereas c e r t a i n v e n o m toxins a n d p o t a s s i u m ions i n d u c e c a l c i u m release at t h e m u s c l e fiber l e v e l a n d n o t o n t h e m o t o r e n d plates.

Hence: i n

a c c o r d w i t h e a r l i e r concepts ( see below ), c a l c i u m appears to b e c r i t i c a l l y i n v o l v e d i n t h e s t a b i l i z a t i o n of receptor c o n f o r m a t i o n , a n d its l i b e r a t i o n b y nucleators w o u l d serve t h e d u a l p u r p o s e o f s t a b i l i z i n g t h e s p u r r e d p h y s i c a l state a n d o f a c t i v a t i n g e n z y m e s c o n t r o l l i n g g l y c o g e n o l y s i s a n d the c o n t r a c t i o n of t h e m u s c l e

fibers.

It w o u l d b e of interest,

therefore,

to d e s i g n d r u g s ( o r p r o t e i n r e a g e n t s ) w h i c h c a n s p e c i f i c a l l y i n f l u e n c e c a l c i u m interactions w i t h receptors.

U p to n o w , o n l y n o n - s p e c i f i c chelat-

i n g agents h a v e b e e n u s e d , a n d these m o s t l y as a n a l y t i c a l tools.

Interest-

ingly, the proposal was recently

receptor

advanced

inhibitors of the alkylating variety (quaternary b l o c k a c a l c i u m b i n d i n g site

(see

below),

that c e r t a i n

(33)

a z i r i d i n i u m salts) m a y

thus c r e a t i n g a p o t e n t i a l l y

v a l u a b l e p r e c e d e n t i n t h e field. I n o u r o w n laboratories, t h e w o r k i n g h y p o t h e s i s that t h e a n i o n i c s i t e - r i c h c h a i n of A C h E

m a y be conformationally homologous

to t h e

A C h r e c e p t o r (34, 35, 37) has n o w b e e n e x p l o r e d sufficiently to a l l o w a p a r t i a l r e s o l u t i o n of t h e l o n g s t a n d i n g controversies r e g a r d i n g t h e r e l a t i o n of the e n z y m e to the receptor. bearing i n mind.

T h e following highlights m a y b e w o r t h

T h e e n z y m e i n c l u d e s t w o p h y s i c a l l y different r e g i o n s ,

one w h i c h carries u n s p e c i f i c esteratic a c t i v i t y , a n d another w h i c h carries at least f o u r ( a n d p r o b a b l y m o r e )

a n i o n i c sites, t w o of w h i c h c a n b e

c o v a l e n t l y m a s k e d b y t h e a l k y l a t i n g agent N , N - d i m e t h y l - 2 - p h e n y l a z i r i dinium chloride ( D P A ) creased

(36, 37).

T h e D P A - m o d i f i e d e n z y m e has i n -

esteratic a c t i v i t y t o w a r d n e u t r a l substrates

i n d o p h e n y l acetate o r I P A — b u t none w h a t s o e v e r moles o f

1 4

(38,

39)—such

as

toward A C h . T w o

C - D P A r e a c t p e r active s u b u n i t , a n d o n e of t h e c o v a l e n t l y -

b o u n d D P A m o l e c u l e s is r e a d i l y r e m o v e d at p H 9 - 9 . 5 , thus l e a d i n g t o a m o n o - D P A e n z y m e species w h i c h has s t i l l h i g h e r a c t i v i t y t o w a r d I P A b u t n o n e at a l l t o w a r d A C h (36).

T h e b i s - D P A e n z y m e is c o n f o r m a -

t i o n a l l y u n r e s p o n s i v e to d e c a m e t h o n i u m a n d d - t u b o c u r a r i n e ( I P A a s s a y ) , b u t t h e m o n o - D P A species is as responsive as t h e n a t i v e e n z y m e to t h e latter d r u g , w h i l e b e i n g u n a f f e c t e d b y t h e f o r m e r ( 3 6 ) . H e n c e , d e p o l a r i z i n g a n d n o n - d e p o l a r i z i n g d r u g s i n t e r a c t w i t h different a n i o n i c sites o n the a n i o n i c c h a i n of A C h E , a t o p o g r a p h i c a l d i s t i n c t i o n w h i c h c o n c e i v a b l y u n d e r l i e s t h e i r d i f f e r e n t m o d e s of a c t i o n o n t h e m o t o r e n d p l a t e receptors


150

DRUG DISCOVERY

( F i g u r e 1 ). T h e postulate that d e p o l a r i z e r s a n d n o n - d e p o l a r i z e r s i n d u c e d i v e r g e n t c o n f o r m a t i o n a l responses

at t h e receptor l e v e l w a s c o n f i r m e d

u s i n g t h e c o n f o r m a t i o n a l responsiveness

o f the a n i o n i c c h a i n of A C h E

as a m o d e l . W h e n A C h E is exposed to m e t h a n e s u l f o n y l

fluoride

(MSF),

the esteratic site u n d e r g o e s i r r e v e r s i b l e s u l f o n y l a t i o n , t h e rate o f w h i c h is m a r k e d l y sensitive to q u a t e r n a r y c h a i n (40).

i o n interactions

changes i n i t i a t e d i n the a n i o n i c c h a i n (41),


w i t h the anionic

T h e s e k i n e t i c effects are thus i n d i c a t i v e o f c o n f o r m a t i o n a l a n d of c o n s i d e r a b l e

w a s t h e finding that w he re a s a l k y l m o n o q u a t e r n a r y (34)

interest

a n d bisquater-

n a r y d e p o l a r i z e r s s t r o n g l y s t i m u l a t e t h e rate of the M S F r e a c t i o n

(42),

n o n - d e p o l a r i z e r s s u c h as d - t u b o c u r a r i n e a n d g a l l a m i n e i n h i b i t t h e reaction b y a non-competitive mechanism

(42).

Moreover, the

strongest

k i n e t i c effects w e r e o b t a i n e d w i t h t h e best d e p o l a r i z e r s o f the m o t o r e n d p l a t e m e m b r a n e . T h e s e m o d e l studies p r o v e b e y o n d q u e s t i o n t h a t r e l a t i v e affinities as s u c h f o r A C h E (as m e a s u r e d b y the A C h assay) are i r r e l e v a n t w h e n the p u r p o s e is to establish the existence of s i m i l a r i t i e s o r differences b e t w e e n the a n i o n i c c h a i n of the e n z y m e a n d the A C h receptors.

I t is

true affinity a n d t h e degree as w e l l as n a t u r e of t h e response w h i c h h a v e a bearing

on drug-receptor

interactions,

as w a s p o i n t e d o u t a b o v e .

Recently, conclusive structural evidence for our anionic chain hypothesis w a s p r o v i d e d b y L e u z i n g e r (43), p r e p a r a t i o n (44)

w h o s h o w e d that h i s c r y s t a l l i n e A C h E

(65,000 daltons p e r a c t i v e s u b u n i t ) is a c t u a l l y m a d e u p

of t w o different chains of e q u a l m o l e c u l a r w e i g h t s a n d c h e m i c a l l y d i s -

Figure 1. Schematic of the probable physical structure of acetylcholinesterase. One physical region carries the esteratic site, which is proximal to one anionic site (Site I); the other region would carry at least four anionic sites, and would be homologous to the acetylcholine receptor of the motor end plate excitable membrane. Sites I and II are masked by DP A, but Site II can be regenerated at alkaline pH. Decamethonium (C ) would interact at least with Sites I and II whereas curare would bind at III and IV, and perhaps at II and III. Most quaternary salt suhstituents bind on the anionic chain [exo-binding (26, 36, 42, 3 5 ) ] , thus projecting away from the esteratic region. 10


6.

BELLEAU

Receptor

151

Mechanisms

sociatable b y exposure to m e r c a p t o e t h a n o l i n u r e a s o l u t i o n . I n a g r e e m e n t w i t h h y p o t h e s i s , o n l y one of the separated chains carries l a b e l e d p h o s p h o r u s after t r e a t m e n t of the n a t i v e e n z y m e w i t h r a d i o a c t i v e d i i s o p r o p y l fluorophosphonate

(DFP).

T h e s e n e w observations

thus c o n f i r m o u r

h y p o t h e s i s that A C h E is a n o n - s p e c i f i c esterase o n w h i c h a c o n f o r m a t i o n a l l y r e s p o n s i v e a n i o n i c c h a i n a c t i n g as a n A C h - p r o t o r e c e p t o r has b e e n g r a f t e d (27),

(Figure 1).

I n d e e d , a s o m e w h a t analogous c o n f o r m a t i o n a l


responsiveness of the A C h r e c e p t o r has b e e n c h e m i c a l l y d e m o n s t r a t e d b y R a n g a n d R i t t e r (45).

I n a d d i t i o n , b o t h the e n z y m e a n d the r e c e p t o r

are m o d i f i e d b y t h i o l reagents (43, 46),

c o n t r a r y to e a r l i e r i m p r e s s i o n s .

F r o m the p o i n t of v i e w of d r u g research, these n e w advances are of p r a c t i c a l significance i n a n u m b e r of w a y s : the r e c e p t o r surface f o r A C h i n c l u d e s several a n i o n i c sites, a l l of w h i c h a p p e a r to b e i n v o l v e d i n the cooperative

n u c l e a t i o n of the

s p u r r e d p h y s i c a l state of the

receptor.

C l e a r l y these sites m u s t b e d i s t i n g u i s h a b l e b y t h e i r stereoelectronic

re

q u i r e m e n t s so that m a n y structures b e a r i n g o n l y a f o r m a l r e s e m b l a n c e to A C h m a y be a c c o m m o d a t e d , w h i c h accounts f o r the w i d e d i v e r s i t y of d r u g structures w h i c h are c a p a b l e of i n d u c i n g c u r a r i z a t i o n or d e p o l a r i z a tion.

T h e a l k a l o i d d - t u b o c u r a r i n e is c e r t a i n l y a distant r e l a t i v e of A C h ,

a n d it is d o u b t f u l that i t c o u l d h a v e b e e n d e s i g n e d r a t i o n a l l y u s i n g the A C h structure as a m o d e l . A s it turns out, i n fact, the m o d e l of the A C h i n t e r a c t i o n w i t h the esteratic c h a i n of the e n z y m e , o n w h i c h several i n t e r pretations

of

structure-activity

relationships

are

still

based

(48)

is

erroneous since m o s t A C h analogs i n t e r a c t m a i n l y w i t h the a n i o n i c c h a i n ( e x o - b i n d i n g ) (37, 41, 42)

( F i g u r e 1 ) , the t o p o g r a p h y of w h i c h has o n l y

b e g u n to b e e x p l o r e d (36, 45). ficities

U n t i l the d e t a i l e d t o p o g r a p h i e s a n d s p e c i

of s u c h large b i n d i n g surfaces are u n d e r s t o o d , n o v e l effector

inhibitor prototypes unpredictable.

endowed with

specificity w i l l

remain

or

essentially

It seems c l e a r that the c l a s s i c a l concept, w h i c h r e q u i r e s

antagonist m o l e c u l e s to o c c u p y the agonist b i n d i n g sites p r o p e r , s h o u l d b e u s e d w i t h extreme c a u t i o n i n v i e w of the m u l t i p l i c i t y of b i n d i n g sites a n d the w i d e o c c u r r e n c e of e x o - b i n d i n g at the a c t i v e site l e v e l

(11).

W e have recently established ( B . Belleau, A . Paturet, a n d M . Saucier, i n p r e p a r a t i o n ) that e r y t h r o c y t e - b o u n d A C h E behaves as a g e n u i n e a l l o steric e n z y m e d i s p l a y i n g " e x p l o s i v e " c o o p e r a t i v e b e h a v i o r w h i c h suggests that i t p r o b a b l y p l a y s a r o l e i n the c o n t r o l of m e m b r a n e c o n f i g u r a t i o n . A phase t r a n s i t i o n m u s t be first n u c l e a t e d b y a p p r o p r i a t e effectors

before

the esteratic center c a n a c c e p t the substrate A C h . W e c o n c l u d e t h a t the r e g u l a t o r y units of m e m b r a n e - b o u n d A C h E possess the b i o p h y s i c a l q u a l i fications

for a receptor-like role on excitable membranes.

ADRENERGIC SYSTEMS. O v e r the past f e w years, s i g n i f i c a n t advances i n o u r u n d e r s t a n d i n g of the in-situ

c h e m i s t r y of b o t h the a- a n d β-recep-

tors for c a t e c h o l a m i n e s h a v e b e e n m a d e .

W e s h a l l e x a m i n e b r i e f l y some


152

DRUG DISCOVERY

of the h i g h l i g h t s as t h e y p e r t a i n to the s t r u c t u r a l a n d c o n f o r m a t i o n a l p r o p e r t i e s of these receptors. The

It appears m o r e a n d m o r e l i k e l y that c e r t a i n classes

α-Receptor.

o f C N S d r u g s m a y act p a r t l y b y i n f l u e n c i n g the α-receptors ( 4 9 ) .

Hence,

the n e e d to a c h i e v e b e t t e r c o m p r e h e n s i o n of d r u g a c t i o n at this l e v e l . B e c a u s e m e m b r a n e - b o u n d A T P a s e s are s o m e h o w i n v o l v e d i n a c t i v e trans p o r t across

membranes

(such

as the

Na ,K -activated ATPase) +

much

+


s p e c u l a t i o n o n the p o s s i b l e i n v o l v e m e n t of A T P a n d A T P a s e s as c o m p o n e n t s of the α-receptor system has b e e n m a d e (2, 41, 5 0 ) .

T h e tempta

t i o n to v i s u a l i z e A T P as f o r m i n g p a r t of the b i n d i n g sites f o r c a t e c h o l a m i n e s has n o d o u b t b e e n r e i n f o r c e d b y the k n o w n r o l e of A T P i n the b i n d i n g of c a t e c h o l a m i n e s

i n the storage granules

(51),

as w e l l as its

i n v o l v e m e n t as substrate f o r the h o r m o n e - s e n s i t i v e a d e n y l c y c l a s e s the c o n t r a c t i l e p r o t e i n s . stereochemistry

A tentative c o n n e c t i o n b e t w e e n

and

catecholamine

a n d receptor-bound A T P was envisaged several

years

ago ( 2 ) , b u t the first d e t a i l e d m o l e c u l a r t h e o r y of c a t e c h o l a m i n e a c t i o n as b a s e d o n the " A T P - r e c e p t o r " p i c t u r e was a d v a n c e d b y B l o o m a n d Goldman

(50).

A s f a r as the w r i t e r is a w a r e , t h e y w e r e the

first

to

present cogent a r g u m e n t s f o r the c e n t r a l r o l e of c a l c i u m as a l i n k b e t w e e n r e c e p t o r responses

a n d m e t a b o l i c events, a n i d e a w h i c h has since l e d

others to suggest that c a l c i u m m a y i n d e e d act as a s e c o n d u n i v e r s a l messenger

(besides c y c l i c A M P ) o f m e m b r a n e events

(52).

I n spite of the elegance of B l o o m a n d G o l d m a n ' s p r o p o s a l , v a r i o u s aspects of t h e i r m o d e l s m a y b e q u e s t i o n e d .

F o r instance, the a s s u m e d

c e n t r a l r o l e of A T P as a p r i m a r y r e a c t i o n site f o r c a t e c h o l a m i n e s

denies

a p r i m a r y i n v o l v e m e n t of the r e c e p t o r p r o t e i n itself. A s f a r as is k n o w n , e n z y m e i n h i b i t o r s a n d activators ( w i t h the e x c e p t i o n of m e t a l i o n chelat i n g agents ) act o n p r o t e i n b i n d i n g sites rather t h a n t h e i r cofactors. drug N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline ( E E D Q )

selective p r o t e i n reagent d e v o i d of c h e m i c a l affinity f o r n u c l e o t i d e s i n f a c t behaves (55).

as a selective i r r e v e r s i b l e i n h i b i t o r of the

The

(53),

a

(54),

α-receptors

P a r t l y f o r these reasons w e h a v e c o n c e i v e d a n a l t e r n a t i v e m o d e l

w h e r e b o t h c a l c i u m a n d p r o t e i n g r o u p s w o u l d serve as b i n d i n g sites f o r catecholamines

and inhibitors (56).

It w a s p r o p o s e d that the c a l c i u m

site w o u l d b e essential o n l y f o r c a t e c h o l a m i n e b i n d i n g a n d n o t f o r an tagonists.

Recent experimental evidence was obtained w h i c h

this h y p o t h e s i s ( 5 7 ) .

supports

H o w e v e r , o u r m o d e l also s p e c i f i e d that the r e c e p t o r

m i g h t be a c a l c i u m - m a g n e s i u m d e p e n d e n t A T P a s e o n w h i c h the t e r m i n a l p h o s p h a t e of A T P w o u l d c o m e i n contact w i t h the a m m o n i u m f u n c t i o n of c a t e c h o l a m i n e s so that catalysis of A T P c l e a v a g e w o u l d b e s o m e h o w i n v o l v e d i n the n u c l e a t i o n of a phase t r a n s i t i o n as w e l l as c a l c i u m trans l o c a t i o n at the m e m b r a n e l e v e l .

[ T h i s p a r t of the m o d e l m a y

appear

unnecessary, a n d p e r h a p s too s p e c u l a t i v e , b e c a u s e the n u c l e a t i o n of a


6.

BELLEAU

Receptor

153

Mechanisms

phase t r a n s i t i o n n e e d not b e a n A T P - r e q u i r i n g process.]

In

contrast,

r a p i d r e p o l a r i z a t i o n m a y d e p e n d o n e n e r g y s u p p l i e s i n the m e m b r a n e , b u t this c a n h a r d l y c o n c e r n the c a t e c h o l a m i n e receptors p e r se.

What

m a y a p p e a r w o r t h r e t a i n i n g , h o w e v e r , is the m o d e l of the c a l c i u m - p r o t e i n c o m p l e x i n c a t e c h o l a m i n e n u c l e a t i o n of phase transitions i n v i e w of the n e w l y e s t a b l i s h e d p a r t i c i p a t i o n of c a l c i u m i n the d e p o l a r i z a t i o n of the m o t o r e n d p l a t e receptors.

I n d e e d , it c o u l d b e s h o w n that c a l c i u m is


r e q u i r e d f o r n o r e p i n e p h r i n e ( N E ) to n u c l e a t e the s p u r r e d state of t h e receptor, a p h y s i c a l f o r m w h i c h is c h e m i c a l l y less r e a c t i v e t h a n the r e s t i n g conformation

toward phenoxybenzamine

(an

alkylating

agent)

(57).

M o r e o v e r , i n the absence of c a l c i u m , N E c o u l d not p r o t e c t against i n a c t i v a t i o n of the r e c e p t o r b y the latter a l k y l a t i n g d r u g , i n a c c o r d w i t h the model

(57).

T h e reverse s i t u a t i o n , w h e r e the s p u r r e d state w o u l d b e c h e m i c a l l y m o r e r e a c t i v e t h a n the b r a c e d r e s t i n g state, thus b e c o m e s e q u a l l y p l a u s i b l e since the r e a c t i v i t y of a b i n d i n g site m a y as easily b e i n c r e a s e d b y a c h a n g e of c o n f o r m a t i o n .

I n fact, this has b e e n o b s e r v e d f o r A C h E

w e l l as the m o t o r e n d p l a t e receptor.

Therefore,

evidence could

as be

o b t a i n e d w h i c h c l e a r l y confirms this e x p e c t a t i o n as regards the a d r e n e r g i c α-receptor. K a l s n e r d i s c o v e r e d that E E D Q , w h i c h i n d u c e s the f o r m a t i o n of an e x t r e m e l y stable a m i d e b o n d at the a c t i v e site l e v e l of the r e c e p t o r (55),

has its p o t e n c y m a r k e d l y e n h a n c e d b y l o w concentrations of v a r i o u s

p h e n e t h y l a m i n e s i n c l u d i n g N E , as w e l l as s e r o t o n i n a n d e v e n h i s t a m i n e (58). motor

It seems e v i d e n t that as for the A C h E p r o t o r e c e p t o r c h a i n a n d the e n d p l a t e receptor,

some

additional anionic binding

sites

for

agonist structures m u s t b e present o n the α-receptor surface so t h a t i n t e r actions w i t h these sites s t r o n g l y enhance the r e a c t i v i t y of the

EEDQ

target site b y w a y of a n i n d u c e d c o n f o r m a t i o n a l c h a n g e i n the r e c e p t o r (Figure 2).

Independent

e v i d e n c e f o r the presence

of at least

two

a n i o n i c sites o n the α-receptor w a s p r o v i d e d b y the f o l l o w i n g o b s e r v a tions: (a)

the b i s a l k y l a t i n g agent I was r e c e n t l y s h o w n t o act as a v e r y

p o t e n t i r r e v e r s i b l e b l o c k e r of the α-receptor (59),

this b e i n g the first t i m e

that a b i s q u a t e r n a r y " c u r a r e a n a l o g " is f o u n d to d i s p l a y h i g h affinity f o r this r e c e p t o r system, a n d ( b )

v e r y l o w concentrations of t h e a l k y l a t i n g

agent D P A , w h i c h h a v e no i n f l u e n c e o n the tissue response to N E , w i l l nevertheless

m a r k e d l y alter

benzamine action, receptor

the

c h e m i c a l characteristics

of

phenoxy

recovery being strongly accelerated

D P A is i n i t i a l l y present o n the surface (33).

when

T h i s also suggests that D P A

alkylates a n accessory a n i o n i c site a n d as a result, the r e c e p t o r c o n f o r m a t i o n is a l t e r e d i n a m a n n e r w h i c h renders the p h e n o x y b e n z a m i n e — r e c e p t o r b o n d m u c h m o r e h a b i l e ( a l t e r n a t i v e l y , the D P A r e c e p t o r species

may

expose a different a c t i v e site to p h e n o x y b e n z a m i n e ). It is of c o n s i d e r a b l e significance t h e n that l i k e the A C h E p r o t o r e c e p t o r c h a i n a n d the e n d


154

DRUG DISCOVERY

p l a t e receptor, the a d r e n e r g i c α-receptor s h o u l d also possess m o r e t h a n one a n i o n i c site c a p a b l e of i n t e r a c t i n g w i t h a d r e n e r g i c amines a n d other modifiers so that c o n f o r m a t i o n a l changes

m a y be

i n d u c e d as c a n

r e a d i l y r e v e a l e d b y the c h e m i c a l a c c e s s i b i l i t y a n d r e a c t i v i t y of specific sites.

be

other

O n e is t e m p t e d to c o n c l u d e that these three r e c e p t o r sys

tems are b i o p h y s i c a l l y r e l a t e d .

This conclusion m a y be more

sensible

t h a n i t appears since i t accounts r a t h e r w e l l f o r the p u z z l i n g f a c t that


d r u g s ( m o s t l y r e c e p t o r antagonists ) d o n o t d i s c r i m i n a t e too w e l l b e t w e e n the A C h - a n d the c a t e c h o l a m i n e α-receptors.

T h e s e n e w advances

cer

t a i n l y offer m o r e r e a l i s t i c g u i d e l i n e s t h a n the o l d e r m o d e l s i n t h e g e n e r a l area of 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 ( see below ).

The

β-receptor.

T h a t the e n z y m e a d e n y l cyclase is i n t i m a t e l y asso

c i a t e d w i t h b i o g e n i c a m i n e a n d p e p t i d e h o r m o n e a c t i o n o n ^-receptors is now

firmly

e s t a b l i s h e d , a n d there is n o d o u b t that c y c l i c A M P is the

m e d i a t o r r e s p o n s i b l e f o r the a d a p t i v e responses of a v a r i e t y of target cells (60, ence

61). of

E v i d e n c e has also a c c u m u l a t e d that cyclase is u n d e r t h e i n f l u regulatory

subunits

d e p e n d e n t specificities (60,62).

or

discriminators endowed

with

tissue-

A role f o r c a l c i u m , p o s s i b l y as a s e c o n d

messenger of s i g n a l r e c e p t i o n , at the m e m b r a n e l e v e l has b e e n d e m o n strated (52).

W e h a v e r e c e n t l y s h o w n that b r a i n a d e n y l c y c l a s e m a y b e

c a l c i u m d e p e n d e n t , a l t h o u g h it suffers i n h i b i t i o n at h i g h (63).

concentrations

H e n c e , it appears that c a l c i u m is p r o b a b l y a n essential c o m p o n e n t

of a l l the r e c e p t o r systems f o r n e u r o t r a n s m i t t e r s . I n the l i g h t of these n e w d e v e l o p m e n t s , it b e c o m e s m o r e a n d m o r e e v i d e n t t h a t t h e earlier v i s u a l i zations of a d i r e c t i n t e r a c t i o n of c a t e c h o l a m i n e s w i t h c y c l a s e - b o u n d A T P as a m o d e l f o r β-receptor a c t i v a t i o n (2, 50, 64)

m a y not be applicable

since r e g u l a t o r y subunits p r o b a b l y constitute the sites of b i n d i n g a n d p r e s u m a b l y the receptors

themselves.

r e l a t i o n s h i p s a m o n g β-receptor

Consequently, structure-activity

antagonists are n o l o n g e r r e a d i l y i n t e r

p r é t a b l e i n terms of the o l d e r m o d e l s a l t h o u g h i t r e m a i n s to b e p r o v e d that antagonists interact w i t h the agonist b i n d i n g sites themselves. v i e w of o u r

finding

In

that e x o - b i n d i n g p r e d o m i n a t e s i n other systems, i t


6.

BELLE AU

Receptor

ι


155

Mechanisms

π

Figure 2. The receptor surface for norepinephrine (NE) is shown to include two anionic sites (A); phenethylamines and related structures (including hista mine) interact at Site II and thus induce a conformation change (B) favoring NE binding at Site I, which in turn leads by way of another conformation change to the active, spurred state of the receptor (C). In the Β state, the reac tion at Site I with EEDQ is facilitated by the increased proximity of a surface nucleophile (-NH ) to the mixed anhydride intermediate (D E). Carbamylation of this nucleophile as in Ε leads to irreversible inactivation of the receptor. 2

appears l i k e l y that t h e same c o n c e i v a b l y a p p l i e s to β-receptor antagonists; o t h e r w i s e F i s h e r ' s l o c k - a n d - k e y fit p r i n c i p l e w o u l d b e m e a n i n g l e s s since b o t h agonists a n d antagonists w o u l d h a v e to share i d e n t i c a l b i n d i n g sites. B e c a u s e β-receptors are m a i n l y c o n c e r n e d w i t h a c t i v a t i o n a n d i n h i b i t i o n of a d a p t i v e m e t a b o l i c responses r a t h e r t h a n w i t h t h e d i r e c t g e n e r a t i o n o f a c t i o n p o t e n t i a l s , i t is p r o b a b l e that those b i o p h y s i c a l p a r a m e t e r s w h i c h i n t e r v e n e u n i q u e l y i n n e u r o t r a n s m i t t e r actions o n e x c i t a b l e m e m b r a n e s m a y n o t b e a p p l i c a b l e to t h e β-receptors. T h i s c o u l d a c c o u n t f o r t h e f a c t that most n o n - d i s c r i m i n a t i n g d r u g s w h i c h are k n o w n to i n h i b i t t h e r e sponses of e x c i t a b l e m e m b r a n e s h a v e little, i f a n y , effect o n t h e β - r e c e p tors. R e c e n t l y , h o w e v e r , i t has b e e n c l a i m e d t h a t c e r t a i n antidepressants ( w h i c h a r e potentiators of a d r e n e r g i c amines ) i n f l u e n c e t h e u r i n a r y levels of c y c l i c A M P b y a m e c h a n i s m p r e s u m a b l y i n v o l v i n g c y c l i c A M P - p h o s phodiesterase i n h i b i t i o n ( 6 5 , 66).

H o w e v e r , i t is d i f f i c u l t to see h o w t h e

a c c u m u l a t e d b r a i n c y c l i c A M P c o u l d traverse t h e c e l l m e m b r a n e s , t h e blood-brain

b a r r i e r , t h e l i v e r a n d k i d n e y s a m o n g o t h e r tissues, a n d


156

DRUG DISCOVERY

a p p e a r i n the u r i n e as b r a i n c y c l i c A M P w h i c h has e s c a p e d t h e a c t i o n of p h o s p h o d i e s t e r a s e as a result of a n t i d e p r e s s a n t i n h i b i t i o n of the latter. T h e firm c o n c l u s i o n is r e a c h e d that t h e c a t e c h o l a m i n e b i n d i n g surfaces of the a- a n d /^-receptors are d i f f e r e n t since m o s t antagonists of the f o r m e r d o not i n h i b i t the latter.

It w i l l b e i n t e r e s t i n g to see w h e t h e r

m o l e c u l a r m a n i p u l a t i o n s of the c y c l i c A M P s t r u c t u r e w i l l l e a d to n e w u s e f u l d r u g s . T h e prospects m a y a p p e a r g o o d since c y c l i c A M P is i n -


v o l v e d i n g l y c o g e n o l y s i s , l i p o l y s i s , steroidogenesis, m e m b r a n e

permea-

b i l i t y , t r a n s l a t i o n , t r a n s c r i p t i o n , a n d as a n a c t i v a t o r of specific p r o t e i n kinases (67).

T h i s latter p r o p e r t y suggests that i t m a y b e i n v o l v e d at

some m e t a b o l i c stage i n the p h e n o m e n o n of m e m o r y

fixation.

I n v i e w of

the s e n s i t i v i t y of v a r i o u s /^-receptors to p e p t i d e h o r m o n e s , m u c h m o r e attention m a y h a v e to b e d e v o t e d to the synthesis of p e p t i d e agonists a n d antagonists since i m p r o v e d s p e c i f i c i t y of a c t i o n m a y b e e x p e c t e d .

The Theory of Sequential Control Sites I n a p r o j e c t e d p a p e r , a c o m p r e h e n s i v e d i s c u s s i o n of the n a t u r e a n d r o l e of w h a t w e t e n t a t i v e l y define as s e q u e n t i a l c o n t r o l sites o n r e c e p t o r surfaces ( a n d some r e g u l a t o r y e n z y m e s ) w i l l be p r e s e n t e d . W e s h a l l here b e c o n c e r n e d o n l y w i t h those g e n e r a l aspects w h i c h are e s p e c i a l l y relev a n t to the p r o b l e m of 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 a m o n g d r u g s . R e c e n t e v i d e n c e s u m m a r i z e d a n d i n t e g r a t e d a b o v e makes i t clear that at least three r e c e p t o r systems f o r c a t i o n i c n e u r o t r a n s m i t t e r s i n c l u d e on t h e i r e x p o s e d surfaces s e v e r a l a n i o n i c b i n d i n g sites w h e r e c o n f o r m a t i o n a l changes m a y be i n i t i a t e d or b l o c k e d . T h e s e findings are at v a r i a n c e w i t h the p r e v i o u s i d e a that o n l y one a c t i v e center is r e s p o n s i b l e f o r the o b s e r v e d effects i n d u c e d b y n e u r o t r a n s m i t t e r s .

T h e systems s t u d i e d i n -

c l u d e the m o t o r e n d p l a t e r e c e p t o r a n d the p u t a t i v e p r o t o r e c e p t o r c h a i n of A C h E , w h i c h b o t h c a r r y at least f o u r d r u g - b i n d i n g a n i o n i c sites p e r a c t i v e u n i t , a n d the N E r e c e p t o r , w h i c h carries at least t w o ( a n d p r o b a b l y m o r e s u c h sites, i n v i e w of its a p p a r e n t b i o p h y s i c a l k i n s h i p to the other two

systems).

critical w i t h

I n a n y event, the exact n u m b e r of a n i o n i c sites is n o t r e g a r d to

the

basic

mechanistic

implications.

The

first

q u e s t i o n w h i c h comes to m i n d i s : w h y s h o u l d there b e s e v e r a l t o p o g r a p h i c a l l y d i s t i n c t a n i o n i c sites f o r n e u r o t r a n s m i t t e r s w h e n o n l y one s h o u l d be necessary to n u c l e a t e the s p u r r e d p h y s i c a l state of these receptors?

C o n s i d e r a t i o n s of a b i o p h y s i c a l n a t u r e m a y p r o v i d e the basis of

an a n s w e r to this q u e s t i o n .

F i r s t , i t c a n b e a g r e e d that the v e l o c i t y of

n e r v e i m p u l s e t r a n s m i s s i o n across t h e s y n a p t i c g a p s h o u l d o c c u r at the m a x i m u m s p e e d a t t a i n a b l e , w h i c h i d e a l l y w o u l d r e q u i r e the t r a n s m i t t e r to t r a v e l at a rate a p p r o a c h i n g that of d i f f u s i o n - c o n t r o l l e d processes. H e n c e , the t r a n s m i t t e r s h o u l d b e m i n i m a l l y h i n d e r e d b y the n a t u r a l d i f -


6.

BELLEAU

Receptor

157

Mechanisms

f u s i o n b a r r i e r s to its k e y sites of a c t i o n o n the p o s t - s y n a p t i c surface.

receptor

N e a r the r e c e p t o r surface the p r o b l e m of t i m i n g a n d efficiency

of p r o d u c t i v e captures c a n b e h a n d l e d s i m p l y b y r e d u c i n g the d i m e n s i o n a l i t y i n w h i c h d i f f u s i o n takes p l a c e , f r o m t h r e e - d i m e n s i o n a l to t w o w h i c h has

been

g i v e n a s o l i d q u a n t i t a t i v e t h e o r e t i c a l basis b y A d a m a n d D e l b r i i c k

d i m e n s i o n a l surface

d i f f u s i o n , a f u n d a m e n t a l process

(68).

T h e s e authors p o i n t e d out that " i n the i n t e r m e d i a t e r a n g e of

distances


of a f e w m i c r o n s a n e w p r i n c i p l e m a y be i n v o l v e d i n that the d i f f u s i n g m o l e c u l e s r e a c h t h e i r d e s t i n a t i o n area not d i r e c t l y b y free d i f f u s i o n i n t h r e e - d i m e n s i o n a l space b u t b y s u b d i v i d i n g the d i f f u s i o n process

into

successive stages of l o w e r s p a t i a l d i m e n s i o n a l i t y . " A c c o r d i n g l y , i f

the

effector m o l e c u l e sticks loosely to some sites, d i f f u s i o n a l o n g the surface w i l l b e f a v o r e d ( F i g u r e 3 ) so that the c a t c h at some other k e y sites w i l l be s i g n i f i c a n t l y i m p r o v e d , a consequence advantage

for synaptic membrane

of c o n s i d e r a b l e e v o l u t i o n a r y

surfaces

a n d t h e i r receptors

since

shorter r e a c t i o n times a n d e c o n o m i c use of l o w t r a n s m i t t e r concentrations are e n s u r e d . B u c h e r (60)

has p o i n t e d out that surface d i f f u s i o n m a y w e l l

c o n t r i b u t e to the h i g h t u r n o v e r n u m b e r s of m e m b r a n e - b o u n d e n z y m e s , a n d the c o n c e p t w a s i n d e e d a p p l i e d e v e n e a r l i e r b y T r u r n i t (70) system A C h E - A C h .

to the

It is a p p a r e n t t h e n that a n e t w o r k c o m p r i s i n g s e v e r a l

a n i o n i c sites o n the p r o t o r e c e p t o r c h a i n of A C h E m a y c o n t r i b u t e enor m o u s l y to the h i g h rate of c a t c h at a n y one site, p r o v i d e d that the c a t i o n i c m o l e c u l e s d o not i n t e r a c t too s t r o n g l y w i t h c e r t a i n a n i o n i c sites as t h e y o c c u r o n the r e s t i n g state of the receptor surface.

F o r this to h a p p e n , a

l o w e r degree of c o m p l e m e n t a r i t y of the n a t u r a l effector or substrate w i t h c e r t a i n sites m a y be necessary;

o t h e r w i s e surface d i s p l a c e m e n t s

might

b e c o m e too s l u g g i s h . It m a y be c o n v e n i e n t t h e n to speak of s e q u e n t i a l c o n t r o l sites as a n a t u r a l means of h a n d l i n g the p r o b l e m of t i m i n g a n d efficiency i n the p r o d u c t i v e c a t c h of nucleators o n the surface ( F i g u r e 3 ).

Θ 3-Dimensional

Diffusion

2-Dimensional Diffusion

a

a

Figure 3. Schematic of the efficiency of effector ( + ) capture by a surface carrying multiple anionic sites. Transfer from one site to the other involves two-dimensional translocations, thus leading to a much improved efficiency and timing of catch at any given site.


158

DRUG DISCOVERY

W e l l - d e v e l o p e d s e q u e n t i a l c o n t r o l sites, w h e r e c o n f o r m a t i o n a l changes can

be i n d u c e d , s h o u l d b e e s p e c i a l l y p r e v a l e n t a m o n g r e c e p t o r

surfaces

for neurotransmitters i n v i e w of the r a p i d i t y w i t h w h i c h i n t e r n e u r o n a l c o m m u n i c a t i o n s m u s t b e e s t a b l i s h e d i n the C N S as a n e x a m p l e . In

p r i n c i p l e , f o r effective n u c l e a t i o n a n d o p t i m u m s t a b i l i z a t i o n of

the s p u r r e d state of the receptors, o n l y one strategic a n i o n i c site m a y n e e d interact w i t h the n u c l e a t o r , b u t i t seems m u c h m o r e p r o b a b l e that


w e a k e r interactions w i t h some c o n t r o l sites m a y c o n t r i b u t e i n a c o o p e r a tive m a n n e r to the s t a b i l i t y of the s p u r r e d state. T h e i m m e d i a t e e n v i r o n m e n t of the a n i o n i c sites w i l l o b v i o u s l y differ f r o m one site to another, and their respective c h e m i c a l reactivity w i l l be altered b y conformational changes, thus p r o v i d i n g a n e x p l a n a t i o n f o r the recent observations ( s u m m a r i z e d a b o v e ) w i t h E E D Q ( 5 8 ) , c u r a r i z i n g agents (42), α-blockers of the a l k y l a t i n g v a r i e t y (33).

and adrenergic

In addition, and most impor

t a n t l y , the s e q u e n t i a l c o n t r o l site m o d e l a l l o w s the p r e d i c t i o n t h a t h i g h concentrations of nucleators m a y saturate some u n o c c u p i e d sites as t h e y o c c u r i n the s p u r r e d state of the r e c e p t o r .

T h i s w o u l d cause the latter

to suffer c o n f o r m a t i o n a l b l o c k a d e or f r e e z i n g ( F i g u r e 4 ) , as w o u l d b e reflected, f o r instance, i n the p h e n o m e n a of d e s e n s i t i z a t i o n , t a c h y p h y l a x i s , etc.

I n d e e d , a n excess of A C h w i l l i n d u c e " c u r a r i z a t i o n " of the e n d p l a t e

m e m b r a n e , as w o u l d b e e x p e c t e d i f a d d i t i o n a l A C h m o l e c u l e s saturate the c o n t r o l sites of the r e c e p t o r w h e n i n the s p u r r e d or a c t i v a t e d state.

ΘΘΘ,

Resting

State

Active

State

Locked Active State

Figure 4. Schematic of a formal conformational equilibrium between the resting and active state of the excitable membrane receptor of the motor end plate. In the latter state two strategic sites are occupied, and two control sites are free; when saturation of the latter is enforced by an excess of effector mole cules, the active state is changed to a more stable "frozen" conformation, leading to prolonged depolarization of the membrane. Topographical Symmetry vs. Molecular Quasi-Symmetry.

The mi

c r o s c o p i c e n v i r o n m e n t of e a c h s e q u e n t i a l c o n t r o l site w i l l o b v i o u s l y h a v e its o w n characteristics, a n d i n v i e w of the p o s t u l a t e d l o w e r c o m p l e m e n t a r i t y of the n e u r o t r a n s m i t t e r f o r these s e q u e n t i a l c o n t r o l sites, t h e struc t u r a l r e q u i r e m e n t s f o r antagonist b i n d i n g m a y b e m u c h less r i g o r o u s t h a n at strategic sites, a n d s t r o n g b i n d i n g s h o u l d b e f a v o r e d w h e n t h e d r u g


6.

BELLE AU

Receptor

159

Mechanisms

structure is s t e r e o e l e c t r o n i c a l l y c o m p a t i b l e w i t h t h e m i c r o e n v i r o n m e n t o f a n y o n e of t h e c o n t r o l sites. H e n c e , m a n y structures b e a r i n g o n l y a m o d est

analogy

( i f a n y at a l l ) t o t h e n e u r o t r a n s m i t t e r s

m a y preferably

interact s t r o n g l y w i t h t h e c o n t r o l sites r a t h e r t h a n t h e strategic ones w h e r e the n e u r o t r a n s m i t t e r

achieves

a "lock-and-key

fit."

Consequently, the

antagonists w i l l p e r t u r b t h e r e c e p t o r c o n f o r m a t i o n , a n d i f c o o p e r a t i v i t y is also

achieved,

v e r y h i g h potencies

m a y occasionally

be

observed.


A s s u m i n g that a d r u g c a n interact w i t h m o r e t h a n o n e c o n t r o l area at a time, the receptor

conformation

m a y be changed

or stabilized

more

effectively since a greater d e g r e e of o v e r - a l l c o m p l e m e n t a r i t y w o u l d b e achieved.

O f t h e m u l t i t u d e of orientations

w h i c h a n effector s u c h as

A C h c a n assume at a n y t w o v i c i n a l s e q u e n t i a l sites, t h e r e exist t w o w h e r e the m o l e c u l e s w i l l b e a r r a n g e d i n t a i l - t o - t a i l f a s h i o n ( F i g u r e 5 ) . T h i s is w h a t w o u l d o b t a i n , f o r instance, i f d i s p l a c e m e n t f r o m o n e site t o a n o t h e r n e i g h b o r i n g site is a c c o m p l i s h e d t h r o u g h a 1 8 0 ° r o t a t i o n a l m o t i o n . F o r this to h a p p e n , t h e t w o adjacent c o n t r o l areas w h e r e t h e m o l e c u l e s a r e interacting might be topographically enantiomeric physical chiralities.

o r possess o p p o s i n g

A c c o r d i n g l y , effector m o l e c u l e s possessing

suitable

Figure 5. Schematic of sequential control sites on the surfaces of the motor end plate receptor and the protoreceptor chain of acetylcholinesterase. Each circle encompasses an area sufficiently large to accommodate one molecule of acetylcholine. The arrows represent the shortest possible paths of surface displacements from one site to the other. In two positions of the effector, a tail-to-tail arrangement is possible when two molecules are bound on two vicinal binding areas. This raises the possibility that the two adjacent areas might possess suitable elements of topographical symmetry or dissymmetry (the planes being represented by dotted lines). The prediction may then be allowed that symmetrical, doubly charged effectors (such as succinyldicholine) might display complementarity with two areas at a time. Hence, each half of the effector molecule could hear a mirror image relationship to the other (meso configurations) for optimal interaction with two adjacent areas at a time (see text and Figure 6).


160

DRUG DISCOVERY

elements of s y m m e t r y m i g h t a l l o w f o r s i m u l t a n e o u s

quasi-complemental

interactions w i t h t w o c o n t r o l areas. T h i s m a y e n c o u r a g e tighter a n d m o r e selective b i n d i n g , as w e l l as i n c r e a s e d p o t e n c y i f the c o n t r o l sites c o o p e r a t i v e l y i n the s t a b i l i z a t i o n of a g i v e n r e c e p t o r c o n f o r m a t i o n . f o l l o w i n g observations theory.

offer s u b s t a n t i a l s u p p o r t f o r this

act The

topographical

Succinyldicholine, a symmetrical molecule resulting from

the

t a i l - t o - t a i l f u s i o n of t w o A C h m o l e c u l e s , is one of the most p o t e n t a n d


best k n o w n d e p o l a r i z e r s of the m o t o r e n d p l a t e m e m b r a n e as w e l l as the most effective c o n f o r m a t i o n m o d i f i e r of the a n i o n i c p r o t o r e c e p t o r c h a i n of A C h E .

T h e postulated topographically enantiomeric relation between

the adjacent c o n t r o l areas ( F i g u r e 6 )

i n v o l v e d i n the b i n d i n g of this

Figure 6. Interaction of βηοοίηι^Ι-β,β'-άΐηιβ^ΙαιοΗοΙίηβ with two neighboring anionic areas. If the latter display a topographical plane of symmetry or dis symmetry (dotted line), the meso isomer should interact optimally, but not the or Ό,Ό-enantiomers. This model agrees with experiment (see text). effector is s u p p o r t e d b y the f a c t that, whereas the D,D- a n d L , L - s u c c i n y l β,β'-dimethyldocholine

are essentially i n a c t i v e i n b o t h systems, the meso

i s o m e r is f a i r l y a c t i v e ( a b o u t o n e - t h i r d the a c t i v i t y of s u c c i n y l d i c h o l i n e ) b o t h at the r e c e p t o r a n d e n z y m e levels ( u n p u b l i s h e d results ) ( F i g u r e 6 ). M o r e o v e r , a n i m p r e s s i v e n u m b e r of potent c u r a r i z i n g agents are

either

s y m m e t r i c a l or q u a s i - s y m m e t r i c a l m o l e c u l e s ( s u c h as the toxiferins, t u b o c u r a r i n , etc).

H o w e v e r , as n o t e d above, some of the c o n f o r m a t i o n a l ^

sensitive c o n t r o l sites m a y be less d e m a n d i n g t h a n t h e strategic from

the

actions

stereoelectronic

standpoint,

site(s)

a n d , accordingly, i o n - i o n inter

[ w h i c h s u p p l y m o s t of the b i n d i n g energy ( 7 ) ]

w i t h molecules

d i s p l a y i n g l o w e r degrees of s y m m e t r y w i l l also be a l l o w e d , w h i c h ex p l a i n s w h y s u c h a vast n u m b e r of b i s q u a t e r n a r y ions are k n o w n to possess c u r a r i z i n g a c t i v i t y to some degree.

A c o r o l l a r y of c o n s i d e r a b l e i m p o r

tance is that a n y r e c e p t o r system w h i c h , l i k e the m o t o r e n d p l a t e r e c e p t o r


6.

BELLEAU

Receptor

161

Mechanisms

or the p r o t o r e c e p t o r c h a i n of A C h E , is c a p a b l e of i n t e r a c t i n g w i t h s i m i l a r l y d i v e r s e d r u g structures m a y also possess s e q u e n t i a l c o n t r o l b i n d i n g sites w h e r e c o n f o r m a t i o n a l p e r t u r b a t i o n s of one k i n d or another m a y b e induced.

A c c o r d i n g l y , at least t w o other r e c e p t o r

systems

would

also

i n c l u d e s u c h sites : the a d r e n e r g i c α-receptor, f o r w h i c h c o n c r e t e e v i d e n c e was a l r e a d y g i v e n f o r the presence o f accessory sites o n its surface, a n d the analgesic r e c e p t o r , w h i c h is k n o w n to r e s p o n d to as w i d e a v a r i e t y


of s t r u c t u r a l types as the c u r a r e receptor. F o r the n e u r o t r a n s m i t t e r

norepinephrine, symmetry

considerations

s i m i l a r to those a p p l i e d a bove to the s u c c i n y l d i c h o l i n e b i n d i n g sites a l l o w the p r e d i c t i o n that q u a s i - s y m m e t r i c a l l y d i s p o s e d b i n d i n g areas f o r aro m a t i c rings m a y b e present o n t h e receptor surface

(Figure 7), and if

the c o n f o r m a t i o n a l a n d e l e c t r o n i c p r o p e r t i e s of the d r u g are c o m p a t i b l e w i t h the d e t a i l e d t o p o g r a p h y , u n p r o d u c t i v e p h y s i c a l states m a y b e r e a d i l y i n d u c e d i n the receptor.

T h e s e q u e n t i a l c o n t r o l areas b e i n g q u a s i - s y m

m e t r i c a l l y d i s p o s e d , the basis of a n e x p l a n a t i o n f o r the w e l l - e s t a b l i s h e d

Figure 7. Schematic of the receptor sur face for norepinephrine (NE). Site II is selective for phenethylamine patterns (in cluding histamine and serotonin), and Site I for catecholamines. Hence, the vicinal control areas include aromatic binding sites, possibly separated by a plane of topographical dissymmetry. Interactions at Site II cause a conformational change ichich enhances the EEDQ reaction at Site I (see Figure 2). The spatial arrange ment of the aromatic ring binding areas could well fit the structural pattern of the quasi-symmetrical "diphenylmethane" se ries of drugs (dotted lines).



162

DRUG DISCOVERY

Figure 8. General structure-type, based on the "magic" diphenylmethane pattern of symmetrical skeleton as found in many drugs affecting cholinergic, adrenergic, and histaminergic receptors r e c u r r e n c e of the " m a g i c " d i p h e n y l m e t h a n e p a t t e r n i n a w i d e v a r i e t y o f d r u g s m a y b e at h a n d ( F i g u r e 8 ) . A m o n g d r u g s c u r r e n t l y i n use w h i c h are b a s e d

on the diphenylmethane pattern, one

antihistamines,

neuroleptics,

finds

stimulants, sympatholytics,

antidepressants, parasympatho-

l y t i c s , analgesics, etc. B e c a u s e of the less specific n a t u r e of some of t h e c o n t r o l sites, m a n y of these d r u g s are n o t receptor specific.

Specificity

m a y b e a c h i e v e d o n l y b y c a p i t a l i z i n g o n t h e subtle c o n f o r m a t i o n a l a n d t o p o g r a p h i c a l differences f r o m the other. elsewhere.

w h i c h must distinguish one receptor

surface

C o n c r e t e m o d e l e v i d e n c e f o r this v i e w w i l l b e p u b l i s h e d

Suffice i t to say that this is w h a t is m o s t p r o b a b l y

accom-

p l i s h e d w h e n the m e d i c i n a l c h e m i s t i n t r o d u c e s c o n f o r m a t i o n a l

restric-

tions ( s u c h as b r i d g i n g ) i n t h e d i p h e n y l m e t h a n e s k e l e t o n o r w h e n h e s l i g h t l y alters s y m m e t r y b y i n c o r p o r a t i n g r i n g substituents

(Figure 7).

O n e area of r e s e a r c h w h i c h appears n o t to h a v e b e e n e x p l o r e d w o u l d b e the d e s i g n i n g of n o v e l s y m m e t r i c a l o r q u a s i - s y m m e t r i c a l m o l e c u l e s b a s e d o n t h e f u s i o n of c a t e c h o l a m i n e - h k e

r i n g s i n v a r i o u s t a i l - t o - t a i l patterns

i n the m a n n e r s u c c e ssf u lly a p p l i e d to c u r a r i z i n g agents. I n d e e d , w e h a v e a l r e a d y a p p l i e d the p r i n c i p l e suc c e ssf u lly i n t h e case of t h e p o t e n t s y m metrical bisalkylating adrenergic blocker ( I ) (59) described above. F i n a l l y , o n e of the most perverse concatenations of r e c e p t o r p r o p e r ties is f o u n d i n the area of n a r c o t i c analgesics a n d t h e i r antagonists. N o t h e o r y of 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 has s u r v i v e d v e r y l o n g i n this field,

a l t h o u g h r e c e n t l y some i n t e r e s t i n g studies of t h e subtle c o n f i g u r a -

t i o n a l s p e c i f i c i t y o f t h e r e c e p t o r h a v e b e e n i n i t i a t e d b y Portoghese

(71).

T h e o n l y p o i n t w h i c h m a y b e w o r t h e m p h a s i z i n g at this t i m e is t h a t t h e extreme c o n f o r m a t i o n a l d i v e r s i t y of d r u g s a c t i n g at this l e v e l is s t r o n g l y r e m i n i s c e n t of the b r o a d a d a p t a b i l i t y of the m o t o r e n d p l a t e

receptor.

T h i s p a r a l l e l i s m suggests that t h e analgesic r e c e p t o r m a y s i m i l a r l y i n c l u d e several s e q u e n t i a l c o n t r o l sites o n its surface w h e r e t h e p r i n c i p l e of t o p o -


6.

BELLE AU

Receptor

163

Mechanisms

graphical quasi-symmetry m a y be operative ( i n d e e d , several strong analgesics b e l o n g to the d i p h e n y l m e t h a n e p a t t e r n of s y m m e t r i c a l s k e l e t o n ) . S o m e p r a c t i c a l i m p l i c a t i o n s of this c o n c e p t i n the d e s i g n of n o v e l classes of p o t e n t i a l analgesics are rather o b v i o u s . W i t h r e g a r d to t h e m o l e c u l a r basis of n a r c o t i c - a n a l g e s i c a n d n a r c o t i c - a n t a g o n i s t actions, the p o s s i b i l i t y of a n a n a l o g y w i t h d e p o l a r i z e r a n d n o n - d e p o l a r i z e r actions o n the m o t o r e n d p l a t e r e c e p t o r m a y deserve c o n s i d e r a t i o n . A t the m o m e n t , it m a y b e


w o r t h w h i l e c o n s i d e r i n g the h y p o t h e s i s that n a r c o t i c analgesics

might

p r o d u c e a p r o l o n g e d , p e r h a p s c o o p e r a t i v e p e r t u r b a t i o n or d i s t o r t i o n of the s p u r r e d p h y s i c a l state of receptors i n the m a n n e r w h i c h c h a r a c t e r i z e s d e p o l a r i z e r s of the e n d p l a t e m e m b r a n e w h e r e a s n a r c o t i c

antagonists

m a y s t a b i l i z e the r e s t i n g p h y s i c a l state of the r e c e p t o r , b y a n a l o g y w i t h t u b o c u r a r i n e - l i k e agents w h i c h b l o c k t h e i r r e c e p t o r w i t h o u t m e m b r a n e depolarization.

T h i s c o u l d a c c o u n t f o r the f a c t t h a t c e r t a i n

narcotic

antagonists c a n also d i s p l a y analgesic a c t i v i t y since the r e c e p t o r w o u l d also b e b l o c k e d , a l b e i t b y a d i f f e r e n t m o l e c u l a r m e c h a n i s m c o n d u c i v e to a different m e m b r a n e c o n f i g u r a t i o n . If this w e r e true, p h y s i c a l d e p e n d ence o n m o r p h i n o i d s m i g h t b e l i n k e d to m e m b r a n e c o l l a p s e b y w a y of a p r o l o n g e d " d e p o l a r i z a t i o n " t y p e of r e c e p t o r c o n f o r m a t i o n a l c h a n g e .

In

a n y event, the s e q u e n t i a l c o n t r o l site t h e o r y f o r n e u r o t r a n s m i t t e r s r e a d i l y accounts f o r the o c c u r r e n c e of m o r p h i n e - l i k e a c t i v i t y i n s e v e r a l types of loosely r e l a t e d classes of structures.

Finally, it may be w o r t h w h i l e con-

s i d e r i n g the p o s s i b i l i t y that c e r t a i n m e n t a l deficiencies or

aberrations

m a y be l i n k e d , not necessarily to alterations i n n e u r o t r a n s m i t t e r a v a i l a b i l i t y , b u t to i n b o r n or a c q u i r e d defects i n the s e q u e n t i a l c o n t r o l sites. T h i s w o u l d o b v i o u s l y b e reflected i n a less effective rate of c a t c h b y the sensitive

membranes

and

decreased

effectiveness

of

interneuronal

communications.

Acknowledgments T h e research of the past f e w years o n w h i c h this p r e s e n t a t i o n is i n p a r t b a s e d was g e n e r o u s l y s u p p o r t e d b y the N a t i o n a l R e s e a r c h C o u n c i l of C a n a d a , the D e f e n c e R e s e a r c h B o a r d of C a n a d a , a n d B r i s t o l L a b o r a tories. Y-H

T h e s k i l l f u l c o l l a b o r a t i o n of V . D i T u l l i o , J - L . L a v o i e , H . T a n i ,

T s a i , E . W ù l f e r t , I. M a c d o n a l d , B . L i p p e r t , a n d D . G o d i n ,

and

M . D i T u l l i o , m a d e this r e s e a r c h p o s s i b l e .

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164

DRUG

DISCOVERY

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November 5, 1970.


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