9 Design of Kinase Inhibitors Conformational and Mechanistic Considerations G E O R G E L. K E N Y O N and R E B E C C A E. R E D D I C K Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143
General aspects of enzymatic reactions catalyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. F i n a l l y , the design of an affinity label for creatine kinase i s reviewed as an example of how such information can be used in the search for agents directed at an enzyme's active s i t e . The d e s i g n o f a d r u g t h a t a c t s by a l t e r i n g t h e a c t i v i t y o f a k i n a s e must b e g i n w i t h t h e s t u d y o f t h e i n t e r a c t i o n s o f t h i s c l a s s o f enzymes w i t h t h e i r n u c l e o t i d e s u b s t r a t e s , their co-substrates and r e g u l a t o r y m o l e c u l e s s u c h as c y c l i c n u c l e o t i d e s . Once such i n t e r a c t i o n s a r e a d e q u a t e l y u n d e r s t o o d , enzymes c a n p o t e n t i a l l y be exploited in r a t i o n a l drug design i n at l e a s t three ways. The most o f t e n u s e d i s t h a t o f enzyme i n h i b i t i o n , the main s u b j e c t o f this chapter. The s e c o n d p o s s i b i l i t y i s f o r t h e enzyme to be i n v o l v e d i n the c o n v e r s i o n of a b i o l o g i c a l l y i n a c t i v e molecule to one w h i c h i s b i o l o g i c a l l y a c t i v e , i . e . , the c o n v e r s i o n of prodrug to drug. The t h i r d p o s s i b i l i t y , w h i c h h a s n o t y e t b e e n e x p l o i t e d , a t l e a s t i n a d i r e c t way, i s t h a t o f a c t i v a t i o n o f e n z y m e s . This c h a p t e r w i l l b r i e f l y c o v e r some i m p o r t a n t requirements and properties of the general enzymatic reaction catalyzed by kinases. There are many e x a m p l e s o f t h e u s e o f a n a l o g s o f the common s u b s t r a t e , ATP, as probes o f the c o n f o r m a t i o n a l and s t e r i c requirements of kinase a c t i v e s i t e s . ATP a n a l o g s t h a t f u n c t i o n a s a f f i n i t y l a b e l s h a v e b e e n u s e d a s t o o l s f o r p i n p o i n t i n g amino a c i d r e s i d u e s p r e s e n t a t or near the a c t i v e s i t e . The k i n a s e s c a n be divided i n t o t h r e e groups b a s e d on t h e t y p e o f n o n - A T P s u b s t r a t e phosphorylated enzymatically. S e l e c t e d k i n a s e s from these three groups w i l l be d i s c u s s e d w i t h r e s p e c t t o s t u d i e s u s i n g substrate analogs and i n h i b i t o r s . T h e s e s t u d i e s c a n be u s e d i n t h e d e s i g n
0097-6156/ 84/ 0251 -0189S06.25/ 0 © 1984 American Chemical Society
190
CONFORMATIONALLY DIRECTED DRUG DESIGN
of s p e c i f i c s u b s t r a t e s or i n h i b i t o r s o f p o t e n t i a l use as drugs. Lastly, the r a t i o n a l e f o r the design of a s p e c i f i c irreversible inhibitor of creatine kinase, namely e p o x y c r e a t i n e , w i l l be r e viewed. Kinases are proteins whose catalytic function i s the t r a n s f e r o f t h e y - p h o s p h o r y l m o i e t y o f ATP t o a p a r t i c u l a r acceptor: R-X-H + A T P * * " ^ = = ±
A D P " + R-X-P0|~ + H 3
+
Both ATP a n d t h e p h o s p h o r y l a c c e p t o r become r e v e r s i b l y a n d s e l e c t i v e l y bound t o t h e enzyme d u r i n g c a t a l y s i s . So f a r , k i n a s e s t h a t h a v e b e e n shown t o r e a c t b y d i r e c t p h o s p h o r y l t r a n s f e r b e t w e e n ATP and t h e c o - s u b s t r a t e s show s t r i c t i n v e r s i o n o f configuration at phosphorus, w h i l e t h o s e w i t h a p h o s p h o r y l a t e d enzyme i n t e r m e d i a t e show r e t e n t i o n o f c o n f i g u r a t i o n a t p h o s p h o r u s ( 1 , 2 ) . All known A T P - u t i l i z i n g enzymes have a r e q u i r e m e n t o f dival e n t m e t a l c a t i o n s f o r a c t i v i t y (3) > a l t h o u g h t h e r o l e s t h a t these_ metal i o n s p l a y have n o t been f u l l y e l u c i d a t e d . Typically Mg and Mn a r e a c t i v a t i n g whereas o t h e r m e t a l i o n s such as C a * and Ba are e i t h e r l e s s a c t i v a t i n g or i n h i b i t o r y (4-6). For a l l k i n a s e s one d i v a l e n t c a t i o n i s n u c l e o t i d e b o u n d . In addition, a few k i n a s e s h a v e b e e n shown t o b i n d a s e c o n d d i v a l e n t c a t i o n that may be e i t h e r a c t i v a t i n g a s w i t h p y r u v a t e k i n a s e (7,8) o r inhibi t i n g a s w i t h cAMP d e p e n d e n t p r o t e i n k i n a s e ( 9 ) • I t i s the p r e s ence o f t h i s p a i r o f c a t i o n s a t the a c t i v e s i t e s o f these latter two enzymes t h a t h a s a l l o w e d d e t a i l e d NMR s t u d i e s t o be carried out and has a i d e d i n the f o r m u l a t i o n o f d e t a i l e d working models of t h e i r a c t i v e - s i t e g e o m e t r i e s (10-13)• These s t u d i e s w i l l be d i s c u s s e d elsewhere i n the chapter. +
+
2
2
2
ATP:
T h e Common
Substrate
ATP a n d numerous ATP a n a l o g s have b e e n e x t e n s i v e l y studied as substrates, i n h i b i t o r s and as v a r i o u s other probes o f the active sites of kinases. E f f o r t s have b e e n made t o p i n p o i n t t h e c o n f o r mational and s t e r i c r e q u i r e m e n t s o f ATP a t t h e a c t i v e site of kinases i n order t o d i s t i n g u i s h between general and s p e c i f i c c h a r a c t e r i s t i c s o f ATP b i n d i n g . ATP T r i p h o s p h a t e C h a i n C o n f o r m a t i o n . Much o f t h e work i n t h e a r e a of ATP t r i p h o s p h a t e c h a i n c o n f o r m a t i o n has been performed by Cleland and co-workers ( K - 1 6 ) . T h e i r s t u d i e s on m e t a l ( I I I ) A T P interactions with k i n a s e s have l e d t o t h e c l a s s i f i c a t i o n o f k i nases a c c o r d i n g t o the s t e r e o c h e m i s t r y o f the polyphosphate chain as it binds to the act i v e s i t e . For the kinases they studied (hexokinase, glycerokinase, creatine kinase, phosphofructokinase, 3-phosphoglycerate kinase, acetate kinase, arginine kinase, adeny l a t e k i n a s e a n d p y r u v a t e k i n a s e ) i t was f o u n d t h a t 3, y - b i d e n t a t e chromium(III)-ATP (CrATP) and n o t a , S , Y - t r i d e n t a t e CrATP is a
9. KEN YON AND REDDICK
Design of Kinase Inhibitors
191
substrate. However, t h e t r i d e n t a t e i s o m e r i s a s t r o n g c o m p e t i t i v e i n h i b i t o r o f some k i n a s e s ( e . g . , c r e a t i n e k i n a s e , p h o s p h o f r u c t o k i nase, 3-phosphoglycerate kinase, andacetate kinase)(14) • I* addition, t h e two s c r e w s e n s e i s o m e r s of S,Y-bidentate CrATP ( F i g u r e 1a) were d i s t i n g u i s h e d b y t h e enzymes (1^.). T h u s , h e x o k i nase, glycerokinase, creatine kinase andarginine kinase are specific f o r t h e A i s o m e r ( F i g u r e 1a-1) whereas pyruvate kinase, adenylate kinase andphosphofructokinase a r e s p e c i f i c f o r the A i s o m e r ( F i g u r e 1a-2). R e c e n t l y , cAMP d e p e n d e n t p r o t e i n k i n a s e h a s a l s o b e e n f o u n d t o be s p e c i f i c f o r t h e A i s o m e r ( 1 7 ) . I n h i b i t i o n s t u d i e s w i t h CrADP c o m p l e x e s a l l o w e d t h e s e w o r k e r s to conclude t h a t hexokinase and g l y c e r o k i n a s e both r e l e a s e MgADP as t h e 3 - m o n o d e n t a t e f o r m , whereas c r e a t i n e k i n a s e , pyruvate k i nase, adenylate kinase, acetate kinase, 3-phosphoglycerate kinase, and phosphofructokinase a l l r e l e a s e MgATP a s t h e b i d e n t a t e form (14)• Creatine kinase was s p e c i f i c a l l y i n h i b i t e d b y i s o m e r I ( F i g u r e 1a-3)(1_6) o f b i d e n t a t e C r A D P , w h i c h i s b e l i e v e d t o be t h e A isomer. Thus, t h e mechanism f o r p h o s p h o r y l t r a n s f e r w i t h r e spect t o t h e n u c l e o t i d e bound m e t a l a p p e a r s t o p r o c e e d according t o two p a t h w a y s . The f i r s t i s t h e r e a c t i o n o f S , y - b i d e n t a t e MgATP to f o r m 3 - m o n o d e n t a t e MgADP a n d t h e s e c o n d i s t h e r e a c t i o n o f t h e b i d e n t a t e MgATP t o f o r m a,3-MgADP ( F i g u r e 1 b ) ( 1 6 ) . 1
Nucleotide Base Conformation. U s i n g NMR d a t a , a relationship between t h e degree o f s p e c i f i c i t y and t h e conformation of bound ATP a t t h e a c t i v e s i t e h a s b e e n shown f o r a number o f ATP u t i l i zing enzymes. Two e x a m p l e s o f t h e s e a r e c A M P - d e p e n d e n t protein k i n a s e a n d p y r u v a t e k i n a s e (18,19)• I t a p p e a r s t h a t enzymes t h a t exhibit higher nucleotide triphosphate s p e c i f i c i t y bind ATP s o t h a t t h e g l y c o s i d i c bond a n g l e ( x ) ( F i g u r e 2a) i s g r e a t l y d i s t o r t e d f r o m i t s f r e e s o l u t i o n v a l u e o f 40-44°. Based on x - r a y d a t a c i t e d in reference (1_8) h e x o k i n a s e a l s o a p p e a r s t o c o n f o r m to this trend. 8 - B r A T P ( F i g u r e 2b) i s a n ATP a n a l o g i n w h i c h t h e g l y c o s i d i c bond a n g l e (x) i s r e s t r i c t e d s u c h t h a t t h e s y n c o n f o r m a t i o n o f t h e base i s g r e a t l y p r e f e r r e d (19i20). T h i s compound h a s b e e n f o u n d t o be a n a l t e r n a t e s u b s t r a t e , a l b e i t a r a t h e r p o o r o n e , f o r s e v e r a l kinases (pyruvate k i n a s e , hexokinase, phosphofructokinase, and a d e n y l a t e kinase)(22,23). I s o e n z y m e s sometimes d i s p l a y d i f f e r e n t i a l s p e c i f i c i t i e s f o r t h i s a n a l o g (22). 8 , 5 - C y c l o - A M P and 8 , 5 ! cyclo-ADP are analogs locked i n the a n t i conformation (Figure 2c)(24). They a r e s u b s t r a t e s f o r a d e n y l a t e k i n a s e and pyruvate kinase, r e s p e c t i v e l y (25). I n t h e case o f a d e n y l a t e k i n a s e , 8 , 5 ' c y c l o - A M P i s a c t u a l l y a b e t t e r s u b s t r a t e t h a n AMP i t s e l f , support i n g t h e i d e a t h a t t h e enzyme p r e f e r e n t i a l l y b i n d s AMP i n t h e a n t i conformation. !
Non-hydrolyzable ATP a n a l o g s s u c h a s a d e n y l y l imidodiphosp h a t e (AMP-PNP, F i g u r e 2d) a n d 3 , Y - a d e n y l y l m e t h y l e n e b i s p h o s p h o nate ( C H ^ - A T P , F i g u r e 2e) h a v e b e e n u s e d a s i n h i b i t o r s o f kinases b e c a u s e t n e y c a n b i n d i n a s i m i l a r manner t o t h e n a t u r a l s u b s t r a t e
192
CONFORMATIONALLY DIRECTED DRUG DESIGN
2) R = AMP
1) R = AMP 3) R = Adenosine (
D C I P O
X
p
O
p - o - p ^ ^ p ^ ^Ade ;
C
X
°
?
°
^
O
X — P O-P
1
R, *
\J
O
/
°
°
O
P
v
Ade
^
V
(b) Figure 1. a) Structural isomers of 3,Y-bidentate M(III)ATP and a,3-bidentate M(III)ADP complexes; b) Proposed mechanisms of kinases.
9.
KENYON AND
193
Design of Kinase Inhibitors
REDD1CK
0 O O 1 B II -OPOPOPOCH2I I I \—J Base OH O H
(a) NH
-vj
2
O O O U II II •OPOPOPO I I
x
0-0-0
f
y Y
OH O H
OH OH
(b)
(c) NH
NH
2
O O O O^N II II II "™ | "| O — P - N - P - O - P - O - i jr^ 1 H 1 1 r N - O - O - O ) Y-f OH O H
2
00
O O O H II II I -0-P-CH -P-0-P-0-i .0^J I I I \ _ 7 -0 -o -0 rn OH O H 2
x
Figure 2. a) G l y c o s i d i c bond angle(x) in nucleotide triphosp h a t e s ; b) 8 - B r A T P ; c ) S ^ ' - C y c l o - A M P ; d) A d e n y l y l imidodip h o s p h a t e ; e) 3 , T - A d e n y l y l methylene bisphosphon a t e ; f) Regions of b u l k t o l e r a n c e e x p l o r e d by Hampton et al(28-31).
194
CONFORMATIONALLY DIRECTED DRUG DESIGN
yet do n o t u n d e r g o t h e p h o s p h o r y l t r a n s f e r r e a c t i o n (2^). These analogs allow close-to-normal binding of the co-substrate and regulatory molecules s o t h a t t h e mode o f b i n d i n g o f other substrates o r c o - f a c t o r s c a n be i n v e s t i g a t e d i n t h e a b s e n c e o f p h o s phoryl transfer. Exploration of B u l k T o l e r a n c e a t ATP S i t e s . Non-covalent type i n h i b i t o r s have a l s o been u s e d t o s t u d y b u l k t o l e r a n c e a r o u n d t h e ATP b i n d i n g s i t e s . I n t h i s v e i n Hampton a n d c o - w o r k e r s h a v e b o t h synthesized a n d t e s t e d a s i n h i b i t o r s a l a r g e number of adenine nucleotide analogs (Figure 2f) t o probe the bulk t o l e r a n c e a t a number o f p o s i t i o n s on t h e p a r e n t compound ( 2 8 - 3 1 ) • These compounds h a v e b e e n u s e d t o s t u d y s y s t e m a t i c a l l y t h e i s o e n z y m e s e l e c t i v i t y of adenylate kinases, hexokinases, thymidine k i n a s e s and pyruvate k i n a s e s w i t h r e s p e c t t o b u l k t o l e r a n c e a t many s i t e s on the ATP m o l e c u l e . Some o f t h e most i s o e n z y m e s p e c i f i c results were o b t a i n e d w i t h p y r u v a t e k i n a s e i s o e n z y m e s K , L a n d M u s i n g ADP derivatives. H e r e 3 - 0 M e - A D P was f o u n d t o i n h i b i t p y r u v a t e k i n a s e preferentially with a r a t i o of i n h i b i t o r y potency of 7.6:6.0:1.0 f o r the K,M and L isoenzymes , r e s p e c t i v e l y . A n o t h e r compound, 8 NHEt-ADP, was s e l e c t i v e f o r the M isoenzyme, giving a ratio of 7 . 1 : 1 . 2 : 1 . 0 f o r t h e M, K a n d L f o r m s , r e s p e c t i v e l y . !
Affinity Labeling o f C a t a l y t i c ATP S i t e s . Residues i n v o l v e d in ATP b i n d i n g a r e p o t e n t i a l l y r e v e a l e d b y t h e u s e o f a f f i n i t y l a b e l s t h a t a r e based on A T P ' s s t r u c t u r e . P e r h a p s t h e most s y s t e m a t i c a l l y s t u d i e d o f t h e s e compounds i s 5'-fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been r e p o r t e d t o l a b e l a t least s i x k i n a s e s (32-4.1). I n the case o f r a b b i t muscle pyruvate k i n a s e such work h a s i n d i c a t e d t h e p r e s e n c e o f a t y r o s i n e r e s i d u e w i t h i n the metal n u c l e o t i d e b i n d i n g s i t e and an e s s e n t i a l c y s t e i n e residue located at or near the f r e e metal b i n d i n g site (32). A similar reagent, 5'-FSBGuanosine, r e v e a l e d t h e p r e s e n c e o f two cysteine r e s i d u e s a t t h e c a t a l y t i c s i t e o f t h i s same e n z y m e , b o t h distinct r e s i d u e s from those m o d i f i e d by 5 ' - F S B A (33i34)« With yeast pyruvate k i n a s e both t y r o s i n e and c y s t e i n e residues were m o d i f i e d b y 5 - F S B A a t t h e c a t a l y t i c s i t e (35)> and w i t h porcine cAMP-dependent p r o t e i n k i n a s e a l y s i n e r e s i d u e was l a b e l e d a t t h e active s i t e (36). !
f
Unfortunately, as y e t t h e r e emerges no c l e a r p a t t e r n of a general r o l e f o r t h e s e r e s i d u e s e i t h e r i n b i n d i n g ATP o r i n c a t a lyzing phosphoryl transfer. Indeed 5'-FSBA i s not necessarily specific f o r t h e c a t a l y t i c ATP s i t e b u t r a t h e r h a s b e e n shown in some cases to bind preferentially to regulatory ATP s i t e s of kinases (37,38). N e v e r t h e l e s s , s u c h l a b e l i n g e x p e r i m e n t s do o f f e r the possibility of c h a r a c t e r i z a t i o n and comparison of peptide fragments from the s i t e modified, whether i t be catalytic or regulatory. A review o f the use o f 5 - F S B A w i t h k i n a s e s and other A T P - u t i l i z i n g enzymes h a s v e r y r e c e n t l y a p p e a r e d ( / £ ) . !
The a f f i n i t y
labeling
reagent ATP-dialdehyde
(Figure
3b)
has
9.
Design of Kinase
KEN YON AND REDDICK
NH
Inhibitors
195
NH
2
I
ii
ii
2
"
ii
7
-OPOPOPO—^.oJ
yOJ OH OH
O
(o)
R
N
O
(b) 1) R = -fCH2>nNHCOCH l; n = 2-8, ]
2
R = H;R = P 0 3 - , P 0 £ 2
NHR
3
2
3
2) R = f(CH hO]h1CH t NHCOCH l
1
]
2
2
2
2
f
n =l 2;R = H;R =P Oi2
f
R
3
3
2
3) R = tCH ^ CONMefCH > NMeCOfCH >nCOCH l 1
R -O-^OJ
2
n
2
m
2
2
f
n = 3,4; m = 3 4 ; R = H ; R = P Oj"
3
2
f
OH OH
3
3
4) R = H ; R =-NH{CH } NHCOCH l, 1
2
2
n
2
n = 2,4,6,8; R = PsOt 3
(c) F i g u r e 3a) 5 - F l u o r o s u l f o n y l b e n z o y l a d e n o s i n e ; b) A T P ; c1-c4) P o t e n t i a l e x o - a c t i v e - s i t e - d i r e c t e d t i g a t e d by Hampton e t a l ( 4 7 - 5 0 ) . 1
Dialdehyde agents inves-
196
CONFORMATIONALLY DIRECTED DRUG DESIGN
also been used f o r the purpose o f i d e n t i f y i n g b a s i c amino acid residues a t or near the r i b o s e b i n d i n g s i t e ( s ) o f c r e a t i n e k i n a s e , pyruvate k i n a s e and p h o s p h o f r u c t o k i n a s e (43-45)• I t appears to l a b e l a l y s i n e r e s i d u e i n each case. W i t h t h e l a t t e r two enzymes it does n o t a c t as a s t r a i g h t f o r w a r d a f f i n i t y l a b e l since more than one r e s i d u e p e r a c t i v e s i t e i s m o d i f i e d . Inactivation with t h e s e two enzymes h a s b e e n f o u n d t o o c c u r i n t h e a b s e n c e o f e i t h e r NaBH. o r NaBH^CN; thus the product o f the r e a c t i o n of t h i s reagent with l y s i n e i s p r o p o s e d t o be a d i h y d r o x y morpholine-like adduct rather t h a n a S c h i f f s base as has been found w i t h other enzymes ( 4 6 ) . Isoenzyme s p e c i f i c i t y f o r o t h e r p a r t s o f t h e ATP b i n d i n g s i t e has a l s o been examined . Thus ATP d e r i v a t i v e s ( F i g u r e s 3 c 1 - 3 c 4 ) b e a r i n g a n i o d o a c e t a m i d e on C - 8 o r N w i t h a s p a c e r arm o f v a r y i n g length ( 2 - 1 9 atoms) consisting of methylene, amide or ether l i n k a g e s were d e s i g n e d t o i n a c t i v a t e s p e c i f i c i s o e n z y m e s (47-50). These exo-active-site-directed r e a g e n t s were t e s t e d for tissue specificity on isoenzymes i n c l u d i n g t h e L , K and M r a t pyruvate kinases, four r a t hexokinases and t h r e e r a t a d e n y l a t e kinases. They were a l s o t e s t e d f o r s p e c i e s s p e c i f i c i t y b y c o m p a r i n g adenylate k i n a s e s from r a b b i t , p i g and carp and a d e n y l a t e kinases, thymidine k i n a s e s , and h e x o k i n a s e s from y e a s t and b a c t e r i a . The most i s o e n z y m e - s p e c i f i c r e s u l t s were o b t a i n e d f o r p y r u v a t e k i n a s e i s o e n z y m e s M,K a n d L w i t h compound 3 c 1 ( n = 8 ) , where t h e L isozyme was i n a c t i v a t e d t o t h e e x t e n t o f 80% w h i l e t h e M a n d K i s o e n z y m e s were n o t a f f e c t e d ( 4 8 ) . Similarly, with adenylate kinase from r a b b i t , c a r p , a n d p i g o n l y t h e r a b b i t m u s c l e enzyme (76% i n a c t i v a t i o n ) was a f f e c t e d by i n t e r a c t i o n w i t h compound 3 c 1 ( n = 6 ) , whereas t h e o t h e r two i s o e n z y m e s r e m a i n e d u n a f f e c t e d ( 4 7 ) . f
6
Nucleic
Acids
As
Cosubstrates
Of t h e k i n a s e s whose n o n - A T P s u b s t r a t e s a r e n u c l e i c a c i d derivatives t h y m i d i n e k i n a s e a n d a d e n y l a t e k i n a s e a r e p e r h a p s t h e most studied. Thymidine k i n a s e i s o f i n t e r e s t because o f the e x i s t e n c e o f a s p e c i f i c c e l l u l a r i s o e n z y m e i n d u c e d by h e r p e s s i m p l e x virusI(HSV-I)(51)• The s t r a t e g y i n d r u g d e s i g n h e r e i s t o s e a r c h f o r compounds t h a t would b o t h a c t as s u b s t r a t e s f o r t h i s enzyme a n d exert t h e i r pharmacologic a c t i v i t i e s as t h e i r phosphorylated p r o ducts (52,53)« Analogs o f thymidine, c y t o s i n e , u r i d i n e , guanidine and a d e n i n e have a l l been i n v e s t i g a t e d . Bulk t o l e r a n c e and p h y s icochemical requirements of nearly a l l possible sites of these a n a l o g s have b e e n s t u d i e d , a n d , as a r e s u l t , a f i g u r e s i m i l a r t o F i g u r e 2 f c o u l d be d r a w n . ( 5 4 - 5 8 ) . F o r many o f t h e s e a c o r r e l a t i o n between t h e a b i l i t y t o f u n c t i o n as s u b s t r a t e s and as anti-HSV-1 a g e n t s has been examined ( 5 5 i 5 7 , 5 8 ) .
9.
KEN YON
AND
REDDICK
Design of Kinase Inhibitors
A c y c l o v i r i s a p a r t i c u l a r l y important alternate thymidine k i n a s e :
197 substrate
for
Because it e x h i b i t s s u c h good a n t i v i r a l a c t i v i t y a l o n g w i t h low host t o x i c i t y , t h e b i n d i n g , s u b s t r a t e and a n t i v i r a l a c t i v i t i e s o f many g u a n o s i n e a n a l o g s d i f f e r i n g i n t h e i r 9 - r i n g s u b s t i t u e n t have been i n v e s t i g a t e d (57). Some compounds ^ h a t a r e c l o s e l y r e l a t e d to a c y c l o v i r , n a m e l y t h o s e d i f f e r i n g by e i t h e r a d d i t i o n o f one o r two m e t h y l e n e l i n k a g e s o n e i t h e r s i d e o f t h e e t h e r l i n k a g e , by a branched methyl on t h e d i s t a l s i d e o f t h e e t h e r oxygen, or by s u b s t i t u t i o n of - S or - C H ^ f o r the e t h e r oxygen, were s u b s t r a t e s . In contrast, those e i t h e r w i t h added branched h y d r o c a r b o n groups on t h e p r o x i m a l s i d e o r w i t h a n amino f u n c t i o n a l i t y r e p l a c i n g the acyclic h y d r o x y l were n o t s u b s t r a t e s . Analogs bearing c y c l i c 9 ring substituents on g u a n o s i n e were also tested. Those with ribose a n d d e o x y r i b o s e m o i e t i e s were f o u n d t o h a v e low substrate activity, w h e r e a s t h o s e w i t h a r a b i n o s e ( d i f f e r i n g by h a v i n g o p p o site s t e r e o c h e m i s t r y a t t h e 2 - 0 H ) were f o u n d t o h a v e n o n e . Ade n o s i n e a n d x a n t h i n e d e r i v a t i v e s o f a c y c l o v i r were i n a c t i v e , with the exception of 9-(2-hydroxyethoxymethyl)-2-methylthioadenine. M o n o - o r d i m e t h y l a t i o n o f N-2 o f t h e g u a n o s i n e m o i e t y o r s u b s t i t u t i o n o f N-2 b y t h i o m e t h y l l e f t r e a s o n a b l e a c t i v i t y (38-55%)It i s i n t e r e s t i n g t o n o t e t h a t f o r t h e p y r i m i d i n e b a s e s t h e enzyme i s quite specific f o r the n a t u r a l deoxyribose moiety, whereas for analogs o f the p u r i n e base g u a n o s i n e , which i s quite different from the n a t u r a l s u b s t r a t e , the s e l e c t i v i t y i s a l t e r e d greatly. Thus, it seems t h a t t h e a c y c l o v i r a n a l o g s must be b i n d i n g i n a fundamentally d i f f e r e n t way f r o m t h e n a t u r a l s u b s t r a t e when they exhibit substrate a c t i v i t y . ,
Proteins
and O t h e r P o l y p e p t i d e s
as
Cosubstrate
The next c l a s s of cosubstrates for kinases c o n s i s t s of proteins. Protein k i n a s e s are very important i n the r e g u l a t i o n of cellular processes. I n v e s t i g a t i o n s o f b o t h t h e s e mechanisms a n d t h e r o l e s that t h e p r o t e i n k i n a s e s p l a y m i g h t be a i d e d by t h e u s e o f s e l e c tive inhibitors of these k i n a s e s . One i n d i c a t i o n o f just how little is known about the c h a r a c t e r i s t i c s of protein kinases u b s t r a t e i n t e r a c t i o n s i s t h e f a c t t h a t many o f t h e s e enzymes are named by t h e i r a c t i v a t o r s r a t h e r t h a n by t h e i r s u b s t r a t e s . been
One p r o t e i n k i n a s e , n a m e l y c A M P - d e p e n d e n t p r o t e i n k i n a s e , h a s extensively studied to r e l a t e primary polypeptide structure
198
CONFORMATIONALLY DIRECTED DRUG DESIGN
to substrate activity. The p e p t i d e s e q u e n c e s surrounding the phosphorylation sites (serine residues) i n several protein substrates for c A M P - d e p e n d e n t p r o t e i n k i n a s e s have b e e n determined (59-61). S m a l l p e p t i d e a n a l o g s o f t h e s e have b e e n s y n t h e s i z e d a n d found t o be s u b s t r a t e s (60,62-64)• Systematic substitutions, m o d i f i c a t i o n s and d e l e t i o n s o f i n d i v i d u a l r e s i d u e s i n these small peptides have r e v e a l e d t h a t m u l t i p l e b a s i c r e s i d u e s o n t h e a m i n o terminal side of the phosphorylated serine residue are important i n d e t e r m i n i n g t h e s u b s t r a t e s p e c i f i c i t y o f t h e enzyme ( 6 5 ) . NMR a n d k i n e t i c s t u d i e s have b e e n c o n d u c t e d w i t h t h e hope o f p r o v i d i n g more d e t a i l s a b o u t t h e p o s i t i o n a n d c o n f o r m a t i o n o f t h e polypeptide substrate i n cAMP-dependent p r o t e i n kinase. These have served t o n a r r o w down t h e p o s s i b l e spatial relationships b e t w e e n enzyme bound ATP a n d t h e p h o s p h o r y l a t e d s e r i n e . Thus, a picture of the active s i t e that i s consistent with the available d a t a c a n be drawn ( 1 2 , 1 3 i 6 6 , 6 7 ) . A l t h o u g h t h e s e s t u d i e s have b e e n largely s u c c e s s f u l a t e l i m i n a t i n g some c l a s s e s o f s e c o n d a r y p o l y peptide s t r u c t u r e such as a - h e l i c e s , B - s h e e t s o r a n o b l i g a t o r y Bturn conformation (66), the precise conformation of the substrate i s s t i l l n o t known. The d a t a a r e c o n s i s t e n t w i t h a p r e f e r e n c e f o r certain B-turn structures d i r e c t l y i n v o l v i n g the phosphorylated serine residue. However, t h e y a r e a l s o c o n s i s t e n t w i t h a p r e f e r ence o r r e q u i r e m e n t f o r e i t h e r a c o i l s t r u c t u r e o r some n o n s p e c i fic type o f secondary s t r u c t u r e . Models o f t h e t e r n a r y actives i t e c o m p l e x e s b a s e d on b o t h t h e c o i l a n d t h e BQ , t u r n conformations of one a l t e r n a t e p e p t i d e s u b s t r a t e have been constructed (12). These two m o d e l s a r e c o n s i s t e n t w i t h t h e a v a i l a b l e k i n e t i c a n d NMR d a t a . The number o f d e g r e e s o f r o t a t i o n a l f r e e d o m p r e s e n t i n polypeptide s u b s t r a t e s p r o d u c e s a s t a g g e r i n g number o f possibilities for substrate conformation, e v e n when t h e p e p t i d e i s s m a l l a n d many regular structures are eliminated. One way f u r t h e r t o pinpoint t h e s u b s t r a t e c o n f o r m a t i o n needed f o r o p t i m a l s u b s t r a t e or binding activity i s to use conformationally restricted analogs (20). T h i s i d e a has a l r e a d y been e x p l o i t e d i n t h e use o f p r o l i n e and h y d r o x y p r o l i n e i n some o f t h e s y n t h e t i c p e p t i d e a n a l o g s mentioned earlier. H o w e v e r , no work h a s y e t b e e n done on n o n p o l y p e p tide analogs, and i n p a r t i c u l a r , c o n f o r m a t i o n a l l y r e s t r i c t e d a n a l o g s , b u t t h i s may be a f r u i t f u l a r e a f o r f u t u r e r e s e a r c h . Studies s i m i l a r to the substrate s p e c i f i c i t y studies outlined for cAMP-dependent p r o t e i n k i n a s e h a v e a l r e a d y begun for other p r o t e i n k i n a s e s s u c h as cGMP-dependent p r o t e i n k i n a s e ( 6 8 ) , phosp h o r y l a s e k i n a s e ( 6 9 - 7 1 ) a n d two t y r o s i n e - s p e c i f i c p r o t e i n k i n a s e s (72-75). Small Molecules
as
Cosubstrates
The t h i r d c l a s s o f c o s u b s t r a t e s f o r k i n a s e s e n c o m p a s s e s substrates that are neither n u c l e i c a c i d d e r i v a t i v e s nor p r o t e i n s , but are small molecules which s e r v e many functions, often in energy
9.
KEN YON
AND
REDDICK
Design of Kinase
Inhibitors
199
metabolism. R a t h e r t h a n r e v i e w s u b s t r a t e s p e c i f i c i t y f o r one r e p resentative enzyme o f t h i s c l a s s i t may be more u s e f u l to give e x a m p l e s o f some t h e most r e c e n t work on a few k i n a s e s w i t h s m a l l , non-nucleotide substrates. T h r e e enzymes m e n t i o n e d i n t h i s s e c tion are i n v o l v e d i n g l y c o l y s i s (hexokinase, 3-phosphoglycerate k i n a s e and p y r u v a t e k i n a s e ) a n d one ( c r e a t i n e k i n a s e ) i s involved in maintaining the energy r e s e r v o i r i n muscle c e l l s which use large bursts of energy. C r e a t i n e k i n a s e h a s b e e n t h e most systematically studied o f t h e s e enzymes w i t h r e s p e c t to its bulk tolerance and conformation of the non-ATP co-substrates. This w i l l be d i s c u s s e d i n d e t a i l l a t e r i n t h i s c h a p t e r as a n example o f how t h e s e t y p e s o f s t u d i e s c a n be u s e d t o map o u t t h e a c t i v e s i t e . Hexokinase and p y r u v a t e k i n a s e c o n s t i t u t e p o t e n t i a l targets for chemotherapy s i n c e the predominant isoenzymes i n h i g h l y neoplastic rat t i s s u e i n both cases are the f e t a l r a t h e r than the adult i s o e n z y m e s (^8). Isoenzymes o f t h e s e t a r g e t e d k i n a s e s have b e e n compared a n d c o n t r a s t e d i n t h e i r s p e c i f i c i t y f o r ATP a n a l o g s . B u t t h e s e a r c h f o r a g e n t s s e l e c t i v e f o r one k i n a s e i s o e n z y m e c o u l d a l s o be c o n d u c t e d w i t h a n a l o g s o f t h e n o n - n u c l e o t i d e c o s u b s t r a t e s . These substrates are more l i k e l y t o be selective for single enzymes t h a n a r e ATP a n a l o g s , w h i c h may a f f e c t many k i n a s e s and o t h e r A T P - u t i l i z i n g enzymes. For example, there are s i g n i f i c a n t l y f e w e r enzymes w h i c h u t i l i z e PEP as s u b s t r a t e t h a n A T P . The s u b s t r a t e s p e c i f i c i t i e s o f b o t h mammalian a n d y e a s t h e x o k i n a s e s have been e x t e n s i v e l y s t u d i e d ( 7 6 , 7 7 ) . Nevertheless, work in t h i s a r e a c o n t i n u e s both i n the s e a r c h f o r isoenzyme specific inhibitors and in increasingly detailed investigations of the c a t a l y t i c mechanism. Recently p o t e n t i a l t r a n s i t i o n state analogs P1-(adenosine-5 )-P3-glucose-6 t r i p h o s p h a t e ( A p ^ - g l u c o s e ) and P1(adenosine-5 )-P4-glucose-6 t r i p h o s p h a t e ( A p . - g l u c o s e ) were t e s t e d as i n h i b i t o r s o f f o u r h e x o k i n a s e i s o e n z y m e s . However, t h e y were found t o e x h i b i t l e s s a f f i n i t y f o r t h e enzyme t h a n e i t h e r o f the n a t u r a l substrates alone (78). f
1
Nine o t h e r g l u c o s e a n a l o g s were s y s t e m a t i c a l l y s t u d i e d with y e a s t a n d mammalian b r a i n h e x o k i n a s e s ( 7 9 ) • F i v e of these analogs exhibited s u b s t r a t e a c t i v i t y w i t h the y e a s t isoenzyme, and two were found t o be c o m p e t i t i v e i n h i b i t o r s . Two o f t h e alternate s u b s t r a t e s were s t u d i e d w i t h t h e mammalian enzyme, b u t no s i g n i f i c a n t d i f f e r e n c e s were f o u n d . In a n o t h e r r e c e n t s t u d y i t was c o n c l u d e d t h a t b o t h the pyr a n o s e and the f u r a n o s e forms o f the a l t e r n a t e s u b s t r a t e 5-keto-Df r u c t o s e a c t as s u b s t r a t e s . The p y r a n o s e f o r m c a n be compared t o g l u c o s e l a c k i n g h y d r o x y l a t C-1 and h a v i n g a s e c o n d h y d r o x y l a t C 2 (ketohydrate form). The f u r a n o s e f o r m i s c o m p a r a b l e t o f r u c t o s e but bears an e x t r a h y d r o x y l moiety a t C - 5 . Correcting for the amount o f 5 - k e t o - D - f r u c t o s e i n t h e p y r a n o s e f o r m (98%), a K value for the f u r a n o s e f o r m c o u l d be c a l c u l a t e d t h a t i s more tSan an o r d e r o f magnitude s m a l l e r t h a n t h a t f o r f r u c t o s e . This suggests that t h e r e i s a f a v o r a b l e i n t e r a c t i o n b e t w e e n t h e enzyme a n d the a d d i t i o n a l C-5 h y d r o x y l . However, s i n c e no c o n c l u s i v e d a t a f o r C -
200
CONFORMATIONALLY DIRECTED DRUG DESIGN
5 s p e c i f i c i t y have been r e p o r t e d , more work i s n e e d e d t o assess a c c u r a t e l y the e f f e c t of the e x t r a C-5 h y d r o x y l (80). The x - r a y s t r u c t u r e for yeast hexokinase i s also available. Thus, glucose analogs a r e now b e i n g u s e d to elucidate minute d e t a i l s o f the c a t a l y t i c mechanism. R e c e n t l y D - x y l o s e was u s e d i n crystallographic work t o show t h a t t h e 6 - h y d r o x y m e t h y l g r o u p of the natural substrate i s necessary for substrate-induced closure of t h e a c t i v e - s i t e c l e f t (81_). This induced c l o s u r e , which is observed with glucose b i n d i n g (82), i s b e l i e v e d t o be a p a r t of t h e i n d u c e d f i t mechanism p o s t u l a t e d f o r h e x o k i n a s e (83)• S i n c e t h e d i s c o v e r y t h a t g l y c o l a t e was a n a l t e r n a t e substrate for p y r u v a t e k i n a s e (8^)> s e v e r a l o t h e r a - h y d r o x y a c i d s have also b e e n f o u n d t o be s u b s t r a t e s f o r t h i s enzyme (85). This c l a s s of alternate s u b s t r a t e s p r o v i d e s a new a p p r o a c h t h e p r o b l e m o f subs t r a t e s p e c i f i c i t y f o r p y r u v a t e k i n a s e . 3 - N i t r o l a c t a t e i s one s u c h alternate substrate. I n t e r e s t i n g l y , the phosphorylated product of t h i s r e a c t i o n i n a c t i v a t e s t h e enzyme ( 8 6 ) . However, 3-nitrolactate does n o t behave as a s t r a i g h t f o r w a r d a f f i n i t y label since covalent modification occurs n o n s p e c i f i c a l l y . It is hoped that this new i n f o r m a t i o n may l e a d t o t h e d e s i g n o f a n a f f i n i t y label of t h i s enzyme, f u r t h e r s e r v i n g t o p i n p o i n t amino a c i d g r o u p s a t the a c t i v e s i t e . Recently, it was r e p o r t e d t h a t t h e f a s c i o l i c i d e MK-401 (4amino-6-trichloroethenyl-1,3-benzenedisulfonamide) a c t s by inhibiting both p h o s p h o g l y c e r a t e k i n a s e and p h o s p h o g l y c e r a t e mutase, thus e f f e c t i v e l y blocking g l y c o l y s i s (87,88). This agent is a potent competitive i n h i b i t o r of phosphoglycerate kinase. In f a c t , the K. v a l u e o f 0 . 2 9 mM i s t h r e e - f o l d l o w e r t h a n t h e K v a l u e f o r 1,3-diphosphoglycerate. U s i n g c o m p u t e r - g e n e r a t e d models (89)> the s t r u c t u r e s of the n a t u r a l s u b s t r a t e , 1,3-diphosphoglycerate, and t h e i n h i b i t o r , MK-401, were c o m p a r e d . The s t r u c t u r e s m a t c h e d w e l l when t n e c a r b o n s k e l e t o n o f t h e s u b s t r a t e was c o n f o r m e d t o f i t t h e arrangement of the b e n z y l carbons o f the inhibitor. Thus, this inhibitor may be c o n s i d e r e d a c o n f o r m a t i o n a l l y r e s t r i c t e d analog of the natural substrate. L i m i t e d m o d i f i c a t i o n s of the MK-401 structure at the 6 - p o s i t i o n r e v e a l e d a p o s i t i v e correlation between t h e s i z e o f r i n g s u b s t i t u e n t a n d p o t e n c y a s a n i n h i b i t o r . Affinity Labeling Epoxycreatine
of
Creatine Kinase;
Rationale
for
the Design
of
In the c o u r s e o f s t u d y i n g t h e mechanism o f action of creatine k i n a s e f r o m r a b b i t s k e l e t a l m u s c l e (M.M i s o e n z y m e ) , K e n y o n a n d c o workers (4»9Q) have been i n v o l v e d i n the d e s i g n o f s p e c i f i c i r r e versible i n h i b i t o r s that are a c t i v e - s i t e - d i r e c t e d (affinity labels). Creatine kinase c a t a l y z e s the r e v e r s i b l e t r a n s f e r of a p h o s p h o r y l g r o u p ( t h e e l e m e n t s o f " P O i " ) f r o m ATP t o c r e a t i n e , as shown i n t h e f o l l o w i n g r e a c t i o n :
9.
KEN YON
AND
REDDICK
~0
C-CH -N=*C?+ 9
* in creatine
+ MgATP
Design of Kinase
201
Inhibitors
-ine ^ MgADP + ~ 0 C - C H , kinase" ?
Efforts to s y n t h e s i z e an a f f i n i t y l a b e l s t r u c t u r a l l y r e l a t e d to c r e a t i n e were s t i m u l a t e d by t h e l a c k o f detailed information about the active s i t e of creatine kinase. At the time of this w r i t i n g , t h e c o m p l e t e p r i m a r y amino a c i d s e q u e n c e o f r a b b i t m u s c l e c r e a t i n e k i n a s e has n o t y e t been p u b l i s h e d , a l t h o u g h i t may soon be f o r t h c o m i n g o w i n g t o d e v e l o p m e n t s i n r e c o m b i n a n t DNA m e t h o d o l o gy t h a t s h o u l d p e r m i t i s o l a t i o n and s e q u e n c i n g o f t h e c o m p l e m e n t a r y DNA t o i t s m e s s e n g e r RNA. B u t e v e n when t h e p r i m a r y s e q u e n c e i s known, t h e t y p e o f i n f o r m a t i o n p r o v i d e d by a f f i n i t y l a b e l i n g i s important in p i n p o i n t i n g the s u b s t r a t e b i n d i n g site(s) on the enzyme's s u r f a c e . The a b i l i t y to i n a c t i v a t e c r e a t i n e kinase with high selectivity in vivo should also permit d e t a i l e d investigations concerning the bioenergetics of A T P - u t i l i z a t i o n i n muscle action without the c o m p l i c a t i n g f e a t u r e s o f the A T P - p h o s p h o c r e a t i n e interconversion. This has p r o v i d e d a s e c o n d m o t i v a t i n g f o r c e for f i n d i n g an a f f i n i t y l a b e l f o r the enzyme. D e s i g n i n g s p e c i f i c enzyme i n h i b i t o r s on a r a t i o n a l b a s i s when one d o e s n o t have a d e t a i l e d t h r e e - d i m e n s i o n a l c r y s t a l structure to which to r e l a t e i s a rather sophisticated challenge. Some viable approaches to such a challenge are d i s c u s s e d i n a review chapter by S a n t i and K e n y o n (9]_)• T h i s d i s c u s s i o n w i l l f o c u s on our rationale f o r the d e s i g n of an a f f i n i t y l a b e l for creatine kinase, namely N-(2,3-epoxypropyl)-N-amidinoglycine (epoxycreat i n e ):
Exploration of Bulk Tolerance. Most affinity labels contain f u n c t i o n a l groups added t o the s u b s t r a t e ' s b a s i c s t r u c t u r e . Discerning j u s t where a d d e d b u l k c a n be t o l e r a t e d b y t h e enzyme is therefore crucial information. In the case of c r e a t i n e , i t has been determined (92,93) t h a t the s t r u c t u r e s below, for example, a r e good s u b s t i t u t e s f o r c r e a t i n e i n the c r e a t i n e - k i n a s e reaction (V > 25% t h a t o f c r e a t i n e itself): max" T O
CONFORMATIONALLY DIRECTED DRUG DESIGN
202 H
2
N ^ N H
"°2 v^Nv ^CH C
s
H
2
N ^ N H
2
o c.
3
CH N-ethylN-amidinoglycine
C
"°2 -
-
H
1
D-N-methylN-amidinoalanine
cyclocreatine
On t h e o t h e r h a n d , t h e f o l l o w i n g s t r u c t u r e s t h a t a r e v e r y poor s u b s t r a t e s a t b e s t :
H
°2
L-N-methylN-amidinoalanine
r\
2
N
