- Email: [email protected]
function relationships in the inhibition of thimet oligopeptidase by carboxyphenylpropyl-peptides
V o l u m e 294. n u m b e r 3. 183-186 F E B S 10458 ~ 1991 Federation of European Biochemical Societies 00145793/91/$3._~t)
Dc~:cmlx-r IO91
Structure/function relationships in the inhibition o f thimet oligopcpti,tase by c a r b o x y p h e n y l propyl-peptides C. Graham
Knight
and
Alan
J. Barrett
Der~rcs~ncnl o f Bi¢~chemistrt'. Strangeway$ Research Laboratory. EVorts Causeway. Cambridge C B I 4 R N . U N R e c e i v e d 26 S e p t e m b e r I(~I; revised v e r s i o n received 17 O c t o b e r 1991 Some novel N-[l(RS)-carboxy-3-phenylpropyl]trilaeptide p-aminobcnzoates hawe bexm synthesised as inhibitors o f lhimet oligopeptidas¢ (EC 3.4.24.151. The~e compounds are considered to bind as substrate analogues with the Cpp group in SI and the l~ptide portion in the S" ~ite~. The most Dotc,t inhibitor is Cpp-Ala-Pro-Phe-pAb. which has a K. - 7 nM. Substitution o f (~ly for Ala at Pl" leads to weaker binding which can be ascribed to increa.~d rotational tr~x'dom. Good substrates often have Pro at P2" and Pro is favoured over Ala at this position in the inhihitors, too. When P2" is Pro. Phe is p r e f e r r ~ over Tyr and T i p in P3 +. The p-amlnobenToate group makes an ;ml~,rtant contribution to the binding, probably by forming a .salt bridge, and removal o f the ('-terminal negative 4zhargc results in much less potcnl inhibitors. EC 3.4.24. I S; Fm, ym© inhibition; Subsite specificity
i.
INTRODUCTION
T h i m e t o l i g o p e p t i d a s e ( E C 3 . 4 . 2 4 . 1 5 ) is ~" t h i o l - d e pendent metallo-endopeptidase w i d e l y d i s t r i b u t e d in dells and tissues, and previously known by the names Pz-peptidase, endo-oligopeptidase A and soluble endop e p t i d a s e 24.15 [I]. C h a r a c t e r i s t i c f e a t u r e s o f t h e e n zyme are the inhibition by both chelators and thiolreactive reagents and activation by low concentrations of thiols. The biological function of thimct oligop e p t i d a s e is u n k n o w n , b u t is c a p a b l e i n v i t r o o f g e n e r a t ing or destroying a number of pharmacologically active p e p t i d e s a n d is f o u n d i n t h e h i g h e s t c o n c e n t r a t i o n s in the soluble protein fraction of testis, brain and pituitary [2]. l n h i b i t o r s m a y p r o v i d e t h e b e s t a p p r o a c h t o i n v e s t i g a t i n g t h e p h y s i o l o g i c a l r o l e o f t h e e n z y m e [3]. T h i m e t o l i g o p e p t i d a s e is s e l e c t i v e l y a n d r e v e r s i b l y i n hibited by N-[l(RS)-carboxy-3-phenylpropyi]tripeptide p-aminobenzoates (Cpp-peptide-pAb) [4,5]. T h e m e c h a nism of inhibition may be similar to that proposed for thermolysin and Cpp-Leu-Trp [6], w h e r e X - r a y c r y s t a l lography has revealed the coordination of the carboxyl ol the Cpp group to the Zn atom in the active site of the e n z y m e . I n t h i m e t o l i g o p e p t i d a s e , i n t e r a c t i o n o f t h e 3phenyl ring with the hydrophobic S I subsite also makes a n i m p o r t a n t c o n t r i b u t i o n t o t h e b i n d i n g [4] C p p - p e p -
Abbrevlatlons: Ahx, L-2-aminohexanoic acid; Boc, t-butyloxycarbottyl; C I ~ , N-[It(RSk-¢arboxy-3-phenylpropyli; Drip, dinitrophenyl; Moc. 7-melhoxyooumarin-3
Corresptmdence address: C.O. Knight, Departmem o f Bk~hcmistry, Strangeways Research Laboratory, Worts Causeway, Cambridge CBI 4RN, UK.
Pub/is&ed by E./sev~r Sck,nce P u b / t ~ ' r s B. g,'.
tide-pAb are competitive inhibitors, and since the most potent have peptide structures analogous to those of g o o d s u b s t r a t e s [4], t h e a m i n o a c i d s i d e c h a i n s p r e sumably occupy the same specificity subsites on the e n z y m e . B y c o n v e n t i o n [7], t h e s e s u b s i t e s a r e l a b e l l e d S t o t h e N - t e r m i n a l s i d e o f t h e s c i s s i l e b o n d a n d S" t o t h e C-terminal side, numbering being away from the bond cleaved, the scissile bond. The amino acid residues w h i c h o c c u p y t h e s e su b si t e s a r e l a b e l l e d P a n d P', res p e c t i v e l y . A l t h o u g h it is n o t y e t p o s s i b l e t o d e f i n e t h e c h a r a c t e r i s t i c s o f t h e s u b s i t e s p r e c i s e l y [I], g o o d s u b strates and inhibitors of thimet oligopeptidase comm o n l y h a v e h y d r o p h o b i c r e s i d u e s a t P i a n d P Y [2,4]. In a d d i t i o n to t h e i r use in b i o l o g i c a l e x p e r i m e n t s , inhibitors more potent than those currently available are needed for active site titration of the enzyme, so that kinetic studies can be placed on a quantitative basis. Also needed are inhibitors more resistant to degradat i o n , w h i c h c u r r e n t l y c o m p l i c a t e s t h e i r use in s t u d i e s o f t h e r o l e o f t h i m e t o l i g o p e p t i d a s e in v i v o [3]. W e n o w report our initial studies to investigate the relationships between inhibitor structure and function.
2. E X P E R I M E N T A L 2.]. Maaet4ais Boc-amino acid N-hydroxysuc~nimide esters, p-aminobenzoic acid and L-phenylalanine derivatives ~ from Sigma. Uoc-amino acid p.aminobenzoates were made as de~ribed [8]. Ethyl; 2-keto-4-phenylbutanoat© from Scbeizerhall (South Plainfield, N J 07080) was saponifled with N a O H to yield the sodium salt. Cpl~Ala-Ala-Tyr-pAb was a gift from Dr. M. Orlowski. Mount Sinai School o f Medicine, New York, USA. Moc-Pro-I..eu-Oly-Pro-D-Lys(Dnp) was made by solid phase pepcide ~ t n t h e ~ 19L Rat t,n~t~ mim~ ~ w.as purified by a method similar to that used for the chicken enzyn~ 15|. and ga~e a single band o f "/6 kDa in S D S ? l ~ a m l i ~ ~ doctralphorcsh.
183
.~4.
Votume
number
3
UEBS
2.2. 5;Ynth,'s:.~ ¢,I Cpp-.4la-Pro-Ph*'-p.4~ T h i s w a s m a d e s t e p - w i r e by s t a n d a r d ~ l t s t i o n - p h a ~ mctht~cls [4. I0]. R e a c t i o n s ~ c r t " ft~llowt~J b y H P [ . C o n a C1~4 L'olumn, c l u l c d w i t h tt g r a d i e n t o f 5 100~4;, a L ~ t o m t r i l c c o n t a i n i n g 0 I¢IF t r I f l u o r o w u c t l c a c i d ( I m l , m i n ) o v e r 25 rain. T h t , c o l u l n n ¢ I t i a n t ~Jas m o n i t o r e d at ".10 rim. I ~ - - P h e - p , A b 1384 rag. I r e t o o l ) g a s treatL,~J w i t h trifluolat~llccYic ;lt:iti/ w a t e r I 1 9 I. ~'~'v. :q) m l ) f o r 20 n~it~ at 21 ~'C t o r e m o v e t h e B¢~.- g r o u p T h e a c i d w a s r e m o v e d a t 44)°(" in v a c u o a n d t h e t ~ s i d u e w a s d i s s o l v e d in d i m c t h y l f o m l a m i d c ( S a i l , T o t h i s , ~ t u i i o n w a s addc~l B ~ ' - P r o O N S u ( 3 3 0 rag, I I m m o l L l - h y d r o x y l ~ n ~ o t r i a ~ c ~ l e ( I 35 g, I n t m ~ i ) a n d d i - i s o p r o p y l e t h y l a m i n e (O.7 m l . 4 m m o l L A f t e r s t i r r i n g o v e r n i g h t , a n a l y s i s b y H P L C sho~k~t."d t h e r e a c t i o n t o t h e c o m p l e t e . T h e d i m e t h y l f o r m a m / d ~ w a s e v a p o r a t e d a t 4 0 ° ( " in v a c u o a n d c h l o r o f o r m ( ~ ) m l ) was adld~. A f t e r eglra~liom w i t h ~ t w / v } c i t r i c a c i d (2 ~< ~ m l L w a t e r 120 m l ) a n d s a t u r a l e d N a ( ' l ( 2 0 r o l l t h e c h l o r o f o r m s o l u t i o n w a s dried (M8SO4) and evaporated to leave crude Boc-Pro-Phe-pAh_ This w a s Ptx-rystalli~L'd f r o m e t h i * n o l / w a t e r ( y i e l d i n g 0 . 4 6 g. 95~[-I a n d t r e a t e d w i t h tri~luoroa,cctic a c i d / w a t e r a s d e s c r i b e d a b o v e , T h e r e s i d u e a f t e r e v a p o r a t i o n w a s d i s s o l v e d in d i m e t h y l f o r m a m k l e a n d r e a c t e d with Eloc.Ala.~)NS¢ (2~ mr. I mmoh. I-hydroxybenzotriazote and di-isop~xspylethylamine. As before, the solvent was evaporated after r e a c * i o n o v e r n i g h t a n d t h e r e s i d u e d i s s o l v e d in c h l o r o f o r m , e x t r a c t e d a n d d r i e d . T h e c r u d e E k ~ - A l a - P r o - P h e - p A b w a s 97q~, p u r e by H P L ( " a n a l y s i s a n d w a s t r e a t e d w i t h trifluoroa~i~tic a c i d w i t h o u t f u r t h e r p u r i f i c a t i o n . T h e r e s i d u e a f t e r e v a p o r a t i o n w a s d i s s o l v e d in m e t h a n o l / w a t e r ( 1:1 v/v, ~ m l ) a n d b r o u g h t t o p H 7 w i t h 5 M N a O H . S o d i u m 3 - p h c n y l - 2 - k e t o b u t y r a t e ( I g, 5 r e t o o l ) a n d s o d i u m c y a n o b o r o h y d r i d e (0.32 g. 5 r e t o o l ) w e r e a d d e d a n d She m i x t u r e s t i r r ~ at 2 I~'C. H P L C a n a l y s i s s h o w e d t h a t r e a c t i o n w a s ~.~.~mplete a f t e r 24 h. T h e s o l v e a t s were e v a p o r a t e d , w a t e r (50 ml) w a s a d d e d a n d the ,solution acidified w i t h 6 M H C I ( 1.5 m l ) t o p r e c i p i t a t e C p p - A l a - P r o - P h e - p A b . H C I . T h e c r u d e p r o d t u : t wa,~ w a s h e d w i t h e t h y l a c e t a t e , a n d r e e r y s t a l l i s e d f r o m e t h a n o l a n d w a t e r , y i e l d i n g 0 . 3 3 g ¢49~-), m , p . I S ? 188°C. A n a l y s i s : c a l c u l a t e d f o r C , , H ~ N ~ O ~ . H ( ? I ( M , 6 7 9 . 2 ) C . 6 2 . 7 2 ; H , 6.O4; N . 8 . 6 0 ; t~)und ( ' , 62.98: H, 6.1 I ; N . 8.60. T h e other inhibitors w~re mad~ by s i m i l a r m e t h o d s . The identities o f t h e f inal l , ~ p t i d e p r o d u c t s w e r e c o n f i r m e d b y e l e m e n t a l a n a t y s i s , N M R o r fast a t o m b o m b a r d m e n t m a s s s p e c t r o m e t r y . A l l t h e p h e n y l a lanin~-¢ontaining inhibitors displayed characteristic double peaks on H P L C . a t t r i b u t a b h : to t h e D a e t i a l l y r e s o l v t ' d R a n d S d i a s t e r e o m e r s
141. 2.3, D¢ls,rttaOl¢it~on o l K, vcllut',s Thimet
oligoraepttdase activity was assayed
at
300( `` w i t h
the
quenched fluorc,s~cat f r c p t i d c Mc~.'-Pro-L~u ~ ( ~ l y - P r o - D - L y s ( O n p 1 1 9 1 , in ~,hich the s y m b o l + r e p r e s e n t s t h e s¢issile b o n d . T h e f l u o r l m e t e r Table ! Effi.~ct o f i n h i b i t o r s t r u c t u r t : a t P I ' l"
2'
3'
i
+
4"
Cpp Cpp CPlP CI~ Cpp Cpp Cpp CI~
Phc Ala Phe pAb - Leu Pro Phe pAb Ala Ala PIR - pAb -GIyAI2 -- ~ pAb - Ala ~ Pro - Phe - pAb - G l y -- P r o P h e -- p A b Sar • Ala . PI~ pAb Sat Pro Phe pAb
K, ( n M ) 81 279 30.6 774 7.3 289 15700 3410
-~ 10" _+ 10 _+ 1.0 _+ 16 + 0.3 ± g ± 200 ± 130
The Cpl~peptide-pAb inhibitors nre substrate nnalogues and are t h o u g h t t o b i n d w i t h t h e C l ~ g r o u p in t h e S l s u b t i l e o f t h e e n z y m e . T h e r e s i d u e s h a v e b e e n n u m t m r e d a c c o r d i n g l y , a n d t h e s y m b o l *+' ind i c a t e s t h e b o n d c o r r e s p o n d i n g t o t h e s c i s s i l e b o n d in a s u b s t r a t e . T h e error va|uea are standard errors es:imated by the E~fitter pro-
gram. *Value from
184
13l
LETTERS
December
1991
w a s c o n t r o l l e d , a n d d a t a colh_.cted a n d a n a l y s ~ d , h y u s e o f t h e V I . U S Y S s o f t w a r e D a c k a g ~ [I I ]. Whe e n , w m ¢ w a s p r o - a c t i v a t e d w i t h S m M d i t h i o t h r e i t o l in a s s a y b u f f e r ( 5 0 m M T r i s - H C I , D H 7.g, c o n t a i n Ing tlOSqF Itr U i S ) f o r 5 r a i n at 2 1 " ( a n d t h e n d i l u t e d IO*fold a n d k e p t a t 0 " ( ' . A ~ a y ~ , w e r e m a d e w i t h OR n M e m, y m e . b a s e d o n p r o t e i n c o n c e n t r a t i o n . F o r t h e d e t e r m i n a t i o n o f K,. t h e r a t e o f s u b s t r a t ¢ h y d r o l y ~ i ~ in t h e absem.'¢ o f i n h i b i t o r w a s first r e c o r d e d , a n d t h e n t h e i n h i b i t o r w a s a d d ~ in a n e g l i g i b l e v o l u m e a n d t h e n e w s t e a d y s t a l e w a s m o n i t o r e d R a p i d e q u i l i b r a t i o n w a s o b s e r v e d in a l l c a s e s . A p p a r e n t i n h i b i t i o n c o n s t a n t s A'.=r~., w e r e c a l c u l a t e d b y f i t t i n g I h e f r a c t i o n a l a c t i v i t y d a r n t o II1¢ M l o r r i s o r t L~quation [I 2| b y n o n l i n ~ r r e l g l ~ o s i o n , u s i n g t h e p ¢ o g t a m E n ~ l i t t e r ! I-Ascvier-Biosofl, Cambridge, U K ) . V a l u e s wct'¢ c o r r e c t e d l o r t h e effoet o f s u b s t r a t e b y u s i n g e q u a t i o n ( t ) .
K, = K.~.~/(I ÷ [SFK.)
(l)
A v a l u e o f 16 .u M w a s d c t c r m i n * ~ ! f o r Km. T h e di ff er en t:c~ in K, v a | t t ~ I'~twL~n h t h i b i t o r s w e r e c o n v e r t e d i n t o d i f f e r e n c e s in t h e f r ~ e n e r g y o f b i n d i n g t ~ ( ~ . d h y e q u a t i o n (2),
66G~,.a --. - 2 . 3 0 3 R T - Iogl K,,tK',:)
3. R E S U L T S
AND
(2)
DISCUSSION
3. I. A l a n i n e is f i t v o u r e d in P l "
Good peptide substrates of thimct oligopeptidase c o m m o n l y h a v e h y d r o p h o b i c r e s i d u e s i n PI a n d P3". a n d a r g i n i n e is o f t e n f o u n d in Pl" [2], s u g g e s t i n g t h a t t h e SI" site o f t h e e n z y m e is a c c e s s i b l e t o l a r g e side c h a i n s . T h i s c o n c l u s i o n is s u p p o r t e d b y s t u d i e s w i t h i n h i b i t o r s , s i n c e t h e K, o f C p p - P h ¢ - A l a - P h e - p A b is o n l y 3 - f o l d t h a t o f Cpp-Ala-Phe-pAb [4] ( T a b l e 1). L e u c i n e . h o w e v e r , is f o u n d t o be a n u n f a v o u r a b l e r e s i d u e in P i ' . It h a s b e e n o b s e r v e d t h a t r e p l a c e m e n t o f A l a b y G l y in Pl" t o g i v e C p p - G i y - A l a - P h e - p A b r e s u l t s in a far less p o t e n t i a l i n h i b i t o r [4.5], a n d w e find s i m i l a r l y w e a k b i n d i n g w h e n P2" is P r o ( T a b l e !). in p r i n c i p l e , t h e w e a k e r b i n d i n g o f t h e C p p - G l y - i n h i b i t o r s c o u l d be d u e t o t h e l a c k o f a n i n t e r a c t i o n o f t h e a l a n i n e s i d e c h a i n in t h e SI" p o c k e t o f t h e e n z y m e , o r t o g r e a t e r r o t a t i o n a l freedom allowing the compounds to adopt unfav o u r a b l e c o n f o r m a t i o n s . H o w e v e r . t h e K, d i f f e r e n o e s between Cpp-Ala-Ala-Phe-pAb and Cpp-(31y-Aia-PhepAb. and Cpp-Al,~-Pro-Phe-pAb and Cpp-Gly-l~oP h e - p A b c o r r e s p o n d t o d i f f e r e n c e s in b i n d i n g free e n e r g y A A G b i n d o f ! . 9 a n d 2.2 k c a l / M , r e s p e c t i v e l y . B y c o n t r a s t , t h e free e n e r g y c h a n g e o n t r a n s f e r o f a m e t h y l g r o u p f r o m o c t a n - l - o l t o w a t e r is o n l y 0 . 7 k c a l / M [13], w h i c h sug~oests t h a t t h e h y d r o p h o b i ¢ p o c k e t m o d e l is i n a d e q u a t e t o e x p l a i n t h e d i f f e r e n c e s in g i a n d that t h e i n c r e a s e d flexibility o f t h e g l y ¢ i n e d e r i v a t i v e s is res p o n s i b l e f o r t h e i r p o o r e r b i n d i n g . S u c h flexibility h a s been shown to be responsible for the weaker binding of analogues of Cpp-Ala-Pro (enalaprilat) to peptidyl-dip e p t i d a s e A ( a n g i o t e n s i n c o n v e r t i n g e n z y m e ) [14]. W i t h these compounds, binding could be enhanced as much a s 2 5 0 0 - f o l d b y c o n f o r m a t i o n a l r e s t r i c t i o n , s o it s e e m s reasonable to suggest that restricted rotation around the a l a n i n e N - C a b o n d p l a y s a r o l e in o r i e n t i n g t h e C p p c a r b o x y l g r o u p t o w a r d s t h e Z n a t o m in t h e a c t i v e site
Volume 294, n u m b e r 3
FEBS L E T T E R S
of thimet oligopeptidase, although other interactions p r o b a b l y c o n t r i b u t e t o efficient b i n d i n g . F o r e x a m p l e , in t h e c o m p l e x h e t w c e n t h e r m o l y s i n a n d C p p - L e u - T r p (K, = 50 n M [15]). b o t h o x y g e n s o f the C p p c a r b o x y l g r o u p a r c c o o r d i n a t e d t o t h e zinc, b u t in a d d i t i o n e~ch f o r m s t w o h y d r o g e n b o n d s t o o t h e r r e s i d u e s lining t h e a c t i v e site [6]. S i m i l a r l y . t h e p r o t o n a t e d s e c o n d a r y a m i n o g r o u p forms three h y d r o g e n b o n d s to s u r r o u n d i n g r e s i d u e s [6]. T h e i m p o r t a n c e o f this n i t r o g e n in t h e interaction of Cpp-inhibitors with thimet oligop e p t i d a s e is s h o w n c l e a r l y b y t h e s u b s t i t u t i o n o f sarc o s i n e ( S a r ) f o r G l y ( T a b l e I) w h i c h r e s u l t s in v e r y m a r k e d losses o f a c t i v i t y . 3,2. Binding is f a v o u r e d by proline in P2" Several g o o d substrates by thimet oligopeptidase h a v e P r o in P2". i n c l u d i n g b r a d y k i n i n a n d n e u r o t e n s i n [2]. B z - G l y - P h e + A i a - P r o - P h e - p A b [16]. P z - P r o - L e u + G i y - p r o - D - A r g [! 7] a n d t h e q u e n c h e d f l u o r e s c e n t p e p tide, D n p - P r o - L e u + G l y - P r o - T r p - D - L y s [18]. T h e s e d a t a s u g g e s t t h a t t h e b i n d i n g o f p e p t i d e s t o t h i m e t olig o p e p t i d a s ¢ m a y be f a v o u r e d n o t o n l y b y h y d r o p h o b i c r e s i d u e s in P l a n d P3", b u t a l s o b y P r o in P2". T o invest i g a t e t h e effect o f p r o in this p o s i t i o n , w e h a v e c o m p a r e d s o m e m e m b e r s o f t h e series C p p - A l a - A i a - X - p A b a n d C p p - A l a - p r o - X - p A b , w h e r e X is a n a r o m a t i c o r l a r g e h y d r o p h o b i c a m i n o a c i d ( T a b l e II). S u b s t i t u t i o n o f P r o for Ala leads to slightly tighter binding a n d C p p A l a - P r o - P h e - p A b is t h e m o s t p o t e n t i n h i b i t o r o f t h i m e t o l i g o p e p t i d a s e s o f a r d e s c r i b e d w i t h Ki = 7 n M . O r l o w s k i et al. [4] h a v e o b s e r v e d t h a t t h e b i n d i n g o f C p p - A l a - A l a - T y r - p A b t o t h i m e t o l i g o p e p t i d a s e is twice a s t i g h t a s t h a t o f t h e P h e a n a l o g u e , b u t w e d o n o t see this effect w h e n t h e P2" r e s i d u e is P r o a n d b o t h C p p Ala-Pro-Tyr-pAb and Cpp-Ala-Ala-Tyr-pAb are comp a r a b l e in i n h i b i t o r y a c t i v i t y . By c o n t r a s t , t h e i n h i b i t o r w i t h L e u in P Y s h o w s a m a r k e d d e c r e a s e in i n h i b i t o r y p o t e n c y ( T ~ b l e !1). 3.3. Potent inhibition requires a negalively charged C-
terminus All t h e i n h i b i t o r s d i s c u s s e d s o f a r h a v e b e e n p - a m i n o b e n z o a t e s . S t e w a r t [19] h a s p o i n t e d o u t t h a t t h e p A b g r o u p is s t r u c t u r a l l y r e l a t e d to a d i p e p t i d e a n d this g r o u p s e e m s likely t o m a k e a m a j o r c o n t r i b u t i o n t o the b i n d i n g o f t h e i n h i b i t o r s . ~ ' e e x p l o r e d t h e influence o f Table Effect of inhibitor I
÷
CppCt~ Cl~ Cpp Cpp Cpp
-
l" Ala Ala Ala Ala Aim. Ala
3"
-
2"
---
Ala - Ph¢ Ala - Tyr Pro - Ph¢ Pro - Tyr Pro - Trip Pro - Lee
-
!!
structure
at P2' and
PY
4"
K, (riM)
pAb pAb I~Ab pAb pAb pAb
30.6 14.4 7.3 I 1.8 17.6 76.8
_+ 1 . 0 + 0.5 4- 0 . 3 + 0.4 _-4 1 . 0 + 3.0
December 1991 Table I'~I1/~X-I aft i n h i b i t o r
I
4 I'
Cpp Cpp Cpp Cpp Cpp
Ala Ala Ala Ala Ala
2~
3"
4"
Ala Ala Ala Ala Ala
Phe Phe Phe Phe Phe -
pAb OH NHPh NHPhNO~ NH:
!11 ~lruclu'~
:It P 4 " K, ( n M ) 30.6 155 t860 10~ 5790
~ ± + ± ±
1.0 5 50 40 220
i n t e r a c t i o n s at t h e C - t e r m i n u s with a series o f r e a g e n t s in w h i c h the C p p - A l a - A l a - P h e p e p t i d e p a r t w a s r e t a i n e d ( T a b l e ! I I ) . T h e m o s t p o t e n t i n h i b i t o r r e m a i n s the pa m i n o b e n z o a t e , b u t s u r p r i s i n g l y , r e m o v a l of" t h e p A b g r o u p t o yield t h e Free acid results o n l y in a 5 - f o l d i n c r e a s e in K,, c o r r e s p o n d i n g t o the loss o f 1.0 k c a l / M o f b i n d i n g free e n e r g y . B y c o n t r a s t , r e p l a c e m e n t o f the c a r b o x y l g r o u p b y a h y d r o g e n a t o m to give the unc h a r g e d a n i l i d e results in b i n d i n g w e a k e r b y 2.5 k c a i / M . T h e p - n i t r o a n i l i d e i n h i b i t o r , with t h e d i p o l a r n i t r o g r o u p in p l a c e o f t h e c a r b o x y l a t e , is o n l y m a r g i n a l l y m o r e effective t h a n the anilide. T h e i m p o r t a n t c o n t r i b u l i o n o f t h e C - t e r m i n a l n e g a t i v e c h a r g e t o t h e b i n d i n g is c o n f i r m e d w i t h the a m i d e C p p - A l a - A l a - P h e - N H 2 , w h i c h b i n d s m o r e w e a k l y t h a n its p a r e n t acid b y 2.2 k c a l / M . T h e s e free e n e r g i e s a r e in t h e r a n g e o f t h o s e o b s e r v e d f o r salt b r i d g e s in p r o t e i n s [I 3] a n d suggest t h a t , in a d d i t i o n t o b i n d i n g t o the a c t i v e site Z n , the p - a m i n o b e n z o a t e i n h i b i t o r s h a v e a n i m p o r t a n t elect r o s t a t i c i n t e r a c t i o n with |ysine o r a r g i n i n e side c h a i n s in $ 4 ° o r $5" subsites. 4. C O N C L U S I O N A l t h o u g h t h e s t u d ie s d e s c r i b e d h e r e h a v e r e s u l t e d m
an inhibitor of thimet oligopeptidase that binds more tightly than those described before. C p p - A l a - P r o - P h e p A b b i n d s t o o w e a k l y t o be used as a n a c t i v e site t i t r a n t , a n d a f u r t h e r d e c r e a s e in Ki o f a b o u t 2 0 - f o l d will be n e c e s s a r y t o a c h i e v e this aim. O n e a p p r o a c h t o t i g h t e r i n h i b i t o r s m a y b e t o e x p l o r e a l t e r n a t i v e s t o the C p p g r o u p , p e r h a p s b y i n t r o d u c i n g s u b s t i t u e n t s i n t o the p h e n y l ring, a s t h e 7 0 - f o l d d i f f e r e n c e in i n h i b i t o r y potency between carboxyphenyipropyl and carboxy p h e n y l e t h y l d e r i v a t i v e s o f A l a - A l a - P h c - p A b {4] ill u s t r a t e s t h e d r a m a t i c effects o f small c h a n g e s in s t r u c t u r e in this r e g i o n o f t h e m o l e c u l e . C p p - A l a - P r o - P h e - p A b m a y prove to be a g o o d inhibi t o r f o r in v i v o s t u d i e s o f t h i m e t o l i g o p e p t i d a s e . C p p A l a - A l a - P h e o p A b is c l e a v e d b y n e u t r a l e n d o p e p t i d a s e ( E C 3.4.24.1 I) a t t h e A l a - P h e b o n d [3], w h e r e a s the P r o - P h e b o n d m a y be m o r e r e s i s t a n t t o h y d r o l y s i s . T h i s b o n d m a y be c l e a v e d , h o w e v e r , b y p r o l y l o l i g o p e p t i d a s e ( E C 3.4.21.26) a n d c o n t r o l e x p e r i m e n t s w i t h specific i n h i b i t o r s [20] o f this e n z y m e will be n e c e s s a r y , A n a d v a n t a g e o f the C p p - p e p t i d e - p A b i n h i b i t o r s is 185
Volume
294. number
3
F|~BS L i'.-FFFRS
t h e i r r a p i d b i n d i n g to t h i m c t o l i g o r ~ p t i d a ~ e , s o that e q u i l i b r a t i o n is i n s l a n t a n e o u s o n the 10 rain time scale o f o u r as.~ays. By c o n t r a s t . N-{ p h e n y l e t h y i p h o s p h o n y l ) ( 3 1 y - P r o - A h x , d e s i g n e d as a n i n h i b i t o r o f CIo.vtridium hLgtolyth'u~ c o I l a g e n a ~ [21], r e q u i r e s several m i n u t e s t o r e a c h e q u i l i b r i u m with t h i m e t o|igopeptida.,~e, alt h o u g h the final K, (7 n M L is s i m i l a r {A.J. B a r r e t t a n d M . A . B r o w n . u n p u b l i s h e d o b s e r v a t i o n s ) . Likewise, phosphoramidon ( N-[[(6-deoxy-~-L-ma nnopyranosyl)o x y ] h y d r o x y p h o s p h i n y l ] - L e u - T r p ) is a s l o w b i n d i n g in-
hibitor o f t h e r m o l y s i n [221. w h e r e a s C p p - L e u - T r p b i n d s n o r m a l l y { 15]. ,4¢~nowlcd, g,¢m~,nts: W e a t e ( ' p p - A | a - A l a - T y r - p A b , to e x p e r t technical a s s i s t a n c e i+nd t n t e r p t ¢ t l ~ g the N M R
f r a t e f u ] t o Dr. M. O r t o w s k i l~r p r o v i d i n g l...sut.~e H a n d l o r d a n d H o t l y S p c a t m g for ant: t o l)r, tLCC. ticywo¢~l f o r o b t a i n i n g a i l d nlass sp~.x:tra.
REFERENCES [I] Barrett, A,J. (11)90) Biol. C h e m H o p p e - S e y l c r 371 ( S u p p l . ) , 31 ! 320. 12] O r l o w s k i . M., Reznik, S.. A y a l a , J. a n d Picrottl. A . R (1989) B i o c h e m . J 261. "~51 95R. 13] L a s d u n . A., Res.nik, S.. M o l i a e a u ~ . ( ' . J . a n d Orlow.~k i, M. (19891 J. P h a n n a c o l . [~ap. T h e r , 251, 439 447. 14| O r l o w s k l . M., M i c h a u d . C. a n d M o l i n e a u x . ( ' , J . (1988) E~i~hcniistr~' 27. 597 trY)2.
186
December
1991
15] l l a f r e t l . A . I and Br,~uwn. M A . ( I q g 0 ) Bi oche m . J. 271, 701 706. |61 M o n ; ' i n g o . A |:, ~nd M a t t h e w , , . B.W. (1984) B i o c h e m i s t r y 27, ~,724 572,~ 17l ,%¢h¢'~.'t¢r. I a n d BerBer. A. (1967} Biz~hem. B i o p h y g . Res;. C o i t t nt~m. 27. 157 162. |8] O i i l ~ w s k i . M , M ~ c h a u d . ( " a n d C h u , T_CL ( 1 9 8 3 ) l~ur. J, Bttx:hem. 13~. 8 l 88. [0l l'i~ljar, ILl., K n i g h t , C . G . a n d Bar r et t , A.J, t 1990) A n a l . Biocltcm. 18¢,, 112 ;15. [ ! 0 ] Bodans~'k y, M . a n d l~,¢lans~ky, A. (1984) T h e Practice o f Pcplicl¢ Synthesis. Sprinl~c,r-Verlag. B e r l i n II I1 B a w l i n g s . N , D . a n d Barlrctt. A J . ( I ~ J N ) ) C o m p u t e r A p p l . Bio~ci, 6, l i b 119. 112] M o r r i s o n , J,F. ( 1 % 9 ) B i o c h i m . B i o p h y s . A c t a 185. 209=286. | 13] F c r s h l . A. (1985) E n z y m e S t r u c t u r e a n d M e c h a n i s m , 2nd ¢d.. pp. 29~ 310. [14] T h o r s e t t . E . D . t 1 0 8 6 ) A c t u a l . C h i m . | h e r . 13, 257 268_ i15] N l a y c o c k . A . L . . [ k S o u , ~ a . D , M . . P a y n e . L . G . . ten B r o e k c . J.. W u . M . ~ . a n d Patch¢tt, A . A . (1981) B i o c h f m . Biophy~. R¢~, C o m m u n . 102, 963 969. [10] C h u . T.(~. a n d O r l o w s k i , N~. (!~85} [.~ndocrino|ogy 116, 1 4 1 g 1425. [17] B a r r e t t . A,J, ~nd Ti~ljar, U. (19B9) BicMchem, J 261, 1047 1050. [ l g l K n i g h t . C',(L ( l ~ l l B t o c h e m J 274. 45 48. [191 S t e w a r t . F . H . ( ' . (19K3} Aa~tr. J, ( ' h u m . 36, 1629 If~3X [201 T~,ttru, I~., V o s h i m o t o , T., K o r i y a m a , N . a n d F u r u k a w a , S. (19RSl J. B i o c h c m . 104. 580 586. I211 Dive, V,. Y i o t a k i s , A.. ]~lic'olaou. A. a n d T o m a , I:. ( 1 ~ ) 0 ) E u r J. Bier:hem. 1 9 1 , 6 8 5 693. [22] K a m , C_-M.. N i g h | n o , N, a n d P o w e r s , J . C . t 19"/9) B i o c h e m i s t r y 18, 3033 3038.
Dc~:cmlx-r IO91
Structure/function relationships in the inhibition o f thimet oligopcpti,tase by c a r b o x y p h e n y l propyl-peptides C. Graham
Knight
and
Alan
J. Barrett
Der~rcs~ncnl o f Bi¢~chemistrt'. Strangeway$ Research Laboratory. EVorts Causeway. Cambridge C B I 4 R N . U N R e c e i v e d 26 S e p t e m b e r I(~I; revised v e r s i o n received 17 O c t o b e r 1991 Some novel N-[l(RS)-carboxy-3-phenylpropyl]trilaeptide p-aminobcnzoates hawe bexm synthesised as inhibitors o f lhimet oligopeptidas¢ (EC 3.4.24.151. The~e compounds are considered to bind as substrate analogues with the Cpp group in SI and the l~ptide portion in the S" ~ite~. The most Dotc,t inhibitor is Cpp-Ala-Pro-Phe-pAb. which has a K. - 7 nM. Substitution o f (~ly for Ala at Pl" leads to weaker binding which can be ascribed to increa.~d rotational tr~x'dom. Good substrates often have Pro at P2" and Pro is favoured over Ala at this position in the inhihitors, too. When P2" is Pro. Phe is p r e f e r r ~ over Tyr and T i p in P3 +. The p-amlnobenToate group makes an ;ml~,rtant contribution to the binding, probably by forming a .salt bridge, and removal o f the ('-terminal negative 4zhargc results in much less potcnl inhibitors. EC 3.4.24. I S; Fm, ym© inhibition; Subsite specificity
i.
INTRODUCTION
T h i m e t o l i g o p e p t i d a s e ( E C 3 . 4 . 2 4 . 1 5 ) is ~" t h i o l - d e pendent metallo-endopeptidase w i d e l y d i s t r i b u t e d in dells and tissues, and previously known by the names Pz-peptidase, endo-oligopeptidase A and soluble endop e p t i d a s e 24.15 [I]. C h a r a c t e r i s t i c f e a t u r e s o f t h e e n zyme are the inhibition by both chelators and thiolreactive reagents and activation by low concentrations of thiols. The biological function of thimct oligop e p t i d a s e is u n k n o w n , b u t is c a p a b l e i n v i t r o o f g e n e r a t ing or destroying a number of pharmacologically active p e p t i d e s a n d is f o u n d i n t h e h i g h e s t c o n c e n t r a t i o n s in the soluble protein fraction of testis, brain and pituitary [2]. l n h i b i t o r s m a y p r o v i d e t h e b e s t a p p r o a c h t o i n v e s t i g a t i n g t h e p h y s i o l o g i c a l r o l e o f t h e e n z y m e [3]. T h i m e t o l i g o p e p t i d a s e is s e l e c t i v e l y a n d r e v e r s i b l y i n hibited by N-[l(RS)-carboxy-3-phenylpropyi]tripeptide p-aminobenzoates (Cpp-peptide-pAb) [4,5]. T h e m e c h a nism of inhibition may be similar to that proposed for thermolysin and Cpp-Leu-Trp [6], w h e r e X - r a y c r y s t a l lography has revealed the coordination of the carboxyl ol the Cpp group to the Zn atom in the active site of the e n z y m e . I n t h i m e t o l i g o p e p t i d a s e , i n t e r a c t i o n o f t h e 3phenyl ring with the hydrophobic S I subsite also makes a n i m p o r t a n t c o n t r i b u t i o n t o t h e b i n d i n g [4] C p p - p e p -
Abbrevlatlons: Ahx, L-2-aminohexanoic acid; Boc, t-butyloxycarbottyl; C I ~ , N-[It(RSk-¢arboxy-3-phenylpropyli; Drip, dinitrophenyl; Moc. 7-melhoxyooumarin-3
Corresptmdence address: C.O. Knight, Departmem o f Bk~hcmistry, Strangeways Research Laboratory, Worts Causeway, Cambridge CBI 4RN, UK.
Pub/is&ed by E./sev~r Sck,nce P u b / t ~ ' r s B. g,'.
tide-pAb are competitive inhibitors, and since the most potent have peptide structures analogous to those of g o o d s u b s t r a t e s [4], t h e a m i n o a c i d s i d e c h a i n s p r e sumably occupy the same specificity subsites on the e n z y m e . B y c o n v e n t i o n [7], t h e s e s u b s i t e s a r e l a b e l l e d S t o t h e N - t e r m i n a l s i d e o f t h e s c i s s i l e b o n d a n d S" t o t h e C-terminal side, numbering being away from the bond cleaved, the scissile bond. The amino acid residues w h i c h o c c u p y t h e s e su b si t e s a r e l a b e l l e d P a n d P', res p e c t i v e l y . A l t h o u g h it is n o t y e t p o s s i b l e t o d e f i n e t h e c h a r a c t e r i s t i c s o f t h e s u b s i t e s p r e c i s e l y [I], g o o d s u b strates and inhibitors of thimet oligopeptidase comm o n l y h a v e h y d r o p h o b i c r e s i d u e s a t P i a n d P Y [2,4]. In a d d i t i o n to t h e i r use in b i o l o g i c a l e x p e r i m e n t s , inhibitors more potent than those currently available are needed for active site titration of the enzyme, so that kinetic studies can be placed on a quantitative basis. Also needed are inhibitors more resistant to degradat i o n , w h i c h c u r r e n t l y c o m p l i c a t e s t h e i r use in s t u d i e s o f t h e r o l e o f t h i m e t o l i g o p e p t i d a s e in v i v o [3]. W e n o w report our initial studies to investigate the relationships between inhibitor structure and function.
2. E X P E R I M E N T A L 2.]. Maaet4ais Boc-amino acid N-hydroxysuc~nimide esters, p-aminobenzoic acid and L-phenylalanine derivatives ~ from Sigma. Uoc-amino acid p.aminobenzoates were made as de~ribed [8]. Ethyl; 2-keto-4-phenylbutanoat© from Scbeizerhall (South Plainfield, N J 07080) was saponifled with N a O H to yield the sodium salt. Cpl~Ala-Ala-Tyr-pAb was a gift from Dr. M. Orlowski. Mount Sinai School o f Medicine, New York, USA. Moc-Pro-I..eu-Oly-Pro-D-Lys(Dnp) was made by solid phase pepcide ~ t n t h e ~ 19L Rat t,n~t~ mim~ ~ w.as purified by a method similar to that used for the chicken enzyn~ 15|. and ga~e a single band o f "/6 kDa in S D S ? l ~ a m l i ~ ~ doctralphorcsh.
183
.~4.
Votume
number
3
UEBS
2.2. 5;Ynth,'s:.~ ¢,I Cpp-.4la-Pro-Ph*'-p.4~ T h i s w a s m a d e s t e p - w i r e by s t a n d a r d ~ l t s t i o n - p h a ~ mctht~cls [4. I0]. R e a c t i o n s ~ c r t " ft~llowt~J b y H P [ . C o n a C1~4 L'olumn, c l u l c d w i t h tt g r a d i e n t o f 5 100~4;, a L ~ t o m t r i l c c o n t a i n i n g 0 I¢IF t r I f l u o r o w u c t l c a c i d ( I m l , m i n ) o v e r 25 rain. T h t , c o l u l n n ¢ I t i a n t ~Jas m o n i t o r e d at ".10 rim. I ~ - - P h e - p , A b 1384 rag. I r e t o o l ) g a s treatL,~J w i t h trifluolat~llccYic ;lt:iti/ w a t e r I 1 9 I. ~'~'v. :q) m l ) f o r 20 n~it~ at 21 ~'C t o r e m o v e t h e B¢~.- g r o u p T h e a c i d w a s r e m o v e d a t 44)°(" in v a c u o a n d t h e t ~ s i d u e w a s d i s s o l v e d in d i m c t h y l f o m l a m i d c ( S a i l , T o t h i s , ~ t u i i o n w a s addc~l B ~ ' - P r o O N S u ( 3 3 0 rag, I I m m o l L l - h y d r o x y l ~ n ~ o t r i a ~ c ~ l e ( I 35 g, I n t m ~ i ) a n d d i - i s o p r o p y l e t h y l a m i n e (O.7 m l . 4 m m o l L A f t e r s t i r r i n g o v e r n i g h t , a n a l y s i s b y H P L C sho~k~t."d t h e r e a c t i o n t o t h e c o m p l e t e . T h e d i m e t h y l f o r m a m / d ~ w a s e v a p o r a t e d a t 4 0 ° ( " in v a c u o a n d c h l o r o f o r m ( ~ ) m l ) was adld~. A f t e r eglra~liom w i t h ~ t w / v } c i t r i c a c i d (2 ~< ~ m l L w a t e r 120 m l ) a n d s a t u r a l e d N a ( ' l ( 2 0 r o l l t h e c h l o r o f o r m s o l u t i o n w a s dried (M8SO4) and evaporated to leave crude Boc-Pro-Phe-pAh_ This w a s Ptx-rystalli~L'd f r o m e t h i * n o l / w a t e r ( y i e l d i n g 0 . 4 6 g. 95~[-I a n d t r e a t e d w i t h tri~luoroa,cctic a c i d / w a t e r a s d e s c r i b e d a b o v e , T h e r e s i d u e a f t e r e v a p o r a t i o n w a s d i s s o l v e d in d i m e t h y l f o r m a m k l e a n d r e a c t e d with Eloc.Ala.~)NS¢ (2~ mr. I mmoh. I-hydroxybenzotriazote and di-isop~xspylethylamine. As before, the solvent was evaporated after r e a c * i o n o v e r n i g h t a n d t h e r e s i d u e d i s s o l v e d in c h l o r o f o r m , e x t r a c t e d a n d d r i e d . T h e c r u d e E k ~ - A l a - P r o - P h e - p A b w a s 97q~, p u r e by H P L ( " a n a l y s i s a n d w a s t r e a t e d w i t h trifluoroa~i~tic a c i d w i t h o u t f u r t h e r p u r i f i c a t i o n . T h e r e s i d u e a f t e r e v a p o r a t i o n w a s d i s s o l v e d in m e t h a n o l / w a t e r ( 1:1 v/v, ~ m l ) a n d b r o u g h t t o p H 7 w i t h 5 M N a O H . S o d i u m 3 - p h c n y l - 2 - k e t o b u t y r a t e ( I g, 5 r e t o o l ) a n d s o d i u m c y a n o b o r o h y d r i d e (0.32 g. 5 r e t o o l ) w e r e a d d e d a n d She m i x t u r e s t i r r ~ at 2 I~'C. H P L C a n a l y s i s s h o w e d t h a t r e a c t i o n w a s ~.~.~mplete a f t e r 24 h. T h e s o l v e a t s were e v a p o r a t e d , w a t e r (50 ml) w a s a d d e d a n d the ,solution acidified w i t h 6 M H C I ( 1.5 m l ) t o p r e c i p i t a t e C p p - A l a - P r o - P h e - p A b . H C I . T h e c r u d e p r o d t u : t wa,~ w a s h e d w i t h e t h y l a c e t a t e , a n d r e e r y s t a l l i s e d f r o m e t h a n o l a n d w a t e r , y i e l d i n g 0 . 3 3 g ¢49~-), m , p . I S ? 188°C. A n a l y s i s : c a l c u l a t e d f o r C , , H ~ N ~ O ~ . H ( ? I ( M , 6 7 9 . 2 ) C . 6 2 . 7 2 ; H , 6.O4; N . 8 . 6 0 ; t~)und ( ' , 62.98: H, 6.1 I ; N . 8.60. T h e other inhibitors w~re mad~ by s i m i l a r m e t h o d s . The identities o f t h e f inal l , ~ p t i d e p r o d u c t s w e r e c o n f i r m e d b y e l e m e n t a l a n a t y s i s , N M R o r fast a t o m b o m b a r d m e n t m a s s s p e c t r o m e t r y . A l l t h e p h e n y l a lanin~-¢ontaining inhibitors displayed characteristic double peaks on H P L C . a t t r i b u t a b h : to t h e D a e t i a l l y r e s o l v t ' d R a n d S d i a s t e r e o m e r s
141. 2.3, D¢ls,rttaOl¢it~on o l K, vcllut',s Thimet
oligoraepttdase activity was assayed
at
300( `` w i t h
the
quenched fluorc,s~cat f r c p t i d c Mc~.'-Pro-L~u ~ ( ~ l y - P r o - D - L y s ( O n p 1 1 9 1 , in ~,hich the s y m b o l + r e p r e s e n t s t h e s¢issile b o n d . T h e f l u o r l m e t e r Table ! Effi.~ct o f i n h i b i t o r s t r u c t u r t : a t P I ' l"
2'
3'
i
+
4"
Cpp Cpp CPlP CI~ Cpp Cpp Cpp CI~
Phc Ala Phe pAb - Leu Pro Phe pAb Ala Ala PIR - pAb -GIyAI2 -- ~ pAb - Ala ~ Pro - Phe - pAb - G l y -- P r o P h e -- p A b Sar • Ala . PI~ pAb Sat Pro Phe pAb
K, ( n M ) 81 279 30.6 774 7.3 289 15700 3410
-~ 10" _+ 10 _+ 1.0 _+ 16 + 0.3 ± g ± 200 ± 130
The Cpl~peptide-pAb inhibitors nre substrate nnalogues and are t h o u g h t t o b i n d w i t h t h e C l ~ g r o u p in t h e S l s u b t i l e o f t h e e n z y m e . T h e r e s i d u e s h a v e b e e n n u m t m r e d a c c o r d i n g l y , a n d t h e s y m b o l *+' ind i c a t e s t h e b o n d c o r r e s p o n d i n g t o t h e s c i s s i l e b o n d in a s u b s t r a t e . T h e error va|uea are standard errors es:imated by the E~fitter pro-
gram. *Value from
184
13l
LETTERS
December
1991
w a s c o n t r o l l e d , a n d d a t a colh_.cted a n d a n a l y s ~ d , h y u s e o f t h e V I . U S Y S s o f t w a r e D a c k a g ~ [I I ]. Whe e n , w m ¢ w a s p r o - a c t i v a t e d w i t h S m M d i t h i o t h r e i t o l in a s s a y b u f f e r ( 5 0 m M T r i s - H C I , D H 7.g, c o n t a i n Ing tlOSqF Itr U i S ) f o r 5 r a i n at 2 1 " ( a n d t h e n d i l u t e d IO*fold a n d k e p t a t 0 " ( ' . A ~ a y ~ , w e r e m a d e w i t h OR n M e m, y m e . b a s e d o n p r o t e i n c o n c e n t r a t i o n . F o r t h e d e t e r m i n a t i o n o f K,. t h e r a t e o f s u b s t r a t ¢ h y d r o l y ~ i ~ in t h e absem.'¢ o f i n h i b i t o r w a s first r e c o r d e d , a n d t h e n t h e i n h i b i t o r w a s a d d ~ in a n e g l i g i b l e v o l u m e a n d t h e n e w s t e a d y s t a l e w a s m o n i t o r e d R a p i d e q u i l i b r a t i o n w a s o b s e r v e d in a l l c a s e s . A p p a r e n t i n h i b i t i o n c o n s t a n t s A'.=r~., w e r e c a l c u l a t e d b y f i t t i n g I h e f r a c t i o n a l a c t i v i t y d a r n t o II1¢ M l o r r i s o r t L~quation [I 2| b y n o n l i n ~ r r e l g l ~ o s i o n , u s i n g t h e p ¢ o g t a m E n ~ l i t t e r ! I-Ascvier-Biosofl, Cambridge, U K ) . V a l u e s wct'¢ c o r r e c t e d l o r t h e effoet o f s u b s t r a t e b y u s i n g e q u a t i o n ( t ) .
K, = K.~.~/(I ÷ [SFK.)
(l)
A v a l u e o f 16 .u M w a s d c t c r m i n * ~ ! f o r Km. T h e di ff er en t:c~ in K, v a | t t ~ I'~twL~n h t h i b i t o r s w e r e c o n v e r t e d i n t o d i f f e r e n c e s in t h e f r ~ e n e r g y o f b i n d i n g t ~ ( ~ . d h y e q u a t i o n (2),
66G~,.a --. - 2 . 3 0 3 R T - Iogl K,,tK',:)
3. R E S U L T S
AND
(2)
DISCUSSION
3. I. A l a n i n e is f i t v o u r e d in P l "
Good peptide substrates of thimct oligopeptidase c o m m o n l y h a v e h y d r o p h o b i c r e s i d u e s i n PI a n d P3". a n d a r g i n i n e is o f t e n f o u n d in Pl" [2], s u g g e s t i n g t h a t t h e SI" site o f t h e e n z y m e is a c c e s s i b l e t o l a r g e side c h a i n s . T h i s c o n c l u s i o n is s u p p o r t e d b y s t u d i e s w i t h i n h i b i t o r s , s i n c e t h e K, o f C p p - P h ¢ - A l a - P h e - p A b is o n l y 3 - f o l d t h a t o f Cpp-Ala-Phe-pAb [4] ( T a b l e 1). L e u c i n e . h o w e v e r , is f o u n d t o be a n u n f a v o u r a b l e r e s i d u e in P i ' . It h a s b e e n o b s e r v e d t h a t r e p l a c e m e n t o f A l a b y G l y in Pl" t o g i v e C p p - G i y - A l a - P h e - p A b r e s u l t s in a far less p o t e n t i a l i n h i b i t o r [4.5], a n d w e find s i m i l a r l y w e a k b i n d i n g w h e n P2" is P r o ( T a b l e !). in p r i n c i p l e , t h e w e a k e r b i n d i n g o f t h e C p p - G l y - i n h i b i t o r s c o u l d be d u e t o t h e l a c k o f a n i n t e r a c t i o n o f t h e a l a n i n e s i d e c h a i n in t h e SI" p o c k e t o f t h e e n z y m e , o r t o g r e a t e r r o t a t i o n a l freedom allowing the compounds to adopt unfav o u r a b l e c o n f o r m a t i o n s . H o w e v e r . t h e K, d i f f e r e n o e s between Cpp-Ala-Ala-Phe-pAb and Cpp-(31y-Aia-PhepAb. and Cpp-Al,~-Pro-Phe-pAb and Cpp-Gly-l~oP h e - p A b c o r r e s p o n d t o d i f f e r e n c e s in b i n d i n g free e n e r g y A A G b i n d o f ! . 9 a n d 2.2 k c a l / M , r e s p e c t i v e l y . B y c o n t r a s t , t h e free e n e r g y c h a n g e o n t r a n s f e r o f a m e t h y l g r o u p f r o m o c t a n - l - o l t o w a t e r is o n l y 0 . 7 k c a l / M [13], w h i c h sug~oests t h a t t h e h y d r o p h o b i ¢ p o c k e t m o d e l is i n a d e q u a t e t o e x p l a i n t h e d i f f e r e n c e s in g i a n d that t h e i n c r e a s e d flexibility o f t h e g l y ¢ i n e d e r i v a t i v e s is res p o n s i b l e f o r t h e i r p o o r e r b i n d i n g . S u c h flexibility h a s been shown to be responsible for the weaker binding of analogues of Cpp-Ala-Pro (enalaprilat) to peptidyl-dip e p t i d a s e A ( a n g i o t e n s i n c o n v e r t i n g e n z y m e ) [14]. W i t h these compounds, binding could be enhanced as much a s 2 5 0 0 - f o l d b y c o n f o r m a t i o n a l r e s t r i c t i o n , s o it s e e m s reasonable to suggest that restricted rotation around the a l a n i n e N - C a b o n d p l a y s a r o l e in o r i e n t i n g t h e C p p c a r b o x y l g r o u p t o w a r d s t h e Z n a t o m in t h e a c t i v e site
Volume 294, n u m b e r 3
FEBS L E T T E R S
of thimet oligopeptidase, although other interactions p r o b a b l y c o n t r i b u t e t o efficient b i n d i n g . F o r e x a m p l e , in t h e c o m p l e x h e t w c e n t h e r m o l y s i n a n d C p p - L e u - T r p (K, = 50 n M [15]). b o t h o x y g e n s o f the C p p c a r b o x y l g r o u p a r c c o o r d i n a t e d t o t h e zinc, b u t in a d d i t i o n e~ch f o r m s t w o h y d r o g e n b o n d s t o o t h e r r e s i d u e s lining t h e a c t i v e site [6]. S i m i l a r l y . t h e p r o t o n a t e d s e c o n d a r y a m i n o g r o u p forms three h y d r o g e n b o n d s to s u r r o u n d i n g r e s i d u e s [6]. T h e i m p o r t a n c e o f this n i t r o g e n in t h e interaction of Cpp-inhibitors with thimet oligop e p t i d a s e is s h o w n c l e a r l y b y t h e s u b s t i t u t i o n o f sarc o s i n e ( S a r ) f o r G l y ( T a b l e I) w h i c h r e s u l t s in v e r y m a r k e d losses o f a c t i v i t y . 3,2. Binding is f a v o u r e d by proline in P2" Several g o o d substrates by thimet oligopeptidase h a v e P r o in P2". i n c l u d i n g b r a d y k i n i n a n d n e u r o t e n s i n [2]. B z - G l y - P h e + A i a - P r o - P h e - p A b [16]. P z - P r o - L e u + G i y - p r o - D - A r g [! 7] a n d t h e q u e n c h e d f l u o r e s c e n t p e p tide, D n p - P r o - L e u + G l y - P r o - T r p - D - L y s [18]. T h e s e d a t a s u g g e s t t h a t t h e b i n d i n g o f p e p t i d e s t o t h i m e t olig o p e p t i d a s ¢ m a y be f a v o u r e d n o t o n l y b y h y d r o p h o b i c r e s i d u e s in P l a n d P3", b u t a l s o b y P r o in P2". T o invest i g a t e t h e effect o f p r o in this p o s i t i o n , w e h a v e c o m p a r e d s o m e m e m b e r s o f t h e series C p p - A l a - A i a - X - p A b a n d C p p - A l a - p r o - X - p A b , w h e r e X is a n a r o m a t i c o r l a r g e h y d r o p h o b i c a m i n o a c i d ( T a b l e II). S u b s t i t u t i o n o f P r o for Ala leads to slightly tighter binding a n d C p p A l a - P r o - P h e - p A b is t h e m o s t p o t e n t i n h i b i t o r o f t h i m e t o l i g o p e p t i d a s e s o f a r d e s c r i b e d w i t h Ki = 7 n M . O r l o w s k i et al. [4] h a v e o b s e r v e d t h a t t h e b i n d i n g o f C p p - A l a - A l a - T y r - p A b t o t h i m e t o l i g o p e p t i d a s e is twice a s t i g h t a s t h a t o f t h e P h e a n a l o g u e , b u t w e d o n o t see this effect w h e n t h e P2" r e s i d u e is P r o a n d b o t h C p p Ala-Pro-Tyr-pAb and Cpp-Ala-Ala-Tyr-pAb are comp a r a b l e in i n h i b i t o r y a c t i v i t y . By c o n t r a s t , t h e i n h i b i t o r w i t h L e u in P Y s h o w s a m a r k e d d e c r e a s e in i n h i b i t o r y p o t e n c y ( T ~ b l e !1). 3.3. Potent inhibition requires a negalively charged C-
terminus All t h e i n h i b i t o r s d i s c u s s e d s o f a r h a v e b e e n p - a m i n o b e n z o a t e s . S t e w a r t [19] h a s p o i n t e d o u t t h a t t h e p A b g r o u p is s t r u c t u r a l l y r e l a t e d to a d i p e p t i d e a n d this g r o u p s e e m s likely t o m a k e a m a j o r c o n t r i b u t i o n t o the b i n d i n g o f t h e i n h i b i t o r s . ~ ' e e x p l o r e d t h e influence o f Table Effect of inhibitor I
÷
CppCt~ Cl~ Cpp Cpp Cpp
-
l" Ala Ala Ala Ala Aim. Ala
3"
-
2"
---
Ala - Ph¢ Ala - Tyr Pro - Ph¢ Pro - Tyr Pro - Trip Pro - Lee
-
!!
structure
at P2' and
PY
4"
K, (riM)
pAb pAb I~Ab pAb pAb pAb
30.6 14.4 7.3 I 1.8 17.6 76.8
_+ 1 . 0 + 0.5 4- 0 . 3 + 0.4 _-4 1 . 0 + 3.0
December 1991 Table I'~I1/~X-I aft i n h i b i t o r
I
4 I'
Cpp Cpp Cpp Cpp Cpp
Ala Ala Ala Ala Ala
2~
3"
4"
Ala Ala Ala Ala Ala
Phe Phe Phe Phe Phe -
pAb OH NHPh NHPhNO~ NH:
!11 ~lruclu'~
:It P 4 " K, ( n M ) 30.6 155 t860 10~ 5790
~ ± + ± ±
1.0 5 50 40 220
i n t e r a c t i o n s at t h e C - t e r m i n u s with a series o f r e a g e n t s in w h i c h the C p p - A l a - A l a - P h e p e p t i d e p a r t w a s r e t a i n e d ( T a b l e ! I I ) . T h e m o s t p o t e n t i n h i b i t o r r e m a i n s the pa m i n o b e n z o a t e , b u t s u r p r i s i n g l y , r e m o v a l of" t h e p A b g r o u p t o yield t h e Free acid results o n l y in a 5 - f o l d i n c r e a s e in K,, c o r r e s p o n d i n g t o the loss o f 1.0 k c a l / M o f b i n d i n g free e n e r g y . B y c o n t r a s t , r e p l a c e m e n t o f the c a r b o x y l g r o u p b y a h y d r o g e n a t o m to give the unc h a r g e d a n i l i d e results in b i n d i n g w e a k e r b y 2.5 k c a i / M . T h e p - n i t r o a n i l i d e i n h i b i t o r , with t h e d i p o l a r n i t r o g r o u p in p l a c e o f t h e c a r b o x y l a t e , is o n l y m a r g i n a l l y m o r e effective t h a n the anilide. T h e i m p o r t a n t c o n t r i b u l i o n o f t h e C - t e r m i n a l n e g a t i v e c h a r g e t o t h e b i n d i n g is c o n f i r m e d w i t h the a m i d e C p p - A l a - A l a - P h e - N H 2 , w h i c h b i n d s m o r e w e a k l y t h a n its p a r e n t acid b y 2.2 k c a l / M . T h e s e free e n e r g i e s a r e in t h e r a n g e o f t h o s e o b s e r v e d f o r salt b r i d g e s in p r o t e i n s [I 3] a n d suggest t h a t , in a d d i t i o n t o b i n d i n g t o the a c t i v e site Z n , the p - a m i n o b e n z o a t e i n h i b i t o r s h a v e a n i m p o r t a n t elect r o s t a t i c i n t e r a c t i o n with |ysine o r a r g i n i n e side c h a i n s in $ 4 ° o r $5" subsites. 4. C O N C L U S I O N A l t h o u g h t h e s t u d ie s d e s c r i b e d h e r e h a v e r e s u l t e d m
an inhibitor of thimet oligopeptidase that binds more tightly than those described before. C p p - A l a - P r o - P h e p A b b i n d s t o o w e a k l y t o be used as a n a c t i v e site t i t r a n t , a n d a f u r t h e r d e c r e a s e in Ki o f a b o u t 2 0 - f o l d will be n e c e s s a r y t o a c h i e v e this aim. O n e a p p r o a c h t o t i g h t e r i n h i b i t o r s m a y b e t o e x p l o r e a l t e r n a t i v e s t o the C p p g r o u p , p e r h a p s b y i n t r o d u c i n g s u b s t i t u e n t s i n t o the p h e n y l ring, a s t h e 7 0 - f o l d d i f f e r e n c e in i n h i b i t o r y potency between carboxyphenyipropyl and carboxy p h e n y l e t h y l d e r i v a t i v e s o f A l a - A l a - P h c - p A b {4] ill u s t r a t e s t h e d r a m a t i c effects o f small c h a n g e s in s t r u c t u r e in this r e g i o n o f t h e m o l e c u l e . C p p - A l a - P r o - P h e - p A b m a y prove to be a g o o d inhibi t o r f o r in v i v o s t u d i e s o f t h i m e t o l i g o p e p t i d a s e . C p p A l a - A l a - P h e o p A b is c l e a v e d b y n e u t r a l e n d o p e p t i d a s e ( E C 3.4.24.1 I) a t t h e A l a - P h e b o n d [3], w h e r e a s the P r o - P h e b o n d m a y be m o r e r e s i s t a n t t o h y d r o l y s i s . T h i s b o n d m a y be c l e a v e d , h o w e v e r , b y p r o l y l o l i g o p e p t i d a s e ( E C 3.4.21.26) a n d c o n t r o l e x p e r i m e n t s w i t h specific i n h i b i t o r s [20] o f this e n z y m e will be n e c e s s a r y , A n a d v a n t a g e o f the C p p - p e p t i d e - p A b i n h i b i t o r s is 185
Volume
294. number
3
F|~BS L i'.-FFFRS
t h e i r r a p i d b i n d i n g to t h i m c t o l i g o r ~ p t i d a ~ e , s o that e q u i l i b r a t i o n is i n s l a n t a n e o u s o n the 10 rain time scale o f o u r as.~ays. By c o n t r a s t . N-{ p h e n y l e t h y i p h o s p h o n y l ) ( 3 1 y - P r o - A h x , d e s i g n e d as a n i n h i b i t o r o f CIo.vtridium hLgtolyth'u~ c o I l a g e n a ~ [21], r e q u i r e s several m i n u t e s t o r e a c h e q u i l i b r i u m with t h i m e t o|igopeptida.,~e, alt h o u g h the final K, (7 n M L is s i m i l a r {A.J. B a r r e t t a n d M . A . B r o w n . u n p u b l i s h e d o b s e r v a t i o n s ) . Likewise, phosphoramidon ( N-[[(6-deoxy-~-L-ma nnopyranosyl)o x y ] h y d r o x y p h o s p h i n y l ] - L e u - T r p ) is a s l o w b i n d i n g in-
hibitor o f t h e r m o l y s i n [221. w h e r e a s C p p - L e u - T r p b i n d s n o r m a l l y { 15]. ,4¢~nowlcd, g,¢m~,nts: W e a t e ( ' p p - A | a - A l a - T y r - p A b , to e x p e r t technical a s s i s t a n c e i+nd t n t e r p t ¢ t l ~ g the N M R
f r a t e f u ] t o Dr. M. O r t o w s k i l~r p r o v i d i n g l...sut.~e H a n d l o r d a n d H o t l y S p c a t m g for ant: t o l)r, tLCC. ticywo¢~l f o r o b t a i n i n g a i l d nlass sp~.x:tra.
REFERENCES [I] Barrett, A,J. (11)90) Biol. C h e m H o p p e - S e y l c r 371 ( S u p p l . ) , 31 ! 320. 12] O r l o w s k i . M., Reznik, S.. A y a l a , J. a n d Picrottl. A . R (1989) B i o c h e m . J 261. "~51 95R. 13] L a s d u n . A., Res.nik, S.. M o l i a e a u ~ . ( ' . J . a n d Orlow.~k i, M. (19891 J. P h a n n a c o l . [~ap. T h e r , 251, 439 447. 14| O r l o w s k l . M., M i c h a u d . C. a n d M o l i n e a u x . ( ' , J . (1988) E~i~hcniistr~' 27. 597 trY)2.
186
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15] l l a f r e t l . A . I and Br,~uwn. M A . ( I q g 0 ) Bi oche m . J. 271, 701 706. |61 M o n ; ' i n g o . A |:, ~nd M a t t h e w , , . B.W. (1984) B i o c h e m i s t r y 27, ~,724 572,~ 17l ,%¢h¢'~.'t¢r. I a n d BerBer. A. (1967} Biz~hem. B i o p h y g . Res;. C o i t t nt~m. 27. 157 162. |8] O i i l ~ w s k i . M , M ~ c h a u d . ( " a n d C h u , T_CL ( 1 9 8 3 ) l~ur. J, Bttx:hem. 13~. 8 l 88. [0l l'i~ljar, ILl., K n i g h t , C . G . a n d Bar r et t , A.J, t 1990) A n a l . Biocltcm. 18¢,, 112 ;15. [ ! 0 ] Bodans~'k y, M . a n d l~,¢lans~ky, A. (1984) T h e Practice o f Pcplicl¢ Synthesis. Sprinl~c,r-Verlag. B e r l i n II I1 B a w l i n g s . N , D . a n d Barlrctt. A J . ( I ~ J N ) ) C o m p u t e r A p p l . Bio~ci, 6, l i b 119. 112] M o r r i s o n , J,F. ( 1 % 9 ) B i o c h i m . B i o p h y s . A c t a 185. 209=286. | 13] F c r s h l . A. (1985) E n z y m e S t r u c t u r e a n d M e c h a n i s m , 2nd ¢d.. pp. 29~ 310. [14] T h o r s e t t . E . D . t 1 0 8 6 ) A c t u a l . C h i m . | h e r . 13, 257 268_ i15] N l a y c o c k . A . L . . [ k S o u , ~ a . D , M . . P a y n e . L . G . . ten B r o e k c . J.. W u . M . ~ . a n d Patch¢tt, A . A . (1981) B i o c h f m . Biophy~. R¢~, C o m m u n . 102, 963 969. [10] C h u . T.(~. a n d O r l o w s k i , N~. (!~85} [.~ndocrino|ogy 116, 1 4 1 g 1425. [17] B a r r e t t . A,J, ~nd Ti~ljar, U. (19B9) BicMchem, J 261, 1047 1050. [ l g l K n i g h t . C',(L ( l ~ l l B t o c h e m J 274. 45 48. [191 S t e w a r t . F . H . ( ' . (19K3} Aa~tr. J, ( ' h u m . 36, 1629 If~3X [201 T~,ttru, I~., V o s h i m o t o , T., K o r i y a m a , N . a n d F u r u k a w a , S. (19RSl J. B i o c h c m . 104. 580 586. I211 Dive, V,. Y i o t a k i s , A.. ]~lic'olaou. A. a n d T o m a , I:. ( 1 ~ ) 0 ) E u r J. Bier:hem. 1 9 1 , 6 8 5 693. [22] K a m , C_-M.. N i g h | n o , N, a n d P o w e r s , J . C . t 19"/9) B i o c h e m i s t r y 18, 3033 3038.