Inhibition of peptidyltransferase and possible mode of action of a dipeptidyl chloramphenicol analog

Vol. 122, No. 2, 1984

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 748-754

July 31, 1984

INHIBITION OF PEPTIDYLTRANSFERASEAND POSSIBLE MODEOF ACTION OF A DIPEPTIDYL CHLORAMPHENICOLANALOG Sara C. McFarlan and Robert Vince Department of Medicinal Chemistry, College of Pharmacy, Health Sciences Unit F, University of Minnesota, Minneapolis, Minnesota 55455 Received June 18, 1984

A dipeptidyl chloramphenicol analog, D-threo-2-(L-phenylalanylglycyl)amino3-~-nitrophenyl-l,3-propanediol, has been prepared and examined as an inhibitor of ribosomal peptidyltransferase. The analog is a more effective i n h i b i t o r of poly (U,C) directed protein biosynthesis in an Escherichia coli cell-free system than chloramphenicol and shows inhibitory a c t i v i t y equal to the parent antibiotic in the transpeptidation reaction, These results and the common structural features of puromycin and this compound suggest a model for the binding modes of chloramphenicol and chloramphenicol analogs. This proposal invokes four major binding pockets at the A-site of the peptidyltransferase center.

The antibiotic chloramphenicol is a potent i n h i b i t o r of bacterial protein biosynthesis.

Several studies have demonstrated that this anti-

biotic inhibits peptide bond formation catalyzed by the peptidyltransferase center of the ribosome by binding at, or close to, the A-site ( I ) .

Syn-

thetic derivatives of chloramphenicol, in which the dichloroacetyl group has been replaced by one of several aminoacyl groups, inhibited polyphenylalanine synthesis in a cell-free system from E. coli (2) and also inhibited the puromycin reaction (3).

These observations led to the proposal that

chloramphenicol and i t s aminoacyl derivatives i n h i b i t protein synthesis by acting as analogs of the 3'-terminus of aminoacyl-tRNA (3).

This pro-

posal requires that the dichloroacetyl moiety of the antibiotic simulate the amino acid moiety of puromycin.

A number of independent results

have suggested mutual sites of interaction for chloramphenicol and puromycin with ribosomes.

Equilibrium dialysis studies indicate that puro-

mycin can i n h i b i t chloramphenicol binding to ribosomes (4,5). 0006-291X/84 $1.50 Copyright © 1984byAcademic Press, Inc. Allrightsofreproductionin anyform reserved.

748

Studies

Vol. 122, No. 2, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

with aminoacyl oligonucleotides have shown that both a n t i b i o t i c s i n h i b i t the binding of C-A-C-C-A(Phe) to ribosomes (6).

Structural s i m i l a r i t i e s

between puromycin and the aminoacyl-adenyl terminus of aminoacyl-tRNA suggested the generally accepted postulate that puromycin binds to the acceptor site that is normally occupied by aminoacyl-tRNA (7).

Also, reconsti-

tution experiments with ribosomal proteins demonstrated that the ribosome contains a binding s i t e for chloramphenicol that is located at the acceptor site of the peptidyltansferase center (5).

However, attempts to relate

common structural features between chloramphenicol and puromycin and t h e i r binding modes have not been successful.

Hybrid derivatives of chlorampheni-

col and puromycin have shown limited biological a c t i v i t y (8-10).

Bhuta

et al. have suggested that chloramphenicol resembles a t r a n s i t i o n state for the peptidyltransferase catalyzed reaction of peptidyl-tRNA with aminoacyltRNA or puromycin ( I I ) .

In t h e i r proposal chloramphenicol is regarded as

a retro-inverso analog of the aromatic amino acid amide in puromycin.

How-

ever, the moderate to high i n h i b i t o r y a c t i v i t i e s of some of the aminoacyl chloramphenicol analogs cannot be explained by this proposal.

A theoreti-

cal study using computational techniques related chloramphenicol to ~he peptide backbone in peptidyl-tRNA bound to the P-site.

The major drawback to

this model is the lack of recognition that chloramphenicol is essentially an A-site i n h i b i t o r .

NO2

HO-C-H I

NO 2

O IJ

H-?-NH-C-CHCI 2

CH 2 OH

HO-C-H

O

O

I LI II H - ? - N H - C - C H 2 -NH-C-CH-NH2 i CH2OH CH 2

[~

/

~-'N,>~,N/

HO

W

NHOH

~;=o

CHLORAMPHENICOL

i

NH2 -C-El

1 PUROMYCIN

FIG.l:Structures of chloramphenicol, D-threo-2-(L-phenylalanylglycyl)amino-3-~nitrophenyl-l,3-propanediol (~), and puromycin. 749

Vol. 122, No. 2, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

In the present communication we present a highly active dipeptidyl analog of chloramphenicol

(Fig.l)

and suggest a hypothetical model for the

binding mode of chloramphenicol and chloramphenicol analogs at the A-site of the peptidyltransferase center of the ribosome.

MATERIALS AND METHODS Puromycin dihydrochloride was obtained from ICN Parmaceutical, I n c . , [14C]-L-phenylalanine was obtained from New England Nuclear, Escherichia coli cell paste (B, mid log) was purchased from General Biochemicals. The polynucleotides were obtained from Miles Laboratories, and ATP, GTP, phosphoenolpyruvate, and pyruvate kinase were purchased from Sigma~ Preparation of ribosomes, S-IO0, factors washable from ribosomes, and Ac-[l~C]-L-Phe tRNA was as previously described (12). Radioactive samples were counted in a Beckman l i q u i d s c i n t i l l a t i o n counter. The chloramphenicol analog, D-threo-2-(L-phenylalanylglycyl)amino-3p - n i t r o p h e n y l - l , 3 - p r o p a n e d i o l (~), was obtained as an o f f - w h i t e solid: spectra ( i n f r a r e d , u l t r a v i o l e t , and proton magnetic resonance), and elemental analysis (C,H,N) are consistent with the structure present in Fig. I. Details of the synthetic method w i l l be presented elsewhere. RESULTS AND DISCUSSION A dipeptidyl chloramphenicol analog ( I ) in which the dichloroacetyl group was replaced by a l-phenylalanylglycyl

moiety was evaluated as an

i n h i b i t o r of the peptidyltransferase reaction using puromycin as the substrate.

In this assay, a solution of puromycin and i n h i b i t o r was incubated

with a preformed complex consisting of E. coli ribosomes, poly (U), and N-acetyl-[14C]-L-phenylalanyl-tRNA. this reaction is presented in Fig. 2.

The e f f e c t of chloramphenicol and 1 on The i n h i b i t o r y a c t i v i t i e s

parent a n t i b i o t i c and the dipeptidyl analog are comparable. tion required for 50% i n h i b i t i o n

for the

The concentra-

is 3.2 X IO-6M in both cases.

The i n h i b i -

tion of poly (U,C) directed polyphenylalanine formation in an E, coli c e l l free system with washed ribosomes by chloramphenicol and 1 is presented in Fig. 3,

Examination of the data reveals that ~ i n h i b i t s protein biosynthesis

four times more e f f e c t i v e l y than the parent a n t i b i o t i c . The e x c e l l e n t i n h i b i t o r y a c t i v i t y of 1 in the two ribosomal assays and i t s structural

similarity

to puromycin suggest a possible model for the

binding mode of chloramphenicol and chloramphenicol analogs at the A-site of the peptidyltransferase center of the ribosome. 750

In this model (Fig. 4)

Vol. 122, No. 2~ 1 9 8 4

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

100

100 Chloramphemcol IC5o=3 2x 10-6 M

80

"

Chloramphenicol IC5o=3 5x10 -5 M

8O

x +o-+ .> ,( ae"

1_ IC5o=32X10 "6 M

60

60

40

a~

20

20 I

®

40

-7

-6

-5

-4

Log ['Inhibitor (M)~

-3

®

I

I

i

-7

-6

-5

i~e -4

-3

Log Dnh=bltor (M)-]

FIG.2:Inhibition of the Transpeptidation Reaction by chloramphenicol (©) and 1 ( 0 ) . The N-acetyl-[14C]-~-phenylalanyl-tRNA was bound to the E. coli rTbosomes in a reaction mixture containing 100 mM Tris-Cl (pH 7.5),--TOO-m-~ NH4CI (pH 7,5), 15 mMMgAc2, 0.65 mMd i t h i o t h r e i t o l , 3.20 A260 units of washed E. coli 70S ribosomes, 1.2 mMGTP, 0.5 ~mP/ml of poly (U), 136 ~g of factors--w--aThable from ribosomes, and 1.42 A260 units of N-acetyl-[14C]-L-phenylalanyltRNA (9.81 pmoles, sp. act. 527 pCi/pmole) in a total volume of-O.1 ml. The binding mixture was incubated at 28°C for 10 minutes and then placed in an ice bath. The peptidyltransferase reaction was initiated by the addition of 80 ~L of the incubation cocktail to 20 ~L of puromycin (0.1 mM final conc.) or a mixture of puromycin plus inhibitor. Reactions were incubated at 28°C for a specified time and stopped by the addition of 0.5 M NaOH(0.4 ml) to each tube. The tubes were incubated for an additional 30 minutes at 28°C; 1.0 ml of ethyl acetate was added to each tube and the mixture was vortexed vigorously for one minute. The tubes were centrifuged at 5000 rpm for 1.0 minute and 0.5 ml of the organic layer was removed and added to 10 ml of Triton-X-lOO:toluene-Permablend (1:2) liquid s c i n t i l l a t o r for counting. Counting efficiency was approximately 95%. All counts were corrected by blanks in which substrate was absent. All values represent an average of duplicate determinations. FIG.3:Inhibition of [14C]-~-Polyphenylalanine Formation by chloramphenicol (©) and 1 ( 0 ) . Reactions were performed in a final volume of 50 ~L and contained 80 mMTris-Cl (pH 8.0), 4.0 mM phosphoenolpyruvate, 0.8 mM ATP, 0.04 mMGTP, 40 mMKCI, 12 mM MgAc2, 1.94 mMd i t h i o e r y t h r i t o l , 18 ~L S-100 fraction (0.116 mg protein), I mg/ml tRNA, 29 ~g/ml pyruvate kinase, 0.5 ~Ci/ml of [14C]-~phenylalanine, 4.80 A260 units of E. coli ribosomes, and 2 pmR/ml of poly (U,C) (1.0:1.0). A solution was m~ew-h-ich contained all of the above components except poly (U,C) and the desired concentration of inhibitor. The reaction was initiated by the addition of the above solution to a mixture of poly (U,C) and inhibitor. The reaction was incubated for 15 minutes at 37°C and was terminated by the addition of 0.5 M NaOH(0.2 ml). Further incubation was continued for 15 minutes at 37°C followed by neutralization with 0.5 M HCI (0.2 ml). The incubation mixtures were placed in an ice bath, cold 10% trichloracetic acid (2.0 ml) was added, and the precipitate collected after 30 minutes on a Millipore f i l t e r (Type HA, 0.45 w). The f i l t e r s were washed with cold 5% trichloroacetic acid (3 ml) three times, dried, and placed in counting vials containing 10 ml of a toluene-Permablend liquid s c i n t i l l a t o r and counted in a Beckman LS 6800. Counting efficiency was approximately 95%. All counts were corrected by blanks in which poly (U,C) was absent. All values represent an average of duplicate determinations.

the secondary hydroxyl o f chloramphenicol simulates the amino function of puromycin or aminoacyl tRNA. The primary hydroxyl of chloramphenicol mimics the ribose 2'-hydroxyl of puromycin or aminoacyl-tRNA, and the p-nitrophenyl moiety binds at the same s i t e occupied by the aromatic side

751

Vol. 122, No. 2, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NH 2

o< N O--P=O

7

H2

? 0"7

,o o j..o i

O

II O PUROMYCIN

OH

I

-O--P:O

N

o

NH2

I

~

2 ~ C,~. / O "

..~ ~. lo)c o

2- ,.o:::2s.,.

"~ ~C~c~

OH

I I

./

\

~, o

O=C

AFTER TRANSFER

b (L)

FIG. 4:Binding of the C-C-A terminus of phenylalanyl t-RNA to the A-site of ribosomal peptidyltransferase: before transfer, the a-amine of phenylalanine binds to the site as i l l u s t r a t e d ; after transfer, a peptide bond is formed and the A-site accommodates the second amino acid. The chloramphenicol analog ( i ) orients i t s primary OH in the hydroxyl binding s i t e , the benzylic OH in the amino binding s i t e , and the other moieties overlap with corresponding binding sites as i l l u s t r a t e d . The relationship between puromycin, the aminoacyl t-RNA terminus, and the chloramphenicol analog is also i l l u s t r a t e d .

chain ot puromycin or aminoacyl-tRNA.

This proposal is consistent with

the p r e v i o u s l y observed requirements for A - s i t e binding:

I ) the 2 ' - h y d r o x y l

and free amino groups are e s s e n t i a l for the binding and i n h i b i t o r y a c t i v i t i e s o f puromycin (13);

2) ~-hydroxypuromycin, in which the NH2 has been

replaced by a OH, is a competitive i n h i b i t o r o f puromycin at the A - s i t e on ribosomes (14) and polysomes (15);

3) a puromycin analog with the E-methoxy-

phenyl group replaced by a p - n i t r o p h e n y l moiety p a r t i c i p a t e d in the ribosomal p e p t i d y l t r a n s f e r a s e reaction ( I 0 ) ; and 4) the presence o f a hydrophobic pocket in the A - s i t e t h a t can accommodate aromatic side chains o f puromycin or 3'-O-aminoacyladenosines has been suggested by several groups (16-20). These three A - s i t e binding areas can accommodate chloramphenico! by allowing the dichloroacetamido side chain to p r o j e c t i n t o the aminoacyl binding area.

Thus, the replacement o f the d i c h l o r o a c e t y l with the L - p h e n y l a l a n y l 752

Vol. 122, No. 2, 1 9 8 4

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

glycyl moiety represents a chloramphenicol analog (~) that can be further accommodated by those sites normally occupied by the second amino acid residue of, for example, diphenylalanyl-tRNA, prior to translocation to the P-site. Attempts to vary the distance between the phenylalanyl and the 2-aminol-~-nitrophenyl-l,3-propanediol ity.

moieties of ~ resulted in decreased activ-

For example, when the distance was shortened by removal of the glycyl

group, the corresponding analog, D-threo-2-(L-phenylalanyl)amino-3-p-nitrophenyl-l,3-propanediol,

was two orders of magnitude less active than 1 as

an i n h i b i t o r of polyphenylalanine synthesis.

Similarly, extended analogs

such as D-threo-2-(L-phenylalanyl-~-alanyl)amino-3-p-nitrophenyl-l,3-propanediol and D-threo-2-(L-phenylalanyl-¥-butyryl)amino-3-p--nitrophenyl-l,3propanediol exhibited similar decreases in a c t i v i t y .

These observations are

consistent with the idea that one glycyl group provides the correct distance for the proper orientation of the phenylalanine into the binding site depicted in Fig. 4.

Thus, the i n h i b i t o r y a c t i v i t y of ~may be explained by

i t s a b i l i t y to bind to several areas of the A-site of the peptidyltransferase center.

ACKNOWLEDGMENTS We thank Jay Brownell for assistance in the biological assays. This investigation was supported by Grants CA 13592 and CA 23263 from the National Cancer I n s t i t u t e , Department of Health, Education, and Welfare.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I0.

Vasquez, D. (1974) FEBS L e t t . , suppl. 40, 63-72. Coutsogeorgopoulos, C. (1966) Biochim. Biophys. Acta 129, 214-217. Coutsogeorgopoulos, C. (1967) Fifth Int. Cong. Chemother. 4, 371-378. Lessard, J . L . , and Pestka, S. (1972) J. Biol. Chem. 247, 6909-6912. Nierhaus, D., and Nierhaus, K.H. (1973) Proc. Natl. Acad. Sci. USA 70, 2224-2228. Pestka, S. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 395-410. Yarmolinsky, M., and de la Haba, G.L. (1959) Proc. Natl. Acad. Sci. USA 45, 1721-1729. Vince, R., Almquist, R.G., R i t t e r , C.L., and Daluge, S. (1975) Antimicrob. Agents Chemother. 8, 439-443. Verheyden, J.P.H., Wagner, D., and Moffatt, J.G. (1971) d. Org. Chem. 36, 25O-254. Lee, H.J., Fong, K.L.L., and Vince, R. (1981) J. Med. Chem. 24, 304-308. 753

Vol. 122, No. 2, 1 9 8 4 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS

Bhuta, P., Chung, H.L., Hwang, J., and Zemlicka, J. (1980) J. Med. Chem. 23, 1299-1305. Duquette, P.H., Ritter, C.L., and Vince, R. (1974) Biochemistry 13, 4855-4859. Nathans, D., and Neidle, A. (1963) Nature 197, 1076-1077. Fahnestock, S., Neumann, H., Shashoua, V., and Rich, A. (1970) Biochemistry 9, 2477-2483. Pestka, S., Vince, R., Daluge, S., and Harris, R. (1973) Antimicrob. Ag. Chemother. 4, 37-43. Harris, R.J., and Symons, R.H. (1973) Bioorg. Chem. 2, 286-292. Waller, J.P., Endos, T., Lemoine, F., Gutterman, S., and Sandrin E., (1966) Biochim. Biophys. Acta 119, 566-580. Cerna, J., Chladek, S., Rychlik, I . , and Zemlicka, J. (1970) Biochim. Biophys. Acta 199, 291-293. Raacke, I.D. (1971) Biochem. Biophys. Res. Comm. 43, 168-173. Fong, K.L.L., and Vince, R. (1978) J. Med. Chem. 21, 792-795.

754