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P e r s p e c t i v eJourna l of BlomolecularNM R, 3 (1993) 261 269ESCOMJ-Bio NM R 122

N M R structure-based dru g designS t e p h e n W . F e s i k

Pharmaceuttcal Discovery D ivision D-47G, AP 9 Abbo tt Laboratortes, Abb ott Park, I L 60064, U .S A

Received 3 M arch 1993Accepted 17 Ma rch 1993Keywords." Drug design; Molecularmodeling:3D databa se searching: Heteronuclearmulti-dimensionalNM R

S U M M A R YN M R is a useful tool for rapidly determining the confo rma tions of receptor-bound ligands an d identifyingthose protions of the ligand in conta ct with the receptor. In ad dition, the complete 3D structures of receptorsand ligand/receptor complexes can be ob tained using recently developed he teronuclea r multi-dimensionalN M R techniques. This NM R-de rived structural info rm ation is poten tially useful for aiding in the design ofimproved pharm aceutical agents . Approaches for utilizing the NM R-de rived structural inform ation alongwith the com puta tional tools tha t facilitate this process are discussed.

I N T R O D U C T I O NT h e d i s c o v e r y o f n e w p h a r m a c e u t i c a l a g e n t s is a d i f f ic u l t t a s k . I n a d d i t i o n t o b i n d i n g t i g h t l y

a n d s p e c if i ca l ly t o i t s t a r g e t s i te , t h e n e w c h e m i c a l e n t i t y m u s t b e c h e m i c a l l y a n d m e t a b o l i c a l l ys t a b le , b e r e l a t i v e l y n o n t o x i c , b e a b l e t o g e t t o t h e s i t e o f a c t i o n , a n d b e s y n t h e t i c a l l y a n de c o n o m i c a l l y f e a si b l e t o p r e p a r e . T h e d r u g d i s c o v e r y p ro c e s s b e g i n s b y i d e n t i f y i n g a l e a d c o m -p o u n d t h a t p o s s e s se s t h e d e s i r e d p h a r m a c o l o g i c a l a c t i v i ty , f o l l o w e d b y t h e s y n t h e s is a n d b i o l o g i -c a l t e st i n g o f s e v e ra l a n a l o g s i n s e a r c h o f th e i d e a l d r u g c a n d i d a t e . I n p r a c t i c e , n e w l e a d s a r eg e n e r al l y e i th e r a n a t u r a l h o r m o n e o r e n z y m e s u b s t r at e , o r t h e y a re f o u n d b y r a n d o m l y s c re e n in gn a t u r a l p r o d u c t s o r c h e m i c a l li b r a ri e s . T h e f i n a l d r u g m o l e c u l e i s d i s c o v e r e d b y f i n e - t u n i n g t h e s ei n i ti a l l e a d s b y m a k i n g u s e o f s t r u c t u r e - a c t i v i t y r e l a ti o n s h i p s . T h i s p r o c e s s is ti m e c o n s u m i n g a n do f t e n r e q u i r e s t h e s y n t h e s i s a n d t e s t i n g o f s e v e r a l h u n d r e d c o m p o u n d s .

A no ther app roach fo r d i s cover ing new pharmaceu t ica l agen t s i s s t ruc tu re -bas ed d rug des ign , inw h i c h t h e 3 D s t r u c t u r e o f a r e c e p t o r o r l i g a n d - r e c e p t o r c o m p l e x i s u s e d t o g e n e r a t e n e w l ea d s a n dg u i d e le a d o p t i m i z a t i o n . U s i n g s t r u c tu r e s o b t a i n e d f r o m X - r a y c r y s t a l l o g r a p h y , t h i s a p p r o a c hh a d a i d e d i n t h e d i s c o v e r y o f n o v e l i n h i b i t o r s f o r H I V p r o t e a s e ( B u r e s e t a l. , 1 9 90 ; D e s J a r l a is e ta l . , 1990 ; Er icks o n e t a l . , 1990) , thym idy la te s yn the ta s e (A pp e l t e t a l ., 1991 ; V a rne y e t a l ., 1992),c a r b o n i c a n h y d r a s e ( B a l d w i n e t a l . , 1 9 8 9 ) , a n d p u r i n e n u c l e o s i d e p h o s p h o r y l a s e ( E a l i c k e t a l . ,1 99 1) . I n p r in c i p l e , N M R s p e c t r o s c o p y c o u l d a l s o be u s e d t o o b t a i n s t r u c t u r a l i n f o r m a t i o n f o r0925-2738/$10.00 9 1993 ESCOM Science Publishers B.V.

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262des ign ing new d rug mo lecu l e s (Fes ik , 1989 , 1991 ). In t h i s pe rspec t i ve , t he po t en t i a l o f u s ing N M Rst ruc tu res fo r d rug des ign is d iscussed w i th emphas i s o n t he t ype o f re l evan t s t ruc tu ra l i n fo rma-t io n t h a t c a n b e o b t a i n e d b y m o d e r n h i g h - r e s o lu t io n N M R t e c h ni q u e s a n d t h e c o m p u t a t i o n a ltoo l s t ha t a re ava i l ab l e fo r u t i l i z ing t he NMR-der ived s tuc tu ra l i n fo rma t ion .N M R S T R U C T U R E S O F B O U N D L I G A N D S

T h e N M R s p e c t r a o f d r u g m o l e c u le s b y t h em s e l v e s in s o l u t i o n a r e r e l a ti v e ly s im p l e; h o w e v e r ,i n mos t ca ses i t i s d i f f icu l t t o de r ive any u se fu l s t ruc tu ra l i n fo rm a t ion fo r d rug des ign f rom theseN M R spec t ra . In t he absence o f t he i r recep to rs , d rug mo lecu l e s a re gene ra l l y f lex ibl e and ex i st inseve ra l rap id ly i n t e rconve r t i ng con fo rma t ions , mak ing i t ex t reme ly d i f f i cu l t t o co r re l a t e con fo r -m a t ion and b io log i ca l ac t iv i t y . In con t ra s t , t he 3D s t ruc tu re o f a d rug w hen bo un d to i ts b io log i -ca l s it e o f ac t i on w ou ld be m uch m ore u se fu l i n t he des ign o f new molecu l e s . The re a re seve ra lN M R m ethod s t ha t can be u sed t o de r ive t h is s t ruc tu ra l i n fo rma t ion (Gr i f fey and Red f i e ld , 1987 ;Fes ik e t a l ., 1990b ; O t t i ng and W i i th r i ch , 1990 ; Fes ik , 1991 ). Fo r wea k ly bou nd l i gands , t rans -fe r red nuc l ea r Ove rhause r e f fec ts (NO Es) can be me asu red fo r li gands t ha t exchange rap id ly f romt h e b o u n d t o t h e f r e e s t a te a n d u s e d t o d e t e r m i n e t h e c o n f o r m a t i o n o f a li g a nd w h e n b o u n d t o ama crom olecu l e (A lb rand e t a l. , 1979 ; C lo re e t a l. , 1981 ; C lo re and G rone nbo rn , 1982 ). Ho wev er ,t h e t r an s f e r r e d N O E e x p e r im e n t c a n b e a p p l i e d o n l y to t h e s t u d y o f w e a k l y b o u n d l i g an d s t h a trap id ly exchange on and o f f t he enzyme . Unfo r tuna t e ly , m any o f t he i n te re s t ing l eads b ind t i gh t lyt o t h e ir t a r g e t p r o t e i n s a n d c a n n o t b e s t u d i e d b y tr a n s f e r re d N O E e x p e ri m e n t s.

To de t e rmine t he con fo rm a t ion o f t i gh tly boun d l i gands , i so tope -ed i t i ng (Benda l l e t al ., 1981 ;Ot t i ng e t a l ., 1986 ; Bax and Wei ss , 1987 ; Gr i f fey and Red f i e ld , 1987 ; Ra nce e t a l. , 1987 ; Fes ik e ta l. , 1988 ) o r he t e ronuc l ea r 3D N M R techn iques (Fes ik and Zu ide rw eg , 1988 ; M ar io n e t a l. , 1989 ;K ay e t a l ., 1990a ) cou ld be app l i ed , a l l owing the se lec ti ve de t ec t i on o f p ro tons a t t ached to ani so top i ca l l y l abe l ed d rug m o lecu l e when b ou nd to i ts recep to r p ro t e in (F es ik e t al ., 1988 , 1990a,1991a; Pe t ros e t a l ., 1991, 1992a; W eb er e t a l. , 1991). Al terna t ively , the pro ton N M R signals ofan un l abe l ed l i gand can be se l ec ti ve ly obse rved wh en bo und to an i so top i ca l l y l abe l ed recep to r byusing i so tope-f i l te r ing techniqu es (W ider e t a l . , 1990; Iku ra a nd Bax, 1992; Pe t ros e t a l. , 1992b).

I n a d d i t i o n t o t h e c o n f o r m a t i o n o f t h e b o u n d l i g a nd , i s o t o p e - a id e d N M R e x p e r im e n t s c a n a l s obe u sed t o i den t i fy t hose po r t i ons o f the l i gand tha t a re i n c lo se p rox im i ty t o t he recep to r (Fes iket al ., 1988, 1990a, 1991a; W ide r et al ., 1990 ; Pe tro s et al ., 1991, 1992a; W eb er et al. , 1991). Onewou ld expec t t ha t s t ruc tu ra l changes i n such reg ions wou ld l ead t o l i gands w i th l ower a f f i n i t y .N M R m e t h o d s h a v e a l s o b e e n d e v e l o p e d t o s t u d y l i g a n d - r e c e p t o r c o m p l e x e s , u s in g p a r a m a g n e t -ic agents (Sc hm idt e t a l ., 1982; Ang l is ter e t a l ., 1984; de Jon g e t a l . , 1988; Pe t ros e t a l. , 1990, 1992c;Fes ik e t a l . , 1991b) . Techn iques have been deve loped tha t re ly on t he change i n l ong i tud ina lr e l a x a ti o n r a te s o f t h e s o l v e n t -e x p o s e d p r o t o n s o f t h e b o u n d l ig a n d i n t h e p r e s e n ce o f a p a r a m a g -n e ti c a g e n t a n d h a v e b e e n u s e d t o d e f in e t h e s o l v e n t- e x p o s e d s u rf a c e o f i m m u n o s u p p r e s s a n t swhen bound to t he i r t a rge t p ro t e in s (Fes ik e t a l . , 1991b ; Pe t ro s e t a l . , 1992c ) . Changes i n t hesereg ions o f the l i gand shou ld be mo re pe rm i ssab l e .N M R S T R U C T U R E S O F R E C E P T O R S A N D L I G A N D - R E C E P T O R C O M P L E X E S

Three -d im ens iona l s t ruc tu res o f d rug recep to rs (e.g. , M ichn ick e t a l ., 1991 ; M oo re e t al ., 1991 )

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263an d l i g an d - recep to r co m p lex es (e.g. , Ik u ra e t al ., 1 9 9 2 ; Th e r i au l t e t a l. , 1 9 9 2 ; M ead o w s e t al .,1 99 3) c a n a l s o b e o b t a i n e d b y N M R t o p r o v i d e a d d i ti o n a l, v a l u a b l e s tr u c t u r al i n f o r m a t i o n f o rd r u g d e s ig n . I n d e e d, w i t h t h e d e v e l o p m e n t o f h e t e r o n u c le a r 3 D a n d 4 D N M R t e c h n iq u e s ( F e s i kand Zuid erw eg, 1988; M ari on e t al ., 1989; K ay e t al . , 1990a, 1990b; Clo re e t al . , 1991; Zu iderw ege t a l ., 1 9 9 1) , l a rg e r an d m o re p rec i se N M R s t ru c tu re s can b e d e t e rmin ed t h an p rev io u s ly p o ss ib l e( C l o r e a n d G r o n e n b o r n , 1 99 1) . N e w N M R m e t h o d s a l l o w t h e 1 H , 1 3C , a n d 15N r e s o n a n c e s o frecep to r p ro t e in s t o b e rap id ly a s s ig n ed . In ad d i t i o n , man y mo re NOEs (C lo re e t a l . , 1 9 9 1 ;Mead o ws e t a l . , 1 9 9 3 ) a s we l l a s h e t e ro - an d h o mo n u c l ea r J -co u p l i n g co n s t an t s (Mo n te l i o n e e ta l. , 1 9 8 9 ; K ay an d Bax , 1 9 90 ; Em erso n an d M o n te l i o n e , 1 9 9 2 ; Szy p e rsk i e t a l ., 1 9 92 ; X u e t a l. ,1 99 2) c a n b e o b t a i n e d b y u s i ng r e c e n t ly d e v e l o p e d h e te r o n u c l e a r m u l t i d im e n s i o n a l N M R t e ch -n iq u es . Th ese s t u d i es req u i re u n i fo rm ly 1 5 N- an d 1 3 C-l ab e led recep to rs , wh ich ca n b e read i l yo b t a in ed f ro m e i t h e r b ac t e r i a l (Mu ch mo re e t a l . , 1 9 8 9 ) o r , mo re recen t l y , mammal i an ce l l s(Ha n sen e t a l. , 1 9 92 ) t h a t o v e rex p ress t h e p ro t e in o f i n te re s t.U T I L I Z IN G T H E N M R - D E R I V E D S T R U C T U R A L I N F O R M A T I O N

H o w c a n t h e N M R - d e r i v e d s t r u c tu r a l i n f o r m a t i o n b e u s e d to d e s ig n n e w p h a r m a c e u t i c a la g e n ts ? F r o m l i g a n d - r e c e p t o r N O E s , t h e f u n c t io n a l g r o u p s o f th e l ig a n d t h a t a r e l ik e ly to b eimp o r t an t fo r b in d in g t o t h e recep to r can b e i d en t i f i ed , an d t h e re l a t i v e sp a t i a l o r i en t a t i o n o ft h e se f u n c t io n a l g r o u p s c a n b e d e t e r m i n e d f r o m t h e b o u n d c o n f o r m a t i o n o f t h e li g an d . T h i ss t ru c tu ra l i n fo rm a t io n ab o u t t h e l ig an d , wh ich can b e o b t a in ed i n a s l it tl e a s 1 -2 week s b y u s in gt h e i so t o p e - a i d e d N M R m e t h o d s d i s c u s s e d a b o v e , c o u l d b e u s e d to d e s i g n a n a lo g s w i t h d i f f er e n tmo lecu l a r f ramew o rk s t h a t p o s i t i o n t h e imp o r t an t fu n c t i o n a l g ro u p s o f t h e li g an d i n th e i r ex pe r i-men ta l l y d e t e rmin ed sp a t i a l o r i en t a t i o n . Th ese n ew an a lo g s m ay b in d mo re t i g h t ly t o t h e recep to rdue to en t ro p ic considera t ions , m ay be mor e metabo l ica l ly s tab le , o r eas ier to syn thesize . Por t ionso f t h e l ig an d w h ich a re so lv en t -ex p o sed a re i d en t if i ed o n t h e b as i s o f a l ack o f l i g an d - rec ep to rN O E s o r p a r a m a g n e t i c - i n d u c e d c h a n g e s in t he p r o t o n r e l a x a ti o n r a t e s o f th e b o u n d l i ga n d . T h i sin fo rm a t io n m ay b e u se fu l fo r id en t i fy in g t h e fu n c t i o n a l g ro u p s o f t h e l ig an d t h a t can b e mo d i f i edwi th o u t a f fec t i n g b in d in g a f f i n i t y . Th ese mo d i f i ca t i o n s co u ld imp ro v e t h e p h y s i ca l p ro p e r t i e s o fthe l igand , a id ing i t s t ra nsp ort to the b io log ica l s i te o f ac t ion .

T h e c o m p l e t e 3 D s t r u c tu r e o f th e r e c e p t o r o r l i g a n d - r e c e p t o r c o m p l e x p r o v i d e s a d d i ti o n a li n fo rm a t io n t o a id d ru g d es ign . By ma k in g u se o f N M R- d e r iv ed s t ru c tu re s , t h e s te r i c an d e l ec -t r o n ic p r o p e r t i e s o f th e r e c e p t o r p o c k e t c a n b e m a p p e d a n d u s e d t o d e s ig n m o l e c u l es t h a to p t ima l ly f it i n to th i s s it e. F ro m th e N M R s t ru c tu re o f a recep to r - l i g an d co mp lex , t h e in t e rac t i n gfu n c t i o n a l g ro u p s o f th e recep to r an d l i g an d a re i d en ti f ied , l ead in g t o a b e t t e r ap p rec i a t i o n o f th ein t e rac t i o n en e rg ie s . In ad d i t i o n , n ew fu n c t i o n a l g ro u p s o n t h e recep to r t h a t a re i n c lo se p ro x im -i t y t o t h e l i g an d can b e i d en t i f i ed f ro m th e s t ru c tu re an d u sed t o d es ig n an a lo g s wi th h ig h e raff in i ty or incre ased speci f ic i ty tha t b ind to th i s add i t ional s ite.C O M P U T A T I O N A L T O O L S F O R N M R S T R U C T U R E - B A S E D D R U G D E S I G N

In o rd e r t o u ti li ze t h e N M R- d e r iv ed s t ru c tu ra l i n fo rm a t io n e f fec t iv e ly , co m p u ta t i o n a l t o o l s a rereq u i red . In t h i s sec ti o n , so me o f th ese co m p u ta t i o n a l t o o l s a re d esc r i b ed a lo n g wi th th e NM R -d e r iv ed s t ru c tu ra l i n fo rma t io n t h a t i s u sed b y t h e d i f fe ren t me th o d s .

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t ,

! 9F ig . 1 . N M R s t r uc tu r e o f th e F KB P - a s c o m y c in c o m p le x (Me a d o ws e t al ., 1 9 93 ) a n d lo w- e n e r g y ( - 3 k c a l /mo l ) G RI Dc o n to u r s ( Go o d f o r d , i9 8 5 ) g e n e r a te d w i th a me th y l p r o b e , u s in g th e N M R s t ru c tu re o f th e F KB P a s c o my c in c o mp le xwitho ut the l igand . The ascomycin l igand is shown as a ba l l and s t ick model . Th e low-energy contours cor respondin g tohydrop hobic b ind ing s i tes a re used by the p lpecol iny l r ing (A) and methyl g roup of the pyranose r ing (B) of the ascomyc inl igand .

Molecular graphicsTh ere a re seve ra l co m p u te r p ro g rams t h a t can b e p u rch ased w h ich wil l a l l o w n ew mo lecu le s t o b erap id ly c rea t ed an d d i sp l ay ed . By m an u a l l y a l t e r in g t h e imag es o n t h e co mp u te r sc reen , mo lecu l e sc a n b e d e s i g ne d t h a t s u p e r i m p o s e o n t h e e x p e r i m e n t a ll y d e t e rm i n e d b o u n d c o n f o r m a t i o n o f t h el i g an d o r t h a t ap p ea r t o f i t i n to a recep to r s i t e an d fo rm fav o rab l e h y d ro p h o b ic , e l ec t ro s t a t i c , o rh y d r o g en -b o n d in g l i g a n d - re cep to r i n t e rac t i o n s . Th i s p ro cess is a id ed b y co lo r - co d in g t h e d if fe r -en t p a r t s o f t h e mo lecu l e s , d i sp l ay in g th e v an d e r Wa a l s ' su r faces o f t h e l i g an d an d re cep to r s it e,an d i n t e rac t i v e ly ch eck in g fo r b ad v an d e r Waa l s ' co n t ac t s wh i l e d o ck in g n ew l i g an d s .

Al th o u g h n o t a g rap h i c s p ro g ram in i t s e l f , t h e GRID a lg o r i t h m (Go o d fo rd , 1 9 8 5 ) i s a u se fu lt o o l fo r l o ca t i n g an d v i su a l i z i n g p o t en t i a l l i g an d b in d in g s i t e s . GRID ca l cu l a t e s t h e i n t e rac t i o nen e rg i e s b e tween a p ro b e (e .g . me th y l , c a rb o n y l , h y d ro x y l ) an d t h e recep to r p ro t e in . Wi th am o l e c u l a r m o d e l in g p r o g r a m o n e c a n d i s p l a y a c o n t o u r p l o t o f th e i n t e r ac t io n e n e rg ie s f o r t h ed i f fe ren t p ro b es a lo n g w i th t h e recep to r p ro t e in . N ew an a lo g s a re d es ig n ed b y p l ac in g fu n c t i o n a lg ro u p s a t t h e l o w-en e rg y s i t e s fo r t h e d i f fe ren t p ro b es an d l i n k in g t h em to g e th e r t o fo rm mo le -cu l e s t h a t f i t wi th in t h e av a i l ab l e su r face o f th e rece p to r p o ck e t , wh ich can b e v i su a l ized wi thG R I D b y c o n t o u r in g a t a hi g h -e n e r g y l ev e l ( G o o d f o r d , 1 98 5) . A n e x a m p l e o f t h e G R I D o u t p u ti s sh o w n in F ig . 1 . Th e N M R s t ru c tu re o f th e imm u n o su p p ressa n t , a sco m y c in (b a l l an d s t i ck ),co mp lex ed t o i t s t a rg e t p ro t e in (FKBP) i s sh o wn (Mead o ws e t a l . , 1 9 9 3 ) . In ad d i t i o n , t h e f i g u res h o w s t h e c o n t o u r s c o r r e s p o n d i n g t o t h e f a v o r a b l e b i n d in g s it es f o r a m e t h y l p r o b e g e n e r a t e d b yu s i n g t h e G R I D p r o g r a m ( G o o d f o r d , 1 9 8 5 ) f r o m t h e F K B P s t r u c t u r e w i t h o u t t h e l i g a n d . L o w -en e rg y co n to u rs co r re sp o n d in g t o h y d ro p h o b ic b in d in g s i t e s were o b se rv ed i n reg io n s t h a t a re

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B ( H - D )~ 2 H 2 N ~ ( O - H )l OH

( H y d r o p h o b i c I Ig r o u p ) 0( A : )

F i g . 2 . ( A ) S c h e m a t i c d e p i c t i o n o f t h e s u b s t r u c t u r e s u s e d i n t h e g e o m e t r i c s e a r c h f o r a n o n p e p t i d e i n h i b i t o r o f H I Vp r o t e a s e ( B u r e s e t a l. , 1 9 9 0) . T h e s e a r c h i n g s t r a t e g y w a s b a s e d o n t h e X - r a y c r y s t a l s t r u c t u r e o f a H I V p r o t e a s e - i n h i b i t o rc o m p l e x ( E n c k s o n e t al ., 1 9 9 0) . T h e p o i n t s w e r e d e f i n e d a s a h y d r o g e n b o n d d o n o r ( D ) , w h i c h c o u l d b e e i t h e r a n O H , N H ,o r N H 2 g r o u p , a h y d ro g e n b o n d a c c e p t o r ( A :) , c o r r e s p o n d i n g t o O H , = O . = N H , o r = S , a n d a h y d r o p h o b i c g r o u p . ( B )L e a d i n h i b i t o r s f o u n d i n t h e s e a rc h .

used by the a scom ycin l igand . A s sho wn in F ig . 1 , t he p ipeco l iny l r ing (F ig . 1A) and the methy lg roup o f the pyrano se r ing (F ig . 1B) o f ascomycin f it i n to hydro pho b ic pocke t s .3D geom etric searching

An othe r s t r a t egy fo r f ind ing new l eads is by sea rch ing da taba ses con ta in ing the 3D s t ruc tu reso f kno wn com po und s fo r molecu les tha t f it pa r t i cu la r subs t ruc tu ra l and geom et r i c c r i te r i a ( fo r ar ev iew see M ar t in , 1992). The 3D s t ruc tu res in the da ta base a r e e i the r exper imen ta l ly de te rmined(e.g. , c rys t a l s truc tu res f ro m the C am br idge s t ruc tu ra l da tabas e (Al l en e t a l., 1991)) o r compu ta -t iona l ly genera ted , us ing ru le -based p rogram s such as C O N C O R D (Pear lman , 1987). The c r i t e ri aused to cond uc t the sea rch typ ica l ly cons i s t o f po in t s co r r espo nd ing to a tom s, p ro jec t ions f romatoms, o r t he cen te r o f rings, l i nes o f de f ined l eng th be tw een these po in t s , and ang les defined byt h re e o r f o u r p o in t s . N M R - d e r i v e d s t r u ct u r a l i n f o r m a t i o n c o u l d b e u s e fu l fo r s e tt in g u p t h e 3 Dd a t a b a s e s e a rc h . K n o w l e d g e o f th e f u n c t io n a l g r o u p s t h a t c o n t a c t t h e r e c e p t o r, o b t a i n e d f r o mpro te in - l igan d NO Es, cou ld be used to def ine the po in t s i n the sea rch a long wi th the i r pa r t icu la rchemica l charac te r i s t i c s ( e .g . , hydrogen bond donor o r accep to r , e t c . ) , wh i l e the NM R-de te r -mined b ou nd c onfo rma t ion o f the l igand can def ine the r e la t ive d i s t ances and ang les be tweenthese po in t s ( a toms) . I f t he 3D s t ruc tu re o f the r ecep to r i s know n, a 3D da ta base can be sea rched

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266u s in g p o in t s co r re sp o n d in g t o fav o rab l e b in d in g s i t e s an d t h e re l a t i v e d i s t an ces an d an g l e sb e tw een th ese p o in t s . Th i s i n p u t fo r 3 D s t ru c tu re q u e r i e s can b e ea s i ly g en e ra t ed w i th t h e G R IDp r o g r a m ( G o o d f o r d , 1 9 8 5 ) , w h i c h i s a u s e f u l c o m p l e m e n t a r y t o o l t o 3 D s t r u c t u r e s e a r c h i n ga lg o r i t h ms .

A n e x a m p l e o f a s t r u c tu r e - b a s e d g e o m e t r ic s e a r ch f o r a n o n p e p t i d i c l e a d in h i b i to r o f H I Vp r o t e a s e h a s b e e n r e p o r t e d b y B u r e s e t a l. ( 19 90 ). U s i n g t h e A L A D D I N p r o g r a m ( V a n D r i e e t al.,1 9 89 ), Bu res an d co w o rk e rs sea rch ed sev e ra l 3 D d a t ab as es u s in g t h e sea rch c r i t e r ia d e f i n ed b y anX-r ay c ry s t a l s tru c tu re o f an H IV p ro t ea se - i n h ib i t o r co mp lex . Th e c r it e r ia u sed i n t h e sea rchc o n s i s te d o f a ce n t ra l h y d r o x y l g r o u p , t w o h y d r o g e n b o n d d o n o r s , a h y d r o g e n b o n d a c c e p t o r ,a n d a h y d r o p h o b i c g r o u p s e p a r a t e d b y th e d i st a n ce s s h o w n i n F ig . 2 A . F r o m t h is 3 D g e o m e t r i csea rch , a se r ie s o f d ib en z o p h e n o n e s (F ig . 2 B) were fo u n d w h ich mo d era t e ly (1 0 1 0 0 ~ tM) i n h ib i t-ed HI V p ro t ease an d se rv ed a s a s ta r t in g p o in t fo r l ead o p t im iza t i o n (Bu res e t a l. , 1 9 9 0) .

I n a d d i t i o n t o A L A D D I N ( V a n D r i e e t a l . , 1 9 8 9 ) , w h i c h w a s u s e d i n t h e e x a m p l e d e s c r i b e da b o v e , s e v e r al o t h e r c o m p u t e r p r o g r a m s f o r 3 D g e o m e t r i c s e ar c h in g a r e c o m m e r c i a l ly a v a i la b l e,i n cl u d in g M A C C S - 3 D ( M o o c k e t al., 1 99 0) , 3 D S E A R C H ( S h e r id a n e t a l. , 1 98 9) , C h e m D B S - 3 D(Mu ra l l an d D av ie s , 1 9 90 ), SYB YL /3 D B Un i ty (d i s t r i b u t ed b y Tr ip o s A sso c i a t e s , In c .) , an dCAVEAT (Bar t l e t t e t a l . , 1 9 8 9 ) .3 D s t e r i c sea r ch in g

A n o t h e r a p p r o a c h f o r i d e n ti f y in g n o v e l l e a d c o m p o u n d s i n v o l ve s se a r ch i n g 3 D d a t a b a s e s f o rc o m p o u n d s t h a t s ter ica l ly f it i n to a recep to r p o ck e t . Th i s ap p ro ach req u i re s a 3 D s t ru c tu re o f ar e c e p to r , w h i c h c a n b e d e t e r m i n e d b y N M R , a s w e ll a s a d a t a b a s e o f 3 D s t ru c t u re s . A n e x a m p l eo f a c o m p u t e r p r o g r a m f o r 3 D s t e r ic s e a rc h in g i s t h e D O C K a l g o r it h m ( K u n t z e t al ., 1 98 2) . W i t hD O C K , t h e s e a r c h f o r li g a nd s w i t h c o m p l e m e n t a r y s u r fa c e s to a r e c e p t o r i s a c c o m p l i s h ed i n t h re es t ag es . F i r s t , t h e rec ep to r p o ck e t is fi ll ed b y a se t o f o v e r l ap p in g sp h e re s t h a t i n t e r sec t wi th t h erecep to r su r face , p ro v id in g a n imag e o f t h e sp ace access ib l e t o a li g an d . Nex t , co m p o u n d s f r o mth e d a t ab ase a re rep re sen t ed b y sp h e re s t h a t d e f i n e t h e sp ace o ccu p i ed b y t h e mo lecu l e s . In t h en ex t s t ep , t h e two se t s o f sp h e re s co r re sp o n d in g t o t h e sh ap e o f t h e li g an d an d rece p to r p o ck e t a rem a t c h e d a n d s c o r e d o n t h e b a s is o f t h e ir d e g r ee o f ov e r la p . I m p r o v e m e n t s i n th e D O C K a l go -r i t h m h av e a l l o wed t h e sco r in g o f l i g an d s n o t o n ly o n t h e b as i s o f s t e ri c c ri t e r ia b u t a l so acco rd in gto mo lecu l a r mech an i c s i n t e rac t i o n en e rg i e s (Men g e t a l. , 1 9 9 2) .

T h e D O C K a l g o r it h m h a s b e e n s h o w n t o b e a p o w e r f u l t o o l to g e n e r a te n e w l e a ds f r o m t h e 3 Ds t ru c tu re o f th e recep to r . In d eed , sev e ra l l ead en zy me in h ib i t o r s ac t i v e i n th e mic ro m o la r ran g eh a v e b e e n f o u n d w i t h D O C K ( K u n t z , 1 99 2) . H o w e v e r , le a d o p t i m i z a ti o n h a s p r o v e d t o b e m o r ed i ff i cu l t, o win g t o t h e m an y a ssu m p t io n s u se d i n th e 3 D s t e ri c sea rch es , su ch a s th e co n f o rm a t io no f t h e l ig an d , n eg l ec t o f en t ro p y a n d so lv en t, an d s imp l if i ed ev a lu a t i o n o f t h e i n t e rac ti o n en e rg ie s(Ku n tz , 1 9 92 ). Ap p ro ac h es fo r imp ro v in g 3 D s t e ri c sea rch es b y a l lev i a ti n g t h ese d if f icu l ti e s a re i np ro g ress .3 D s t r u c tu re g en er a t io n

A n o t h e r a p p r o a c h f o r f i n di n g n e w l e a d s i n v ol v e s t h e p i e ci n g t o g e t h e r o f m o l e c u l a r f r a g m e n t sto create ( ra th e r t h an sea rch ) co mp o u n d s t h a t wi l l b in d t o a recep to r wh o se 3 D s t ru c tu re i sk n o w n ( M o o n a n d H o w e , 1 9 9 1 ; N i s h i b a t a a n d I ta i , 1 9 9 1 ; B 6 h m , 1 99 2) . T h e G R O W a l g o r it h m(Mo o n an d Ho we , 1 9 9 1 ) was o r i g in a l l y d es ig n ed fo r t h i s p u rp o se an d au to ma t i ca l l y l i n k s amin o

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26 7a c id s t o g e t h e r t o f o r m p e p ti d e s t h a t c o u l d b in d t o e n z y m e s . W i t h G R O W , a n a m i d e g r o u p ism a n u a l l y p la c e d i n t o a n e x p e r im e n t a l l y d e t e r m i n e d 3 D s t r u c t u re o f a r e c e p t o r a n d e x p a n d e d i n t oa p e p t i d e b y a d d i n g a m i n o a c i d t e m p l at e s . A n o t h e r p r o g r a m , L U D I ( B 6 h m , 1 9 9 2) , i de n ti fi e spo ten t ia l in te rac t ion s i te s f rom the 3D s t ruc tu re o f the recep to r , s elec ts m o lecu la r f ragme n ts f roma l ib ra ry o f o rgan ic m o lecu le s to in te rac t wi th these si te s, and au tom at ica l ly links these p iecestoge the r wi th b r idges se lec ted f rom a l ib ra ry o f mo lecu la r f ragmen ts .C O N C L U S I O N S A N D F U T U R E P E R S P EC T IV E S

N M R is a u s e f u l t o o l f o r r a p id l y d e t e rm i n i n g th e c o n f o r m a t i o n s o f r e c e p t o r - b o u n d l ig a n d s a n diden t i fy ing those po r t ions o f the l igand in con ta c t wi th i ts r ecep to r . The co mple te 3D s t ruc tu reso f recep to rs and l ig and- r ecep to r complexes can be ob ta ined a s a r e su l t o f r ecen t advances inh e t e r o n u c l e a r m u l t i d i m e n s i o n a l N M R t e ch n i q ue s . Wi t h t h e h e l p o f i m p r o v e d c o m p u t e r g r a p h ic sso f tware and recen t ly deve loped s t rateg ie s fo r sea rch ing 3D da tabases , th i s NM R- der iv ed s t ruc -t u r a l i n f o r m a t i o n c o u l d be u s e d to a i d d r u g d e s ig n , a n d f u r t h e r a d v a n ce s in b o t h N M R a n d t h e s ecompu ta t iona l too ls wi l l make th i s app roach even more feas ib le . In the nea r fu tu re , NMRs t ru c t u r es w i ll b e d e t e r m i n e d m o r e r a p i d l y b y a u t o m a t i n g t h e a s s ig n m e n t p r o c e d u r e a n d s t r u c tu r ede te rm ina t ions , and 3D s t ruc tu res wil l be de te rm ined more p rec i se ly by inc lud ing add i t iona ld i s tance , angu la r , and chemica l sh i f t -de r ived re s t ra in t s in to the s t ruc tu re ca lcu la t ions . Thus ,N M R s t ru c t u re s o f l a rg e r m o l e c u l es a n d b i o m o l e c u l a r sy s te m s t h a t h a v e p r e v io u s l y b e e n t o od i f ficu l t to s tud y by NM R wi ll become accessible. The co m pu ta t io na l too ls fo r u s ing the N M R -der ived s t ruc tu ra l in fo rma t ion wi l l a l so improve by sea rch ing 3D da tabases more rap id ly , bya l lowing m ore tha n one co n fo r m a t ion o f the mo lecu le s to be sea rched (Mura l l and D av ies , 1990 ;Smell ie e t a l ., 1991; Gfin er e t a l. , 1992) , and by im prov ing the de novo design s tra tegies . Inadd i t ion , be t te r p rocedures fo r eva lua t ing the mode led compounds wi l l be deve loped tha t t akeent rop y and so lvent in to consi dera t ion (Sti ll e t a l ., 1990; Ku ntz , 1992) . The se advances , cou pledwi th the imp roved capab i l i ty to rap id ly g ene ra te and te s t d iverse mo lecu le s f rom ex tens ive lib ra r -ie s, w il l m a k e N M R s t r u c t u re - b a s e d d r u g d e s ig n a n i m p o r t a n t a p p r o a c h f o r d i s co v e r in g n e wpharmaceu t ica l agen ts .R E F E R E N C E SAlb ran d, J .P. , Birdsall, B. , Feeney, J ., Rob erts, G.C .K. an d Burgen, A.S.V. (1979) Int . J. Biol . Macromo l., 1, 37-41.Allen, F.H ., Davies, J .E., G alloy , J .J ., Johnson, O., Ken nard , O., M acrea, C.F . , Mitchell, E.M., Mitchell, G.F . . Smith,

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