Mechanism of the Antifungal Action of (S)-2-Amino-4-oxo-5

Chapter 39

Mechanism of the Antifungal Action of (S)-2Amino-4-oxo-5-hydroxypentanoic Acid, RI—331 Inhibition of Homoserine Dehydrogenase in Saccharomyces cerevisiae Downloaded by STANFORD UNIV GREEN LIBR on June 22, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0504.ch039

1

H. Yamaki, M. Yamaguchi, and H. Yamaguchi

Institute of Applied Microbiology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113 Tokyo, Japan An antifungal amino acid antibiotic, (S)2-amino -4-oxo-5-hydroxypentanoic acid (RI-331) preferentially inhibited protein biosynthesis in Saccharomyces cerevisiae, by inhibiting biosynthesis of the aspartate family of amino acids ( methionine, isoleucine and threonine ) . This inhibition was effected by impeding the biosynthesis of their common intermediate precursor homoserine. The target enzyme of RI-331 was homoserine dehydrogenase (EC.1.1.1.3). Since such enzyme activity is not present in animal c e l l s , the selective antifungal activity of the antibiotic is thus explained. The antibiotic was active against some plant pathogenic fungi, suggesting the p o s s i b i l i t y that the antibiotic is useful as antifungal agrochemicals.

In our program seaching f o r new a n t i f u n g a l a n t i b i o t i c s , we i s o l a t e d an a n t i f u n g a l amino a c i d a n t i b i o t i c , (S)2-amino4-oxo-5-hydroxypentanoic a c i d coded as RI-331 ( F i g . 1) from c u l t u r e b r o t h of Streptomyces s p . The a n t i b i o t i c has an i n h i b i t o r y e f f e c t a g a i n s t a wide range of yeasts i n c l u d i n g s e v e r a l pathogenic fungi of medical importance such as Candida a l b i c a n s and Cryptococcus neoformans. A l s o the a n t i b i o t i c was e f f e c t i v e i n t r e a t i n g murine systemic candidiasis being highly tolerated by experimental animals when given o r a l l y or i n t r a v e n o u s l y 1

Current address: Research Center for Medical Mycology, Teikyo University, 359 Otsuka, Hachioji City, Tokyo 192-03, Japan

0097-6156/92/0504-0428$06.00/0 © 1992 American Chemical Socifttv

In Synthesis and Chemistry of Agrochemicals III; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

39. YAMAKI ET AL.

(S)-2-AminO'4-oxo-5-hydmxypentanow Acid, RI-331

429

Downloaded by STANFORD UNIV GREEN LIBR on June 22, 2012 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0504.ch039

( 1 ) (Fig. 2 ) . The a n t i b i o t i c t h e r e f o r e i s l i k e l y promising f o r use against systemic c a d i d i a s i s i n humans. We a l s o described here the i n h i b i t o r y a c t i o n of the a n t i b i t o t i c against p l a n t pathogenic f u n g i . Our major concern was t o understand the biochemical basis of the a n t i f u n g a l a c t i o n of the a n t i b i o t i c . In an attempt to c l a r i f y the s e l e c t i v e a n t i f u n g a l a c t i o n , we explored the mechanism of a c t i o n of RI-331 using a s u s c e p t i b l e s t r a i n of Saccharomyces c e r e v i s i a e as an organism t e s t e d . P r e f e r e n t i a l I n h i b i t i o n of P r o t e i n Biosynthesis by RI-331 F i r s t we studied the e f f e c t of the a n t i b i o t i c on biosyntheses of DNA, RNA and p r o t e i n by growing yeast c e l l s . The a n t i b i o t i c p r e f e r e n t i a l l y i n h i b i t e d protein biosynthesis, whereas RNA and DNA biosythses were l e s s s u s c e p t i b l e t o RI-331 (Table I ) . Table I. E f f e c t of RI-331 on Incorporation of Radiolabeled Precursors into Protein, RNA and DNA i n Growing Saccharomyces c e r e v i s i a e

RI-331

(pg/ml)

0 15 150

RI-331

0 15 150

1+

[ C]Asn

17,264 (100) 5,796 ( 34) 1,407 ( 8)

(jug/ml)

14

[ C]Gln

16,773 (100) 5,282 ( 32) 1,032 ( 6)

3

[ H] adenine taken up i n t o RNA DNA

10,457 (100) 9,775 ( 93) 7,014 ( 67)

3,543 (100) 2,930 ( 83) 1,956 ( 55)

SOURCE: Reproduced with permission from r e f . 2. Copyright 1990. The data represent the incorporated r a d i o a c t i v i t y (dpm). Valuues i n bracket represent i n c o r p o r a t i o n as a percentage of the untreated c o n t r o l .



430

SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S III

HO - CH* - C - C H Ο

2

- CH - COOH NH

2

Figure 1. Chemical s t r u c t u r e of RI-331

100 m g / k g (p.o.) t w i c e daily

5 0 m g / k g (p.o.) t w i c e daily

Placebo 10

15

Days a f t e r i n f e c t i o n

Figure 2. Therapeutic e f f i c a c y of o r a l treatment regimens of RI-331 i n mice c a n d i d i a s i s . ( Adapted from ref. 1 ).


39. YAMAKI ET AL.

(S)-2-Amino-4-oxo-5-hydroxypentanoic Acid, RI-331

431


The p r o t e i n s y n t h s i s i n a c e l l - f r e e system prepared from the yeast c e l l s was however r e f r a c t o r y t o the a n t i b i o t i c . These r e s u l t s l e d us t o the p o s s i b i l i t y t h a t RI-331 acts not on the steps of amino a c i d polymerization on ribosomes, but blocks de novo b i o s y n t h e s i s of c e r t a i n amino a c i d (s). T h i s p o s s i b i l i t y was checked by analyzing the composition of i n t r a c e l l u l a r amino a c i d s of the yeast t r e a t e d with RI-331. I n h i b i t i o n of Biosynthesis of the Aspartate Family of Amino A c i d s . In RI-331 t r e a t e d c e l l s , the pool s i z e of threonine, methionine and i s o l e u c i n e were markedly reduced (Table II) . Table I I . Changes i n the Composition of Amino A c i d Pools Induced by RI-331 i n Saccharomyces c e r e v i s i a e

Amino a c i d

Gly Ala Val Leu He Met Cys Phe Tyr Pro His Ser Thr Glu Asp Lys Arg

C o n t r o l (C)

1 .499 5.665 1 .153 0.207 0.315 0.064 0.025 0.096 0.088 1 .075 65.274 1 .619 11 .217 52.523 4.579 2.885 42.149

+ RI-331 (R)

1 .139 12.237 12.949 0.597 0.015 0.007 0.017 0.151 0.939 0.381 33.158 14.804 1 .257 8.552 11.758 3.604 40.592

R/C

0.76 2.16 11 .23 2.88 0.05 0.11 0.68 1 .57 10.67 0.35 0.51 9.14 0.11 0.16 2.57 1 .25 0.96

SOURCE: Reprinted with permission from r e f . 2. Copyright 1990. The value of (C) and (R) represent the amounts o f i n t r a c e l l u l a r amino a c i d expressed as nmol/2x10 c e l l s without and with RI-331(15 pq/ml)-treatment, respectivel y . The value of R/C i n d i c a t e s the r a t i o of (R) t o (C). 8



432

SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S III

I n t e r e s t i n g l y , these three amino a c i d s , together with aspartate, are known t o be metabolized through the same metabolic pathway i n p r o t o t r o p h i c microorganisms (Fig. 3). Then, we asked i f the reduction of these amino a c i d s i n RI-331-treated yeast cells is responsible for the a n t i f u n g a l a c t i o n of the a n t i b i o t i c . To answer t h i s question, the a n t a g o n i s t i c e f f e c t of each amino a c i d on anti-Saccharomyces a c t i o n of RI-331 was tested. The a d d i t i o n of methionine, threonine, i s o l e u c i n e and t h e i r intermediate precursor homoserine to the chemically defined c u l t u r e medium markedly reversed the a n t i f u n g a l a c t i o n of RI-331. Homoserine showed a p a r t i c u l a r l y strong r e v e r s i n g e f f e c t (Table III) . Table I I I . The A n t a g o n i s t i c E f f e c t of Several Amino Acids on the Growth I n h i b i t o r y A c t i v i t y of RI-331 against Saccharomyces c e r e v i s i a e

Amino a c i d added

IC

None (control) Gly Ala Val Leu He Met Phe Pro Ser Thr Homoserine Asp Asn Glu Gin Arg Lys

5 0

of RI-331 (pg/ml)

Degree of reversion

1 .7 2.5 2.5 2.9 2.1 6.3 12.5

1.0 1 .5 1 .4 1 .7 1 .2 3.7

2.8

1 .6 1 .2

2.0 4.8

9.9 50.0 2.0 6.6 2.4

3.7 1 .9 1 .9

7.4

2.8 5.8 29.4

1.2 3.9 1 .4 2.2 1.1 1.1

SOURCE: Reproduced with permission from r e f . 2. Copyright 1990. The value of I C of RI-331 was determined on the b a s i s of the o p t i c a l d e n s i t y of c u l t u r e s . 5 0



39.

YAMAKI E T A L .

(S)'2-Amino--4-oxo-5-hydroxypentanoic Acid, RI-331

2-Ketobutyrate

F i g u r e 3. Biochemical pathway i n v o l v e d i n metabolism of the a s p a r t a t e f a m i l y of amino a c i d s i n p r o t o t r o p h i c fungi and the assumed s i t e of a c t i o n of R I - 3 3 1 . The marks are shown as f o l l o w s : • • — • G e n e r a l , • d e f e c t i v e i n mammals, and e s s e n t i a l amino a c i d s f o r mammals are boxed. A b b r e v a t i o n s : Hser, homoserine; AcHser, acetylhomoserine. ( Adapted from r e f . 2 ) .


433

434

SYNTHESIS A N D CHEMISTRY O F A G R O C H E M I C A L S III


These r e s u l t s t h e r e f o r e suggested that RI-331 acts on some step (s) involved i n the pathway converting aspartate t o homoserine. I n h i b i t i o n of Homoserine Dehydrogenase. In order t o i n v e s t i g a t e d e t a i l s of the t a r g e t s i t e of RI-331 a c t i o n , enzymatic studies were performed by using a c e l l - f r e e preparation from a s t r a i n of S. c e r e v i s i a e p r o t o t r o p h i c i n the pathway responsible f o r converting aspartate t o homoserine. In the enzymatic conversion of aspartate i n t o homoserine, 1 mole of ATP and 2 moles of NADPH are consumed (Fig. 4) . The rate of the r e a c t i o n depending on added aspartate and ATP can be monitored by the decrease i n absorbance a t 340 nm as NADPH disappears. The r a t e of NADPH consumption was s i g n i f i c a n t l y i n h i b i t e d by the a n t i b i o t i c , i n d i c a t i n g the i n h i b i t i o n of conversion of aspartate t o homoserine ( 3 ) . In the conversion o f aspartate t o homoserine three enzymes aspartate kinase, aspartate semialdehyde dehydrogenase and homoserine dehydrogenase are involved (Fig. 4) . To f u r t h e r examine the s e n s i t i v i t y of these enzymes t o the a n t i b i o t i c , we p u r i f i e d these enzymes using mutant s t r a i n s of S. c e r e v i s i a e , blocked i n the pathway from aspartate t o homoserine. Aspartate kinase was p u r i f i e d from the hom2 mutant, S2614C, ( lacking aspartate semialdehyde dehydrogenase ) by the method as described ( 4 ) . Aspartate semialdehyde dehydrogenase was p u r i f i e d from t h e hom6 mutant, STX25-2A, ( l a c k i n g homoserine dehydrogenase ), and homoserine dehydrogenase was p u r i f i e d from the hom2 mutant, S2614C as described ( 5,6 ) . The former two enzymes, aspartate kinase and aspartate semialdehyde dehydrogenase, were r e f r a c t o r y t o the a n t i b i o t i c , and the l a s t enzyme, homoserine dehydrogenase was s i g n i f i c a n t l y s e n s i t i v e t o RI-331. Homoserine dehydrogenase a c t i v i t y i n the forward r e a c t i o n , determined by NADPH dehydrogenation and dependent on the added substrate aspartate semialdehyde (ASA), was s i g n i f i c a n t l y i n h i b i t e d by the a n t i b i o t i c . The i n h i b i t i o n of the enzyme a c t i v i t y was enhanced i f NADP was added t o the r e a c t i o n mixture a t high concentrations r e l a t i v e t o NADPH ( F i g . 5), even although NADP i s not required i n the forward r e a c t i o n . The r a t e o f the forward r e a c t i o n of the c o n t r o l ( without RI-331 ) was not a f f e c t e d by a d d i t i o n of NADP. These r e s u l t s presumably i n d i c a t e that RI-331 i n a c t i v a t e s the enzyme by i n t e r a c t i n g with the enzyme-NADP complex. The i n h i b i t i o n was the mixed type of competitive and noncompetitive i n h i b i t i o n with respect t o ASA with a


39. YAMAKI ET AL


ÇOOH H?N-ÇH

Aspartate

ÇH2


COOH ATP

Aspartate kinase (EC.2.7.2.4) t ÇOOH

H2N-ÇH CH C-0-® 2

Aspartylphosphate

II

Aspartate semialdehyde dehydrogenase (EC. 1.2.1.1 1)

0 ^ -

COOH H2N-CH CH2 CH

NADPH

Aspartate semialdehyde

II

0 Homoserine dehydrogenase (EC.1.1.1.3)

71V

NAD(P)H

RI-331

ÇOOH H2N-ÇH ÇH2 C-CH2OH II

0 MET THR ILE

COOH H2N -CH ÇH2 CH2OH

Homoserine

F i g u r e 4 . B i o c h e m i c a l pathway from a s p a r t a t e t o homoserine, and t h e t a r g e t s i t e o f RI-331 ( Adapted from r e f . 3 ) .


435


436


K i value of 2 mM vs a Km value of 0.05 mM a t a p h y s i o l o g i c a l concentration of NADP (0.2 mM), and the r e a c t i o n was i n h i b i t e d a t high concentrations ( > 0.04 mM ) of the substrate ASA i t s e l f (Fig. 6) . The i n h i b i t i o n o f t h i s forward r e a c t i o n by the a n t i b i o t i c was noncompetitive with respect t o NADP, and t h e a s s o c i a t i o n constant f o r NADP i n the presence of the a n t i b i o t i c revealed two values of 0.4 and 2 mM as analyzed by Dixon-plot (Fig. 7) ( Yamaki, H., J . A n t i b i o t i c s , i n press ) , suggesting t h a t the enzyme might i n t e r a c t with the a n t i b i o t i c i n two ways t o give ternary complexes ( enzyme, NADP and a n t i b i o t i c ) of differing stabilities. Homoserine dehydrogenase was more s e n s i t i v e t o the a n t i b i o t i c i n the reverse r e a c t i o n than i n the forward r e a c t i o n . The i n h i b i t i o n was competitive with respect t o the substrate homoserine showing a K i value of 0.025 mM vs Km value of 17 mM ( F i g . 8) ( Yamaki, H., J . A n t i b i o t i c s , i n press ) . Moreover, p r i o r exposure of the enzyme t o NADP before s t a r t i n g the r e a c t i o n enhanced the extent of i n h i b i t i o n of the enzyme i n t h e reverse r e a c t i o n ( Yamaki, H., J . A n t i b i o t i c s , i n press ) . The p o s s i b i l i t y t h a t the enzyme-NADP complex i s formed f i r s t , then i n t e r a c t s with the a n t i b i o t i c , l e a d i n g t o i n a c t i v a t i o n o f the enzyme (Fig. 9) , can be supported by the high degree of i n h i b i t i o n of the enzyme by t h e a n t i b i o t i c i n the reverse r e a c t i o n i n which enzyme-NADP complex formation i s an e s s e n t i a l step of the r e a c t i o n ( F i g . 8 ) . The experimental data could be explained i f binding of NADP were t o induce a conformational change i n t h e enzyme which f a c i l i t a t e s binding of the a n t i b i o t i c , and causes an accumulation of the i n a c t i v e enzyme-NADP-RI-331 complex i n a n t i b i o t i c - t r e a t e d yeast cells resulting i n decreasing enzyme activity. Growth I n h i b i t i o n of Plant Pathogenic Fungi by RI-331. We found t h a t the a n t i b i o t i c markedly i n h i b i t s the growth of Cladosporium f u l v u s which i s a pathogen o f tomato l e a f mold disease ( F i g . 10), but not t h a t of C h r o l l e r a spp., suggesting that the a n t i b i o t i c selectively inhibits c e r t a i n pathogenic fungi, and i s p o s s i b l y u s e f u l as a n t i f u n g a l agrochemicals. Discussion. A l a r g e number o f amino a c i d analogs have been developed as antimetabolites so f a r , some o f which e x h i b i t a n t i -



39.

YAMAKI E T A L .

(S)-2-Amino-4-oxo--5-hydroxypentanoic Acid, RI-331

437

RI-331 ( m M )

Figure 5. Influence of NADP concentration on the i n h i b i t i o n of homoserine dehydrogenase a c t i v i t y i n the forward r e a c t i o n by RI-331. ( Reproduced from J . A n t i b i o t i c s 1992 45 i n press ) .

1.0 r

0

100

200

1/S(mM" ) 1

Figure 6. Lineweaver-Burg p l o t of the i n h i b i t i o n by RI-331 o f homoserine dehydrogenase i n the forward r e a c t i o n with respect t o the substrate, aspartate semialdehyde. ( Reproduced from J . A n t i b i o t i c s 1992 45 i n press ) .


438



0.4 U

NADP (mM) Figure 7. Dixon-plot of the i n h i b i t i o n by RI-331 o f homoserine dehydrogenase i n the forward r e a c t i o n with respect t o NADP. ( Reproduced from J . A n t i b i o t i c s 1992 45 i n press ) .

ο + RI-331 1 m M

1.0 1/V

0.5

— A + Rl-331 0.1 m M — · 0

0

ι 0.1

0.02 1

1/S(mM )

Figure 8. Lineweaver-Burg p l o t of the i n h i b i t i o n by RI-331 of homoserine dehydrogenase i n the reverse r e a c t i o n with respect t o the substrate, homoserine. ( Reproduced from J . A n t i b i o t i c s 1992 45 i n press ) .


39.

YAMAKI E T AL.



ASA

Figure 9. Scheme of enzyme-substrate complex i n homoserine dehydrogenase r e a c t i o n , and an assumed mode of i n t e r a c t i o n of RI-331 with enzyme. Abbrevations: Enz; homoserine dehydrogenase from S. c e r e v i s i a e , ASA; aspartate semialdehyde, HS; homoserine, (F); forward r e a c t i o n , (R) ; reverse r e a c t i o n . ( Reproduced from J . A n t i b i o t i c s 1992 45 i n press ) .


439


440

SYNTHESIS A N D C H E M I S T R Y OF A G R O C H E M I C A L S I I I

1

1 0

RI-331

1 00

fog/

1000

disk)

F i g u r e 10. The growth i n h i b i t i o n of Cladosporium f u l v u s by RI-331. C. f u l v u s was c u l t u r e d i n agar p l a t e o f Y N B w/o amino a c i d medium and the i n h i b i t i o n zone was detected by paper-disk assay.



39. YAMAKI ET AL.

(S)-2-Amim-4-oxo-5-hydroxypentanoic Acid, RI-331

441

fungal a c t i v i t y . The majority of e f f e c t i v e compounds have been chemically synthesized, although some were n a t u r a l products, i s o l a t e d e i t h e r as a n t i b i o t i c s or substances t o x i c t o animals ( 7,8 ) . We are able t o f i n d out the i n h i b i t o r of b i o s y n t h e s i s of e s s e n t i a l amino acids among the compounds reported p r e v i o u s l y . We are i n t e r e s t e d i n whether the i n h i b i t o r s of homoserine dehydrogenase from E s c h e r i c h i a c o l i which have been reported p r e v i o u s l y , 2-amino-4-oxo-5-chloropentanoate ( 9 ) and β -hydroxyn o r v a l i n e ( 110 ) , could be u s e f u l f o r a n t i f u n g a l agent. I n h i b i t o r of the b i o s y n t h e s i s of e s s e n t i a l amino a c i d s f o r animals other than threonine, i s o l e u c i n e and methionine i s a l s o of our i n t e r s t , asking whether such i n h i b i t o r could be u s e f u l f o r a n t i f u n g a l chemotherapy or as a n t i f u n g a l agrochemicals. Also I t should be worthy of searching f o r a n t i f u n g a l agrochemicals out of a n t i f u n g a l agents of medical use. Acknowledgments We are indebted t o Drs. S. Omura, T. Nagate, H. Fukushima, and C. Yokoo, Taisho Pharmaceutical Co., L t d . , Ohmiya, Saitama Prefecture, Japan, and Dr. H. S a i t o , Teikyo U n i v e r s i t y , H a c h i o j i , Tokyo, Japan f o r t h e i r valuable d i s c u s s i o n of t h i s work. T h i s work was supported by a Japanese A n t i b i o t i c s Research A s s o c i a t i o n Research Grant. Literature Cited. 1. Yamaguchi,H.; Uchida,K., H i r a t a n i , T . , Nagate,T., Watanabe,N. & Omura,S. Ann.N.Y.Acad.Sci.1988,544, 188-190. 2. Yamaguchi,M.; Yamaki,H., Shinoda,T., Tago,Y., Suzuki,H., Nishimura,Τ. & Yamaguchi,Η. J . A n t i b i o t i c s 1990,43,411-416. 3. Yamaki,H.; Yamaguchi,Μ., Imamura,H., Suzuki,H., Nishimura,Τ., Saito,Η. & Yamaguchi,Η. Biochem.Biophys. Res.Commun.1990,168,837-843. 4. Black,S.; Wright,N.G. J.Biol.Chem.1955,213,27-38. 5. Black,S.; Wright,N.G. J.Biol.Chem.1955,213,39-50. 6. Black,S.; Wright,N.G. J.Biol.Chem.1955,213,51-60. 7. Shive,W.; Skinner,C.G. In Amino a c i d analogues; Hochster,R.M. & Quastel,J.H.,Ed.,Metabolic I n h i b i t o r s ; Academic Press: New York,1963, V o l . 1; pp 1-58.


442

SYNTHESIS

AND

CHEMISTRY OF AGROCHEMICALS


8. Fowden,L.; Lewis,D. & H. T r i s t r a m : In Toxic amino a c i d s . Their a c t i o n as antimetabolites; In Advances i n Enzymology; Nord,F.F., Ed.; Interscience P u b l i s h e r s : New York,1967, V o l . 29; pp 89-163. 9. Hirth,C.G.; Veron,M., V i l l a r - P a r a s i , C . , Hurion,N. & Cohen,G.N. Eur.J.Biochem.1975,50,425-430. 10. Cohen,G.N.; Patte,J-C., Truffa-Bachi,Ρ., Sawas,C. & Doudoroff,M. Mecanismes de r e g u l a t i o n des a c t i v i t e s cellulaires chez l e s microorganismes; Colloques Intern.C.N.R.S.: Paris,1965; V o l . 124, pp 243-258. R E C E I V E D April 27,

1992


III

Mechanism of the Antifungal Action of (S)-2-Amino-4-oxo-5

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