[Boschke F.L.] Medicinal Chemistry

7/25/2019 [Boschke F.L.] Medicinal Chemistry

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T op ics in urrent hem istry

F o r t s c h ri t te d e r C h e m i s c h e n F o r s c h u n g

Medicinal hemistry

Springer Verlag

B e r l i n H e i d e l b e r g N e w Y o r k 9 7 7


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This series presents critical reviews of the p resent position and future trends in mo dern chemical

research. It is addressed to all research and industrial chemists who wish to keep abreast of ad-

vances in their subject.

As a rule, contr ibutio ns are specially commissi oned. The editors and pub lishers will, however , al-

ways be pleased to receive suggestions and supp lementary information. Papers are accepted for

Topics in Current Chemist ry in English.

I S B N 3 - 5 4 0 - 0 8 3 6 6 - 9 S p r i n g e r - V e rl a g B e rl i n H e i d e l b e r g N e w Y o r k

I S B N 0 - 3 8 7 - 0 8 3 6 6 - 9 S p r i n g e r - V e r l a g N e w Y o r k H e i d e l b e r g B e rl i n

Library of Congress Cataloging in Publication Data. Hahn, Fred Ernest, 1916- Medicinal chemistry. (Topics

in current chemistry ; v. 72) Bibliography: p. 1. Chemistry, Pharmaceutical--Addresses, essays, lectures.

I. Wehrli, Walter, 1935- joint author. II. Orth, Dieter R., 1938- joint author. III . Title. IV. Series.

QD1.F58 vol. 72 [RS410] 540 ,8s [615 .19] 77-24573

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned.

specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying

machine or similar means, and storage in data banks. Under 54 of the German Copyright Law where copies

are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by

agreement with the publisher.

by Springer-Verlag Berlin Heidelberg 1977

Printed in Germany

The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a

specific statement, that such names are exempt from the relevant protective laws and regulations and therefore

free for general use.

Typesetting and printing: Schwetzinger Verlagsdruckerei GmbH, 6830 Schwetzingen. Bookbinding: Konrad

Triltsch, Graphischer Betrieb, 8700 Wiirzburg

2152/3140-543210


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onten ts

M o d e s o f A c t i o n o f A n t i m i c r o b i a l A g e n t s

F r e d E . H a h n

Ansa my c ins : Chem is t ry , B iosyn thes is and B io log i ca l Ac t iv i t y

W a l t e r W e h r l i

21

S y n t h e se s a n d A c t i v it y o f H e t e r o p r o s t a n o i d s

D i e t e r O r t h a n d H a n s - E c k a r t R a d u n z

51

H y p o l i p id a e m i c A r y l o x y a c e t ic A c i d s

Er i ch Schach t

99

T i l o r o n e H y d r o c h l o r id e : T h e D r u g P r o fi le

P r a k a s h C h a n d r a a n d G e o r g e J . W r i g h t

125

A u t h o r I n d e x V o l u m e s 2 6 - 7 2 1 49


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E d i t o r ia l B o a r d :

Pro f . Dr . M ic hae l J. S . D e w ar

Prof . Dr . K l a u s H a f n e r

Pr o f . D r . E d g a r H e i l b r o n n e r

Prof . Dr . S h 6 I t 6

Prof . Dr . J e a n M a r i e L e h n

Prof . Dr . K u r t N i e d e n z u

Prof . Dr . K lau s Sc h i l le r

Prof . Dr . G e org W i t t i g

M a n a g i n g E d i t o r :

D r . Fr ie dr ic h L . B osc hk e

S p r i n g e r - V e r l a g

D e p a r t m e n t o f C h e m i s tr y , T h e U n i v e r s it y o f T e x a s

A u s t i n , T X 7 8 7 1 2 , U S A

I n s t it u t fi ir O r g a n i s c h e C h e m i e d e r T H

Pe t e r s e n s t r a l 3e 1 5 , D - 6 1 00 D a r ms t a d t

Ph y s i k a l i s c h - Ch e m i s c h e s I n s t it u t d e r Un i v e r s i t i it

K l i n g e l b e r g s t r a Be 80 , CH - 4 00 0 B a s e l

D e p a r t m e n t o f C h e m i s t ry , T o h o k u U n i v e r s i ty ,

S e n d a i, J a p a n 9 8 0

I n s t i t u t d e C h i mi e , Un i v e r s i t 6 d e S t r a s b o u r g , 4 , r u e

B l a i s e Pa s c a l , B . P . 2 9 6 /R 8 , F - 6 7 00 8 S t r a s b o u r g - Ce d e x

Un i v e r s i t y o f Ke n t u c k y , C o l l e g e o f A r t s a n d Sc i e n c e s

D e p a r t m e n t o f C h e m i s t r y , L e x i n g to n , K Y 4 0 5 0 6 , U S A

I n s t i t u t f i ir Ph y s i k a l i s c h e Ch e m i e d e r Un i v e r si t~ i t

I m N e u e n h e i m e r F e l d 7 , D - 6 9 0 0 H e i d e l b e r g 1

I n s t i t u t fi ir O r g a n i s c h e Ch e m i e d e r Un i v e r si t~ i t

I m N e u e n h e i m e r F e l d 27 0 , D - 6 9 0 0 H e i d e l b e r g 1

Sp r i n g e r - V e r l a g , Po s t f a c h 1 05 2 80 ,

D - 6 9 0 0 H e i d e l b e r g 1

Po s t f a c h 1 05 2 80 - D - 6 9 00 H e i d e l b e r g 1

T e l e p h o n e 0 6 2 2 1 ) 4 8 7 -1 - T e l e x 0 4 - 6 1 7 2 3

H e i d e l b e r g e r P l a t z 3 D - 1 0 0 0 B e r l i n 3 3

T e l e p h o n e 0 3 0 ) 8 2 2 0 0 1 - T e l e x 0 1 - 8 3 3 1 9

S p r i n g e r - V e r l a g 1 7 5, F i f t h A v e n u e N e w Y o r k , N Y 1 0 0 1 0

N e w Y o r k I n c . T e l e p h o n e 4 7 7 - 8 2 0 0


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M o d e s o f c t i o n o f n t im i c r o b ia i g e n t s

P r o f e s s o r D r . F r e d E . H a h n

Wal t e r Reed Army Ins t i t u t e o f Resea rch Wash ing ton D.C. 20012 U.S .A.

T a b l e o f C o n t e n t s

l I .

HI

I V .

~

V I .

V I I . R e f e r e n c e s

I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . 2

W h a t C o n s t i t u t e s t h e M o d e o f A c t i o n ? . . . . . . . . . . . . 2

T h e T e s t O r g a n i s m s a n d D r a g E f f e c t s U p o n T h e m . . . . . . . . 4

T h e S t r a t e g y o f M o d e o f A c t i o n S t u d i e s . . . . . . . . . . . 6

1 . I n h i b i t i o n o f D N A S y n t h e s i s . . . . . . . . . . . . . . 7

2 . I n h i b i t i o n o f R N A S y n t h e s i s . . . . . . . . . . . . . . 9

3 . I n h i b i t i o n o f P r o t e i n S y n t h e s i s . . . . . . . . . . . . . . 1 0

4 . I n h i b i t i o n o f C e l l W a l l B i o s y n t h e s i s . . . . . . . . . . . . 12

5 . I n t e r fe r e n c e w i t h t h e S t ru c t u r a l a nd F u n c t i o n a l I n t e g r i ty o f M e m b r a n e s 1 3

R e s e a r c h o n M e c h a n i s m s o f A c t i o n . . . . . . . . . . . . . 16

Conclusions

. . . . . . . . . . . . . . . . . . . . . 1 7

18


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F. E. Hahn

I . I n t r o d u c t i o n

A c t i o n s o f c h e m o t h e r a p e u t i c d r u g s h a v e b e e n i n v e s t ig a t e d f o r m o r e t h a n t h r e e

d e c a de s . T h e b i b l i o g r a p h y o f s o u r c e a r t ic l e s in t h i s r e s e a r c h f i e ld c o m p r i s e s m o r e

t h a n 1 0 , 0 00 o r i g in a l p a p e rs ; a n u m b e r o f t e x t b o o k s a n d m o n o g r a p h c o l l e c t i o n s h a v e

b e e n p u b l i s h e d 1 7 ) . T h e p u r p o s e o f t h is p r e s e n t a r ti c le c a n n o t b e a n a t t e m p t a t

r e v ie w i n g e v e n a s m a ll p a r t o f t h e l a rg e li t e r a tu r e o n m o d e s a n d m e c h a n i s m s o f

a c t i o n o f c h e m o t h e r a p e u t i c d r u g s. W h a t i s i n t e n d e d , i n s t e a d , i s t o p r e s e n t s o m e o f th e

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

th i s f i e ld o f s c i ence .

W h e n t h e a u t h o r o f t h is e ss a y w a s c o n f r o n t e d i n 1 9 4 9 w i t h t h e p r o b l e m o f e l u c i d a t-

in g t he m o d e o f a c t i o n o f c h l o r a m p h e n i c o l , t h e r e e x i s te d v i r tu a l ly n o m e t h o d o l o g -

i ca l o r c o n c e p t u a l g u i d e l in e s w h i c h c o u l d b e a p p l i e d t o t h i s ta s k . A r e v i e w o n t h e

m o d e o f a c t io n o f s u l f o n a m i d e s i n 1 9 4 3 8 ) h a d o c c u p i e d 8 7 p a g e s , c i t e d 2 9 1 r e f e r -

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

o f th e b e t t e r u n d e r s t o o d m o d e s o f c h e m o t h e r a p e u t i c d r u g a c ti o n .

The r ea son fo r t he se i n i t i a l d i f f i cu l t i e s we re c l ea r ly t ha t s c i en t i s t s d id no t know

c o n c e p t u a l l y w h a t t o l o o k f o r , n o r m e t h o d o l o g i c a ll y h o w t o l o o k . A r e vi e w o f t h e

m e c h a n i s m o f a c t i o n o f t h e t e t r a c y c l in e s , a s l a t e as 1 9 6 7 , o f f e r e d 6 p a g e s o f t a b u -

l a t i o n o f a c t i v i t i e s w h i c h h a d b e e n f o u n d t o b e in h i b i t e d b y t h e s e a n t i b i o t ic s 9)

W h ile i t i s n o t u n u s u a l t o r e a d t h a t t h e a d v a n c e s i n t h e l a s t 1 5 y e a r s a r e m e t h o d o l o g -

i ca l i n n a t u r e , i t is n o t a l w a y s a p p r e c i a t e d t h a t a n s w e r s b e g a n t o c o m e f o r t h o n l y

a f t e r i nves t i ga to r s had l e a rned t o a sk pe r t i nen t q ues t i ons .

I I . W h a t C o n s t i t u t e s t h e M o d e o f A c t i o n ?

I t is a p p r o p r i a t e , t h e r e f o r e , t o r e p r i n t h e r e t h e p o s t u l a t e s o f t h i s a u t h o r w h i c h w e r e

o f f e r e d in a s y m p o s i u m l e c tu r e o n M o d e s o f A c t i o n o f A n t i b io t i cs a t t h e F o u r t h

I n t e r n a t i o n a l C o n g r e s s o f B i o c h e m i s t r y i n 1 9 5 8 ~o)

T h e p r o b l e m o f e lu c id a t in g t h e m o d e o f g r o w t h i n h i b it io n o f a n a n ti b i o ti c is . . . .

n o t s i m p l y o n e o f d is c o ve r in g s o m e a c t io n u p o n s o m e p h y s i o lo g i c a l p r o c es s o r

b i ochem i ca l reac ti on . M an y such f i nd i ngs co m e up dur i ng ex t en de d i nve s ti ga ti ons

a n d a r e n o t i n f r e q u e n t ly m i s i n t e rp r e t e d a s m o d e s o f a c ti o n. T h e r e a l p r o b l e m is

concep t ua l , v i z. , e l im i na t i on o f un i m po r t an t o r s econdary e f f e c t s and i den t i f ica t i on

o f t he p r i m a ry proces s o r r eac t i on w hose i nh i b i t i on is o ri gir ia lly r e spons ib l e f o r

g r o w t h i n h i b i t i o n .

W e have ad op t ed a s e t o f c r ite ria wh i ch a phys i o l og ica l p roces s o r b i oche m i ca l

r eac t ion m us t f u l f i l l i n o rder t o b e cons i de red t he key p roces s whose i nh i b i t i on l eads

t o t he overa l l r e su l t o f g ro wt h i nh i b it ion .

1 . T h e i n h i b i t e d r e a c ti o n m u s t b e o f v i ta l n e c e ss it y f o r t h e e c o n o m y o f t h e m i c r o b i a l

ce l l

2 . The i nh i b i t ion m us t be p ro du ced spec if ica l ly i n o rgan i sm s whose grow t h i s suscep -

t i b l e t o t he a c t i on o f t he d rug.


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Modes of A ction of Antimicrobial Agents

3 . T h e i n h i b i t io n m u s t b e p r o d u c e d b y a n a n t i b i o t ic c o n c e n t r a t i o n t h a t is o f t h e

s a m e o r d e r a s t h e g r o w t h . i n h i b i t o r y c o n c e n t r a t i o n r an ge .

4 . T h e d e g r e e o f i n h i b i ti o n m u s t a p p r o a c h a n a l l- o r- n o ne e f f e c t .

5 . T h e i n h i b i ti o n m u s t d e p e n d u p o n t h e s p e c if i c c h e m i c a l s t r u c t u r e o f t h e a n t i-

b i o t i c m o l e c u l e i n p r e c i s e l y th e s a m e m a n n e r a s d o e s t h e g r o w t h - i n h i b i t o r y e f f e c t. '

H e r e a r e som e se l ec t ed exam pl e s o f re sea r ch s t ud ie s w h i ch d i d no t cons i de r t he

above pos t u l a te s . I n 1949 a pap e r w as pub l i shed w h i ch r e po r t e d i nh i b i t ions o f

bac t e r i a l e st e ra se s by ch l o r am phen i co l 1~ ). N o t h o u g h t w a s g iv e n t o t h e r e q u i r e m e n t

t ha t t he i nh i b i t ed r eac t i on m us t be v i t a l ly i m po r t an t t o g r o w t h o f suscep t i b le cells .

O n e y e a r l a te r a p p e a r ed a p a p e r w i t h t h e t i tl e , o n t h e m e c h a n i s m o f a c t io n o f

a u r e o m y c i n t 2 ). H e r e t h e e f f e c t o f c h l o r t e t ra c y c l i n e w a s s t u d ie d i n a m a m m a l i a n

l iv e r m i t o c h o n d r i a l s y s t e m i n w h i c h t h e d r u g i n t e r f e r e d w i t h o x i d a ti v e p h o s p h o r y l a t i o n .

I t w as co m p l e t e l y m i ssed t ha t l ive r c e l ls ar e no t t a r ge t o r gan i sm s f o r t he an t i b i o t i c

ac t i on o f t e t racyc l i ne s . Th i s v i o l a te s the s econd p os t u l a t e ,

viz . ,

t he i nh i b i t i on m us t

be p r odu ced spec i fi c a ll y i n o rgan i sm s w hose g r ow t h i s suscep t ib l e t o t h e d r ug un de r

s t u d y . W i th i n a s h o r t t i m e , i t w a s s h o w n 1 3 . 1 4 ) t h a t c h l o r t e t r a c y c l in e b in d s M g 2 + b y

c h e l a t io n a n d t h a t t h e i n h i b i t i o n o f o x id a t iv e p h o s p h o r y l a t i o n w a s t h e s im p l e

r e s u lt o f m a g n e s i u m d e f i c ie n c y . E v e n r e c e n t l y , a p a p e r w a s p u b l is h e d w h i c h r e p o r t e d

e f f e c t s o f t h e a n ti m a la r ia l s, c h l o r o q u i n e a n d p r i m a q u i n e , o n p o l y p e p t i d e s y n th e s is

i n a c e l l - fr ee sys t em f r om ra t l iv e r i s )

O n e o f t h e m o s t f r e q u e n t l y n e g l e c te d r u le s i n t h e i n v e s ti g a ti o n o f m o d e s o f a c t i o n

p e r t a in s t o t h e u s e o f e x ce s si ve d r u g c o n c e n t r a t io n s . I n a s t u d y o f t h e e f f e c t s o f

ch l o r t e t r acy c l i ne on a (m am m al i an ) D - am i no ac i d ox i dase 16 ), t he a n t i b i o t i c con -

cen t r a t i on w as 1 .2 x 1 0 - 3M w h i ch is 575 tag/m l , r e . m o r e t han t w o o r de r s o f

m agn i t ude h i ghe r t han t he g r ow t h - i nh i b i t o r y concen t r a t i ons f o r suscep t i b l e bac t e r i a .

T h e l i te r a t u re is r e p l e te w i t h r e p o r t s o f m i n o r i n h i b i t io n s o f s o m e b i o c h e m i c a l

o r p h y s io l o g ic a l p r o c e s s b y c h e m o t h e r a p e u t i c d r u g s. I n m y o w n l a b o r a t o r y i t w a s

f ou nd t ha t qu i nac r i ne caused a sl igh t i nh i b i t i on o f p r o t e i n b i osyn t hes i s i n suscep ti b l e

bac t e r i a 17) w h i ch su bseque n t l y w as ve r i f ied in a c e l l -f r ee po l y U sys t em , po l ym er i z i ng

phen y l a l an i ne 18 ) . S i nce i t w as a l so e s t ab l ished , how eve r , t ha t t he d r ug is a s tr ong

i nh i b i t o r o f D N A b i osyn t hes i s 17) and t ha t t h i s e f f e c t a cco un t s f o r t he bac t e r i c i da l

ac t i on o f qu i nac r i ne , t he s l igh t e f f ec t on p r o t e i n b i osyn t hes i s w as no t m i s t aken a s

t h e m o d e o f a c t io n .

A n i n t er e s ti n g b o d y o f i n v e s ti g at io n s w a s p u b l is h e d o n t h e e f f e c t s o f c h l o r t e t r a c y -

c l ine on bac t e r i a l n i tr o - r educ t a se s (r ev i ew ed i n9 ) ) . S i nce a r educ t a se en zy m e i so la t ed

f r om ch l o r t e t r acyc l i ne - r e s i s tan t bac t e r ia w as l es s s ens it ive t o t he an t i b i o t i c , one cou l d

h a v e h o p e d t h a t t h e s e s t u di e s w e r e i n so m e m a n n e r d i r e c te d t o w a r d s t h e m o d e o f

a c t i o n o f c h l o r t e tr a c y c l i n e . H o w e v e r , a n t ib i o t ic a l ly i n a c ti v e d e g r a d a t i o n p r o d u c t s

o f ch l o r t e t r acy c l i ne a lso i nh i b i t ed n i t r o - r educ t a se s f r om bac t e r i a 19 ). Th i s w as i n

d i s ag r e e m e n t w i t h t h e l as t p o s tu l a t e t h a t t h e o b s e r v e d in h i b i t io n m u s t d e p e n d u p o n

t he spec i f i c chem i ca l s t r uc t u r e o f t he a n t i b i o t i c m o l ecu l e in p r ec i s e l y t he s am e

m a n n e r a s d o e s t h e g r o w t h - in h i b i t o r y e f f e c t . In o u r e x t e n s iv e s t ud i e s o n t h e m o d e

o f a c t i o n o f c h l o r a m p h e n i c o l , w e c a r r ie d o u t r o u t i n e l y c o n t r o l e x p e r i m e n t s w i t h

t he a n t i b i o t i c a l ly non- ac t ive enan t i om er o f t he d r ug i n o r de r t o d i s al low non- spec i f i c

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

3


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F. E. Hahn

An i n t e r e s t ing exce p t i on t o t h e abs o l u t e va l i d it y o f t he t i f t h po s t u l a t e i s t he

cons i de r ab le ac t i v i t y o f ch l o r am phen i co l de r i vat ive s in ce l l -f r ee m ode l s ys t em s o f

p r o t e i n s yn t hes i s when t he s e de r i va t ive s a r e s ubs t i t u t ed w i t h am i no acy l r e s idues

i ns t ead o f w i t h d i ch l o r oace t y l a s i s t he an t i b i o t i c i t s e lf r ev . in 20 ) ). Th i s ha s been

t r a c e d t o t h e n e c e s s i ty o f t h e d i c h l o r o a c e t y l g r o u p i n g in a id in g i n t h e p e r m e a t i o n

o f t he an t i b i o t i c t h r o ug h t he bac t e r i a l enve l ope 21 ). Th e am i no acy l de r iva t ive s have

ve r y l ow an t i bac t e r ia l a c t i v i t y 20). Pe r m ea t i on f a i lu r e s o f a c t i no m y c i n D, m ac r o l i de s

and d i s t am yc i n A wi t h r e s pec t t o ce r t a i n f am i l ie s o f bac t e r i a occ l ude t he a c t i on o f

t he s e an t i b i o t ic s on t he i r i n tr aceUul ar d r ug r ecep t o r s and t a r ge t r e ac t i ons bu t c an be

o v e r c o m e e x p e r i m e n t a l l y b y m e a s u r es w h i c h r e n d e r t e s t o r g a ni sm s p e r m e a b l e .

I II . T e s t O r g a n i s m s a n d D r u g E f f e c t s U p o n T h e m

Si nce quan t i t a t i ve d r ug ac t i ons on t he v i ab i li t y an d / o r g r ow t h o f m i c r oo r gan i s m s a r e

t he ba s ic r e f e r ence pa r am e t e r s o f all m od e o f a c t i on s t ud i e s , t he s e l ec t ion o f a t e s t

o r gan is m and t he d e t e r m i na t i on o f t he e f f ec t s o f a d rug upo n i t a r e t he e s s en t i al

f i rs t s teps i n a ll i nves ti ga ti ons on m od es and m ech an i s m s o f chem ot he r ape u t i c d r ugs .

I f t he d r ug un de r s t udy i s a r ea s onab l y b r oad s pec t r um an t i bac t e r ia l agen t , i t i s

ea s y t o s e l ec t a non - pa t hog en i c t e s t bac t e r i um wh i ch l ends i ts e l f t o ex t ens i ve

l abo r a t o r y i nves t iga t ions . M any s uch s t ud i e s have t he r e f o r e be en ca r r i ed ou t w i t h

p r o t o t r o p h i c s tr a in s ofEscherichia coli whi ch g r ow i n m i ne r a l m ed i um s upp l i ed

w i t h a s o u r c e o f c a r b o n . W i th a v i e w t o a n t ic i p a t e d t e st in g f o r t h e i n c o r p o r a t i o n o f

bu i l d ing b l ocks i n t o t he m a c r om ol ecu l e s o f a te s t o r gan i s m , it c an be advan t ageous

t o s e l ec t a u x o t r o p h i c m u t a n t s w h i c h m a y r e q u ir e a c e r t a in a m i n o a c id s u c h as

p h e n y l a l a n in e ) o r t h y m i n e f o r g r o w t h . A f r e q u e n t l y u se d t e s t s t r a in is

E. coli

T A U -

wh i ch r equ i r e s t hym i ne , u r ac i l and a r g i n ine .

C h e m o t h e r a p e u t i c d r u g s w i t h a n e x t r e m e l y n a r r o w s p e c t r u m o f a c t i v i ty s u c h a s

i s on iaz i d o r p . am i nos a l i cy l ic a c id w h i ch ac t exc l us i ve l y on

Mycobacteria are

di f -

f i cu l t t o s t udy , becaus e n o c onv en i en t t e s t o r gan i s m i s avai lab l e.

A s pec i a l p r ob l em a r os e wi t h r e s pe c t t o t he an t i p l a s m od i a l d r ugs ch l o r o qu i n e

Res o ch i n ) and qu i nac r i ne At eb r i n ) . P l a s m od i a can no t be p r opaga t ed i n ce l l- f ree

l if e le s s m ed i a and t he l i m i t ed ava i lab i l it y o f e r y t h r o cy t i c p l as m od i a l cu l t u r e s s eve r e ly

l im i t s b i ochem i ca l o r m o l ecu l a r ph a r m aco l og i ca l s t ud i e s on t he ac t i on o f an t i m a l ar ia l s .

T h e a c t io n s o f c h l o r o q u i n e a n d q u i n a c ri n e w e r e , t h e r e f o r e , s t u d ie d i n

Bacillus

megaterium o r E. coli, respec t ive ly 22)

Th e s e l ec ti on o f bac t e r i a a s t e s t o r gan is m s f o r t he i nves t iga t i on o f an t i m a l a r ia l

d r ugs d i d , a t f i r s t , no t go un cha l l enged an d r equ i r ed a s c i en t if i c j u s t i f i c a t i on wh i ch

was g iven as fo l low s 22).

Owing to the near universality o f the molecular processes, fundamental to

microbial growth and replication, the choice of test organisms or cell-free test

systems is dictated not so much by the pathogenic properties of specific micro-

organisms but rather by considerations of methodological feasibility and of the

opportunity of physical and conceptual isolation of a given phenomenon to be

studied. In no instance in which a problem of the mechanism [of action] of a

chemotherapeutic drug has been solved, was such a solution accomplished through


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Modes of Action o f A ntimicrobial Agents

work on a ma]or pathogenic target organism or has required that the study be

extended to such pathogens. In this respect the investigative perspective in molecular

pharmacology differs from that in pathology, immunology or medical microbiology

which must be concerned with the pathogenic, immunogenic or diagnostic properties

of specific etiological agents of communicable diseases.

lt wouM be erroneous to consider an investigation o f the inhibition of major

biosynthetic pathways or of the mechanistic details of such inhibitions merely a

model until such a time when a confirmatory analogy study was carried out with

intact o f fractionated cells of a pathogenic microorganism for no deeper reason than

that the given chemotherapeutic molecule can be used to cure clinical illness produced

by the growth and replication of such an organism. Essentially, any microorganism

that is subject to growth inhibition by critical concentrations of a given chemothera-

peutic drug is, for that very reason, a suitable test organism in mode of action studies.

Thi s r ea s on ig ha s been f u l l y j u s t i f i ed i n t he s ense t ha t s ubs equ en t s t ud i e s on t he

a c t i o n o f c h l o r o q u i n e o n p l a s m o d i a h a v e c o n f i r m e d t h e r e s u lt s o f p r e c e d i n g w o r k 2 2)

wh i ch us ed ba c t e r i a a s t e s t o r gan is m s ( r ev .23 ) ). The r e a r e i n s tances , how eve r , i n wh i ch

no s u i tab l e t e s t o r gan is m can be f o un d des p i t e a s ys tem a t i c s ea rch f o r i t . I n t he

l abo r a t o r y o f t h i s wr i t e r , no t e s t bac t e r i um was d i s cove r ed wh i ch was s us cep t i b le

t o r ea s on ab l y l ow con cen t r a t i o ns o f qu i n i ne and ce r t a i n new i nves ti ga ti ona l an t i .

m a l a r ia l d r ugs cou l d no t be s t ud i ed i n bac t e r i a l cu l t u r e s becaus e o f t he i r l im i t ed

s o l ub i l it y in aqueo us m ed i a .

A m o n g t h e m a n y m i c ro b i o lo g i c a l m e t h o d s o f d e m o n s t r a t in g g r o w t h i n h i b i t io n s

s ta n d s o u t t h e d e t e r m i n a t i o n o f d e c re a s e s i n

growth rates

b y g r a d e d d r u g c o n c e n -

t r a ti o n s . G r o w t h r a t e s o f c u l tu r e s w h i c h h a v e e n t e r e d t h e e x p o n e n t i a l p h a s e o f

m u l t i p l i c a t i on ( m eas u r ed e i t he r t u r b i d i m e t r i c a l l y , by e l ec t r on i c coun t i ng s uch a s i n

a C o u l t e r C o u n t e r , o r b y p l a t in g a n d c o l o n y c o u n t s ) a r e s y s t e m a t i c a ll y r e d u c e d ,

y i e l d i ng fam i l ie s o f g r o wt h cu r ves wi t h dec r ea s i ng s l opes . Th e r eg r es s ion coe f f i c i en t s

o f s u c h e x p o n e n t i a l g r o w t h c u r v e s ca n b e e x p r e s s e d i n p e r c e n t s o f i n h i b i t io n b y

( l oga r i thm i ca l ly ) g r aded d r ug con cen t r a t i o ns , and t he s e pe r cen t ages , conve r t ed t o

t h e i r p r o b i t s , a re l in e a r f u n c t i o n s o f t h e l o g a r i th m o f d r u g c o n c e n t r a t i o n a n d p e r m i t

i n t e r p o l a t i o n t o t h e 5 0 p e r c e n t i n h i b i to r y c o n c e n t r a t i o n , I D s o w h i c h is t h e m o s t

p r ec i se m eas u r e o f an t i bac t e r i a l po t en cy 24 ). Fu r t he r m or e , t he I Ds o i n m o l a r con -

c e n t r a t i o n r e p r e s e n t s t h e d i s s o c ia t io n c o n s t a n t o f a d r u g - r e c e p to r c o m p l e x w h i c h is

f o r m ed and i t s r e c i p r oca l va l ue i s t he a ppa r en t a s s oc i a ti on cons t an t . Th i s does no t

s p e c i fy i f t h e r e c e p t o r is o n t h e c e ll su r fa c e w h e r e i t s o c c u p a n c y p r e c e d e s e n t r y o f

t he d r ug i n t o t he ce l l o r i s i n t r ace l lu l a r and caus a l l y r e spons i b l e f o r t he m o l ecu l a r

m e c h a n i s m o f a c t i o n o f t h e d r ug .

T h i s t y p e o f k i n e t i c a n al y si s o f g r o w t h i n h i b it i o n w o r k s w i t h p r e d o m i n a n t l y

bac t e r i o s t a t i c d r ugs s uch a s ch l o r am phen i co l o r t he t e t r ac yc l i ne s25) bu t ha s been

e x t e n d e d t o t h e s t u d y o f t h e b a c t e ri c id a l a n t i b i o ti c , s t r e p t o m y c i n26).

B r o u g h t t o l o g ic a l c o n c l u s io n , it w a s s h o w n t h a t t h e p r o b i t t r a n s f o r m a t i o n s o f

b a c t e ri a l g r o w t h i n h i b it i o n a n d i n h i b i ti o n o f D N A b i o s y n t h e si s b y N i t ro a k r i d i n

3 5 8 2 w e r e s u p e r p o sa b l e w h i le t h e s a m e f u n c t i o n s f o r i n h i b i ti o n o f R N A a n d p r o t e i n

b i os y n t hes e s we r e s upe r pos ab l e upo n eac h o t he r bu t i nd i ca t ed a le s s e r s us cep t i b i l i ty

o f t h e t e s t o r ga n is m . T h i s l e d t o t h e c o n c l u s i o n t h a t t h e m o d e o f a n t i b a c t e ri a l a c t io n

o f t he n i t r oac r i d i ne was i ts i nh i b i t i on o f DN A b i os yn t hes i s 27) .


10/157

F. E. Hahn

K i n e t ic a n a ly s is o f g r o w t h i n h i b i t io n s b y g r a d e d c o n c e n t r a t i o n s o f a c h e m o t h e r a -

peu t i c d r ug f a i led wi t h pen i c i l li n 2 s ). Up t o a c e r t a i n t h r e s ho l d d r ug co nce n t r a t i o n

and f o r a f rac t i on o f one d oub l i ng t i m e , t he an t i b i o t i c had l i t tl e i n f luence t u r b i -

d i m e t r i c a l l y ) on g r o w t h r a t e s ; be yo nd t he s e c ri t ic a l t i m e and c onc en t r a t i on l i m i ts ,

t he t e s t cu l t u r e un de r w en t r ap i d l y si s,

i e

m o r p h o l o g i c a l d e s t r u c t i o n o f t h e b a c t e ri a l

cell s. T he s am e p r ob l em can l og i ca ll y be p r ed i c t ed f o r a ll d r ugs w h i ch i n t e r f e r e wi t h

t he i n t eg r i t y o f t he ce ll wa l l, r e s u l ti ng i n l y s is and phys i ca l d i s a s sem bl y o f t he t e s t

ceils.

T h e c o n c e n t r a t i o n o f c h o i c e f o r m o d e o f a c t i o n s t u d ie s is th e l o w e s t d r u g c o n -

c e n t r a t i o n w h i c h i n h ib i ts g r o w t h e n t i r e l y . T h is c a n e i t h e r b e e s t im a t e d b y e x t r a p o l a-

t i o n o f t h e p r o b i t - t ra n s f o r m e d l o g d o s ag e r e s p o n se c o r r e l a t io n o r b y d e t e r m i n a t i o n

o f t h e M I C , L e. m i n im a l i n h i b i t o r y c o n c e n t r a t i o n , b y t h e m e t h o d o f s eria l t w o f o l d

d i l u ti o n o f d r u g - co n t a in i n g m e d i u m i n t e s t t u b e s , i n o c u l a ti o n o f t h e s e m e d i a a n d

v is ua l obs e r va t i on o f g r ow t h a f t e r i ncub a t i on ove r n i gh t .

I V . T h e S t r a t e g y o f M o d e o f A c t i o n S t u d ie s

At t he ea r l y beg inn i ng o f m od e o f a c t ion s t ud i e s , t he f i e l d was wi t ho u t a s ys t em a t i c

a p p r o a c h a n d , h e n c e , r e l e g a te d t o e m p i r ic a l i n q u i ry i n w h i c h s c o r e s o f e n z y m e

r eac t i ons and phys i o l og i ca l p r oces s e s had t o be t e s t ed f o r s us cep t i b i l it y t o a che m o -

t h e r a p e u t i c d r u g u n d e r i n v es t ig a t io n i n th e h o p e t h a t s o m e m a j o r e f f e c t w o u l d b e

unco ve r ed wh i ch cou l d be co ns i de r ed s i gn i fi c an t. An ea r l y r ev iew on s u l f onam i des s)

a n d a l o n g t a b u l a t io n o f a c t io n s o f t e tr a c y c l i n e s9 ) a re i nd i ca t ive o f t he r e s u l ts wh i ch

wer e em pi r i ca ll y ob t a i ned .

A r e v i ew o f t h e m o d e o f a c t i o n f ie l d to d a y l e ad s t o t h e c o n c l u s i o n th a t t h e r e

ex i s t s a ve r y l i n i ted n um ber o f i n t e r f e r e nces wi t h phys i o l og i ca l p r oces s e s o f m i c r o -

b ia l ce il s w hich resul t in ce l l de a th or in inh ib i t ions o f ce l lu la r rep l i ca t ion . Reg ardless

o f t he und e r l y i ng m ech an i s t i c rea s ons , t he r e a r e m a i n l y f ive ca tego r i e s o f a c t i on

w h i c h l e a d t o c h e m o t h e r a p e u t i c p o t e n c y .

1 . I nh i b i t i on o f DN A s yn t hes i s ,

2 . i nh i b i t i on o f RN A s yn t hes i s ,

3 . i nh i b i t i on o f p r o t e i n s yn t hes i s,

4 . i nh i b i t i on o f c e l l wa l l s yn t hes is and

5 . i n t e r f e r e n c e w i t h t h e s t ru c t u r a l a n d f u n c t i o n a l i n t e g r i ty o f c e ll m e m b r a n e s .

Subs t ances s uch a s t he an t i b i o t ic s o f t he an t i m y c i n f am i l y wh i ch i n t e r f e r e wi t h

e l ec t r on t r ans p o r t i n t he r e s p i r a t o r y cha i n a re gene r a l ly t ox i c and , f o r t h i s r e a s on ,

u n s u i t e d a s c h e m o t h e r a p e u t i c d ru g s. T h e o n l y p r a c t i ca l u se o f a n t i m y c i n is t h a t a s

a f i sh poi son .

A f t e r t he s e l ec t ion o f a s u it ab le t e s t o r gan i s m , t he s t r a t egy o f m o de o f a c t i on

s t ud i e s i nvo lves ba s ica l ly t he t e s t i ng o f t he c om po un d und e r s t ud y f o r it s e f f ec t s

on each o f t he f i ve p r oces s e s l i st ed above . Th i s r equ i r e s inva r i ab l y t he s t udy o f

ac t i ve l y g r owi ng cu l t u r e s o r , be t t e r , o f o r gan is m s wh i ch a r e i ncuba t ed i n a m ed i um

wh i ch s up por t s g r ow t h and m u l t ip l i c a t i on . B ac t e r ic i da l d r ugs , f o r exam pl e , pen i c il li n


11/157

Modes of Action of Antimicrobial Agents

and strepto my cin have no lethal effects on non-growing bacterial suspensions and

actions on macrom olecular syntheses can, of course, no t be investigated in restin g

organisms, i e in suspensions of bacteria in buffered salt solutions devoid of nu trients.

The resting cell technique was mo stly used in man ome tric experiments o n gas

exchan ges in cell suspen sions, oxidizing a v ariety o f sub strates 28).

1 Inhib i tion o f DNA Synthesi s is usually signalled by bactericidal actio ns o f the

drug under investigation: organisms which can n ot replicate their chromo som es

cann ot produce a viable progeny. In addition, inhibitions of DNA synthesis freque ntly

give rise to long filamento us forms of bacteria. I t is believed that the c omp letion o f

one round of chromosomal DNA biosynthesis produces a signal which actuates

cross septation and cytokinesis. In its absence, the cells grow longer but do no t divide.

Figure 1 shows such f i laments ofE .

coli

after exposure to 2 10 -4 M quinacrine for

24 hou rs 17) and Fig. 2 shows another microscopic field pho tograp hed in the fluores-

cence microscope an d dem onstrating the fluorescent staining of the bacterial

f i laments by the drug.

Fig. 1. Filaments ofEscherichia coli formed during 24 hour s of growth in the presence of

9 9 17)

2 x 10 -4 M quma crme

One classical and early examp le of selective inh ibitio n of DN A bio synth esis is

shown in Fig. 3 for the a nitbiotic, mi tom yci n C 29). A co ncen tration o f 0.1/ag/m l

(3 x 10 -7 M) completely inhibited DNA sy nthesis in E col i B, while RNA synthesis,

prote in synthesis and gro wt h meaning turbidity,

i e

cell mass increase, continued.

However, after the experimental period of on ly 90 min , the num ber o f viable bacteria

had decreased by 85 per cent. By that time, bacterial filaments were visible under

the microscope.

With the introdu ction of radiochemical method s, DNA biosynthesis and i ts

inhibition is usually followed either by measuring th e incorpo ration o f radioactive

thym ine into thy mine auxo trophs of bacter ia or the incorporat ion of radioactive

thym idin e into proto troph ic organisms. In the la tter instance, it is practical to include

in the ex perimental m edium a large excess of non-radioactive deoxyad enosin e in order


12/157

F E Hahn

Fig. 2. Same object as in Fig. 1 but photog raphed under the fluorescen ce microscop e

0.3 0 Cont ro l /~ /~

- . . . . . MC 0 .1 pg /m l / ~ /

S /

0 t I ,~. U. / U

;I ,~o '~ ./. ~ i x

~ - . _~.--- v ~ ~ z

(l.> . / / ~ j - . ~ - / - . ~ - . - / 0

o ~o ~ "" ~" J' " o~.D- -- ~ - o 06

->

( ~ j ~ - -

i

J 0 0 4

f ~ /

~.~:- ,< __ x . . . . . . . . . . . . i ~_

.02 o

0 l i I I 0

0 30 60 90

Zncubation t ime in minutes

Fig. 3. Selective inhibition of DN A biosynthesis

in E colt

by 0 1 pg/ml of mitomy cin C 29


13/157


t o d e c r ea s e t h e h y d r o l y s i s o f t h e l a be l le d p r e c u r s o r b y a d e o x y n u c l e o s i d a s e w h i c h

bac t e r i a exc r e t e i n t o t he i r g r owt h m ed i um . Wi t hou t t h i s p r ecau t i on t he l abe l l ed

com po un d i n t he m ed i um i s qu i ck l y d i s si pa t ed and i nco r p o r a t i on l eve ls o f f ow i ng

t o a n e x h a u s t i o n o f t h y m i d i n e .

A l ar ge n u m b e r o f a n t i b i o ti c a n d s y n t h e t i c i n h i b it o r s o f D N A b i o s y n t h e si s f o r m

c o m p l e x e s w i t h D N A a n d a c t a s te m p l a t e p o i s o n s f o r t h e r e p l i c a ti o n o f c h r o m o s o m a l

DN A 6, 7 ). Thes e s ubs t ances a r e p r e f e r en t i a l i nh i b i t o r s o f eu ca r y o t i c c e l ls s uch a s

p r o t ozoa o r neop l a s t i c c e l l s . The on l y an t i bac t e r i a l chem ot he r apeu t i c d r ug wh i ch

is a s e lec t ive i nh i b i t o r o f DNA b i os yn t hes i s is na l id i x i c ac i d . Th i s s yn t he t i c com -

p o u n d d o e s n o t f o r m c o m p l e x e s w i t h D N A b u t a c ts in a n u n k n o w n m a n n e r w h i c h

a p p e a r s t o i n v o lv e a p e r t u r b a t i o n o f t h e i n t e r a c t io n o f D N A w i t h a b a c t e r ia l m e m b r a n e

s ys t em w hi ch i s i n s t r um en t a l i n DNA r ep l i ca t i on 3~

I n b a c t e ri a , D N A c o m p l e x i n g d ru g s o r e x p e r i m e n t a l D N A c o m p l e x e r s h a v e b e e n

f ou nd s e l ec t ive l y t o i nh i b i t t he r ep l i c a t ion o f p l a s m i d i c , e s pec i a ll y o f R- f ac t o r , D NAs

and , hen ce , t o e l i m i na t e r e s i st ance de t e r m i na n t s and r e s t o r e an t i b i o t i c s ens i t iv i t y t o

s uch o r gan i sm s 31 ). Th i s ha s op ened t he p r o s pec t o f deve l op i ng a s peci a l c a t ego r y o f

d r ugs f o r t he co m bi na t i o n ( wi t h an t i b i o t i c s ) t r e a t m en t o f Gr am - nega t i ve bac t e r i a l

infec t ions 32) .

2 . I n h i b it io n o f R N A S y n t h e si s us ua l l y ha s bac t e r i o s t a t i c cons eq uence s s ince i t

does no t r e s u l t i n s eve r e ly unba l anc ed b i os yn t hes i s and g r ow t h . When RN A b i os yn -

t he s is is t u r ned o f f by an i nh i b i t o r , t he ce l l c an no t r ep l en i s h m es s enge r RNA wh i ch ,

i n m i c r oo r gan i s m s , i s s ub j ec t t o r ap i d t u r no ve r . T he p r og re s s ive de f i c i ency i n

m es s enge r RNA r e s u l ts in a p r og re s s ive decay i n t he r a t e o f p r o t e i n b i os yn t hes i s .

F o r e x a m p l e , in c u l t u r e s of Bacillus subtilis, pr o t e i n s yn t hes i s had cea s ed en t i r e l y

a f t e r one f o u r t h o f a doub l i ng t i m e w hen R NA s yn t hes i s was s pec i fi c a ll y and

c o m p l e t e l y i n h i b it e d b y a c t i o n o m y c i n D 33 . S i n ce t h e i n i ti a ti o n o f a n e w r o u n d o f

DN A b i os yn t hes i s requ i r e s the a d h o c s y n th e s is o f i n i t ia t o r p r o t e i n ( s ) , D N A

biosynthes i s comes to a s t ands t i l l a f t e r a l ag as the resul t of the fa i lure of in i t i a tor

p r o t e i n s yn t hes is . Th i s i n t e r e s t ing s equen ce o f even t s w as f ir s t de s c r i bed b y Ki r k 34)

f o r t h e a c t io n o f a c t i n o m y c i n D i n S t aph y l ococ cus aureus .

A ct i nom yc i n D is a se l ec ti ve i nh i b i t o r o f RN A s yn t hes i s bu t i ts u s e in cance r

che m ot he r ap y is s eve r e ly l i m i t ed owi ng t o i t s t ox i c i t y . I t is t ho ug h t t ha t t he t w o

cyc l i c pep t i de m o i e t ie s o f t he an t i b i o t i c whos e t h r ee - m em ber ed a r om a t i c r ing s ys t em

is in t e r ca l a te d i n t o d o u b l e s t ra n d e d D N A o c c l u d e t h e m i n o r g r o o v e o f d u p l e x D N A

a n d i m p e d e t h e p r o g re s s io n o f t h e e n z y m e , R N A p o l y m e r a s e .

A n i m p o r t a n t f a m i l y o f i n h i b i to r s o f p r o c a r y o t i c R N A s y n th e s is a r e t h e a n t i b io t i cs

o f t he ans am yc i n s er ie s whos e p r o t o t yp e i s r i f am pi c i n . I t inh i b i t s bac t e ri a l RNA

s y n t h es i s a t th e e x t r e m e l y lo w c o n c e n t r a t i o n o f 0 . 0 1 / a g / m l ( 1 . 4 1 0 - 8 M ) b y b i n d in g

t o bac t e r ia l RN A po l ym er a s e a s ).

W i t h th e e x c e p t i o n s o f m i t o m y c i n C a n d a c t i n o m y c i n D w h i c h a r e , a t g r o w t h -

i n h i b i t o r y c o n c e n t r a t io n s , s p e c if ic i n h ib i to r s o f D N A o r R N A b i o s y n t h e s es , o t h e r

D N A - c o m p l e x i n g c h e m o t h e r a p e u t i c d r u g s in h i b it b o t h c a te g o r ie s o f b i o s y n t h e s e s

a l t h o u g h u s u al ly t o d i f f e r e n t e x t e n t s w h i c h d e p e n d n o t o n l y o n t h e d r u g c o n c e n -

t r a t i ons bu t a ls o on t he t e s t o rgan i s m . Fo r exam pl e , qu i nac r i ne i s a p r e f e r en t i a l

i n h i b i to r o f D N A b i o s y n th e s i s in E. col i 17 , whi le in Baci l lus cereus t he d r ug ac t ed


14/157

F. E. Hahn

pr ed om i nan t l y o n RN A b i osyn t hes i s 36 ). While such spec i e s - depend en t spec i fi c it i es

i n t he ac t i ons o f D N A - com pl ex i ng d rugs a re f a i r ly gene r a l ly r ecogn i zed , t he und e r l y i ng

reasons a re no t c lear . In in vi tro s tu d i es o n i n h i b i ti o n s o f D N A a n d R N A p o l y m e r a s e

r eac t i ons by qu i nac r i ne , t he t w o dosage re sponse co r r e l a t i ons w e r e no t s i gn if i c an tl y

d i f f e r en t f r om each o t h e r 22 ).

3 I n h i b i t io n o f P r o t e in S y n t h e s is is a m o s t p r e d o m i n a n t m o d e o f a c t i o n a m o n g

an t i b i o ti c s . The r e a r e m or e t han 20 s ing le an t i b i o t ic s o r con gene r i c f am i l i es o f

an t i b i o t ic s w h i ch ac t i n t h i s m an ne r . W i th t he ex cep t i on o f t he am i nog l ycos i de s w h i ch

a r e bac t e r ic i da l d rugs , i nh i b i t ions o f p r o t e i n syn t hes i s p r oduc e bac t e r i o s t a si s . I n t e r -

e s ti n g ly , t h e re is n o s y n t h e t i c c o m p o u n d a m o n g c li ni ca l c h e m o t h e r a p e u t i c d r u g s

w h i c h i n h i b it p r o t e i n b i o s y n th e s i s, a l th o u g h a n u m b e r o f e x p e r i m e n t a l a m i n o a c i d

an t i m e t abo l i t e s a f f ec t p r o t e i n syn t hes i s by i nh i b i t i ng am i no acy l t r ans f e r RN A

syn t he t a se s .

Ch l o r am p hen i co l and ch l o r t e t r acyc l i ne w e r e the f i r s t an t i b i o t i c s recogn i zed t o

be i nh i b i t o r s o f p r o t e i n syn t hes i s on t he ba sis o f f ind i ngs t ha t t hey i nh i b i t ed

i n d u c e d e n z y m e s y n t h e s e s in b a c t e r ia af ter i n d u c t i o n h a d o c c u r r e d a n d s y n t h e s e s

h a d b e e n u n d e r w a y f o r c o n s id e r a b le p e r i o d s o f t im e

37 .

I nh i b i t i ons o f p r o t e i n syn t hes i s w e r e subsequen t l y con f i r m ed by chem i ca l ana l yse s

f o r p r o t e i n i n g r ow i ng b ac t e r i a l cu l t u r e s 38 ' 39 . Fi gur e 4 39 shows the spec i f i c

i n h i b it i o n o f p r o t e i n b i o s y n t h e si s b y c h l o r a m p h e n i c o l in E co l i a n d t h e c o n t i n u a t i o n

o f R N A ( r i b o s e ) a n d D N A b i o s y n t h e s is f o r a n e x p e r i m e n t a l p e r i o d o f 5 0 m i n u t e s .

S i m i l a r r e su l ts have been t yp i ca l l y ob t a i ned w i t h o t he r i nh i b i t o r s o f p r o t e i n b i osyn -

thesis .

Whi le t he syn t hes i s o f D N A l eve ls o f f f o r r ea sons d i scus sed i n t he p r eced i ng

s e c t io n , R N A s y n t h es i s in c h l o r a m p h e n i c o l - e x p o s e d b a c t e r ia c a n c o n t i n u e f o r

e x t e n d e d p e r i o d s o f t im e . S o m e o f th i s e x c es s R N A is tr a n s f e r R N A o r i s f o u n d

i n i ncom pl e t e ( w i t h r e spec t t o p r o t e i n ) r i bosom e l i ke pa r t ic l e s ( r ev . i n4~ bu t

m u c h o f t h e b a c t er ia l R N A , a c c u m u l a ti n g d u r i n g i n h i b i ti o n o f p r o t e i n s y n t h e s is

i s messen ger

R N A 4 1 .

When bac t e r i a a r e r e lea sed f r om i nh i b i t i on o f p r o t e i n

s y n th e s is , g r o w t h d o e s n o t r e su m e i m m e d i a t e l y in m i n m a l m e d i u m . I n s t e a d , t h e r e

is co n s i d e ra b l e m e ss e n g er R N A b r e a k d o w n a n d e x c r e t i o n o f i t s p r o d u c t s i n t o t h e

m ed i um 4~ , 42) be f o r e ba l anced g r o w t h beg i ns again . H ow eve r , i f the an t i b i o t i c - fr ee

o r gan i sm s a r e r e suspen ded i n am i no- ac i ds con t a i n i ng m ed i a 43 ' 44 ), no b r eak dow n

of RN A is obse r ved and t he o r gan i sm s re sum e p r o t e i n syn t hes i s w i t hou t de l ay , soon

a c c o m p a n i e d b y R N A s y n t h es i s in t h e m a n n e r c h a r a c te r is t ic o f b a l a n c ed g r o w t h 4 4).

E v i d e n t l y , t h e u n b a l a n c e d s y n th e s is o f e x c e s s R N A d u r i n g i n h i b it i o n o f p r o t e i n

synthes i s i s not bac te r ic ida l .

T h e b a c t e r i o s t a t i c e f f e c t o f c h l o r a m p h e n i c o l h a s b e e n in v e s ti g a te d q u a n t i t a ti v e l y4s).

A d d i t io n o f t h e a n t i b io t i c t o c u l t u re s o f E . co l i B/ r g r ow i ng expo nen t i a l l y in b r a i n -

h e a r t i n f u s io n b r o t h w i t h a g e n e r a ti o n t i m e o f 2 1 . 9 0 m i n w a s f o l lo w e d b y c o n -

s i de rab l e f u r t he r i nc rea se in t o t a l c e l l num ber ( a s m easu r ed i n a Co u l t e r co un t e r )

and an i n i t ia l inc r ea se in t he nu m ber o f co l on y- f o r m i ng bac t e r i a w h i ch t he n

pr og r e s si ve l y dec r ea sed by 75 pe r c en t du r i ng con t i nue d incub a t i on f o r a t o t a l

o f 13 . 7 gene r a t i on t i m es ( F i g . 5 ) . D i v i si on o f c e ll s i n ch l o r am p hen i co l - con t a i n i ng

cu l t u r e s w as obse r ved unde r t he m i c r osco pe 4s ) . I t is pos s ib l e t ha t bac t e r i o s t a s i s

10


15/157

60

12


5 0

- 30

s

< 2 0 c .

::L

1 0 - 6 M )

L A c t i o n s o n e u k a r y o t e s :

Cl ea r ev i dence ex i st s to p r ove t ha t ansam yc i ns such a s r i f am pi c i n have no e f f ec t

o n e u k a r y o t i c R N A p o l y m e r a s e s , b e t h e y o f n u c le a r , m i t o c h o n d r i a l o r c h l o r o p la s t ic

35


39/157

W. Wehrli

o r ig i n . Ce r t a i n r epo r t s c l a im i ng inh i b i t i on o f m i t ocho ndr i a l o r ch l o r op l a s t i c RN A

p o l y m e r a s e a r e d o u b t f u l b e c a u s e o f t h e e x p e r i m e n t a l c o n d i t io n s u s e d o r t h e h i g h

d r ug c o n c e n t r a t i o n n e e d e d a s c o m p a r e d t o t h a t r e q u i r e d f o r in h i b i t io n o f t h e b a c -

t e r ia l enzym e . R i f am yc i ns w i t h l i poph i l ic s ide cha i ns and so m e de r iva t ive s o f s t r ep t o -

va r ic i n and ge l dan am y c i n have been f ou nd t o i nh i b i t i nd i s c r i m i na t e l y a l ar ge nu m be r

o f b o t h D N A a n d R N A p o l y m e r a s e s o f b a c t e r ia l , e u k a r y o t i c a n d v i r al o ri g in . T h e

1 0 0 - 1 0 0 0 0 - f o l d h i g h e r d r u g c o n c e n t r a t i o n n e e d e d f o r i n h ib i t io n , a s w e ll as th e

l ack o f e nzy m e spec i f i c i t y , de f i n i t e l y d i s t ingu i shes t h i s e f f ec t f r om t he i nh i b i t i on o f

b a c t e ri a l R N A p o l y m e r a s e .

H E f f e c t s o n R N A t u r n o u t v ir us es :

A s h a s a l r e a d y b e e n m e n t i o n e d , s o m e l ip o p h i li c r if a m y c i n s a n d s o m e s t r e p t o -

v a ri ci ns a n d g e l d a n a m y c i n s a f f e c t th e g r o w t h o f c e ll s t r a n s f o r m e d b y R N A t u m o u r

v i ruse s o r t he RN A - depen den t D N A p o l ym er ase ( reve r se t r ansc r ip t a se ) cha r ac t e r i s ti c

o f t he se v ir u se s . A ga i n , h igh d r ug co ncen t r a t i ons a r e need ed t o p r odu ce an e f f e c t

a n d o n l y p a r t i al , b u t n e v e r a b s o l u te , s e l e c ti v it y o f e n z y m e i n h i b i ti o n h a s b e e n f o u n d .

I l L E f f e c t s o n D N A v ir u se s a n d la rg er i n f e c t i o u s a g e n ts b e l o n gi n g t o t h e g e n u s o f

C h l a my d i a e :

Cer t a i n ansam yc i n de r i va ti ve s , such a s r i f am pi c i n , i nh i b i t t he g r ow t h o f the se

o r ga n is m s a t h i g h d r u g c o n c e n t r a t i o n . T h e u n d e r l y i n g m e c h a n i s m o f a c t i o n is p o o r l y

u n t e r s t o o d a t p r e s e n t , b u t i t d o e s n o t s e e m t o b e r e l a te d t o R N A o r D N A s y n th e s is .

3 . 2 . E f f e c t s o n B a c t e r ia

3 . 2 . 1 . I n t e r a c t i o n w i t h D N A - d e p e n d e n t R N A P o l y m e r a s e

T h e p o t e n t a n t i b a c te r i a l a c ti v i ty o f t h e r i fa m y c i n s , s tr e p t o v ar i ci n s a n d t o l y p o m y c i n s

is a c o n s e q u e n c e o f t h e s p e c i fi c i n h i b i t io n o f D N A - d e p e n d e n t R N A p o l y m e r a s e , th e

e n z y m e r e sp o n s i bl e fo r m o s t o f t h e t ra n s c r i p ti o n o f D N A t o R N A . T h e i n t e r a c t i o n

b e t w e e n t h e s e a n s a m y c i n s a n d t h e e n z y m e h as a c c o r d i n g ly b e e n s t u d i e d i n g r e a t

de t a il . Because o f it s e a sy ava i lab i li ty , ri f am pi c i n ha s b een used m os t o f t e n a s t he

m o d e l c o m p o u n d i n s u c h s t ud i e s, b u t i t i s r e a s o n a b le t o a s s u m e t h a t t h e d a t a o b -

t a i ned w i t h r i f am pi c i n a lso ho l d t r ue , a t l e a s t qua l i t a t ive l y , f o r t he o t he r r i f am yc i ns ,

t h e s t r e p t o v a ri c in s a n d t o l y p o m y c i n s .

R i f a m p i c i n w a s f ir s t s h o w n b y H a r t m a n n

e t a l

s4) t o have a spec i f ic inh i b i t o r y

e f f e c t o n R N A p o l y m e ra s e f ro m

E col i

L a t e r , o t h e r a c t iv e a n s a m y c i n s w e r e f o u n d

a n d R N A p o l y m e r a s e s f r o m a l a rg e v a r ie t y o f b a c t e r i a o t h e r t h a n

E co i l

p r o v e d to b e

s e ns it iv e t o t h e d r u g. M o r e r e c e n t l y , a n R N A p o l y m e r a s e f r o m

E co l i

c o n t a i n in g o n l y

o n e s u b u n i t a n d p r o b a b l y i n v o lv e d i n t h e i n i t i a ti o n o f D N A r e p l ic a t io n ( d n a G g e n e

pr o du c t ) ha s been show n t o be r e s i s t an t t o r i f am pi c i n s s ). Th i s ho l ds t r u e a l so f o r t he

va r ious m a m m al i an R N A po l ym er ase s . I n con t r a s t t o non - spec i f i c i nh i b i t o r s o f tr an -

s c r ip t i o n s u c h a s a c t i n o m y c i n a n d m i t o m y c i n , r i f a m p i c i n i n t e r a c ts s p e c i f ic a l ly w i t h

t he bac t e r i a l enzym e i ts e l f. Wi t h t he a i d o f 1ac_ l abel led ri f am pi c i n i t co u l d be show n

t h a t t h e d r u g f o r m s a v e r y s ta b le c o m p l e x w i t h t h e e n z y m e in a m o l a r ra t i o o f

1 : 156 , 57 ). Th e d i s soc i a t ion con s t an t o f t h i s com pl ex i s

1 0 9 M

a t 37 ~ and

36


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Ansamycins - Chem istry Biosynthesisand BiologicalActivity

2 . 7 x 10 - 1~ M a t 0 ~ s a ' sg ). The se r e l a t ive l y l ow cons t an t s exp l a i n w hy ansam yc i ns ,

a n d i n p a r t i c u la r r if a m p i c in , i n h ib i t b o t h t h e a c t iv i ty o f R N A p o l y m e r a s e a n d t h e

g r o w t h o f b a c t e ri a a t v e r y l o w d r u g c o n c e n t r a t i o n s . E x p e r i m e n t s w i t h r if a m p i c in -

r e s i s tan t m u t an t s hav e show n 6~ 61 ) tha t t he 15 subu n i t o f R N A po l ym er ase con t a i ns

t h e b i n d i n g s it e f o r t h e d r ug . I t c o u l d b e d e m o n s t r a t e d , h o w e v e r , th a t b i n d i n g t o t h e

e n z y m e p r e c u r s o r a 2 ~ is v e r y w e a k 6 2 . To f o r m t he spec i fi c d rug - b i nd i ng s it e , the

f ou r subun i t s a2/3 /3 ', a s t hey o ccu r in t he co r e enz ym e , a r e r equ i r ed , a , t he subun i t

n e c e s s ar y f o r s p e c if ic p r o m o t e r r e c o g n i t io n , a f f e c t s o n l y w e a k l y t h e a n s a m y c i n

bind ing si tes 8).

3 . 2 . 2 . M o d e o f A c t i o n

I n h i b i t io n o f R N A p o l y m e r a s e is a d ir e c t c o n s e q u e n c e o f t h e b i n d i n g o f t h e d r u g t o

t h e e n z y m e : o n t h e o n e h a n d , e n z y m e i n h i b i ti o n o n l y o c c u r s, w h e n t h e a n s a m y c i n

is b o u n d , a n d o n t h e o t h e r h a n d , d r u g b in d i n g n e v e r s e em s t o o c c u r w i t h o u t c a u si n g

i nh i b i t i on .

T w o a s p e c ts o f t h e m o d e o f a c t i o n m u s t t h e r e f o r e b e d i st in g u i sh e d :

a ) w h a t d e t e r m i n e s a n d i n f l u e n c e s t h e b i n d in g o f a n s a m y c i n s t o R N A p o l y m e r a s e

b ) w h i c h f u n c t i o n o f R N A p o l y m e r a s e is i m p a i r e d w h e n a n s a m y c i n s a r e b o u n d

t o t h e e n z y m e .

R e c e n t i n v e st ig a t io n s o f t h e r a t e o f b i n d in g o f r if a m p i c in t o R N A p o l y m e r a s e

a t va ri ous s tages o f t ransc r i p t i on have show n t ha t nuc l e i c ac ids bo un d t o t he enz ym e

s t r ong l y dec r ea se the on - r a t e o f t he an t i b i o t i c t o t he enz ym e sa). R i f am pi c i n b i nds

5 0 0 t im e s s l o w e r to a s p e c if ic R N A p o l y m e r a s e - 1 7 D N A c o m p l e x t h a n t o t h e f r e e

e n z y m e . T h e r a t e o f d r u g b in d i n g t o a t e r n a r y e n z y m e - D N A - R N A c o m p l e x , as i t

occu r s du r ing R N A cha i n e l onga t i on is a t le a s t 5 o r de r s o f m agn i t ude s l ow er . These

d i r ec t k i ne t i c m easu r em e n t s a r e i n l ine w i t h ea r l i e r obse r va t i ons t ha t D N A can

p r o t e c t R N A p o l y m e r a s e a g a in s t in a c t i v a ti o n b y r i fa m p i c i n 6 3 - 6 s ) a n d t h a t a n s a m y -

t i n s i nh i b i t R N A cha i n i n i t ia t i on , bu t n o t e l onga t i on 66). Th e r a t e o f enzy m e i nh ib i -

t i o n b y a n s a m y c i n s t h e r e f o r e p a ra ll e ls t h e o n - r a t e o f d r u g b i n d i n g a n d c a n v a r y

g r ea t l y depend i ng o n t he s tage o f t r ansc r i p t i on .

F o r a l o n g t i m e t h e r e w a s n o c le a r p i c t u r e a s t o w h i c h f u n c t i o n o f t h e R N A

p o l y m e r a s e w a s i m p a i r e d b y a n s a m y c i n s. V e r y r e c e n t l y , M c C lu r e a n d c o l l a b o r a t o r s

c l a r if i ed t he p r ob l em i n a s e ri e s o f ve r y e l egan t expe r i m en t s 67) , pe r sona l com m u ni ca -

t i o n . U p o n a n a ly z i n g t h e i n i t ia t i o n o f R N A s y n t h es i s o n t h e P R p r o m o t e r o f p h ag e ),,

McC l u re f oun d t ha t r i fam p i c i n le ads t o ab o r t i ve i n i t i a t i on , i e t o t h e f o r m a t i o n o f

t h e d i n u c l e o t i d e p p p A p U , a n d i n h i b it s th e f o r m a t i o n o f th e s e c o n d p h o s p h o d i e s t e r

b o n d . A m o d e l i s p r o p o s e d i n w h i c h r i f a m p i c in b i n d i n g t o R N A p o l y m e r a s e s t er i-

t a l l y b l o c k s t h e t r a n s l o c a t i o n o f p p p A p U . T h e d r u g d o e s n o t a f f e c t th e r e c o g n i t io n

o f t h e s p e c if ic p r o m o t e r b y t h e e n z y m e , si n ce t h e o n l y d i n u c l e o t id e f o r m e d i s

pppA pU , w h i ch co r r e spon ds t o t he f i rs t t w o base s o f t he gene s ta r t ing a t PR-

3 . 2 . 3 . S t r uc t u r e - A c t i v i t y Re l a t i ons

Th e an t i bac t e ri a l a c t i v i t y o f t he a nsam y c i ns va ri e s t o a la r ge ex t en t . I n m os t c a se s ,

an t i bac t e r i a l a c t i v i t y pa r a ll e ls t he i nh i b i t i on o f t he bac t e r i a l RN A po l ym er ase and

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w . Wehrli

c a n t h e r e f o r e b e p r e d i c t e d t o s o m e e x t e n t o n t h e b a s is o f s t r u c tu r e - a c ti v i ty st u d ie s

w i t h t he enzy m e . Th e ava i lab i l it y o f m an y na t u r a l an d s em i syn t he t i c de r iva t ive s ,

e spec i al l y i n t he r i f am yc i n and s t r ep t ova r i c i n g r oups , ha s m ade i t pos si b le t o ge t an

i d e a o f t h e s t r u c tu r a l p a r a m e t e r s n e c e s sa r y t o i n h ib i t R N A p o l y m e r a s e . F o r t h e

r i fam yc i ns , som e O f t he m a i n f ea t u r e s a r e t he f o l l ow i ng6s 69) .

1 . F r e e h y d r o x y l o r k e t o g r o u p s a t C l a n d C 8 ;

2 . u n b r o k e n

a n s a b r i d g e ;

3 . f r ee hy dr o xy l g r oups a t C21 and C23 .

A na l ys i s by X - r ay6 9) a n d N M R s p e c t r o s c o p y7 ~ s h o w s th a t t h e r i f a m y c i n m o l e -

cu l e is ve r y r ig i d (F i g . 2 ) . Th e f ou r oxy gen f u nc t i ons a t C l , Cs , C21 and C2a a l l l ie

on t he s am e s i de o f t he m o l ecu l e . Be i ng r equ i r ed f o r enzy m e i nh i b i t i on i t i s r e a son -

a b le t o a s s u m e t h a t t h e y a r e in v o l v e d i n t h e b i n d i n g o f t h e d r u g t o t h e e n z y m e .

H o w e v e r , k i n e t i c s tu d i e s o f t h e i n t e r a c t io n o f r if a m p i c i n w i t h R N A p o l y m e r a s e i n

v a ri o us s o l v en t s i n d i c at e t h a t t h e b o n d s f o r m i n g t h e d r u g - e n z y m e c o m p l e x a r e m o s t -

l y o f a l i poph i li c na t u r e s8) . La r ge pa r ts o f t he a n s a r i ng t hus s eem t o p a r t i c i pa t e i n

t h e b i n d i n g t o t h e e n z y m e a s w e l l a s t h e c h r o m o p h o r e w h i c h p o s si b ly i n t e r a c ts w i t h

an a r om a t i c am i no ac i d , si nce r i f am pi c i n show s a cha r ac t e r is t i c ba t hoc h r om i c sh i f t

w h e n b o u n d t o t h e e n z y m e . T h e n a t u r e o f t h e i n t e ra c t io n s b e t w e e n t h e d r ug a n d

t h e e n z y m e c o u l d t h e r e f o r e c o n c e iv a b l y b e r e p r e s e n t e d b y t h e m o d e l s h o w n i n

F ig . 14 , in w h i ch t he en zym e cl oses on t he d r ug m ol ecu l e f r om t h r ee s ides . I n such

a m o d e l , p o s i t io n s 3 a n d 4 o n t h e c h r o m o p h o r e d o n o t t a k e p a r t i n th e b i n d in g .

Th i s w ou l d be co ns i s t en t w i t h t he f i nd i ng t ha t chem i ca l subs t i t u t i ons a t C- 3 and

C- 4 o f t he m o l ecu l e i n m an y ca se s have l i tt l e e f f ec t on i t s in t e r ac t i on w i t h t h e en -

z y m e 6 8 , 7 1). E f f e c t s o n e u k a r y o t i c a n d v ir al e n z y m e s o b s e r v e d a f t e r t h e i n t r o d u c t i o n

of subs t i t uen t s i n t he se pos i t i ons a re m os t p r oba b l y no t due t o spec i fi c b i nd i ng such

as is f ound w i t h t he ba c t e r i a l enzy m e .

A l t h o u g h m a n y f a c t s ar e k n o w n a b o u t t h e s t r u c t u r e .a c t i v i ty re l a ti o n s o f a n s a m y -

c i ns , som e p r ob l em s r em a i n unso l ved . R i f am y c i n W, f o r i n s t ance , (F i g . 6 ) does no t

r i fc

hromol

Fig. 14. M odel o f the

in teract ion

between rffampicin and RNA polymerase

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Ansamycins - Chem istry Biosynthesisand BiologicalActivity

s e e m t o i n h i b i t R N A p o l y m e r a s e l l) , a l th o u g h t h e s t r u c tu r a l r e q u i r e m e n t s m e n t i o n e d

above a r e f u l fi l led . On t he o t h e r hand , dam ava r i c i n C , wh i ch i s a s s um e d t o be a p r e -

cu r s o r o f s t r ep t ova r i c i n C and is chem i ca l l y ve r y s i m il a r t o r i f am yc i n W, i s a pow er -

f u l i n h i b i to r o f t h e e n z y m e23) . Mor eove r , am ong t he s t r ep t ova r i c in s , s t r ep t ova r i c i n J

( ace t a t e a t C - 21 ) and s t r ep t ova r i c i n E ( ke t o g r oup a t C . 21 ) s t i ll show s om e ac t i v i t y ,

whe r ea s t he co r r e s po nd i ng r i f am yc i n - C- 21- ace t a t e and r i f am yc i n Y a r e i nac t ive 2~

T h e s t r u c tu r e - a c t iv i t y r e l a ti o n s o f t h e r i fa m y c i n s a n d t h e s t re p t o v a r ic i n s t h e r e f o r e

do no t s eem t o be i den t i ca l .

A n o t h e r p u z z l e is p o s e d b y n a p h t h o m y c i n . T h e f a c t t h a t i t d o e s n o t i n h i b it R N A

p o l y m e r a s e c a n b e e x p l a i n e d b y t h e a b s e n c e o f a h y d r o x y l g r o u p a t C - 8. H o w e v e r , i t

s hows an t i bac t e r i a l a c t i v i t y aga ins t g r am - pos it ive s t ra i n s t ha t i s cou n t e r a c t ed by SH-

con t a i n i ng s ubs t ances s uch a s cys t e i n2 s). N a p h t h o m y c i n i s t h e r e f o r e a n e x a m p l e o f

a n a n s a m y c i n w i t h a n t i b a c te r i al a c t iv i t y n o t d u e t o i n h i b i t i o n o f R N A p o l y m e r a s e .

T h e s t r u c t u r e o f th e a n s a m y c i n s d e t e rm i n e s n o t o n l y t h e i r a c t iv i t y o n R N A

pol ym er a s e , bu t a l s o o t h e r i m p or t an t cha r ac t e r is t i c s s uch a s t he i r ab i l i t y t o pen e t r a -

t e i n t o b a c t e r ia a n d t h e i r p h a r m a c o k i n e t i e s a n d a b s o r p t i o n i n t h e h o s t . T o c i t e

j u s t a f ew exam pl e s : r i f am yc i n B , con t a i n i ng a f r ee ca r b oxy l i c a c i d g r oup , ha s no

an t i bac t e r i a l a c t i v i ty , a l t hough i t i nh i b i t s RN A po l ym er a s e a s s t r ong l y a s r if am pi c i n .

Dam ava r i c in C behaves s i m i la r l y t o r i f am y c i n B , whe r ea s i ts 6 - m e t hy l e t he r i nh i b i t s

R N A p o l y m e r a s e t o a l es se r e x t e n t , b u t h a s g o o d a n t i b a c te r ia l a c t i v i ty2a). Ri fam pi -

c i n owes it s wi des p r ead c l i n ica l u s e t o t h e f ac t t ha t , i n con t r a s t t o m os t o t he r

r i f am yc i n de r i va ti ve s , i t i s we l l abs o r bed wh en g i ven o r a ll y .

3 . 2 . 4 . N a t u r e o f Ans am yc i n Res i s t ance in Mut an t s

M ut an t s r e s i s t an t t o r i f am yc i ns have been i s o l a t ed f r o m a va r i e t y o f m i c r o -o r gan i sm s .

T h e m u t a t i o n r a te o f E . c o l i i s i n t he r eg i on o f 10 - a , and t he co nve r s i on is appa r en t l y

due t o a one - s t ep m u t a t i o n . T he m u t an t s ana l yzed s o f a r m ap a t a s ing le pos i t i on

( 7 9 m i n u t e s ) o n t h e E c o i l c h r o m o s o m e 7 2 ). R N A p o l y m e r a s e h as b e e n p r e p a r e d

f r o m r i f a m y c i n -r e s is t a n t m u t a n t s o f E . c o l i a n d S a u r e u s I n bo t h ca s e s i t was f ou nd

t h a t t h e e n z y m e d i f f e r e d f r o m t h e c o r r e s p o n d i n g e n z y m e o f t h e s e ns it iv e s tr a in i n

t ha t i t is no l onge r inh i b i t ed by r i f am pi c i n and , i n add i t i on , cou l d no l onge r b i nd t he

drugS6, 73, 74). Res i s tance to r i fam pic in was th ere for e caused b y an a l t e ra t io n in th e

s t r u c t u re o f t h e e n z y m e , p r o b a b l y b y t h e r e p l a c e m e n t o f o n e s in gle a m i n o a c i d . I n a

nu m be r o f r e s is t an t m u t an t s , t he s i te o f t he m u t a t i o n was l oca t ed i n t he f l s ubun i t 6~ 61).

Res i s t ance t o r i f am pi c i n i s no t an a l l - o r . no t h i ng phenom enon . Mut an t s c an be

s e l ec t ed t ha t a r e r e si s tan t t o va r i ous con cen t r a t i ons o f t he d r ug . As t he bac t e r i a

b e c o m e m o r e r e s i st a n t t o r i f a m p i c i n , t h e s e n s i ti v it y o f t h e c o r r e s p o n d i n g R N A p o l y -

m er a s e dec r ea s e s , and t he enzym e- an t i b i o t i c com pl ex b ecom es le ss s t ab l e 7 s) . Hence ,

t he deg r ee t o w h i ch t he en zym e and t h e an t i b i o t i c f i t e ach o t h e r va r ie s wi de l y , i nd i-

ca t ing t ha t t he r e a r e m an y pos s ib i li ti e s f o r t he l oca t i on and t he n a t u r e o f t he am i no

a c id s u b s t i tu t i o n i n t h e e n z y m e m o l e c u le . T h e s e su b s t i tu t i o n s w o u l d b e e x p e c t e d t o

occu r m a i n l y i n the ~ s ubun i t , b u t m u t a t i on s i n o t he r s ubun i t s s uch a s ~ o r t~ can no t

be ex c l uded , s ince , a s d is cus sed above , t he co r e en zy m e 0~2 /~ ha s t o be p r e s e n t f o r

t he f o r m a t i on o f t he s pec i fi c d rug - b i nd ing s i te .

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w . Wehrli

The pos sib le occur rence o f ansam yc in re s i st an t m u ta n t s in wh ich a t the s ame

t i m e t h e c o r r e ct f u n c t i o n i n g o f t h e R N A p o l y m e r a s e h a s b e e n a f f e c t e d po s es a n

i n t e re s t in g p r o b le m . R e c e n t l y s u c h m u t a n t s h a v e b e e n f o u n d i n ac tobac i l l u s case i

which were re s is t an t to r i famp ic in and in the s ame muta t io na l even t had deve loped

a u x o t r o p h y f o r g l u t am i n e76). A r i fampic in re s is t an t mu tan t o fE .

co l i

W has been

fou nd to con ta in an a l t e ra t ion in some pa rame te r s regu la t ing the a rg ln ine b iosyn-

the si s 77). Such p le io t rop ic e f fec t s cou ld be o f g rea t he lp in e luc ida t ing the d i f fe ren t

m odes o f the regu la t ion o f gene expre ss ion .

As wou ld be exp ec ted f r om the i r s imi la r m echan i sms o f ac t ion , r e s i st ance to

s tr e p to v a r ic i ns a n d t o l y p o m y c i n s d e ve lo p s in a w a y a n a lo g o u s t o t h a t f o u n d w i t h

the r i famy cins , an d c ross -res is tance is observed be tw een a l l thre e group s o f ant ib io-

tics78, 79)

So fa r no enzy m e has been fou nd th a t c an inac t iva te ansamyc ins e i the r by c leav -

age o r by chem ica l m od i f i ca t ion .

3 .2 .5 . O the r Ef fec t s on Bac te ri a

As has a l ready been m en t ion ed , na ph th om ye in inh ib i t s g rampos i t ive bac ter i a ,

a l t h o u g h i t d o e s n o t i n h i b it R N A p o l y m e r a s e . A n a n t a g o n is m b e t w e e n n a p h t h o m y -

c in and v i t amin K has been obse rved2 a) , b u t t h e u n d e r l y i n g m o d e o f a c t i o n i s n o t

k n o w n .

The tw o s t ruc tu ra l ly re l a t ed an t ib io t i c s s t rep to lyd ig in and t i r an dam yc in shou ld

b e m e n t i o n e d a t t h is p o i n t . T h e y d o n o t b e l o n g t o t h e a n s a m y c i n s, b u t s h o w s o m e

s tr ik ing s imi la r it ies in the i r ch em ica l s t ruc tu re (Fig . 10). S urpr is ingly eno ugh , these

two com pou nds a l so inh ib i t RN A po lymerase by b ind ing to th e 13 subun i t , a l thoug h

h i g he r d r u g c o n c e n t r a t io n s a re r e q u i re d a n d t h e ir m o d e o f a c t io n d i f fe r s f r o m t h a t

o f t h e a n s a m y c i n s6~ so -a2 ) . Gene t i c s tud ie s o f E .

eo l i

h a v e sh o w n t h a t t h e l o ci f o r

r i f ampic in and s t rep to lyd ig in re si s tance m ap ve ry c lose ly toge the r s a) . I t cou ld be

pos tu la t ed tha t the b ind ing s i te s fo r s t r ep to lyd ig in and r i f am pic in pa r t i a lly ove rl ap .

3 . 3. E f f e c ts o n E u k a r y o t e s

The r i f am yc ins , s t r ep tova r i c ins and to ly pom yc in s wh ich inh ib i t bac te r ia l RN A po ly -

m e r a se , d o n o t i n g e n er a l a f f e c t e u k a r y o t i c R N A p o l y m e r a se s , w h e t h e r t h e y a re o f

nuc lea r , mi tocho ndr ia l o r ch lo rop la s ti c o r ig in s2) . The an t ibac te r i a l a c t ion o f the se

ansam ycins resides in the recogn i t ion of a spec i f ic b indin g s i te on th e b ac te r ia l

enzy m e tha t i s absen t in euk a ryo t i c enzym es . Ce r t a in ansam yc ins such a s lipoph i li c

r i famyc in de riva tive s a t h igh c once n t ra t ions show some ac t iv i ty on a l arge va r i e ty o f

m amm al ian DNA and RN A po lymerase s a s we l l as on r i f ampic in - re si s tan t bac te r i a l

RN A po lymerase s2, 84 -87). Even enz ym es o the r than po lymerase s , such a s g lu tama te -

oxa loace ta te - t ransaminase (GO T) , g lu tama te -pyruva te -t ransaminase (GPT) and a lka -

l ine-phosphatases , and p olyp hen yla lan ine synthes is in a cel l- free sys te m are inhib i ted

to some ex ten t sT s s ). Th i s inh ib i t ion is due to non-spec i fi c b ind ing o f the d rug to

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Ansam ycins - Ch emistry Biosyn thesisand Biological Activity

t h es e e n z y m e s , w h e r e b y t h e n u m b e r o f d ru g m o l e c u le s b o u n d d e p e n d s o n t h e ir c o n -

c e n t r a t i o n a n d c a n a m o u n t t o s e v e ra l h u n d r e d s p e r m o l e c u l e o f t h e e n z y m e88).

S t ud i e s o f s ed i m en t a t i on and v i s cos it y show t h a t l i poph i li c r i f am yc i ns f o r m l a rge

aggr ega t es a t t he s am e co ncen t r a t i ons a s t hose l e ad i ng t o en zy m e i nh i b i t i on ( un -

p u b l i sh e d o b s e r v a t i o n ) . T h e s e d a t a c l e a r l y d e m o n s t r a t e t h a t t h e i n h i b i t o r y a c t i o n

o n t h e s e e n z y m e s c a n n o t b e r e l a te d t o t h e v e r y s p e c if ic i n t e r a c t i o n o f s o m e a n s a m y -

c in s w i t h b a c t e r ia l R N A p o l y m e r a s e , w h i c h o c c u r s a t 1 0 0 0 t o I 0 0 0 0 t i m e s lo w e r

con cen t r a t i ons and i n a ve r y w e l l de f i ned s t o i ch i o m e t r i c r a t i o o f 1 : 1 .

N eve r t he l e s s, und e r ce r t a i n cond i t i ons som e ansam yc i ns s eem t o a f f ec t t he

g r o w t h o f e u k a r y o t i c c ells . M e d o f f a n d c o l l a b o r a to r s89 9o), for ins ta nce , hav e

r e p o r t e d t h a t r i f a m p i c in t o g e t h e r w i t h a m p h o t e r i c i n B i n h i b it e d t h e g r o w t h o f c e r -

t a i n yeas t s such a s S. cerevisiae and C. albicans w h e r e a s n e i t h e r a n t i b i o t ic a l o n e h a d

a n y e f f e c t a t t h e c o n c e n t r a t i o n s t e s t e d . T h i s o b s e r v a t io n h a s b e e n c o n f i r m e d b y

o t h e r a u t h o r s w i t h a v a r i e t y o f d i f f e r e n t f u n g i 9 1 - 9 4 ) a n d e v e n w i t h m o u s e L - ce ll s9s).

T h e u n d e r l y i n g m e c h a n i sm o f a c t i o n i s r a t h e r p u z z li n g . O n o n e h a n d , t h e r e s ee m s t o

be som e i nd i ca t i on t ha t R N A syn t hes i s i s i nh i b i t ed , w h i ch ha s l ed t o t he conc l us i on

t h a t a m p h o t e r i c in B f a c i l it a te d t h e p e r m e a t i o n o f r i fa m p i c i n t h r o u g h t h e c e l l m e m -

br ane , a l low i ng r i fam p i c i n t o ac t on RN A syn t hes i s m u ch a s i t does i n b ac t e r i a 89).

O n t he o t he r hand , c a r e f u l s tud i e s have show n t ha t ne i t he r t he va r i ous nuc l ea r 96)

n o r m i to c h o n dr ia 1 9 7 ) R N A p o l y m e r a s e s o f y e a s t a r e i n h i b i t e d , e v e n b y v e r y h i g h

c o n c e n t r a t io n s o f r i fa m p i c i n .

Th e r e su l ts ob t a i n ed i n exp e r i m e n t s w i t h a f r ag il e Sac char om yces cerev is iae

m u t a n t a r e e v e n m o r e p u z z l in g ; i n c o n t r a s t t o t h e w i l d t y p e i t w a s f o u n d t o b e i n h i b -

i t e d b y r if a m p i c in a l o n e a n d e v e n s e em s t o c o n t a i n a n R N A p o l y m e r a s e s e ns it iv e t o

t he d r ug , w he r ea s t he ana l ogous enz ym e f r o m t he w i l d t yp e is r if am pi c i n - r e s is t an t 98).

S i m i la r re su l t s have bee n o b t a i ne d w i t h ve s i cu l ar s tom a t i t i s v ir u s, an R N A v i r us con -

r a in i n g a n e n d o g e n o u s R N A t ra n s c ri p ta s e . H e r e a g a in , b o t h t h e g r o w t h o f t h e m u t a n t

s t r a in an d i t s en zy m e a r e d r ug s ens it ive , bu t t he w i l d t y pe is d r ug r e s i s tan t 99 ). Fu r t he r

s t ud i e s a re need ed t o c l a r i f y t he s i t ua t i on , and i n th i s r e spec t de t e r m i na t i ons o f t he

r a t e o f m u t a t i on t o d r ug r e s is t ance m i gh t be o f g r ea t va lue .

V e r y i n t e r e s ti ng e f f ec t s on e uka r yo t i c c e l ls r e su l t a f t e r t r e a t m en t w i t h an sam y-

c in s c o n ta i n i n g a b e n z e n e c h r o m o p h o r e , e s p ec i a ll y w i t h c o m p o u n d s o f t h e m a y t a n -

s ine g r oup w h i ch a r e t he on l y ansam y c i ns f ound so fa r t o oc cu r i n p l an ts .

M a y t a n s i n e a n d s o m e o f i ts c o n g e n e r s a ff e c t t h e g r o w t h o f K B c e ll s a t c o n c e n -

t r a t i ons a s low a s 10 - 4 t o I 0 - s / l g / m l a2 ). Th ey i r reve r s i b ly inh i b i t c e l l d iv i s ion i n

t he eggs o f s ea u r ch i ns and c l am s , c aus ing t he d i s appea r ance o f t he m i t o t i c appa r a t us .

T h e y i n h i b it t h e p o l y m e r i z a t i o n o f t u b u l i n in vi tro 100) and thu s ac t s imi la r ly to the

v i nca a lka l o ids such a s v i nc ri s ti ne . Bac t e r ia l RN A po l ym er ase i s no t a f f ec t e d up t o a

con cen t r a t i on o f 60 / ~g / m l 1~ D esp i t e it s h i gh l y tox i c e f f ec t s on ce ll s in vi tro m a y -

t ans i ne and o t he r m ay t ans i no i ds a r e h i gh l y po t en t an t i t um our d r ugs w i t h a r e l a t i ve l y

l o w t o x i c i t y in r ive . Th e r ea son f o r t he i r spec if i c a c t i on r em a i ns unc l ea r , bu t d i f fe r -

e n c e s i n th e c e l l s u r fa c e a f f e c ti n g d r u g a tt a c h m e n t a n d p e r m e a t i o n m i g h t b e o f

i m p or t ance . S t r uc t u r e - ac t i v i t y s t ud ie s have revea l ed t he c r uc i a l r o l e o f a f r ee hy dr ox y

gr oup i n pos i t i on 9 and an e s t e r g r oup i n pos i t i on 335, 36 ) ( s ee F ig . 9 ) . May t ans i ne

and o t h e r m a y t ans i no i ds e s t e r if i ed a t C-3 have a po t e n t an t i l eukaem i c e f f ec t in t he

~ tg /k g d o s e ra n g e a n d a r e v e r y c y t o t o x i c , w h e r e a s c o m p o u n d s w i t h o u t a n e s t e r s u c h

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w. W ehtli

a s m a y t a n s i n o l , m a y s i n e , m a y s e n i n e a n d a ls o g e l d a n a m y c i n l a c k a n a n t i t u m o u r

e f f e c t a n d h a v e a c y t o t o x i c i t y 3 - 5 o r d e rs o f m a g n i t u d e l ow e r . C o n v e rs io n o f t h e

f r e e C - 9 h y d r o x y l g r o u p t o t h e C - 9 e t h y l e t h e r y i e l d s a n i n a c t iv e c o m p o u n d . A s t o

t h e m e c h a n i s m o f a c t i o n , it h a s b e e n p o s t u l a t e d t h a t a f t e r s p e c i f ic b i n d i n g t o t h e

t a r g e t p r o t e i n ( s ) , m a y t a n s i n e a c t s a s a n a l k y l a t i n g a g e n t o f f r e e th i o - o r a m i n o - g r o u p s ,

p r e f e r a b l y t h r o u g h C - 9 .

3 .4 . E f f e c t s o n R N A T u m o u r V i r u s es

O n c o g e n i c R N A v i ru s e s c o n t a i n a n R N A - d e p e n d e n t D N A p o l y m e r a s e (r e v e rs e t r a n -

s c r ip t a s e ) th a t is ch a r a c t e r is t i c o f t h i s g r o u p o f v i ru s e s a n d s e e m s t o b e o f i m p o r t a n c e

fo r v i ra l p ro l i f e r a t i on an d t he t r an s fo rm a t ion o f i ts hos t c e l l 1~ lO3). S ince RN A

t u m o u r v i ru s e s m i g h t b e i m p l i c a t e d i n s o m e h u m a n c a n c e r s , i n h i b i to r s o f re v e r se

t r a n s c r ip t a s e c o u l d b e v e r y v a l u a b le r e se a r c h t o o l s i n d e f in i n g t h e e x a c t f u n c t i o n o f

t h is e n z y m e i n v i r al ca r c in o g e n e s is a n d t h e y m i g h t e v e n b e o f c l in i c al v a lu e . T h e e a r l y

f ind ing t ha t c e r t a in l i poph i l ic r i f am yc in de r iva t i ve s i nh ib i t r eve r se t r ansc r ip t a se a nd

h a v e s o m e e f f e c t s o n t r a n s f o r m e d c e ll s 1 ~ 1 0s ) l e d t o g r e a t e f f o r t s b o t h t o s y n t h e s iz e

n e w a n s a m y c i n d e r iv a t iv e s a n d t o a n a l y s e t h e m i n a v a r ie t y o f b i o l o g ic a l s y s t e m s s s2,106)

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

s y n th e s iz e d , i n m o s t c a se s th r o u g h s u b s t i t u t io n o f t h e c h r o m o p h o r e . M a n y o f t h e m

i n h i b i t re v e r se t r a n s c ri p t a se t o s o m e e x t e n t , b u t n o n e o f t h e s u b s t a n c e s s o f a r t e s te d

a c t s i n a n a b s o l u t e l y se l e ct iv e w a y a n d a t c o n c e n t r a t i o n s c o m p a r a b l e t o t h o s e t h a t

i n h i b i t b a c t e r i a l R N A p o l y m e r a s e . I n f a c t , a s h a s a l r e a d y b e e n d i sc u s se d i n t h e p r e -

c e e d i n g s e c ti o n , m a n y o t h e r n u c l e i c a c id p o l y m e r a s e s o f b a c t e r i a l, e u k a r y o t i c a n d

v i r al o r i g in , a s we l l a s o the r e nzy m es such a s phosph a t a se s , a r e a l so i nh ib i t ed t o a

s imi l a r ex t e n t by t he se de r iva t ive s . Fo r a l l the se e nzy m es , inc lud ing r eve r se t r an -

s c r ip t a s e, t h e d r u g c o n c e n t r a t i o n s r e q u i r e d t o b r i n g a b o u t t h is e f f e c t a r e r e la t i v e ly

h i g h ( > 1 / a g [m l ) . A s in t h e c a se o f e u k a r y o t i c p o l y m e r a s e s t h e r e is n o c l e a r ly d e f i n e d

s t o i c h i o m e t r y o f b i n d i n g t o r e v e rs e t r a n s c r i p ta s e i n d e p e n d e n t o f t h e d r u g c o n c e n t r a -

t i on .

I t c a n t h e r e f o r e b e c o n c l u d e d t h a t i n h i b i ti o n o f r ev e r se t r a n s c r ip t a s e c a u s e d b y

a n s a m y c i n s w i t h a m o d i f i e d c h r o m o p h o r e i s n o t d u e t o a s t r o n g a n d d e f i n e d in t e r -

a c t io n b e t w e e n t h e d r u g a n d e n z y m e , b u t r a t h e r re s u lt s f r o m t h e w e a k b i n d in g o f a

v a r y i n g n u m b e r o f a g g r e g a te d d r u g m o l e c u l e s t o a r e l a t iv e l y u n d e f i n e d p r o t e i n r e g i o n

o c c u r r i n g in m a n y e n z y m e s . T h e r e i s l it t le c h a n c e t h a t f u r t h e r c h e m i c a l m o d i f i c a t i o n

o f t h e a n s a m y c i n c h r o m o p h o r e w i ll l e a d to c o m p o u n d s w i t h a s e le c ti v e a c t i v i t y o n

r e v er s e tr a n s c r i p ta s e a t l o w c o n c e n t r a t i o n s .

3 . 5 . E f f e c t s o n D N A V i r u s es a n d L a r g e r I n f e c t i o u s A g e n t s s u c h a s C h l a m y d i a e

M o s t s t u d i e s a b o u t t h e e f f e c t s o f a n s a m y c i n s o n D N A v i r u se s h a v e b e e n m a d e w i t h

vacc in i a v i ru s s l) . I t ha s bee n fo un d tha t som e de r iva t i ve s , such a s r if am p ic in , i nh ib i t

t h e g r o w t h o f t h i s vi ru s . T h e r e is n o d o u b t , h o w e v e r , t h a t t h i s i n h i b i t i o n i s n o t d u e

t o a b l o c k i n R N A s y n t h e s is , a s w a s f o u n d i n b a c t e ri a , b u t a p p a r e n t l y t h e a s s e m b l y

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Ansam ycins - Ch emistry Biosyn thesisand BiologicalActivity

o f i m m at u r e v i ru s pa r t i c le s is a f f ec t e d 1~176 A s in t he ca se o f t he RN A v i r uses ,

ve r y la r ge concen t r a t i ons o f an t i b i o t i c a r e needed t o i nh i b i t t he v i r u s g r ow t h . Thus ,

c l in ica l app l ica t ion i s no t p oss ib le .

[Boschke F.L.] Medicinal Chemistry

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