Studies on binding of toromycin, an antitumor antibiotic, to DNA

Vol.

136,

May

14,

BIOCHEMICAL

No. 3, 1986

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Pages 885-890

1986

STUDIES ON BINDING OF TOROMYCIN, AN ANTITUMOR ANTIBIOTIC, Kazuo

1 Joho , Masakazf and Tikam Jain

1 9 Ken-ichi

Shishido

Uramoto',

Kiyoshi

1 Laboratory

of Natural Products Chemistry, Tokyo Institute Nagatsuta, Yokohama, Kanagawa 227, Japan 2Laboratory of Antibiotics, Riken Institute, Wako-shi, Saitama 351, Japan 3Research and Development Division, Smith Kline and French Laboratories, Philadelphia,

Received

March

TO DNA Isono',

of Technology,

PA19101

17, 1986

Toromycin, an antitumor, bactericidal and antiviral compound, was found to bind to DNA in such a way as to interfere with the dissociation of double helix at an elevated temperature. The antibiotic did not introduce strand scission into DNA. Single-strand-specific nuclease Sl-susceptibility of negatively supercoiled DNA was not influenced by its binding. The antibiotic was shown to bind to both of the alternating purine-pyrimidine copolymers, poly(dG-dC):poly(dG-dC) and poly(dA-dT):poly(dA-dT). The unique C-glycoside molecule of toromycin interacted with single-stranded DNA, but was found to have no affinity for RNA. D 1986 Academic Press, Inc. In 1971, from

Hatano

Streptomyces

reported Horii

et al.

the details et al.

reported

collinus

subsp.

of the

established

isolation

of the

et a1.(5)

an antibiotic,

gilvotanareus,

for

stereochemistry (one

of the

isolated

authors its

spectroscopic

Simplex (8)

from

unequivocally paper)

virus). V(4).

to interact

Nakano Gilvocarcin with

on the more detailed

and his

was evidenced

colleagues

also

double-stranded

and

Recently,

Jain

independently

species(designated

et al.(Z)

as AAC-324)

of chemical

and

and gilvocarcin

to be active

against

and DNA viruses(vaccinia reported

the antitumor

V are

DNA by intercalative of the binding

paper

we wish

the experimental

that

binds

to DNA in such a way as to interfere

Gram-positive

virus

and Herpes

activity

by Wei et a1.(7)

In this it

to report

have

size

Streptomyces

the structure

toromycin

V has been suggested characteristic

from

on the basis

that

the ring

and Takahashi

Fig.1).

mycoplasma et al.

for

analysis.

of Streptomyces

was shown by Hatano

mycobacteria,

established

crystallographic

group

named toromycin(2).

et a1.(4)

V, isolated

B21085

this

they

except

Nakano

and stereochemistry It

which

of toromycin gilvocarcin

a strain

structure

of the antibiotic Subsequently,

residues(3).

the same antibiotic(see

Toromycin

carcin

they

in this

data(6).

undoubtedly bacteria,

sugar

by means of X-ray

toromycin

and deduced

which

isolation

of B21085,

the structure

and stereochemistry described

the

albescens(1).

way.

of gilvo-

and Tomita We have

et al.

studied

of toromycin(gilvocarcin results with

which

lead

dissociation

V).

conclusion of double

0006-291X/86 885

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Copyright 0 1986 rights of reproduction

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by Academic Press. Inc. in any form reserved.

Vol.

136,

No. 3, 1986

8lOCHEMlCAL

a.1.

helix coiled

at an elevated

actinomycin-D, MATERIALS

Chemical

temperature.

DNA by the binding ethidium

AND

structure

change

is different

and adriamycin,

RESEARCH

COMMUNICATIONS

of toromycin.

Conformational

of toromycin bromide

BIOPHYSICAL

of negatively

from a typical

that

super-

by the binding

of

intercalator.

AND METHODS

Antibiotic. Toromycin was isolated and purified according to the method described in Ref. 6, dissolved in 67% dimethylsulfoxide containing 12% methanol and kept at -2O'C. Nucleic acids. Plasmid pTP-4(9), pNSl(10) and pBR322(11) were prepared by a standard method for obtaining a negatively supercoiled DNA. 1 DNA was prepared Alternating from Escherichia coli M65(hcI 85,S7) by the method given in Ref.12. purine-pyrimidine copolymers, poly(dG-dC) :poly(dG-dC) and poly(dA-dT):poly(dA-dT) were purchased from Pharmacia P-L Biochemicals. Single-stranded DNA of coliphage Ml3 was a generous gift from Mr. H. Kono of Tokyo Institute of Technology. Transfer RNA mixture was purchased from Wako Pure Chemical Industries Ltd., Japan and further purified by extensive extraction with phenol. All the nucleic acids were dissolved in TE buffer(10 mM Tris-HCl(pH 7.5)-0.5 mM EDTA) and kept at 4'C. Treatment of DNA with toromycin. Two ng of substrate DNA was incubated with the indicated amount of toromycin in 20 ~1 of TE buffer at 37'C for 15 min. Sl nuclease digestion. To the above reaction mixture(20 nl), one unit of Sl and sodium acetate buffer(pH 5.0), NaCl, &SO,. and MnCl, were added in 30 ~1 of total vol. to give a final cont. 45 mM, 70qmM, 1 mM bnd 1 mM, respectively. The incubation was performed at 37'C for 30 min. Sl nuclease provided by Sankyo Co., Japan was further purified as reported previously(l3), One unit of Sl activity is defined as the amount of the enzyme that converts 50% of 2 pg of single-stranded DNA to acid-soluble form under the above conditions. The Sl reaction was stopped by addition of 2.5 ~1 of 1 M Tris-HCl(pH 8.5) and 2.5 1~1 of 0.2 M EDTA. Agarose gel electrophoresis. Agarose horizontal slab gels(15 x 20 x 0.3 cm) were DreDared and DNA samnles, after addition of one tenth vol. of 0.05% bromphenol biue-80% glycerol,*were electrophoresed in Tris-acetate buffer(50 mM Tris-HCl, 20 mM sodium acetate, 18 mM NaCl and 2 mM EDTA, pH 8.2) under the conditions described in the legends to figures. The DNA bands were stained with ethidium bromide(EtBr), visualized using short wave-length UV light and then, photographed. Analysis of melting profile of DNA. About 10 pg of DNA was incubated without or with 1.25 pg of toromycin in 500 ~1 of TE buffer at 37°C for 15 min and change in OD26 was measured upon elevation of temperature at a rate of 1 deg. C/min in %eckman spectrophotometer DU-8 type. RESULTS AND DISCUSSION Fig.Z-a DNA treated

shows with

the agarose

various

amounts

gel

electrophoretic of toromycin. 886

patterns

of plasmid

The electrophoretic

pTP-4

mobility

Vol.

136,

No. 3, 1986

BIOCHEMICAL

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3.2. Agarose gel electrophoretic patterns of plasmid pT -4 DNA treated with toromycin(a) and Sl nuclease-digest of the toromycin-trea 9 ed pTP-4 DNA(b). In both panels, pTP-4 DNA was incubated with the following amounts of toromycin(M.W. 494). Lane 1, without the drug; 2, 1; 3, 2.5; 4. 10; 5, 25; 6, 100 ng of the drug. The reaction mixtures were adjusted to Sl-reaction buffer, and untreated or treated with Sl and then, subjected to electrophoresis. Electrophoresis was performed in 1% agarose gel at 3-4 V/cm for 2-4 hrs. Capital letters N, L and S are nicked, unit-length linear and supercoiled forms of native pTP-4 DNA, respectively.

of the DNA decreased cating

direct

the nicked strand

interaction DNA did

scissions

of partial result

not

increase

into

has been

also

obtained

reagents

EtBr

decrease

on the

opposite

unbasepaired mobilities Fig.4).

These

A similar Intercalating

known

to relax

reported

that

all of the

sites

seem to suggest

occur

the

the

mole-

depends

on

toromycin-bound

forms

the

In the figure, of pTP-4

decrease

the toromycin-bound DNA is partly due to the resulting from the binding of the antibiotic. 887

that,

influenced

by the binding

still exist even after the if the binding of toromycin

DNA to inhibit

effectively.

and linear

seems to show that

DNA is linear

DNA virtually

DNA was not

of supercoiled

does not

circular

a full-length

limited condition at which about oneThe results shown in Fg.Z-b demon-

the unbasepaired

data

relaxation

of the nicked This

the

of pTP-4

that

the nicked

yielding supercoiled

DNA was linearized.

indicating

sites

single-

Sl-susceptibility

of the DNA. Consequently,

Sl under

the Sl-susceptibility binding.

previously

and then,

at the nick

turns with

pTP-4

intercalative,

have

of

the possibility

V(7,8). are well

nuclease

of negatively

of superhelical

that

of gilvocarcin

sites

strand

DNA was digested

antibiotic is

into

Sl-susceptibility

strate

to introduce

suggested

DNA by toromycin.

and adriamycin et al.

the amount

superhelicity introduces localized unwinding of Sl can cleave on either one of the two DNA molecules.

once at the unbasepaired

of toromycin,

The figure

single-strand-specific

cleaved

of native

was shown not

Since

indi-

A negative

strands

the number

of toromycin,

DNA(7,8).

supercoiled

in the binding

base pairs

cule(l5).

drug

DNA. Shishido

DNA(14).

with

Fig.5).

of negatively

helical

half

, the

DNA(see

supercoiled

supercoiled

pTP-4

antibiotic

such as actinomycin-D,

negatively

of the concentration

of the

relaxation

reagents these

as a function

DNA also

formation the

electrophoretic

decreased(see

of the electrophoretic change

in overall

of the also

mobility charge

of DNA

of

Vol.

136,

BIOCHEMICAL

No. 3, 1986

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

.--O.&l -

2 8 0.50 -

obo-

a.?. Effect of toromycin on melting profiles of poly(dA-dT):poly(dA-dT)(a) and 7, DNA(b) upon elevation of temperature. ); toromycin-bound DNA(-------). Toromycin itself has an Native DNA(absorbance at 260 nm(b7% of that of DNA under the conditions used here), but it was constant over the range of temperature indicated. Thus, the absorbance of the samples containing toromycin was normalized to that of DNA alone.

Fig.3

shows

the effect

alternating

purine-pyrimidine

temperature

before

that

toromycin

indicating

on dissociation

and rapidly

chilled,

heating

that

seem to indicate

that

helix

upon heating. in TE buffer

the resulting helix

Fig.4

DNA sample

of the

linear

helix

The results

revealed

of the DNAs, inhibitory

effect

The unit-length at 80°C for

linear 10 min

1 and 1').

Under

this

DNA was tested.

double-stranded

Of

DNA remained

2 and 2' , and lanes

3 and 3').

is

by toromycin

in part

of

was electrophoresed.

toromycin-bound

cooling(lanes

by elevation

shows the

was observed(lanes

of the

double

of x DNA and

temperature

was heated

majority

and rapid

toromycin.

of double nicks)

of double

was the fact

with

in the melting helix.

the heat-denaturability

even after results

shift

of double

and then

dissociation

condition, interest

random

profiles

poly(dA-dT):poly(dA-dT)

the treatment

an upward

the stabilization

of toromycin

on melting

copolymer

and after

induces

pNS1 DNA(possessing Complete

of toromycin

combined

The

at 80°C in TE buffer. The effects of various DNA were examined to define pBR322 DNA was incubated with

the competitor

phoresed.

nucleic 1 and 2).

mers poly(dG-dC):poly(dG-dC) reincrease their

with

amount

and the

mobility

of toromycin

resulting

electrophoretic

3-7 and 8-12,

of the copolymers.

DNA samples

mobility

respectively), It

under

of pBR322 decreased

The additions of alternating and poly(dA-dT):poly(dA-dT)

of the decreased

amounts(lanes to both

a fixed acids

The electrophoretic

toromycin(lanes

drug

DNAs and tRNA on the binding of toromycin to plasmid the binding specificity of the drug(see Fig.5).

was moreover

888

upon

the coexistence were

electro-

the binding

purine-pyrimidine were reflected

of

copolyby a

of pBR322 as a function

indicating evidenced

the binding that

toromycin

of

of the also

Vol.

136,

No. 3, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

1 2 13 4

5 6 718

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9 10 11

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12 113 1L 15 1617h319

20 2122

Q.3. Agarose gel electrophoretic patterns of the toromycin-bound. linear pNS1 DNA before or after heat-denaturation. Unit-length linear pNS1 DNA possessing random nicks was prepared as follows. Thirty ug of supercoiled pNS1 DNA was digested with pancreatic DNase I(Sigma Chem. Co.) under the limited condition at which more than 80% of the DNA was converted to nicked form and the rest was linearized. The reaction mixture was extracted with phenol, precipitated with ethanol and dissolved in TE buffer. The DNase I-treated DNA was digested with restriction endonuclease -11 which cleaves pNSl DNA at a single unique site(l0). and extracted with phenol, precipitated with ethanol and dissolved in TE buffer. l&II was purchased from Takara Shuso Co., Japan and used according to the supplier's instruction. The unit-length linear pNS1 DNA obtained was incubated with the following amounts of toromycin. Lanes 1 and 1'. without the drug; 2 and 2', 50; 3 and 3'. 200 ng of the drug. The toromycin-bound DNA was subjected to electrophoresis before (lanes l-3) or after(1ane.s l'-3') heat-denaturation. The heat-denaturation and electrophoresis were done as in text and the legend to Fig.2, respectively. a.?. Influence of various nucleic acids on the binding of toromycin to plasmid pBR322. pBR322 DNA and the following amounts of competitor nucleic acids were incubated with 25 ng of toromycin and electrophoresed in 0.7% agarose gel under the conditions as in the legend to Fig.2. All of the lanes contained pBR322 DNA. In lanes 1, 7, 12. 17 and 22, the drug was omitted. Lanes 1 and 2, without competitor. Lanes 3, 0.2; 4, 0.5; 5, 1; 6, 2; 7, 2 ug of poly(dG-dC):poly(dG-dC). Lanes 8, 0.2; 9, 0.5; 10, 1; 11, 2; 12. 2 ug of poly(dA-dT):poly(dA-dT). Lanes 13, 0.4; 14, 1; 15, 2; 16, 4; 17, 4 ug of Ml3 DNA. Lanes 18, 0.4; 19, 1; 20, 2; 21, 4; 22, 4 Dg of tRNA. Capital letters N and S are nicked and supercoiled forms of native pBR322, respectively. The upper S is supercoiled form of native, dimeric pBR322. Majority of poly(dA-dT):poly(dA-dT) and all tRNA ran off the gels.

interacts drug

with

single-stranded

exhibited These

tivity

no interaction observations

for

secondary

DNA. However,

the

tRNA(lanes

seem to indicate structure

drug

DNA of coliphage with

are not

does exhibit

that

13-17).

the base-specificity

involved

a clear

M13(lanes

However,

the

18-22). and selec-

in the binding

of toromycin

deoxyribose-specificity

in its

to

binding. The mechanism vinylic,

lactonic,

of the binding of toromycin is not and phenolic moieties of toromycin, 889

clear. Structurally, in our opinion,

the are not

Vol.

136,

active

enough

The inhibitory is not

observed

suggests

that

linking

to make covalent effect

bond

of toromycin

at the temperature the possible

mechanism

intercalative further

BIOCHEMICAL

No. 3, 1986

examplified

BIOPHYSICAL

higher

than

of toromycin

more detailed

not

to DNA does not analysis

Fig.1).

of double-stranded

92'C(data

binding

COMMUNICATIONS

condition(see

on heat-denaturation

by mitomycin-C(l6,17), A

RESEARCH

at the experimental

mode of its

mechanism(7,8).

the mechanism

AND

but is

shown). involve

rather required

DNA

This cross-

suggests to probe

binding.

ACKNOWLEDGEMENTS The authors for providing Ml3 preparation of Sl assistance to the

are grateful DNA, Sankyo nuclease and analysis of

to Mr. Co. for Dr. K. melting

H. Kono of Tokyo Institute of Technology their help during the course of the Suzuki of Riken Institute for technical profile of DNA.

REFERENCES 1. Hatano,K., Higashide,E., Kameda,Y., Horii,S. and Shibata,M.(1971) Abstracts of Papers of Annual Meeting of the Agricultural Chemical Society of Japan, l-4 April, pp.363. 2. Hatano,K., Higashide,E., Shibata,M., Kameda,Y., Horii,S. and Mizuno,K.(1980) Agric. Biol. Chem. 64, 1157-1163. 3. Horii,S., Fukase,H., Mizuta,E., Hatano,K. and Mizuno,K.(1980) Chem. Pharm. Bull. 8, 3601-3611. 4. Nakano,H., Matsuda,Y., Ito,K., Ohkubo,S., Morimoto,M. and Tomita,F.(1981) J. Antibiotics 2, 266-270. 5. Takahashi,K., Yoshida,M., Tomita,F. and Shirahata,K.(1981) J. Antibiotics 2, 271-275. 6. Jain,T.C., Simolike,G.C. and Jackman,L.M.(1983) Tetrahedron 2, 599-605. 7. Wei,T.T., Byrne,K.M., Warnick-Pickle,D. and Greenstein,M.(1982) J. Antibiotics 35, 545-548. Takahashi,K. and Tamaoki,T.(1982) J. Antibiotics 35, 1038-1041. 8. Tomita,F., Sasatsu,M. and Hamashima,H.(1978) Microbios. Lett. 5, 55-59. 9. Kono,M., 10. Noguchi,N., Shishido,K., Ando,T. and Kono,M.(1983) Gene _?1, 105-110. 11. Bolivar,F., Rodrigues,R.L., Greene,P.J., Betlach,M.C., Heyneker,H.L., Boyer, H.W., Crosa,J.H. and Falkow, S.(1977) Gene 2, 95-105. 12. Goldberg,A.R. and Howe,M.(1969) Virology 2, 200-202. 13. Shishido,K.(1979) Agric. .Biol. Chem. 63, 1093-1102. 14. Shishido,K., Sakaguchi,R. and Nosoh,Y.(1984) Biochem. Biophys. Res. Comm. 124, 388-392. 15. Shishido,K. and Ando,T.(1982) Single-strand-specific nucleases(S.M. Linn & R.J. Roberts eds.) NUCLEASES pp.155-185, Cold Spring Harbor Laboratory. 16. Iyer,V.N. and Szybalski,W.(1963) Proc. Natl. Acad. Sci. U.S.A. 50, 355-362. 17. Iyer,V.N. and Szybalski,W.(1964) Science 3, 55-58.