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Chapter 21. Structure-Activity Relationships of Calmodulin Antagonists
Chapter 2 1 . 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 of Calmodulin A n t a g o n i s t s Walter C. P r o z i a l e c k Department o f P h y s i o l o g y and Pharmacology P h i l a d e l p h i a C o l l e g e of O s t e o p a t h i c M e d i c i n e 4150 C i t y Avenue, P h i l a d e l p h i a , P a . 1 9 1 3 1
I n t r o d u c t i o n - C a l m o d u l i n (CM) i s a w i d e l y - d i s t r i b u t e d , Ca2+-binding prot e i n h a v i n g a m o l e c u l a r w e i g h t o f a p r o x i m a t e l y 17,000. S i n c e i t s d i s c o v e r y i n 1970 as an a c t i v a t o r o f a C a 5 + - s e n s i t i v e form o f p h o s p h o d i e s t e r a s e , CM h a s b e e n shown t o r e g u l a t e a v a r i e t y o f Ca2+-dependent enzymes a n d p r o c e s s e s l v 2 . C o n s i d e r a b l e e v i d e n c e now s u g g e s t s t h a t CM i s t h e p r i n c i p a l m e d i a t o r o f t h e e f f e c t s of Ca2+ i n most e u k a r y o t i c c e l l s . F o r d i s c u s s i o n s of t h e b i o c h e m i c a l p r o p e r t i e s and t h e p h y s i o l o g i c a l f u n c t i o n s of CM, t h e r e a d e r i s r e f e r r e d t o a n y o f s e v e r a l e x c e l l e n t r e v i e w s . 3-9 S i n c e CM p l a y s a f u n d a m e n t a l r o l e i n c e l l b i o l o g y , a g e n t s t h a t i n h i b i t i t s a c t i v i t y s h o u l d h a v e i m p o r t a n t p h a r m a c o l o g i c a l e f f e c t s . An und e r s t a n d i n g of t h e mechanisms by w h i c h t h e s e a g e n t s a l t e r CM a c t i v i t y may h e l p t o e x p l a i n t h e i r p h a r m a c o l o g i c a l a c t i o n s o r may s u g g e s t new a p p r o a c h es f o r m o d i f y i n g v a r i o u s p h y s i o l o g i c a l o r p a t h o l o g i c a l p r o c e s s e s . In a d d i t i o n , t h e d e v e l o p m e n t o f s e l e c t i v e CM a n t a g o n i s t s may p r o v i d e a u s e f u l means f o r f u r t h e r s t u d y i n g t h e b i o l o g i c a l r o l e s of CM. I n t h e mid 1 9 7 0 ' s Weiss and c o - w o r k e r s r e p o r t e d t h a t t h e p h e n o t h i a z i n e a n t i s y c h o t i c s i n h i b i t e d t h e CM-induced a c t i v a t i o n of p h o s p h o d i e s t e r ase. S u b s e q u e n t s t u d i e s h a v e shown t h a t a v a s t a r r a y o f compounds b e l o n g i n g t o d i v e r s e c h e m i c a l and p h a r m a c o l o g i c a l c l a s s e s c a n i n h i b i t t h e a c t i o n s o f CM i n a v a r i e t y of b i o c h e m i c a l s y s t e m s . 8,12913 Table 1 lists t h e c l a s s e s of a g e n t s t h a t h a v e b e e n shown t o i n h i b i t o n e o r more a c t i o n s o f CM. The p u r p o s e o f t h i s r e v i e w i s t o b r i e f l y summarize t h e g e n e r a l mechanisms b y which t h e s e a g e n t s m o d i f y t h e a c t i v i t y o f CM a n d t o examine i n d e t a i l t h e s t r u c t u r a l factors t h a t enable drugs t o i n t e r a c t d i r e c t l y w i t h CM. S e v e r a l a s e c t s o f t h e pharmacology o f CM h a v e b e e n r e v i e w e d e l s e w h e r e . 8,12913,y2953 Mechanisms of P h a r m a c o l o g i c a l l y A l t e r i n g CM A c t i v i t y - Most i f n o t a l l o f t h e e f f e c t s of CM r e s u l t from t h e a c t i v a t i o n , o r o c c a s i o n a l l y i n h i b i t i o n , of s p e c i f i c enzymes. 3-6 F i g u r e 1 i l l u s t r a t e s t h e g e n e r a l mechanism by which CM r e g u l a t e s most Ca2+-dependent enzymes. N e i t h e r Ca2+, n o r CM a l o n e i s a c t i v e . However, e a c h CM m o l e c u l e c a n b i n d up t o 4 C a 2 + i o n s . 3 - 7 , 5 0 , 5 4 The b i n d i n g of C a 2 + a l t e r s t h e c o n f o r m a t i o n o f CM, 3-7 i n c r e a s i n g i t s h e l i c a l c o n t e n t 54-56 a n d e x p o s i n g h y d r o p h o b i c r e g i o n s . 57758 I n t h i s conf o r m a t i o n , t h e Ca2+-CM complex c a n b i n d t o r e g u l a t o r y s i t e s o n t a r g e t enzymes and t h r o u g h a n unknown mechanism a l t e r t h e i r a c t i v i t i e s . 3-6,59 A l t h o u g h c o n t r o v e r s i a l , some e v i d e n c e s u g g e s t s t h a t v a r i o u s C M - s e n s i t i v e enzymes may r e q u i r e d i f f e r e n t s t o i c h i o m e t r i c amounts o f Ca2+ and CM f o r a c t i v i t y 5 6 947 9 60-63
.
T h e r e a r e s e v e r a l mechanisms b y w h i c h d r u g s m i g h t a c t t o i n h i b i t t h e a c t i o n s of CM. 8 , 5 2 A g e n t s m i g h t a c t b y : (A) d e c r e a s i n g t h e c o n c e n t r a t i o n o f a v a i l a b l e Ca2+ a n d p r e v e n t i n g t h e f o r m a t i o n o f t h e a c t i v e Ca2+-CM comp l e x , (B) b i n d i n g t o CM a n d a l t e r i n g i t s a b i l i t y t o b i n d Ca2+, (C) b i n d i n g
ANNUAL REPORTS I N MEDICINAL C H F N I S T R Y 4 I I
Copyright D 1983 by Academ~cPress. Ine All righis of reproductiontn any form r e w v e d . ISBN 0- I20405 I 8 4
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t o t h e Ca2+-CM complex and m o d i f y i n g i t s a c t i v i t y , (D) b i n d i n g t o t h e CMr e c o g n i t i o n s i t e o n t h e CM-sensitive enzyme a nd t h u s p r e v e n t i n g t h e i n t e r a c t i o n o f the Ca2+-CM complex w i t h t h e enzyme, (E) i n t e r a c t i n g , e i t h e r Table 1
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Classes o f A g e n t s t h a t I n h i b i t t h e A c t i o n s o f Ca lm odulin
Pharmacological Class a -adrenergic
antagonist
6 -adrenergic
Chemical Class benzyl- 6 -chloroethylamine ergot alkaloid alkylaminonaphthy1 ether benzodiazepine
mtagonist antianxiety agent antiarrhythmic1,4-dihydropyriantianginal agent dine dimethoxyphenylacet o n i t r i l e antidepressant dibenza zepine dibenzocycloheptadiene antidiarrheal diphenylalkylpiperidine antihistamine phenothiazine antimalarial aminoacridine cinchona alkaloid antipsychotic butyrophenone benzocycloheptap idoisoquinoline dicnzodiazmine diphenylbutfi piperidine phenothiazine
-
cancer chemotherapeutic agent local anesthetic
neuropeptide smooth muscle relaxant miscellaneous agents
*Probable Mechanism
**Selected
phenoxybenzamine
C
14,15
dihydroergotamine propranolol
C C
14 16,17
diazepam
C,D
18,19
felodipine
A,C,E
20,21
verapamil
A,C,E
21
Mpramine amitriptyline
C
C
11,22 11
loperamide
C
23
promethazine quinacrine quinme haloperidol butaclml
C
C C C C
11,22 16,17,22 17 11,12,24 24
clozapine penfluridol
C
12,24
trifluoperazine and analogs chlorprothixene adriamycin vinblast ine
C
11,12,22
C C C
12,22,24
tetracaine dibucaine phenacaine lidocaine B -endorphin me1itt in
C C C C C C
16 ,17,27 16,17,27 16,17,27 27 12,28-30 30,31
prenylamine
C
32
W-7 and analogs
C
32-34
EGTA
A
C
8 ,I1 35-40
Triton X-100
C
41
DDT
C,D
42
chlorpromazine-linked 04
D
43
Mg2’
theophylline R-24571
B
E C
44-47 48-50 51
proadifen (SKF-525A)
C
16,17
Representative Agent
thioxanthene anthracycline vinca alkaloid benzoic es t er cinchoninamide phenylacetamidine phenylamide opioid peptide insect venom peptide diphenylpropylamine naphthalenesfulfypide Ca chelator 04-binding prot e in polpxyethylene detergent chlorinated ethane insecticide modified metal ion methylxanthene miconazoleanalogue B -aminoethyldiphenylpentenoate
calcineurin
C
References
11,12,24
2q
15,26
*Probable mechanism refers t o l e t t e r s shown i n Figure 1, as discussed in the text. ‘“References a r e for each chemical class, not only the representative agent.
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F i g u r e 1 Mechanisms f o r t h e A c t i v a t i o n and I n h i b i t i o n of CM-Sensitive Enzymes
a RESPONSE
c o m p e t i t i v e l y o r non-competitively, w i t h t h e c a t a l y t i c p o r t i o n of the CMs e n s i t i v e enzyme and a l t e r i n g i t s a c t i v i t y , (F) i n t e r a c t i n g w i t h t h e ternary Ca2+-CM-enzyme complex.
2+
Agents t h a t a c t by d e c r e a s i n g t h e c o n c e n t r a t i o n of Ca (Mechanism A) i n c l u d e c h e l a t i n g a g e n t s (e.g. EDTA and EGTA) and t h e s o - c a l l e d Ca2+channel b l o c k e r s (e.g. verapamil and n i f i d i p i n e ) , which i n h i b i t t h e i n f l u x of e x t r a c e l l u l a r Ca2+(see Chapter 9 and r e f e r e n c e s 6 4 , 6 5 ) . S i n c e t h e y do n o t i n t e r a c t d i r e c t l y w i t h CM, t h e s e a g e n t s d i s p l a y l i t t l e s p e c i f i c i t y f o r CM-regulated systems. For example, t h e Ca2+-chelators i n h i b i t a l l biochemical a c t i o n s of C a 2 + , n o t o n l y i t s i n t e r a c t i o n w i t h CM. It should be noted t h a t t h e r e is some evidence t h a t c e r t a i n Ca2+ channel b l o c k e r s might i n t e r a c t w i t h CM i t s e l f . 20,21
L i t t l e i s known about a g e n t s t h a t i n t e r a c t w i t h CM and a l t e r i t s a b i l i t y t o bind Ca2+ (Mechanism B ) . Such a g e n t s might a c t by b i n d i n g d i r e c t l y t o t h e CaZ+-binding s i t e s on CM o r by b i n d i n g t o some o t h e r s i t e on t h e molecule and inducing conformational changes i n t h e Ca2+-binding regions. Although a v a r i e t y o f d i - and t r i v a l e n t c a t i o n s can i n t e r a c t w i t h Ca2+ b i n d i n s i t e s on CM, 50,66 and t o v a r y i n g d e g r e e s m i m i c t h e a c t i o n s of CaZf66,8$it is less c l e a r whether t h e s e a g e n t s can a n t a g o n i z e o r potent i a t e t h e e f f e c t s of Ca2+. S e v e r a l s t u d i e s have shown t h a t M 2+ i n h i b i t s t h e a c t i v a t i o n of CM-dependent enzymes by competing w i t h Ca2' f o r i o n b i n d i n g s i t e s on CM. 44-47 However, La3+, which enhanced t h e Ca2+-mediated binding of CM t o r a t s t r i a t a l p a r t i c l e s , had no e f f e c t on t h e a c t i v a t i o n of a d e n y l a t e c y c l a s e by Ca2+ and CM. 67 R e c e n t l y , s e v e r a l metals i n c l u d i n g Hg, Cd, Zn, Co and S r have been shown t o i n h i b i t t h e CM-induced a c t i v a t i o n of p h o s p h o d i e s t e r a s e , a l t h o u g h t h e mechanism u n d e r l y i n g t h i s e f f e c t h a s y e t t o be determined. 88 R e s u l t s of s t u d i e s examining t h e NMR spectrum of CM s u g g e s t t h a t t h e b i n d i n g of p h e n o t h i a z i n e a n t i p s y c h o t i c s by t h e Ca2+CM complex ( s e e below) a l t e r s t h e Ca2+ b i n d i n g domains of CM. 68369 Howe v e r , t h e s i g n i f i c a n c e of t h i s i s u n c l e a r s i n c e t h e b i n d i n g of t h e drugs themselves r e q u i r e s Ca2+ 8,12 and t h e i n h i b i t o r y e f f e c t s of t h e d r u g s cannot b e overcome by i n c r e a s i n g t h e c o n c e n t r a t i o n of Ca2+. 11 By f a r , t h e l a r g e s t group of CM a n t a g o n i s t s c o n s i s t s of t h o s e a g e n t s t h a t i n t e r a c t d i r e c t l y w i t h t h e Ca2+-CM complex (Mechanism C) Accordingl y , t h i s group of compounds will s e r v e as t h e f o c u s of t h i s review.
.
Thus f a r , l i t t l e a t t e n t i o n h a s been given t o a g e n t s t h a t act a t t h e CM-binding s i t e s on CM-sensitive enzymes (Mechanism D). However, r e c e n t f i n d i n g s suggest t h a t t h e s e may p r o v i d e u s e f u l s i t e s f o r pharmacological i n t e r v e n t i o n . CM t h a t had been i r r e v e r s i b l y l i n k e d t o chlorpromazine by
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u l t r a v i o l e t i r r a d i a t i o n ( s e e below) i n h i b i t e d t h e a c t i v a t i o n of phosphod i e s t e r a s e By n a t i v e CM. 43 The chlorpromazine-CM complex, which w a s una b l e t o a c t i v a t e phosphodiesterase, a p p a r e n t l y competed w i t h CM f o r a regu l a t o r y s i t e on t h e enzyme, s u g g e s t i n g t h a t i t may be p o s s i b l e t o develop a new c l a s s of CM a n t a g o n i s t s t h a t are d i r e c t e d a t t h e CM-binding s i t e s on CM-sensitive enzymes. Since t h e r e is some evidence t h a t v a r i o u s CM-sensit i v e enzymes may have somewhat d i f f e r e n t CM-binding sites,5,6,60-62 a g e n t s d i r e c t e d a t t h e s e s i t e s might d i s p l a y g r e a t e r s e l e c t i v i t y t h a n a g e n t s t h a t i n t e r a c t w i t h CM i t s e l f . Like t h e Ca2+-chelators, a g e n t s t h a t i n t e r a c t w i t h t h e c a t a l y t i c p o r t i o n s of CM-sensitive enzymes (Mechanism D; e.g. methylxanthine phosp h o d i e s t e r a s e i n h i b i t o r s ) should be considered as i n d i r e c t CM a n t a g o n i s t s , s i n c e they do n o t i n t e r a c t w i t h CM o r i t s b i n d i n g s i t e s . These a g e n t s are r e l a t i v e l y n o n - s p e c i f i c because they can i n h i b i t t h e non-stimulated form of t h e enzyme as w e l l as t h e CM-stimulated form. 48-50 I n a d d i t i o n , t h e y can i n h i b i t t h e a c t i v a t i o n of t h e enzyme b a g e n t s o t h e r t h a n CM, o r i n h i b i t CM-insensitive forms of t h e enzyme. %8 No a g e n t s t h a t i n t e r a c t s p e c i f i c a l l y w i t h t h e t e r n a r y Ca2+-CM-enzyme complex (Mechanism F) have been d e s c r i b e d , although a g e n t s t h a t a c t on t h e enzyme o r CM i t s e l f might remain bound a f t e r t h e t e r n a r y complex i s f o r m e d .
It i s , of c o u r s e , p o s s i b l e t h a t c e r t a i n a g e n t s may act by more t h a n one of t h e mechanisms d e s c r i b e d above. For example i t h a s r e c e n t l y been suggested t h a t t h e a n t i p s y c h o t i c a g e n t s t r i f l u o p e r a z i n e and p e n f l u r i d o l i n h i b i t t h e CM-induced a c t i v a t i o n of Ca2+-transport ATPase by i n t e r a c t i n g w i t h b o t h CM and t h e enzyme i t s e l f ?g ,70,89The benzodiazepine a n t i a n x i e t y a g e n t s n o t o n l y bind d i r e c t l y t o CM, l8 b u t a l s o i n t e r a c t w i t h t h e CMs e n s i t i v e p r o t e i n k i n a s e of r a t b r a i n membranes. 19 The a n t i a r r y h t h m i c and a n t i a n g i n a l agent nimodipine i n h i b i t s t h e CM-induced a c t i v a t i o n of phosp h o d i e s t e r a s e b i n t e r a c t i n g w i t h b o t h CM and w i t h t h e c a t a l y t i c p o r t i o n of t h e enzyme.
L
R e v e r s i b l e Binding of Drugs t o CM - Of t h e drugs t h a t i n t e r a c t d i r e c t l y w i t h CM o r t h e CaZ+-CM complex, t h e p h e n o t h i a z i n e a n t i p s y c h o t i c s have been s t u d i e d most e x t e n s i v e l y . These a g e n t s bind t o two d i s t i n c t classes of sites on CM: a c l a s s of s p e c i f i c , h i g h - a f f i n i t y , CaZ+-dependent s i t e s ; and a c l a s s of n o n - s p e c i f i c , low a f f i n i t y , CaZ+-independent s i t e s . 18971-73 The Ca2+-dependent s i t e s appear t o b e t h e pharmacologically important s i t e s s i n c e t h e r e i s an e x c e l l e n t c o r r e l a t i o n between t h e Ca2+-dependent binding of v a r i o u s drugs and t h e i r anti-CM p o t e n c i e s . 8,18 There are 2-3 CaZ+-dependent binding s i t e s p e r CM molecule, w i t h t h e most p o t e n t phenot h i a z i n e s d i s p l a y i n g d i s s o c i a t i o n c o n s t a n t s i n range of 1-10 uM. 12,18 S e v e r a l o t h e r classes of drugs a l s o bind t o t h e s e same CaZ+-dependent sites on CM. Ligand b i n d i n g s t u d i e s have shown t h a t t h e d i p h e n y l b u t y l p i p e r i d i n e and butyrophenone a n t i p s y c h o t i c s , l2,I8 t r i c y c l i c a n t i d e p r e s sants, benzodiazpine a n t i a n x i e t y a g e n t s , l2 3 l8 n e u r o p e p t i d e s , 28 and naphthalenesulfonamide smooth muscle r e l a x a n t s 32 bind t o CM i n a Ca2+dependent manner and can compete w i t h p h e n o t h i a z i n e s f o r s i t e s on CM. The v a r i o u s a e n t s do, however, e x h i b i t markedly d i f f e r e n t a f f i n i t i e s f o r t h e p r o t e i n . 82318 I n t e r e s t i n g l y , t h e more p o t e n t a g e n t s show c e r t a i n s t r u c t u r a l s i m i l a r i t i e s which may e n a b l e them t o bind t o s i m i l a r r e c e p t o r sites on CM. 13922 It should be emphasized t h a t t h e b i n d i n g s t u d i e s j u s t des c r i b e d do not completely r u l e out t h e p o s s i b i l i t y t h a t a p a r t i c u l a r a g e n t may bind t o s e v e r a l sites on CM, some of which might d i f f e r from t h e phenothiazine b i n d i n g s i t e s .
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Among t h e a g e n t s t h a t i n t e r a c t w i t h t h e Ca2+-CM complex are several n a t u r a l l y - o c c u r r i n g p r o t e i n s and p e p t i d e s . These i n c l u d e a Ca2+-binding p r o t e i n c a l l e d c a l c i n e u r i n , 39 a h e a t s t a b l e p r o t e i n , 35 m y e l i n b a s i c t e i n , 38 h i s t o n e , 38 B-endorphin, 28-30 d y n o r p h i n , 2 9 ~ 3 0s u b s t a n c e P 2 v o and t h e b e e venom p e p t i d e melittin?0'31Although t h e p h y s i o l o g i c a l s i g n i f i c a n c e of t h e i n t e r a c t i o n o f t h e s e endogenous compounds w i t h CM i s n o t y e t known, they may f u n c t i o n a s endogenous m o d u l a t o r s of CM a c t i v i t y and may t h e r e b y p r o v i d e p o t e n t i a l s i t e s of drug a c t i o n .
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Although t h e b i n d i n g of t h e phenoI r r e v e r s i b l e Binding of Drugs t o CM t h i a z i n e s and r e l a t e d compounds t o CM can b e r e v e r s e d by removing Ca2+ o r by d i a l y z i n g a g a i n s t an e x c e s s of competing d r u g , 18,71-73 several a g e n t s have been shown t o i n t e r a c t i r r e v e r s i b l y w i t h CM. Upon i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t o r t r e a t m e n t w i t h peroxidase-hydrogen p e r o x i d e , t h e p h e n o t h i a z i n e a n t i p s y c h o t i c s chlorpromazine and t r i f l u o p e r a z i n e b i n d irrev e r s i b l y t o CM, presumably t h r o u g h a f r e e r a d i c a l mechanism. 12974 L i k e t h e r e v e r s i b l e b i n d i n g of t h e s e a g e n t s t o CM, t h e i r r e v e r s i b l e b i n d i n i s enhanced by Ca2+. The Ca2+-dependent b i n d i n g is s a t u r a b l e w i t h one Ca5+dependent drug b i n d i n g s i t e p e r molecule. The i r r e v e r s i b l e b i n d i n g of t h e p h e n o t h i a z i n e s t o CM r e s u l t s i n t h e i r r e v e r s i b l e i n a c t i v a t i o n of CM. 7 4 CM t h a t had been i r r e v e r s i b l y l i n k e d t o chlorpromazine o r t r i f l u o p e r a z i n e by u l t r a v i o l e t i r r a d i a t i o n w a s u n a b l e t o a c t i v a t e p h o s p h o d i e s t e r a s e , l29 7 4 a l t h o u g h t h e drug-CM complex d i d i n h i b i t the a c t i v a t i o n of p h o s p h o d i e s t e r a s e by n a t i v e CM. 43 E a r l e t a l . l4 r e p o r t e d t h a t t h e a - a d r e n e r g i c a n t a g o n i s t s phenoxybenzamine and dibenamine a l s o i n h i b i t e d CM i r r e v e r s i b l y , p r o b a b l y by bindi n g d i r e c t l y t o CM i n a Ca2+-dependent manner. These a g e n t s g e n e r a t e ethyleneimmonium and carbonium i o n i n t e r m e d i a t e s t h a t might b i n d i r r e v e r s i b l y t o e l e c t r o n - r i c h f u n c t i o n a l groups on CM. 7 5 S e v e r a l a l k y l a t i n g a g e n t s t h a t are n o t a - a d r e n e r g i c a n t a g o n i s t s d i s p l a y e d l i t t l e anti-CM a c t i v i t y 1 4 915
.
Along w i t h t h e s e s t u d i e s showing t h a t c e r t a i n d r u g s can i r r e v e r s i b l y i n a c t i v a t e CM, some e v i d e n c e s u g g e s t s t h a t i t may a l s o be p o s s i b l e t o i r r e v e r s i b l y a c t i v a t e CM-sensitive enzymes. Andreasen et a l . d e s c r i b e d a p r o c e d u r e f o r p r e p a r i n g azido-CM t h a t r e t a i n e d most of t h e b i o l o g i c a l a c t i v i t y of n a t i v e CM. 7 6 Upon i r r a d i a t i o n w i t h U.V. l i g h t , the azido-CM formed c o v a l e n t l i n k a g e s w i t h s e v e r a l CM-sensitive enzymes and CM-binding p r o t e i n s and i r r e v e r s i b l y a c t i v a t e d t h e CM-sensitive ATPase o f human erythrocytes. F a c t o r s I n f l u e n c i n g t h e I n t e r a c t i o n of Drugs w i t h CM: S t r u c t u r e - A c t i v i t y S t u d i e s - The s p e c i f i c s t r u c t u r a l f a c t o r s t h a t e n a b l e d r u g s t o b i n d t o CM are p o o r l y understood. It h a s been s u g g e s t e d t h a t t h e a b i l i t i e s of v a r i o u s d r u g s t o i n t e r a c t w i t h CM may b e c l o s e l y r e l a t e d t o t h e i r a b i l i t i e s t o r ~t o~ p a r t i t i o n between a l i p i d p h a s e and a n a q u e o u s p h a s e , 2 4 3 2 7 ~ ~ 4o 3 t h e i r a b i l i t i e s t o s t a b i l i z e membranes. 77-79 These f i n d i n g s are c o n s i s t e n t w i t h t h o s e showing t h a t hydrophobic r e g i o n s o f CM are i n v o l v e d i n t h e b i n d i n g of d r u g s . 57-59 Y 80 According t o proposed models, t h e b i n d i n g of Ca2+ i n d u c e s c o n f o r m a t i o n a l changes i n CM, exposing a hydrophobic domain which can t h e n bind l i p o p h i l i c d r u g s o r serve as an i n t e r f a c e f o r t h e b i n d i n g of CM t o i t s t a r g e t enzymes. 57-59 However, t h e r e is a l s o e v i d e n c e s u g g e s t i n g t h a t o t h e r f o r c e s b e s i d e s hydrophobic i n t e r a c t i o n s may b e i n v o l v e d i n t h e b i n d i n g of d r u g s t o CM. Many h i g h l y l i p o p h i l i c d r u g s a r e o n l y weak i n h i b i t o r s of CM and c e r t a i n h y d r o p h i l i c d r u g s are v e r y p o t e n t CM a n t a g o n i s t s . 13922 S t u d i e s on the pH-dependence of t h e b i n d i n g of t r i f l u o p e r a z i n e t o CM s u g g e s t t h a t i o n i c
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f o r c e s may p l a y an i m p o r t a n t r o l e i n t h i s i n t e r a c t i o n . 1 2 , 1 3 F i n a l l y , rec e n t s t r u c t u r e - a c t i v i t y s t u d i e s have shown t h a t v a r i o u s classes of CM i n h i b i t o r s e x h i b i t c e r t a i n s t r u c t u r a l s i m i l a r i t i e s . l 3 y Z 2 I n many c a s e s , s l i g h t m o d i f i c a t i o n s i n chemical s t r u c t u r e can g r e a t l y a l t e r t h e a b i l i t y of a compound t o b i n d t o CM and i n h i b i t i t s a c t i v i t y . Such s t r u c t u r a l s p e c i f i c i t y i s s u g g e s t i v e of s p e c i f i c drug-receptor i n t e r a c t i o n s and n o t simple hydrophobic bonding. To more p r e c i s e l y determine t h e s t r u c t u r a l f a c t o r s t h a t e n a b l e d r u g s t o i n t e r a c t w i t h CM, i t i s n e c e s s a r y t o s y s t e m a t i c a l l y e v a l u a t e t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of r e p r e s e n t a t i v e chemical c l a s s e s of CM antago n i s t s . To d a t e , o n l y a few such s t u d i e s have been performed. T h e i r res u l t s are summarized below. S e v e r a l s t u d i e s examining t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of t h e p h e n o t h i a z i n e s have been r e p o r t e d . M o d i f i c a t i o n s of t h e promazine s t r u c t u r e (1) included r i n g s u b s t i t u t i o n , 1 1 , 1 3 , 2 2 , 2 3 , 7 7 , 8 1 l e n g t h of side-chain,-22 s t r u c t u r e of s i d e - c h a i n amine, 22 ,82 o x i d a t i o n s t a t e of s u l f u r , 11,22,81 and attachment of an a d d i t i o n a l a r o m a t i c r i n g 82 (2-11). _Within a series of r i n g - s u b s t i t u t e d promazine d e r i v a t i v e s , good c o r r e l a t i o n was found between o r t a n o l / b u f f e r p a r t i t i o n c o e f f i c i e n t s and IC50 v a l u e s f o r i n h i b i t i o n of CM a c t i v i t y , i n d i c a t i n g t h a t t h e hydrophob i c i t y of t h e p h e n o t h i a z i n e n u c l e u s may be an important d e t e r m i n a n t of potency. By c o n t r a s t , no such c o r r e l a t i o n w a s found among a series of promazine a n a l o g s i n which t h e s i d e - c h a i n was modified, a l t h o u g h v a r y i n g t h e n a t u r e and p o s i t i o n of t h e s i d e c h a i n amino group d i d i n f l u e n c e potency.22 A l i k e l y e x p l a n a t i o n f o r t h e s e f i n d i n g s is t h a t both hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s p l a y a r o l e i n t h e b i n d i n g of p h e n o t h i a z i n e s t o t h e Ca2+-CM complex. 1 3 , z 2 Although o n l y l i m i t e d i n f o r m a t i o n i s now a v a i l a b l e , t h e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s of t h e dibenzaze i n e s (12-14) - - appear t o be s i m i l a r t o t h o s e of t h e p h e n o t h i a z i n e s . 1 1 9 1 2 * z 3 The t h i o x a n t h e n e a n t i p s y c h o t i c s ( 1 5 ) c o n t a i n a n e x o c y c l i c double bond
I
'
c72
2
Crz 742
/ N\
CH3
CH3
-4
pounds were s l i g h t l y more p o t e n t CM i n h i b i t o r s t h a n t h e i r analagous phenothiaz i n e s . 12,22,24 Although geometric i s o m e r s can exist a c r o s s t h e double bond, t h e r e w a s l i t t l e evidence of s t e r e o specificity i n the interaction of these a g e n t s w i t h CM. 12,18,24 The implica-
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tions of this lack of stereospecificity have been considered elsewhere.8 ~ 1 21,3 ~ 2 4 The diphenylbutylpiperidine antipsychotics pimozide (16) and penfluridol (=)are among the most potent CM antagonists. 1 3 ~ m ~ 2 Both 4 aromatic rings appear to be necessary for high potency, since the compound a pimozide analog lacking one phenyl group, was only 116 as R-6033 (g), potent as pimozide. 22 Similarly, the butyrophenone antipsychotic haloperidol @), which contains only a single aromatic ring,is a relatively weak CM antagonist. 12~13,22 Hidaka and his co-workers have studied the structure-activit relationships of the naphthalenesulfonamide class o f CM The effect on potency of ring substitution and modifying the length of the side chain 34983984 and position of the sulfonamide function 33934 have been examined. Within a series of 1-naphthalenesulfonamides, good correlation was found between octanolfbuffer partition coefficients and anti-CM potencies. However, the significance of this correlation was reduced when data from the corresponding 2-substituted series was factored in ,34 indicating that the position of the amino group as well as hydrophobicity may be important in determining potency.
antagonist^?^^^^^^^,^^
(z),
Compound R 24571, calmidazolium is a derivative of the antimycotic agent miconazole and appears to be an especially potent and selective CM antagonist. It is about 500 times more potent than trifluoperazine in inhibiting the activation of several M-dependent enzymes, but displays little affinity for several neurotransmitter and hormone receptors to which other CM inhibitors bind.s1 I n addition, R 24571 has been shown to be more potent in preventing the activation of Ca2+ transport ATPase by CM than in preventing the activation of the enzyme by other agents. 59
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Although i t i s n o t y e t clear whether t h e p o l y p e p t i d e c l a s s of CM i n h i b i t o r s bind t o t h e s a m e s i t e s on CM t o which t h e p h e n o t h i a z i n e s b i n d , t h e i r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s s u g g e s t t h a t b o t h hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s a r e involved i n t h e i r b i n d i n g t o CM. Of several p e p t i d e s t h a t bound w e l l t o CM,29 o r d i s p l a y e d anti-CM a c t i v i t y , 30 a l l contained a r e g i o n of b a s i c amino a c i d r e s i d u e s i n c l o s e proximity t o s e v e r a l hydrophobic r e s i d u e s . General S t r u c t u r e C h a r a c t e r i s t i c s of CM I n h i b i t o r s - Although t h e v a r i o u s CM-binding- drugs - Belong- t o d i f f e r e n t chemical c l a s s e s . many e x h i b i t c e r t a i n s t r u c t u r a l similarities. The e s s e n t i a l f e a t u r e s of t h e s e a g e n t s are a l a r g e hydrophobic r e g i o n c o n s i s t i n g of a t least two a r o m a t i c r i n g s , e i t h e r a d j a c e n t t o each o t h e r , o r j o i n e d a t one o r two p o s i t i o n s , and a s i d e c h a i n amino group t h a t i s a t l e a s t 4 atoms removed from t h e aromatic r i n g s . 1 3 $22 The geometric conformation of t h e r i n g s t r u c t u r e does n o t appear t o be an important determinant of anti-CM a c t i v i t y because compounds having d i f f e r e n t r i n g s t r u c t u r e s , such a s t h e diphenylbutylpiperidines, phenot h i a z i n e s , t h i o x a n t h e n e s , dibenzazepines, and naphthalenesulfonamides, can a l l i n t e r a c t w i t h CM. A more important f a c t o r appears t o b e t h e hydrophob i c i t y of t h e r i n g s t r u c t u r e ; s u b s t i t u e n t s t h a t i n c r e a s e hydrophobicity i n c r e a s e a n t i CM potency, whereas t h o s e t h a t d e c r e a s e hydrophobicity dec r e a s e potency. 13 22 9 34 9
The t y p e of amino group does n o t seem t o b e c r i t i c a l s i n c e primary, secondary, t e r t i a r y , p i p e r a z i n y l and i m i d i a z o l e amines i n h i b i t CM. A l l t h a t seems necessary i s t h a t t h e amino group c a r r y a p o s i t i v e charge and that i t b e s e p a r a t e d from t h e hydrophobic r e g i o n of t h e drug molecule by 4 o r more atoms, a l t h o u g h t h e d i s t a n c e r e q u i r e d f o r o p t i m a l a c t i v i t y is not y e t known. 13,22934 T h i s i s s u e i s complicated by t h e f a c t t h a t amino groups a t t h e end of l o n g , f l e x i b l e a l k y l c h a i n s could assume a v a r i e t y of d i f f e r e n t p o s i t i o n s t o a l l o w b i n d i n g t o CM, o r t h e long a l k y l c h a i n s might undergo hydrophobic i n t e r a c t i o n s w i t h CM. I n t e r e s t i n g l y , t h e amino-acrid i n e d e r i v a t i v e q u i n a c r i n e , which h a s a v e r y h y d r o p h i l i c s i d e c h a i n , and t h e thioxanthene a n t i p s y c h o t i c s , which have t h e i r s i d e c h a i n amino groups held i n f i x e d p o s i t i o n s , are p o t e n t CM i n h i b i t o r s , 22,24 s u g g e s t i n g t h a t t h e p o s i t i o n of t h e amino group, r a t h e r t h a n t h e h y d r o p h o b i c i t y of t h e s i d e c h a i n , determines potency. This model f o r t h e i n t e r a c t i o n of drugs w i t h CM a p p l i e s only t o a g e n t s t h a t i n t e r a c t w i t h t h e p h e n o t h i a z i n e binding s i t e s . It i s p o s s i b l e t h a t d r u g s having somewhat d i f f e r e n t s t r u c t u r a l c h a r a c t e r i s t i c s might bind t o d i f f e r e n t s i t e s on CM. Agents such as DDT and T r i t o n X-100 have s t r u c t u r e s t h a t d i f f e r from t h e proposed model. A t p r e s e n t , i t i s not known whether t h e s e a g e n t s bind t o t h e same s i t e s on CM t h a t t h e p h e n o t h i a z i n e s do. 41942 R e s u l t s of NMR and f l u o r e s c e n c e spectroscopy s t u d i e s have provided some evidence f o r t h e e x i s t e n c e of s e v e r a l t y p e s of drug binding s i t e s on CM. 15,20921 E f f e c t s of Drug Binding on t h e Conformation and A c t i v i t y of CM - The mechanism by which drug b i n d i n a l t e r s t h e a c t i v i t y of CM i s n o t completely understood. N M R 2°,68,69 ,86,86and f l u o r e s c e n c e spectroscopy 15,21929 d a t a i n d i c a t e t h a t t h e b i n d i n g of drugs induces changes i n t h e conformation of CM. S t u d i e s examining t h e b i n d i n g of 1 2 5 I - l a b e l l e d CM t o membrane s i t e s have shown a t drug b i n d i n g d e c r e a s e s t h e a f f i n i t y of CM f o r i t s t a r g e t enzymes. 62r@These d a t a s u g g e s t t h a t drug b i n d i n g a l t e r s t h e conformation of t h e Ca2+-CM complex and d e c r e a s e s i t s a b i l i t y t o i n t e r a c t w i t h recogn i t i o n sites on CM-sensitive enzymes.
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The l o c a t i o n of t h e p h e n o t h i a z i n e b i n d i n g sites on CM i s n o t known. I t h a s been s u g g e s t e d t h a t t h e d r u g s may b i n d t o a hydrophobic r e g i o n of CM t h a t s e r v e s as an i n t e r f a c e f o r t h e b i n d i n g of CM t o i t s t a r g e t enzymes. 57-59 R e s u l t s of I H NMR s t u d i e s i n d i c a t e t h a t m e t h i o n i n e and p h e n y l a l a n i n e r e s i d u e s n e a r Ca2+-binding domains 11, 111 and I V of CM may play an important r o l e i n binding phenothiazines. 80986 Other r e s u l t s s u g g e s t t h a t a c i d i c r e s i d u e s on CM are i n v o l v e d i n b i n d i n g b a s i c d r u g s . l 2 3 l 3 These d a t a s u g g e s t t h a t drug b i n d i n g s i t e s on CM may c o n s i s t of a hydrophobic r e g i o n , c o n t a i n i n g m e t h i o n i n e r e s i d u e s , i n c l o s e p r o x i m i t y t o a n a c i d i c r e s i d u e s u c h as a s p a r t i c o r g l u t a m i c a c i d . Head -et a l . r e p o r t e d t h a t a cyanogen bromide-cleavage fragment of CM, c o n t a i n i n g r e s i d u e s 77-124, w a s a b l e t o b i n d p h e n o t h i a z i n e s i n a Ca2+-dependent manner. 85 The r e c e n t f i n d i n g s t h a t c e r t a i n d r u g s can b i n d i r r e v e r s i b l y t o CM should a i d i n f u r t h e r i d e n t i f y i n g drug b i n d i n g s i t e s on CM. 1 4 s 7 0 References 1. W.Y. Cheung, Biochem. Biophys. Res. Commun., 2, 533 (1970). 2. S. Kakiuchi and R. Yamazaki, Biochem. Biophys. Res. Commun. 1104 (1970). 3. C.O. Brostrom and D.J. Wolff, Biochem. Pharmacol. 30, 1395 (1981). 4. W.Y. Cheung, Science (Washington. D.C.), 207, 19 (1980). C.B. Klee, T . R . Crouch and P.G. Kichman, Annu. Rev. B€oc!em. S , 484 (1980). 5. 6. C.B. Klee, i n "Calcium and Cell Function", Vol. 1, W.Y. Cheung, Ed., Academic Press, New York, N.Y. (1980) p. 59. T.C. Vanaman, in "Calcium and Cell Function: Vol. 1, W.Y. Cheung, Ed., Academic Press, 7. New York, N.Y. (1980), p . 41. 8. B. Weiss and T.L. Wallace, in "Calcium and Cell Function", Vol. 1, W.Y. Cheung, Ed., Academic Press, New York, N.Y. (1980) p. 329. 9. J . Krebs, Cell Calcium, 2, 295, (1981). 10 * B. Weiss, R. Fertel, R. Figlin and P. Uzunov, Mol. Pharmacol. 615 (1974). 11. R.M. Levin and B. Weiss, Mol. Pharmacol. 2, 5 8 1 (1976). 12. B. Weiss, W. Prozialeck, M. Cimino, M.S. Barnette and T.L. Wallace, Ann. N.Y. Acad. Sci., 356, 319 (1980). 2217 (1982). 13. B. Weiss, W.C. Prozialeck and T.L. Wallace, Biochem. Pharmacol. 1 4 . C.Q. Earl, W.C. Prozialeck and B. Weiss, Fed. Proc. Fed. Am. Sac. Exp. Biol., 1565 (1982). 15. K. Watanabe and W.I. West, Fed. Proc. Fed. Am. SOC. Exp. B i o l . , 41, 2292 (1982). 16. M. Volpi, R . I . Sha'afi, P.M. Epstein, D.M. Andrenyak and M.B. Feinstein, Proc. Nat!. Acad. Sci. USA., 795 (1981). 363 (1981). 17. M. Volpi, R . I . Sha'afi and M.B. Feinstein, Mol. Pharmacol., 18. R.M. Levin and B. Weiss, J . Pharmacol. Exp. Ther., 208, 454 (1979). 19. R . J . DeLorenzo, S. Burdette and J. Holderness, Science, 213, 546 (1981). 20 * S.-L. Brostrgm, B. Ljung, Sven Mzrdh, S. Forsen and E. Thulin, Nature (London), 777 (1981). 21. P.M. Epstein, K. Fiss, R. Hachisu and D.M. Andrenyak, Biochem. Biophys. Res. Commun., 105, 1142 (1982). 22. W.C. Prozlaleck and B. Weiss, J . Pharmacol. Exp. Ther., 222, 509 (1982). 23. J.H. Zavecz, T.E. Jackson, G.L. Limp and T.O. Yellin, Eur. J. Pharmacol., 78, 375, (1982). 24. J.A. Norman, A.H.Drummond and P. Moser, Mol. Pharmacol., 16. 1089 (1979). 25. N. Katoh, B.C. Wise, R.W. Wrenn, J.F. Kuo, Biochem. J . , 199, (1981). 26. K. Watanabe, E.F. Williams, J . S . Law and W.L. West, Biochem. Pharmacol., 30, 335 (1981). 27. T. Tanaka and H. Hidaka, Biochem. Biophys. Res. Commun., ,&I 447 (1981). 28. M. Sellinger-Barnette and B. Weiss, Mol. Pharmacol., 86 (1982). 29. D.A. Malencik and S . R . Anderson, Biochemistry, 3480 (1982). in press. 30. M. Sellinger-Barnette and B. Weiss, Adv. Cyclic Nucleotide Res., 31. M. Comte, Y. Maulet and J.A. Cox. Biochem. J . 269 (1983). 32. H. Hidaka, T. Yamaki, M. Naka, T. Tanaka, H. Hayashi and R. Kobayashi, Mol. Pharmacol., 17, 66 (1980). 5 7 1 (1981). 33. H. Hidaka, M. Asano and T. Tanaka, Mol. Pharmacol., 34. T. Tanaka, T. Ohmura and H. Hidaka, Mol. Pharmacol., 22, 403 (1982). 35. R.K. Sharma, E. Wirch and J.H. Wang, J . Biol. Chem., 3575 (1978). 36. R.W. Wallace, T.J. Lynch, E.A. Tallant, and W.Y. Cheung, J . Biol. Chem., 254, 377, (1978). 37. F.L. Larsen, B.U. Raess, T . R . Hinds and F.F. Vincenzi, J . Supramol. Struct., 2, 269 (1978). 227 (1980). 38. R.J.A. Grand and S.V. Perry, Biochem. J., 39. C.B. Klee, T.H. Crouch and M.H. Krinks, Proc. Nat. Acad. Sci. USA, 6270 (1979). 40. T. Itano, R. Itano and J.T. Penniston, Biochem. J., 455 (1980).
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16,
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.
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_-
I n t r o d u c t i o n - C a l m o d u l i n (CM) i s a w i d e l y - d i s t r i b u t e d , Ca2+-binding prot e i n h a v i n g a m o l e c u l a r w e i g h t o f a p r o x i m a t e l y 17,000. S i n c e i t s d i s c o v e r y i n 1970 as an a c t i v a t o r o f a C a 5 + - s e n s i t i v e form o f p h o s p h o d i e s t e r a s e , CM h a s b e e n shown t o r e g u l a t e a v a r i e t y o f Ca2+-dependent enzymes a n d p r o c e s s e s l v 2 . C o n s i d e r a b l e e v i d e n c e now s u g g e s t s t h a t CM i s t h e p r i n c i p a l m e d i a t o r o f t h e e f f e c t s of Ca2+ i n most e u k a r y o t i c c e l l s . F o r d i s c u s s i o n s of t h e b i o c h e m i c a l p r o p e r t i e s and t h e p h y s i o l o g i c a l f u n c t i o n s of CM, t h e r e a d e r i s r e f e r r e d t o a n y o f s e v e r a l e x c e l l e n t r e v i e w s . 3-9 S i n c e CM p l a y s a f u n d a m e n t a l r o l e i n c e l l b i o l o g y , a g e n t s t h a t i n h i b i t i t s a c t i v i t y s h o u l d h a v e i m p o r t a n t p h a r m a c o l o g i c a l e f f e c t s . An und e r s t a n d i n g of t h e mechanisms by w h i c h t h e s e a g e n t s a l t e r CM a c t i v i t y may h e l p t o e x p l a i n t h e i r p h a r m a c o l o g i c a l a c t i o n s o r may s u g g e s t new a p p r o a c h es f o r m o d i f y i n g v a r i o u s p h y s i o l o g i c a l o r p a t h o l o g i c a l p r o c e s s e s . In a d d i t i o n , t h e d e v e l o p m e n t o f s e l e c t i v e CM a n t a g o n i s t s may p r o v i d e a u s e f u l means f o r f u r t h e r s t u d y i n g t h e b i o l o g i c a l r o l e s of CM. I n t h e mid 1 9 7 0 ' s Weiss and c o - w o r k e r s r e p o r t e d t h a t t h e p h e n o t h i a z i n e a n t i s y c h o t i c s i n h i b i t e d t h e CM-induced a c t i v a t i o n of p h o s p h o d i e s t e r ase. S u b s e q u e n t s t u d i e s h a v e shown t h a t a v a s t a r r a y o f compounds b e l o n g i n g t o d i v e r s e c h e m i c a l and p h a r m a c o l o g i c a l c l a s s e s c a n i n h i b i t t h e a c t i o n s o f CM i n a v a r i e t y of b i o c h e m i c a l s y s t e m s . 8,12913 Table 1 lists t h e c l a s s e s of a g e n t s t h a t h a v e b e e n shown t o i n h i b i t o n e o r more a c t i o n s o f CM. The p u r p o s e o f t h i s r e v i e w i s t o b r i e f l y summarize t h e g e n e r a l mechanisms b y which t h e s e a g e n t s m o d i f y t h e a c t i v i t y o f CM a n d t o examine i n d e t a i l t h e s t r u c t u r a l factors t h a t enable drugs t o i n t e r a c t d i r e c t l y w i t h CM. S e v e r a l a s e c t s o f t h e pharmacology o f CM h a v e b e e n r e v i e w e d e l s e w h e r e . 8,12913,y2953 Mechanisms of P h a r m a c o l o g i c a l l y A l t e r i n g CM A c t i v i t y - Most i f n o t a l l o f t h e e f f e c t s of CM r e s u l t from t h e a c t i v a t i o n , o r o c c a s i o n a l l y i n h i b i t i o n , of s p e c i f i c enzymes. 3-6 F i g u r e 1 i l l u s t r a t e s t h e g e n e r a l mechanism by which CM r e g u l a t e s most Ca2+-dependent enzymes. N e i t h e r Ca2+, n o r CM a l o n e i s a c t i v e . However, e a c h CM m o l e c u l e c a n b i n d up t o 4 C a 2 + i o n s . 3 - 7 , 5 0 , 5 4 The b i n d i n g of C a 2 + a l t e r s t h e c o n f o r m a t i o n o f CM, 3-7 i n c r e a s i n g i t s h e l i c a l c o n t e n t 54-56 a n d e x p o s i n g h y d r o p h o b i c r e g i o n s . 57758 I n t h i s conf o r m a t i o n , t h e Ca2+-CM complex c a n b i n d t o r e g u l a t o r y s i t e s o n t a r g e t enzymes and t h r o u g h a n unknown mechanism a l t e r t h e i r a c t i v i t i e s . 3-6,59 A l t h o u g h c o n t r o v e r s i a l , some e v i d e n c e s u g g e s t s t h a t v a r i o u s C M - s e n s i t i v e enzymes may r e q u i r e d i f f e r e n t s t o i c h i o m e t r i c amounts o f Ca2+ and CM f o r a c t i v i t y 5 6 947 9 60-63
.
T h e r e a r e s e v e r a l mechanisms b y w h i c h d r u g s m i g h t a c t t o i n h i b i t t h e a c t i o n s of CM. 8 , 5 2 A g e n t s m i g h t a c t b y : (A) d e c r e a s i n g t h e c o n c e n t r a t i o n o f a v a i l a b l e Ca2+ a n d p r e v e n t i n g t h e f o r m a t i o n o f t h e a c t i v e Ca2+-CM comp l e x , (B) b i n d i n g t o CM a n d a l t e r i n g i t s a b i l i t y t o b i n d Ca2+, (C) b i n d i n g
ANNUAL REPORTS I N MEDICINAL C H F N I S T R Y 4 I I
Copyright D 1983 by Academ~cPress. Ine All righis of reproductiontn any form r e w v e d . ISBN 0- I20405 I 8 4
204
Sect. IV
- M e t a b o l i c Diseases,
Endocrine Function
B a i l e y , Ed.
t o t h e Ca2+-CM complex and m o d i f y i n g i t s a c t i v i t y , (D) b i n d i n g t o t h e CMr e c o g n i t i o n s i t e o n t h e CM-sensitive enzyme a nd t h u s p r e v e n t i n g t h e i n t e r a c t i o n o f the Ca2+-CM complex w i t h t h e enzyme, (E) i n t e r a c t i n g , e i t h e r Table 1
-
Classes o f A g e n t s t h a t I n h i b i t t h e A c t i o n s o f Ca lm odulin
Pharmacological Class a -adrenergic
antagonist
6 -adrenergic
Chemical Class benzyl- 6 -chloroethylamine ergot alkaloid alkylaminonaphthy1 ether benzodiazepine
mtagonist antianxiety agent antiarrhythmic1,4-dihydropyriantianginal agent dine dimethoxyphenylacet o n i t r i l e antidepressant dibenza zepine dibenzocycloheptadiene antidiarrheal diphenylalkylpiperidine antihistamine phenothiazine antimalarial aminoacridine cinchona alkaloid antipsychotic butyrophenone benzocycloheptap idoisoquinoline dicnzodiazmine diphenylbutfi piperidine phenothiazine
-
cancer chemotherapeutic agent local anesthetic
neuropeptide smooth muscle relaxant miscellaneous agents
*Probable Mechanism
**Selected
phenoxybenzamine
C
14,15
dihydroergotamine propranolol
C C
14 16,17
diazepam
C,D
18,19
felodipine
A,C,E
20,21
verapamil
A,C,E
21
Mpramine amitriptyline
C
C
11,22 11
loperamide
C
23
promethazine quinacrine quinme haloperidol butaclml
C
C C C C
11,22 16,17,22 17 11,12,24 24
clozapine penfluridol
C
12,24
trifluoperazine and analogs chlorprothixene adriamycin vinblast ine
C
11,12,22
C C C
12,22,24
tetracaine dibucaine phenacaine lidocaine B -endorphin me1itt in
C C C C C C
16 ,17,27 16,17,27 16,17,27 27 12,28-30 30,31
prenylamine
C
32
W-7 and analogs
C
32-34
EGTA
A
C
8 ,I1 35-40
Triton X-100
C
41
DDT
C,D
42
chlorpromazine-linked 04
D
43
Mg2’
theophylline R-24571
B
E C
44-47 48-50 51
proadifen (SKF-525A)
C
16,17
Representative Agent
thioxanthene anthracycline vinca alkaloid benzoic es t er cinchoninamide phenylacetamidine phenylamide opioid peptide insect venom peptide diphenylpropylamine naphthalenesfulfypide Ca chelator 04-binding prot e in polpxyethylene detergent chlorinated ethane insecticide modified metal ion methylxanthene miconazoleanalogue B -aminoethyldiphenylpentenoate
calcineurin
C
References
11,12,24
2q
15,26
*Probable mechanism refers t o l e t t e r s shown i n Figure 1, as discussed in the text. ‘“References a r e for each chemical class, not only the representative agent.
Chap. 21
SAR of Calmodulin A n t a g o n i s t s
Prozialeck
205
-
F i g u r e 1 Mechanisms f o r t h e A c t i v a t i o n and I n h i b i t i o n of CM-Sensitive Enzymes
a RESPONSE
c o m p e t i t i v e l y o r non-competitively, w i t h t h e c a t a l y t i c p o r t i o n of the CMs e n s i t i v e enzyme and a l t e r i n g i t s a c t i v i t y , (F) i n t e r a c t i n g w i t h t h e ternary Ca2+-CM-enzyme complex.
2+
Agents t h a t a c t by d e c r e a s i n g t h e c o n c e n t r a t i o n of Ca (Mechanism A) i n c l u d e c h e l a t i n g a g e n t s (e.g. EDTA and EGTA) and t h e s o - c a l l e d Ca2+channel b l o c k e r s (e.g. verapamil and n i f i d i p i n e ) , which i n h i b i t t h e i n f l u x of e x t r a c e l l u l a r Ca2+(see Chapter 9 and r e f e r e n c e s 6 4 , 6 5 ) . S i n c e t h e y do n o t i n t e r a c t d i r e c t l y w i t h CM, t h e s e a g e n t s d i s p l a y l i t t l e s p e c i f i c i t y f o r CM-regulated systems. For example, t h e Ca2+-chelators i n h i b i t a l l biochemical a c t i o n s of C a 2 + , n o t o n l y i t s i n t e r a c t i o n w i t h CM. It should be noted t h a t t h e r e is some evidence t h a t c e r t a i n Ca2+ channel b l o c k e r s might i n t e r a c t w i t h CM i t s e l f . 20,21
L i t t l e i s known about a g e n t s t h a t i n t e r a c t w i t h CM and a l t e r i t s a b i l i t y t o bind Ca2+ (Mechanism B ) . Such a g e n t s might a c t by b i n d i n g d i r e c t l y t o t h e CaZ+-binding s i t e s on CM o r by b i n d i n g t o some o t h e r s i t e on t h e molecule and inducing conformational changes i n t h e Ca2+-binding regions. Although a v a r i e t y o f d i - and t r i v a l e n t c a t i o n s can i n t e r a c t w i t h Ca2+ b i n d i n s i t e s on CM, 50,66 and t o v a r y i n g d e g r e e s m i m i c t h e a c t i o n s of CaZf66,8$it is less c l e a r whether t h e s e a g e n t s can a n t a g o n i z e o r potent i a t e t h e e f f e c t s of Ca2+. S e v e r a l s t u d i e s have shown t h a t M 2+ i n h i b i t s t h e a c t i v a t i o n of CM-dependent enzymes by competing w i t h Ca2' f o r i o n b i n d i n g s i t e s on CM. 44-47 However, La3+, which enhanced t h e Ca2+-mediated binding of CM t o r a t s t r i a t a l p a r t i c l e s , had no e f f e c t on t h e a c t i v a t i o n of a d e n y l a t e c y c l a s e by Ca2+ and CM. 67 R e c e n t l y , s e v e r a l metals i n c l u d i n g Hg, Cd, Zn, Co and S r have been shown t o i n h i b i t t h e CM-induced a c t i v a t i o n of p h o s p h o d i e s t e r a s e , a l t h o u g h t h e mechanism u n d e r l y i n g t h i s e f f e c t h a s y e t t o be determined. 88 R e s u l t s of s t u d i e s examining t h e NMR spectrum of CM s u g g e s t t h a t t h e b i n d i n g of p h e n o t h i a z i n e a n t i p s y c h o t i c s by t h e Ca2+CM complex ( s e e below) a l t e r s t h e Ca2+ b i n d i n g domains of CM. 68369 Howe v e r , t h e s i g n i f i c a n c e of t h i s i s u n c l e a r s i n c e t h e b i n d i n g of t h e drugs themselves r e q u i r e s Ca2+ 8,12 and t h e i n h i b i t o r y e f f e c t s of t h e d r u g s cannot b e overcome by i n c r e a s i n g t h e c o n c e n t r a t i o n of Ca2+. 11 By f a r , t h e l a r g e s t group of CM a n t a g o n i s t s c o n s i s t s of t h o s e a g e n t s t h a t i n t e r a c t d i r e c t l y w i t h t h e Ca2+-CM complex (Mechanism C) Accordingl y , t h i s group of compounds will s e r v e as t h e f o c u s of t h i s review.
.
Thus f a r , l i t t l e a t t e n t i o n h a s been given t o a g e n t s t h a t act a t t h e CM-binding s i t e s on CM-sensitive enzymes (Mechanism D). However, r e c e n t f i n d i n g s suggest t h a t t h e s e may p r o v i d e u s e f u l s i t e s f o r pharmacological i n t e r v e n t i o n . CM t h a t had been i r r e v e r s i b l y l i n k e d t o chlorpromazine by
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u l t r a v i o l e t i r r a d i a t i o n ( s e e below) i n h i b i t e d t h e a c t i v a t i o n of phosphod i e s t e r a s e By n a t i v e CM. 43 The chlorpromazine-CM complex, which w a s una b l e t o a c t i v a t e phosphodiesterase, a p p a r e n t l y competed w i t h CM f o r a regu l a t o r y s i t e on t h e enzyme, s u g g e s t i n g t h a t i t may be p o s s i b l e t o develop a new c l a s s of CM a n t a g o n i s t s t h a t are d i r e c t e d a t t h e CM-binding s i t e s on CM-sensitive enzymes. Since t h e r e is some evidence t h a t v a r i o u s CM-sensit i v e enzymes may have somewhat d i f f e r e n t CM-binding sites,5,6,60-62 a g e n t s d i r e c t e d a t t h e s e s i t e s might d i s p l a y g r e a t e r s e l e c t i v i t y t h a n a g e n t s t h a t i n t e r a c t w i t h CM i t s e l f . Like t h e Ca2+-chelators, a g e n t s t h a t i n t e r a c t w i t h t h e c a t a l y t i c p o r t i o n s of CM-sensitive enzymes (Mechanism D; e.g. methylxanthine phosp h o d i e s t e r a s e i n h i b i t o r s ) should be considered as i n d i r e c t CM a n t a g o n i s t s , s i n c e they do n o t i n t e r a c t w i t h CM o r i t s b i n d i n g s i t e s . These a g e n t s are r e l a t i v e l y n o n - s p e c i f i c because they can i n h i b i t t h e non-stimulated form of t h e enzyme as w e l l as t h e CM-stimulated form. 48-50 I n a d d i t i o n , t h e y can i n h i b i t t h e a c t i v a t i o n of t h e enzyme b a g e n t s o t h e r t h a n CM, o r i n h i b i t CM-insensitive forms of t h e enzyme. %8 No a g e n t s t h a t i n t e r a c t s p e c i f i c a l l y w i t h t h e t e r n a r y Ca2+-CM-enzyme complex (Mechanism F) have been d e s c r i b e d , although a g e n t s t h a t a c t on t h e enzyme o r CM i t s e l f might remain bound a f t e r t h e t e r n a r y complex i s f o r m e d .
It i s , of c o u r s e , p o s s i b l e t h a t c e r t a i n a g e n t s may act by more t h a n one of t h e mechanisms d e s c r i b e d above. For example i t h a s r e c e n t l y been suggested t h a t t h e a n t i p s y c h o t i c a g e n t s t r i f l u o p e r a z i n e and p e n f l u r i d o l i n h i b i t t h e CM-induced a c t i v a t i o n of Ca2+-transport ATPase by i n t e r a c t i n g w i t h b o t h CM and t h e enzyme i t s e l f ?g ,70,89The benzodiazepine a n t i a n x i e t y a g e n t s n o t o n l y bind d i r e c t l y t o CM, l8 b u t a l s o i n t e r a c t w i t h t h e CMs e n s i t i v e p r o t e i n k i n a s e of r a t b r a i n membranes. 19 The a n t i a r r y h t h m i c and a n t i a n g i n a l agent nimodipine i n h i b i t s t h e CM-induced a c t i v a t i o n of phosp h o d i e s t e r a s e b i n t e r a c t i n g w i t h b o t h CM and w i t h t h e c a t a l y t i c p o r t i o n of t h e enzyme.
L
R e v e r s i b l e Binding of Drugs t o CM - Of t h e drugs t h a t i n t e r a c t d i r e c t l y w i t h CM o r t h e CaZ+-CM complex, t h e p h e n o t h i a z i n e a n t i p s y c h o t i c s have been s t u d i e d most e x t e n s i v e l y . These a g e n t s bind t o two d i s t i n c t classes of sites on CM: a c l a s s of s p e c i f i c , h i g h - a f f i n i t y , CaZ+-dependent s i t e s ; and a c l a s s of n o n - s p e c i f i c , low a f f i n i t y , CaZ+-independent s i t e s . 18971-73 The Ca2+-dependent s i t e s appear t o b e t h e pharmacologically important s i t e s s i n c e t h e r e i s an e x c e l l e n t c o r r e l a t i o n between t h e Ca2+-dependent binding of v a r i o u s drugs and t h e i r anti-CM p o t e n c i e s . 8,18 There are 2-3 CaZ+-dependent binding s i t e s p e r CM molecule, w i t h t h e most p o t e n t phenot h i a z i n e s d i s p l a y i n g d i s s o c i a t i o n c o n s t a n t s i n range of 1-10 uM. 12,18 S e v e r a l o t h e r classes of drugs a l s o bind t o t h e s e same CaZ+-dependent sites on CM. Ligand b i n d i n g s t u d i e s have shown t h a t t h e d i p h e n y l b u t y l p i p e r i d i n e and butyrophenone a n t i p s y c h o t i c s , l2,I8 t r i c y c l i c a n t i d e p r e s sants, benzodiazpine a n t i a n x i e t y a g e n t s , l2 3 l8 n e u r o p e p t i d e s , 28 and naphthalenesulfonamide smooth muscle r e l a x a n t s 32 bind t o CM i n a Ca2+dependent manner and can compete w i t h p h e n o t h i a z i n e s f o r s i t e s on CM. The v a r i o u s a e n t s do, however, e x h i b i t markedly d i f f e r e n t a f f i n i t i e s f o r t h e p r o t e i n . 82318 I n t e r e s t i n g l y , t h e more p o t e n t a g e n t s show c e r t a i n s t r u c t u r a l s i m i l a r i t i e s which may e n a b l e them t o bind t o s i m i l a r r e c e p t o r sites on CM. 13922 It should be emphasized t h a t t h e b i n d i n g s t u d i e s j u s t des c r i b e d do not completely r u l e out t h e p o s s i b i l i t y t h a t a p a r t i c u l a r a g e n t may bind t o s e v e r a l sites on CM, some of which might d i f f e r from t h e phenothiazine b i n d i n g s i t e s .
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Among t h e a g e n t s t h a t i n t e r a c t w i t h t h e Ca2+-CM complex are several n a t u r a l l y - o c c u r r i n g p r o t e i n s and p e p t i d e s . These i n c l u d e a Ca2+-binding p r o t e i n c a l l e d c a l c i n e u r i n , 39 a h e a t s t a b l e p r o t e i n , 35 m y e l i n b a s i c t e i n , 38 h i s t o n e , 38 B-endorphin, 28-30 d y n o r p h i n , 2 9 ~ 3 0s u b s t a n c e P 2 v o and t h e b e e venom p e p t i d e melittin?0'31Although t h e p h y s i o l o g i c a l s i g n i f i c a n c e of t h e i n t e r a c t i o n o f t h e s e endogenous compounds w i t h CM i s n o t y e t known, they may f u n c t i o n a s endogenous m o d u l a t o r s of CM a c t i v i t y and may t h e r e b y p r o v i d e p o t e n t i a l s i t e s of drug a c t i o n .
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Although t h e b i n d i n g of t h e phenoI r r e v e r s i b l e Binding of Drugs t o CM t h i a z i n e s and r e l a t e d compounds t o CM can b e r e v e r s e d by removing Ca2+ o r by d i a l y z i n g a g a i n s t an e x c e s s of competing d r u g , 18,71-73 several a g e n t s have been shown t o i n t e r a c t i r r e v e r s i b l y w i t h CM. Upon i r r a d i a t i o n w i t h u l t r a v i o l e t l i g h t o r t r e a t m e n t w i t h peroxidase-hydrogen p e r o x i d e , t h e p h e n o t h i a z i n e a n t i p s y c h o t i c s chlorpromazine and t r i f l u o p e r a z i n e b i n d irrev e r s i b l y t o CM, presumably t h r o u g h a f r e e r a d i c a l mechanism. 12974 L i k e t h e r e v e r s i b l e b i n d i n g of t h e s e a g e n t s t o CM, t h e i r r e v e r s i b l e b i n d i n i s enhanced by Ca2+. The Ca2+-dependent b i n d i n g is s a t u r a b l e w i t h one Ca5+dependent drug b i n d i n g s i t e p e r molecule. The i r r e v e r s i b l e b i n d i n g of t h e p h e n o t h i a z i n e s t o CM r e s u l t s i n t h e i r r e v e r s i b l e i n a c t i v a t i o n of CM. 7 4 CM t h a t had been i r r e v e r s i b l y l i n k e d t o chlorpromazine o r t r i f l u o p e r a z i n e by u l t r a v i o l e t i r r a d i a t i o n w a s u n a b l e t o a c t i v a t e p h o s p h o d i e s t e r a s e , l29 7 4 a l t h o u g h t h e drug-CM complex d i d i n h i b i t the a c t i v a t i o n of p h o s p h o d i e s t e r a s e by n a t i v e CM. 43 E a r l e t a l . l4 r e p o r t e d t h a t t h e a - a d r e n e r g i c a n t a g o n i s t s phenoxybenzamine and dibenamine a l s o i n h i b i t e d CM i r r e v e r s i b l y , p r o b a b l y by bindi n g d i r e c t l y t o CM i n a Ca2+-dependent manner. These a g e n t s g e n e r a t e ethyleneimmonium and carbonium i o n i n t e r m e d i a t e s t h a t might b i n d i r r e v e r s i b l y t o e l e c t r o n - r i c h f u n c t i o n a l groups on CM. 7 5 S e v e r a l a l k y l a t i n g a g e n t s t h a t are n o t a - a d r e n e r g i c a n t a g o n i s t s d i s p l a y e d l i t t l e anti-CM a c t i v i t y 1 4 915
.
Along w i t h t h e s e s t u d i e s showing t h a t c e r t a i n d r u g s can i r r e v e r s i b l y i n a c t i v a t e CM, some e v i d e n c e s u g g e s t s t h a t i t may a l s o be p o s s i b l e t o i r r e v e r s i b l y a c t i v a t e CM-sensitive enzymes. Andreasen et a l . d e s c r i b e d a p r o c e d u r e f o r p r e p a r i n g azido-CM t h a t r e t a i n e d most of t h e b i o l o g i c a l a c t i v i t y of n a t i v e CM. 7 6 Upon i r r a d i a t i o n w i t h U.V. l i g h t , the azido-CM formed c o v a l e n t l i n k a g e s w i t h s e v e r a l CM-sensitive enzymes and CM-binding p r o t e i n s and i r r e v e r s i b l y a c t i v a t e d t h e CM-sensitive ATPase o f human erythrocytes. F a c t o r s I n f l u e n c i n g t h e I n t e r a c t i o n of Drugs w i t h CM: S t r u c t u r e - A c t i v i t y S t u d i e s - The s p e c i f i c s t r u c t u r a l f a c t o r s t h a t e n a b l e d r u g s t o b i n d t o CM are p o o r l y understood. It h a s been s u g g e s t e d t h a t t h e a b i l i t i e s of v a r i o u s d r u g s t o i n t e r a c t w i t h CM may b e c l o s e l y r e l a t e d t o t h e i r a b i l i t i e s t o r ~t o~ p a r t i t i o n between a l i p i d p h a s e and a n a q u e o u s p h a s e , 2 4 3 2 7 ~ ~ 4o 3 t h e i r a b i l i t i e s t o s t a b i l i z e membranes. 77-79 These f i n d i n g s are c o n s i s t e n t w i t h t h o s e showing t h a t hydrophobic r e g i o n s o f CM are i n v o l v e d i n t h e b i n d i n g of d r u g s . 57-59 Y 80 According t o proposed models, t h e b i n d i n g of Ca2+ i n d u c e s c o n f o r m a t i o n a l changes i n CM, exposing a hydrophobic domain which can t h e n bind l i p o p h i l i c d r u g s o r serve as an i n t e r f a c e f o r t h e b i n d i n g of CM t o i t s t a r g e t enzymes. 57-59 However, t h e r e is a l s o e v i d e n c e s u g g e s t i n g t h a t o t h e r f o r c e s b e s i d e s hydrophobic i n t e r a c t i o n s may b e i n v o l v e d i n t h e b i n d i n g of d r u g s t o CM. Many h i g h l y l i p o p h i l i c d r u g s a r e o n l y weak i n h i b i t o r s of CM and c e r t a i n h y d r o p h i l i c d r u g s are v e r y p o t e n t CM a n t a g o n i s t s . 13922 S t u d i e s on the pH-dependence of t h e b i n d i n g of t r i f l u o p e r a z i n e t o CM s u g g e s t t h a t i o n i c
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f o r c e s may p l a y an i m p o r t a n t r o l e i n t h i s i n t e r a c t i o n . 1 2 , 1 3 F i n a l l y , rec e n t s t r u c t u r e - a c t i v i t y s t u d i e s have shown t h a t v a r i o u s classes of CM i n h i b i t o r s e x h i b i t c e r t a i n s t r u c t u r a l s i m i l a r i t i e s . l 3 y Z 2 I n many c a s e s , s l i g h t m o d i f i c a t i o n s i n chemical s t r u c t u r e can g r e a t l y a l t e r t h e a b i l i t y of a compound t o b i n d t o CM and i n h i b i t i t s a c t i v i t y . Such s t r u c t u r a l s p e c i f i c i t y i s s u g g e s t i v e of s p e c i f i c drug-receptor i n t e r a c t i o n s and n o t simple hydrophobic bonding. To more p r e c i s e l y determine t h e s t r u c t u r a l f a c t o r s t h a t e n a b l e d r u g s t o i n t e r a c t w i t h CM, i t i s n e c e s s a r y t o s y s t e m a t i c a l l y e v a l u a t e t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of r e p r e s e n t a t i v e chemical c l a s s e s of CM antago n i s t s . To d a t e , o n l y a few such s t u d i e s have been performed. T h e i r res u l t s are summarized below. S e v e r a l s t u d i e s examining t h e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of t h e p h e n o t h i a z i n e s have been r e p o r t e d . M o d i f i c a t i o n s of t h e promazine s t r u c t u r e (1) included r i n g s u b s t i t u t i o n , 1 1 , 1 3 , 2 2 , 2 3 , 7 7 , 8 1 l e n g t h of side-chain,-22 s t r u c t u r e of s i d e - c h a i n amine, 22 ,82 o x i d a t i o n s t a t e of s u l f u r , 11,22,81 and attachment of an a d d i t i o n a l a r o m a t i c r i n g 82 (2-11). _Within a series of r i n g - s u b s t i t u t e d promazine d e r i v a t i v e s , good c o r r e l a t i o n was found between o r t a n o l / b u f f e r p a r t i t i o n c o e f f i c i e n t s and IC50 v a l u e s f o r i n h i b i t i o n of CM a c t i v i t y , i n d i c a t i n g t h a t t h e hydrophob i c i t y of t h e p h e n o t h i a z i n e n u c l e u s may be an important d e t e r m i n a n t of potency. By c o n t r a s t , no such c o r r e l a t i o n w a s found among a series of promazine a n a l o g s i n which t h e s i d e - c h a i n was modified, a l t h o u g h v a r y i n g t h e n a t u r e and p o s i t i o n of t h e s i d e c h a i n amino group d i d i n f l u e n c e potency.22 A l i k e l y e x p l a n a t i o n f o r t h e s e f i n d i n g s is t h a t both hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s p l a y a r o l e i n t h e b i n d i n g of p h e n o t h i a z i n e s t o t h e Ca2+-CM complex. 1 3 , z 2 Although o n l y l i m i t e d i n f o r m a t i o n i s now a v a i l a b l e , t h e s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s of t h e dibenzaze i n e s (12-14) - - appear t o be s i m i l a r t o t h o s e of t h e p h e n o t h i a z i n e s . 1 1 9 1 2 * z 3 The t h i o x a n t h e n e a n t i p s y c h o t i c s ( 1 5 ) c o n t a i n a n e x o c y c l i c double bond
I
'
c72
2
Crz 742
/ N\
CH3
CH3
-4
pounds were s l i g h t l y more p o t e n t CM i n h i b i t o r s t h a n t h e i r analagous phenothiaz i n e s . 12,22,24 Although geometric i s o m e r s can exist a c r o s s t h e double bond, t h e r e w a s l i t t l e evidence of s t e r e o specificity i n the interaction of these a g e n t s w i t h CM. 12,18,24 The implica-
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tions of this lack of stereospecificity have been considered elsewhere.8 ~ 1 21,3 ~ 2 4 The diphenylbutylpiperidine antipsychotics pimozide (16) and penfluridol (=)are among the most potent CM antagonists. 1 3 ~ m ~ 2 Both 4 aromatic rings appear to be necessary for high potency, since the compound a pimozide analog lacking one phenyl group, was only 116 as R-6033 (g), potent as pimozide. 22 Similarly, the butyrophenone antipsychotic haloperidol @), which contains only a single aromatic ring,is a relatively weak CM antagonist. 12~13,22 Hidaka and his co-workers have studied the structure-activit relationships of the naphthalenesulfonamide class o f CM The effect on potency of ring substitution and modifying the length of the side chain 34983984 and position of the sulfonamide function 33934 have been examined. Within a series of 1-naphthalenesulfonamides, good correlation was found between octanolfbuffer partition coefficients and anti-CM potencies. However, the significance of this correlation was reduced when data from the corresponding 2-substituted series was factored in ,34 indicating that the position of the amino group as well as hydrophobicity may be important in determining potency.
antagonist^?^^^^^^^,^^
(z),
Compound R 24571, calmidazolium is a derivative of the antimycotic agent miconazole and appears to be an especially potent and selective CM antagonist. It is about 500 times more potent than trifluoperazine in inhibiting the activation of several M-dependent enzymes, but displays little affinity for several neurotransmitter and hormone receptors to which other CM inhibitors bind.s1 I n addition, R 24571 has been shown to be more potent in preventing the activation of Ca2+ transport ATPase by CM than in preventing the activation of the enzyme by other agents. 59
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B a i l e y , Ed.
Although i t i s n o t y e t clear whether t h e p o l y p e p t i d e c l a s s of CM i n h i b i t o r s bind t o t h e s a m e s i t e s on CM t o which t h e p h e n o t h i a z i n e s b i n d , t h e i r s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s s u g g e s t t h a t b o t h hydrophobic and e l e c t r o s t a t i c i n t e r a c t i o n s a r e involved i n t h e i r b i n d i n g t o CM. Of several p e p t i d e s t h a t bound w e l l t o CM,29 o r d i s p l a y e d anti-CM a c t i v i t y , 30 a l l contained a r e g i o n of b a s i c amino a c i d r e s i d u e s i n c l o s e proximity t o s e v e r a l hydrophobic r e s i d u e s . General S t r u c t u r e C h a r a c t e r i s t i c s of CM I n h i b i t o r s - Although t h e v a r i o u s CM-binding- drugs - Belong- t o d i f f e r e n t chemical c l a s s e s . many e x h i b i t c e r t a i n s t r u c t u r a l similarities. The e s s e n t i a l f e a t u r e s of t h e s e a g e n t s are a l a r g e hydrophobic r e g i o n c o n s i s t i n g of a t least two a r o m a t i c r i n g s , e i t h e r a d j a c e n t t o each o t h e r , o r j o i n e d a t one o r two p o s i t i o n s , and a s i d e c h a i n amino group t h a t i s a t l e a s t 4 atoms removed from t h e aromatic r i n g s . 1 3 $22 The geometric conformation of t h e r i n g s t r u c t u r e does n o t appear t o be an important determinant of anti-CM a c t i v i t y because compounds having d i f f e r e n t r i n g s t r u c t u r e s , such a s t h e diphenylbutylpiperidines, phenot h i a z i n e s , t h i o x a n t h e n e s , dibenzazepines, and naphthalenesulfonamides, can a l l i n t e r a c t w i t h CM. A more important f a c t o r appears t o b e t h e hydrophob i c i t y of t h e r i n g s t r u c t u r e ; s u b s t i t u e n t s t h a t i n c r e a s e hydrophobicity i n c r e a s e a n t i CM potency, whereas t h o s e t h a t d e c r e a s e hydrophobicity dec r e a s e potency. 13 22 9 34 9
The t y p e of amino group does n o t seem t o b e c r i t i c a l s i n c e primary, secondary, t e r t i a r y , p i p e r a z i n y l and i m i d i a z o l e amines i n h i b i t CM. A l l t h a t seems necessary i s t h a t t h e amino group c a r r y a p o s i t i v e charge and that i t b e s e p a r a t e d from t h e hydrophobic r e g i o n of t h e drug molecule by 4 o r more atoms, a l t h o u g h t h e d i s t a n c e r e q u i r e d f o r o p t i m a l a c t i v i t y is not y e t known. 13,22934 T h i s i s s u e i s complicated by t h e f a c t t h a t amino groups a t t h e end of l o n g , f l e x i b l e a l k y l c h a i n s could assume a v a r i e t y of d i f f e r e n t p o s i t i o n s t o a l l o w b i n d i n g t o CM, o r t h e long a l k y l c h a i n s might undergo hydrophobic i n t e r a c t i o n s w i t h CM. I n t e r e s t i n g l y , t h e amino-acrid i n e d e r i v a t i v e q u i n a c r i n e , which h a s a v e r y h y d r o p h i l i c s i d e c h a i n , and t h e thioxanthene a n t i p s y c h o t i c s , which have t h e i r s i d e c h a i n amino groups held i n f i x e d p o s i t i o n s , are p o t e n t CM i n h i b i t o r s , 22,24 s u g g e s t i n g t h a t t h e p o s i t i o n of t h e amino group, r a t h e r t h a n t h e h y d r o p h o b i c i t y of t h e s i d e c h a i n , determines potency. This model f o r t h e i n t e r a c t i o n of drugs w i t h CM a p p l i e s only t o a g e n t s t h a t i n t e r a c t w i t h t h e p h e n o t h i a z i n e binding s i t e s . It i s p o s s i b l e t h a t d r u g s having somewhat d i f f e r e n t s t r u c t u r a l c h a r a c t e r i s t i c s might bind t o d i f f e r e n t s i t e s on CM. Agents such as DDT and T r i t o n X-100 have s t r u c t u r e s t h a t d i f f e r from t h e proposed model. A t p r e s e n t , i t i s not known whether t h e s e a g e n t s bind t o t h e same s i t e s on CM t h a t t h e p h e n o t h i a z i n e s do. 41942 R e s u l t s of NMR and f l u o r e s c e n c e spectroscopy s t u d i e s have provided some evidence f o r t h e e x i s t e n c e of s e v e r a l t y p e s of drug binding s i t e s on CM. 15,20921 E f f e c t s of Drug Binding on t h e Conformation and A c t i v i t y of CM - The mechanism by which drug b i n d i n a l t e r s t h e a c t i v i t y of CM i s n o t completely understood. N M R 2°,68,69 ,86,86and f l u o r e s c e n c e spectroscopy 15,21929 d a t a i n d i c a t e t h a t t h e b i n d i n g of drugs induces changes i n t h e conformation of CM. S t u d i e s examining t h e b i n d i n g of 1 2 5 I - l a b e l l e d CM t o membrane s i t e s have shown a t drug b i n d i n g d e c r e a s e s t h e a f f i n i t y of CM f o r i t s t a r g e t enzymes. 62r@These d a t a s u g g e s t t h a t drug b i n d i n g a l t e r s t h e conformation of t h e Ca2+-CM complex and d e c r e a s e s i t s a b i l i t y t o i n t e r a c t w i t h recogn i t i o n sites on CM-sensitive enzymes.
Chap. 2 1
S A R o f Calmodulin A n t a g o n i s t s
Prozialeck
211
The l o c a t i o n of t h e p h e n o t h i a z i n e b i n d i n g sites on CM i s n o t known. I t h a s been s u g g e s t e d t h a t t h e d r u g s may b i n d t o a hydrophobic r e g i o n of CM t h a t s e r v e s as an i n t e r f a c e f o r t h e b i n d i n g of CM t o i t s t a r g e t enzymes. 57-59 R e s u l t s of I H NMR s t u d i e s i n d i c a t e t h a t m e t h i o n i n e and p h e n y l a l a n i n e r e s i d u e s n e a r Ca2+-binding domains 11, 111 and I V of CM may play an important r o l e i n binding phenothiazines. 80986 Other r e s u l t s s u g g e s t t h a t a c i d i c r e s i d u e s on CM are i n v o l v e d i n b i n d i n g b a s i c d r u g s . l 2 3 l 3 These d a t a s u g g e s t t h a t drug b i n d i n g s i t e s on CM may c o n s i s t of a hydrophobic r e g i o n , c o n t a i n i n g m e t h i o n i n e r e s i d u e s , i n c l o s e p r o x i m i t y t o a n a c i d i c r e s i d u e s u c h as a s p a r t i c o r g l u t a m i c a c i d . Head -et a l . r e p o r t e d t h a t a cyanogen bromide-cleavage fragment of CM, c o n t a i n i n g r e s i d u e s 77-124, w a s a b l e t o b i n d p h e n o t h i a z i n e s i n a Ca2+-dependent manner. 85 The r e c e n t f i n d i n g s t h a t c e r t a i n d r u g s can b i n d i r r e v e r s i b l y t o CM should a i d i n f u r t h e r i d e n t i f y i n g drug b i n d i n g s i t e s on CM. 1 4 s 7 0 References 1. W.Y. Cheung, Biochem. Biophys. Res. Commun., 2, 533 (1970). 2. S. Kakiuchi and R. Yamazaki, Biochem. Biophys. Res. Commun. 1104 (1970). 3. C.O. Brostrom and D.J. Wolff, Biochem. Pharmacol. 30, 1395 (1981). 4. W.Y. Cheung, Science (Washington. D.C.), 207, 19 (1980). C.B. Klee, T . R . Crouch and P.G. Kichman, Annu. Rev. B€oc!em. S , 484 (1980). 5. 6. C.B. Klee, i n "Calcium and Cell Function", Vol. 1, W.Y. Cheung, Ed., Academic Press, New York, N.Y. (1980) p. 59. T.C. Vanaman, in "Calcium and Cell Function: Vol. 1, W.Y. Cheung, Ed., Academic Press, 7. New York, N.Y. (1980), p . 41. 8. B. Weiss and T.L. Wallace, in "Calcium and Cell Function", Vol. 1, W.Y. Cheung, Ed., Academic Press, New York, N.Y. (1980) p. 329. 9. J . Krebs, Cell Calcium, 2, 295, (1981). 10 * B. Weiss, R. Fertel, R. Figlin and P. Uzunov, Mol. Pharmacol. 615 (1974). 11. R.M. Levin and B. Weiss, Mol. Pharmacol. 2, 5 8 1 (1976). 12. B. Weiss, W. Prozialeck, M. Cimino, M.S. Barnette and T.L. Wallace, Ann. N.Y. Acad. Sci., 356, 319 (1980). 2217 (1982). 13. B. Weiss, W.C. Prozialeck and T.L. Wallace, Biochem. Pharmacol. 1 4 . C.Q. Earl, W.C. Prozialeck and B. Weiss, Fed. Proc. Fed. Am. Sac. Exp. Biol., 1565 (1982). 15. K. Watanabe and W.I. West, Fed. Proc. Fed. Am. SOC. Exp. B i o l . , 41, 2292 (1982). 16. M. Volpi, R . I . Sha'afi, P.M. Epstein, D.M. Andrenyak and M.B. Feinstein, Proc. Nat!. Acad. Sci. USA., 795 (1981). 363 (1981). 17. M. Volpi, R . I . Sha'afi and M.B. Feinstein, Mol. Pharmacol., 18. R.M. Levin and B. Weiss, J . Pharmacol. Exp. Ther., 208, 454 (1979). 19. R . J . DeLorenzo, S. Burdette and J. Holderness, Science, 213, 546 (1981). 20 * S.-L. Brostrgm, B. Ljung, Sven Mzrdh, S. Forsen and E. Thulin, Nature (London), 777 (1981). 21. P.M. Epstein, K. Fiss, R. Hachisu and D.M. Andrenyak, Biochem. Biophys. Res. Commun., 105, 1142 (1982). 22. W.C. Prozlaleck and B. Weiss, J . Pharmacol. Exp. Ther., 222, 509 (1982). 23. J.H. Zavecz, T.E. Jackson, G.L. Limp and T.O. Yellin, Eur. J. Pharmacol., 78, 375, (1982). 24. J.A. Norman, A.H.Drummond and P. Moser, Mol. Pharmacol., 16. 1089 (1979). 25. N. Katoh, B.C. Wise, R.W. Wrenn, J.F. Kuo, Biochem. J . , 199, (1981). 26. K. Watanabe, E.F. Williams, J . S . Law and W.L. West, Biochem. Pharmacol., 30, 335 (1981). 27. T. Tanaka and H. Hidaka, Biochem. Biophys. Res. Commun., ,&I 447 (1981). 28. M. Sellinger-Barnette and B. Weiss, Mol. Pharmacol., 86 (1982). 29. D.A. Malencik and S . R . Anderson, Biochemistry, 3480 (1982). in press. 30. M. Sellinger-Barnette and B. Weiss, Adv. Cyclic Nucleotide Res., 31. M. Comte, Y. Maulet and J.A. Cox. Biochem. J . 269 (1983). 32. H. Hidaka, T. Yamaki, M. Naka, T. Tanaka, H. Hayashi and R. Kobayashi, Mol. Pharmacol., 17, 66 (1980). 5 7 1 (1981). 33. H. Hidaka, M. Asano and T. Tanaka, Mol. Pharmacol., 34. T. Tanaka, T. Ohmura and H. Hidaka, Mol. Pharmacol., 22, 403 (1982). 35. R.K. Sharma, E. Wirch and J.H. Wang, J . Biol. Chem., 3575 (1978). 36. R.W. Wallace, T.J. Lynch, E.A. Tallant, and W.Y. Cheung, J . Biol. Chem., 254, 377, (1978). 37. F.L. Larsen, B.U. Raess, T . R . Hinds and F.F. Vincenzi, J . Supramol. Struct., 2, 269 (1978). 227 (1980). 38. R.J.A. Grand and S.V. Perry, Biochem. J., 39. C.B. Klee, T.H. Crouch and M.H. Krinks, Proc. Nat. Acad. Sci. USA, 6270 (1979). 40. T. Itano, R. Itano and J.T. Penniston, Biochem. J., 455 (1980).
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