Vol. 67, No. 1, 1975

BIOCHEMICAL

POTENT INHIBITION

AND BIOPHYSICAL

OF ORNITHINE DECARBOXYLASE BY S,y UNSATURATED SUBSTRATE ANALOGS NoSl

Relyea

and Robert

Department

Received

RESEARCH COMMUNICATIONS

September

R. Rando

of Pharmacology

Harvard

Medical

School

Boston,

Massachusetts

02115

11,1975 Summary

Trans-3-dehydro-D, L-ornithine and trans-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of omithine decarbThe KI'S for trans-3-dehydro-L-omithine and trans-1, 4-diamino-2oxylase. butene are 2.2 and 2.0 pM respectively. Both analogs bind much more tightly to the enzyme than either omithine or putrescine. -Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of trans-3-dehydro-D, L-ornithine to the culture medium. The frequent and growth the

has been well

first

marked

step

its

considerable might

of this

reported

to

find

a very

to

test

the

is

a very strong

having

of

by this

by addition is

inhibitor,

Previously,

although it

was found

inhibition

more of the

omithine

Other to a lesser that

than rat

of this

3 times liver

enzyme

(2). 5),

PLP-requiring

(3) be of

cell

two have

growth.

been

o-Methyl

omithine

for

5'-phosphate,

would

enzyme.

that

(4,

stimuli

in rapid

prostate

and

enzyme which

only

of this of rat

it

Thus,

so far

catalyzes

growth

synthesis

decarboxylase

of pyridoxal

questionable.

enzyme.

tested,

division

The rapid

and other

of polyamine been

cell

decarboxylase

inhibitor

inhibitor

an affinity

inhibition

inhibitor

role

rapid

organisms.

hormonal

specific

have

competitive

inhibitor

can be reversed this

which

with

Omithine

as a regulatory

specific

weak

2).

to various

interest

a fair

synthesis

in eukaryotic

importance

is (4),

shows

enzyme

many compounds

oxylase

(1,

synthesis

to show significant

L-omithine

only

documented

possible

be used --in vivo Of the

of polyamine

in polyamine

response

suggests

correlation

D,

decarb-

L-omithine,

but

it

a-Hydrazino-omithine but

so the enzymes

this

inhibition

specificity are

also

of affected

extent.

S,v unsaturated

amino

acids

can be profound

BIOCHEMICAL

Vol. 67, No. 1, 1975

and irreversible This

inhibitors

report

describes

inhibitors these

of certain

the

of pyridoxal

synthesis

linked

appears

promising

the

physiological

roles

decarboxylase

injected

with

of this

specific

activity

oxylase

activity 14

L-omithine-Ilradiochemicals Pig

kidney

diamine

al.

(10).

oxidase

to

(7).

rats

which

This

- mg protein.

had been

enzyme had a

Omithine

decarb-

and Williams-Ashman

New England

Nuclear,

(8).

as were

all

D,

other

method

et al. 40% the

transferase

of Weber were

for

by Weber

et al

(14).

prepared

Trans-3-dehydro-D,

will

this

enzyme this

of yeast

as described

homogenates

one reported

from

Sigma

Chemical

Co.;

activity

was prepared

was assayed enzymatic

by the

as described

method

assay

seen

with

for

decarboxylase,

The 1, 4 diamino-trans-2-butene

activation

of

by Janne

as a micro

S-adenosyl-L-methionine

(12).

method

was

putrescine

over

a

range.

carbamyl

prepared

used was that

activator

to produce

linear

L-Omithine

by the

studies

(9).

The use of

by Harik

and found

properties

by Ono et al.

decarboxylase of

(11).

a potent

considerable

the

of

was obtained 14CJ

Activity

has been described

liver

one of

polyamines.

livers

by Janne

from

S-adenosyl-L-methionine

putrescine,

liver

--in vivo

and the

the

CO /min 2

as described

cadaverine-[

and Williams-Ashman

tested

from

nmoles

C] was purchased

using

Yeast et

unsaturated

used.

was measured

Janne

$,v

(6).

and Methods

- 0.06

was assayed

enzymes

decarboxylase;

enzyme

as described

of 0.04

linked

of several

use in further

was prepared

thioacetamide,

phosphate

omithine

for

Materials Omithine

pyridoxal

RESEARCH COMMUNICATIONS

and interactions

phosphate

inhibitors

delineate

AND BIOPHYSICAL

glycine

be reported of Karlen

in

and Singhal

(13).

For L-omithine

and assayed

L-omithine vinyl

was assayed

elsewhere.

Complete Trans

(17).

393

homogenates The assay

&-transaminase,

as described

was synthesized (16).

crude

by Peraino

by a procedure details

of its

1, 4-diamino-2-butene

of

rat

procedure crude

rat

and Pitot similar synthesis

(15). to and

was prepared

Vol. 67, No. 1, 1975

BIOCHEMICAL

I -10

0

AND BIOPHYSICAL

IO

20 y[L-

30

RESEARCH COMMUNICATIONS

40

Ornithine]

I 60

50

(mM)

The 1 ml assay mixtures each consisted Inhibition by dehydro-ornithine. Fig. 1. of 0.3 units (1 unit = 1 nmole of CO fmin) of enzyme in 0.035 M phosphate buffer, pH 7, containing 0.18 mM PLP and 7 mzi dithiothreitol. The concentrations of Samples were incubated L-omithine-[carboxyl-14C] were varied as indicated. of citric acid. The enzyme for 60 min at 37", then stopped by injection ) is shown, as well as identical samples containing 5 pM (H), standard (n The velocity (v) is 10 pM (M), and 20 uM (o---o ) dehydro-omithine. expressed as nmoles CO2 per 60 min per mg protein.

Results Inhibition

of

Ornithine

Decarboxylase

Trans-3-dehydro-D, is

an effective

competive

was 4.4

of

it

for

the

100 times

pure

(henceforth

inhibitor

of

inhibition

The KI measured enzyme,

is

L-isomer

greater

by trans.-3-Dehydro-D,

L-omithine

substrate-competitive

this

and Discussion

than

rat

observed

pM.

However,

with

in view

that

only

is

pM.

The enzyme

2.2

that

for

the

and thorough

completely

restored

activity.

not

inactivation

competitive

phosphate

can be ruled

with

pyridoxal

concentrations

analog

the

is

affinity

for.this

dialysis

(Fig.

characteristically

2). resulted

The

shown in

Fig.

1.

the

KI

stereospecificity

active,

of

and that analog

is

about

was observed the

the

The inhibition

phosphate

is

strict

Therefore,

out.

decarboxylase.

No time-course

by dehydro-ornithine,

versible

this of

L-omithine.

original

to as dehydro-omithine)

omithine

L-isomer

inhibition

its

referred

liver

likely

L-Ornithine

for

inhibited

enzyme

possibility

of irre-

by dehydro-omithine In fact, in

high

an additional

is

pyridoxal slight

inhibition. Is Dehydro-Omithine The question

a Substrate of

the

possible

for

Omithine

decarboxylation

394

Decarboxylase? of dehydro-omithine

is

of

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

6

‘1”

4

2

1

I

2

4

‘/[PLP] Fig. 2. Competition of pyridoxal phosphate Assay mixtures were similar to those in Fig. ornithine-1-14C, while concentrations of PLP enzyme (o-o), and the sample which contained (m) were slightly inhibited by the excess inhibition by 10 uM 1, 4-diamino-trans-2-butene and 1.0 mM propargylamine 2-butane CM>, PLP.

Table

1

Limits

of possible

electrophoretic isolation

5 nmoles

dismine oxidase inhibition S-adeno sylmethionine decarboxylase activation

**Based

on total

***Based

that

for

75

nmoles

0.5

nmoles

200 nmoles

0.1

nmoles

40 nmoles

* Minimum amount of under experimental

that

of L-isomer rate

Estimated diamine formation*** 0.75

% of substrate activity excluded

nmoles

670%

3.0 nmoles

1.5

1, 4-diamino-trans-2-butene conditions employed.

conversion

on assumption ornithine.

of trans-3-dehydro-ornithine

Maximum possible 1,4-diamino-trans2-butene**

Detection limit*

Method

decarboxylation

(PLP) with various inhibitors. 1, but each contained 0.1 mM Lwere varied. Both the standard 20 UM trans-Zkdehydro-omithine PLP. On the other hand, the (H), 0.5 mM 1, 4-dismino(w) were reversed by excess

nmoles

shown of

of analog

395

17%

7%

to be clearly

trans-3-dehydro-omithine decarboxylation

detectable to diamine.

is

identical

with

Vol. 67, No. 1, 1975

interest

because

the

butene,

is

several

approaches

are

BIOCHEMICAL

also

a highly

along

any diamine

establish

that

of

with

product

methods

substrate

analog,

it

substrate

decarboxylation.

butene

occurs

at

removed

by gel

Inhibition Despite putrescine

strongly

the

very

(7),

1,

weak

the

this

inhibition

very

high

as shown

this

is

with

the

amine

the

to

the

to inhibition. of this

normal

as a sensitive found

assay

that

for

dog kidney

1, 4-diamino-trans-z-

inhibition,

in both

cases,

irreversible

appeared

component

to

(not

is

proved

in Figure

is this

The KI measured more

It

unsaturated

diamine

is

5'-phosphate.

10,000

due,

is not

the

complete

with

(2 mM) of pyridoxal good

than

likely

likely results

from These

inhibitors.

396

it

for

Although

can be reversed 2).

cases

potent

inhibition

prior

complex

complexes

are

is

1,

shows also

reversed

observed

formation thought

by

Also,

propargylamine

in these the

that

to a preferential

(Fig.

and even

that

experi-

explanation.

5'-phosphate

observed

times

by

potent

in these

in part,

substrate,

inhibitor,

The inhibition

and pyridoxal

3.

decarboxylase

to be an extremely

very

competitive

5'-phosphate.

competitive

of omithine

an affinity

a fairly

4-Diamino-2-Butene

inhibition

difference

strongly

inhibition.

here

indicates

trans-conformer,

4-diamino-2-butyne

by pyridoxal

Failure

observed

of

We have

by trans-1,

competitive

concentrations

significant

the

10% that

by both

4-diamino-trans-2-butene

this

While of

I.

was sufficient

of

was used

and a relatively

approaches

any decarboxylation

than

inhibited

However,

These

in Table

basis

be mentioned.

Decarboxylase

inhibitor,

was 2 pM;

binding

formation.

is

which

enzyme.

filtration). Omithine

putrescine.

less

This

a competitive

of

competitive

actual

should

the

1, 4-diamino-2-

the

isolation

there

oxidase

and 1, 4-diamino-2-butyne. of both

ments

if

a rate

of diamine

of

of detection,

was not

show that

was very

consist

any product

by electrophoretic

1, 4-diamino-trans-2-butene oxidase

trans

inhibitor

limits

inhibition

The two enzymatic

diamine

competitive

their

RESEARCH COMMUNICATIONS

decarboxylation,

to measure

formation

The inhibition

this

potent

failed

summarized,

detect

product

AND BIOPHYSICAL

between

to be the

Vol. 67, No. 1,1975

BIOCHEMICAL

AND BIOPHYSICAL

‘/[v 0 rnithine]

RESEARCH COMMUNICATIONS

(mM)

Fig. 3. Inhibition by 1, 4-diamino-trans-2-butene. These inhibition studies The standard enzyme were done under the conditions described in Fig. 1. and 10 PM (M), 1, 4-diamino(n), and samples containing 5 nM (o-0) trans-2-butene.

Effect

of

Dehydro-Omithine

A series L-omithine

of experiments on cultured

be noted

that

these

blasts).

This

known

to be relatively

then

until

oxylase from for

source

hatching, from

rat

liver

high

chick

both

analog

butene addition

L-omithine Also,

4-diamino-2-butyne

cultures,

readily

diffuse

a published in or out

report of

cells

cells.

in muscle

(18).

pectoral

muscle

for

substrate

there

was inhibition

which

was reversed

3 to 11,

(19),

fibrois

declining

from decarb-

was found

to be similar

(Kg=0.15

mM) and its

to that affinity

inhibition

by

by 1, It-diamino-trans-2by high

shown that

so this

of

The ornithine

PLP.

had no observable has since

should

decarboxylase

No irreversible

nM).

(It

numbers

omithine days

of 1 mM 1, 4-diamino-trans-2-butene

muscle

muscle

from

(KI=2.8

of trans-3-dehydro-D,

significant since

embryo

affinity

effect

pectoral

suitable

is highest

in its

the

contained

in chick

was apparent.

and 1,

also

embryo

test

embryo

especially

and it

ll-day

chick

preparations is

Culture

was done to

H-day

trans-3-dehydro-D,

this

the

on Muscle

analog

Although

effect

putrescine probably

on the does not

did not

enter

cells. FIhen concentrations

as low

as 60 ~.IM trans-3-dehydro-D,

397

L-omithine

(30 UM

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

398

RESEARCH COMMUNICATIONS

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

399

RESEARCH COMMUNICATIONS

Vol. 67, No. 1, 1975

BIOCHEMICAL

AND BIOPHYSICAL

400

RESEARCH COMMUNICATIONS

Vol. 67, No. 1, 1975

L-analogue) effect

were

cells,

As shown fold

within

excess

4,

(with

concentrations

the

levels

in these

Removal

considerable Assays

recovery of

rat

showed

inhibited both

of

attempts

the

these

cells

20).

Thus,

Table

II

Inhibition

it

is

carbamyl

of

omithinecarbamyl transferase

1.0

omithine-dtransaminase

7.0 mM (13)

omithine decarboxylase

0.2

mN

(12)

mM

L-omithine

higher

not

be used

amounts medium

for

estimation of material resulted

in

48 hrs.

would

effects

of omithine-metabolizing

KM for Lomithine

Unfortunately,

fresh

and omithine be likely

low

a-trans-

to be significantly used

to be extremely that

inhibitor. of a 40-

trans-3-dehydro-omithine

unlikely

The differ-

by addition

small

transferase

enzymes

are known

highly

about

fewer

with

an accurate

due to the

within

of these

enzymes

to achieve

with

4).

incubation

and so could

by replacement

a distinct had both

(Fig.

somewhat

toxic

cells,

omithine

neither

continued

failed

cultured

concentrations

(15,

Enzyme (rat liver)

liver

that

by the

of

cultures

of L-omithine.

somewhat

of inhibitor

the muscles,

cell

was reversed

were

RESEARCH COMMUNICATIONS

fibers

with

to L-isomer)

Several

available.

aminase

inhibition

omithine

of

differentiated

more striking

reversal.

diamine

plating

The inhibited

fewer

respect

of

AND BIOPHYSICAL

after

48 hrs.

progressively

in Fig.

further

at 3 - 5 hrs

and particularly

ence became

Also,

added

was apparent

total

of

BIOCHEMICAL

or absent

on these

other

(Table

2).

in muscle ornithine-

enzymes

trans-3-dehydroD, L-omithine

Ratio of omfanalog

% inhibition

30 mM

3mM

10

10

100 mM

3mM

33

15

50

50

10 mM

0.2

mM

Fig. 4. Reversible inhibition of muscle cell cultures by trans-3-dehydroL-omithine. Photos 1 a-f show the muscle cells on the 6th day in culture; a - control, b - 1.3 mM L-omithine, c - 60 nM trans-3-dehydro-D, L-omithine d - 60 uM analog + 1.3 mM L-ornithine, e - 120 I.~Manalog, f - 120 (analog), i.lM analog + 1.3 mM L-omithine. Photos 2 a-f show these same six plates 4 days after removal of inhibitor and omithine from medium (10th day in culture).

401

Vol. 67, No. 1, 1975

metabolizing

enzymes

results

are all

fusion

mediated

carboxylase further tool

are

AND BIOPHYSICAL

responsible

for

consistent

with

in some manner

the

the

study

it

of the

by the

reversible

role

of

that the

RESEARCH COMMUNICATIONS

cellular

inhibition

While

seems likely

elusive

observed

reversible

by trans-3-dehydro-omithine. investigation,

for

BIOCHEMICAL

of division

inhibition these

results

in cell

clearly will

The and/or

of ornithine

dehydro-omithine polyamines

inhibition.

derequire

be a fruitful

growth.

Acknowledgements This Career

work

was supported

Development

Award

by N.I.H. GM 00014

grant

number

GM 20366

and by a Research

to R. Rando.

References 1.

Cohen, S.S. (1971), Introduction to the Polyamines, Prentice-Hall, New Jersey. 2. Russell, D.H. (1973), Polyamines in Normal and Neoplastic Growth, Raven Press, New York. 3. Morris, D.R. and Fillingame, R.H., (1974), Ann. Rev. in Biochem., 303-325. 4. Abdel-Monem, M.M., Newton, N.E. and Weeks, C.E. (1974), J. of Med. Chem., 17, 447-451. Polyamines in Normal 5. Harik, S.I., Pasternak, B.W. and Snyder, S.H., (1973), and Neoplastic Growth (ed. by Diane H. Russell), Raven Press, New York, 307-321. 6. Rando, R.R., (1974), Science, 185, 320-324. F. and Takeda, Y., (1972), Biochem. Biophys. 7. Cno, M., Inoue, H., Suzuki, Acta, 284, 285-297. 8. Janne, J. and Williams-Ashman, H.G., (1971), J. Biol. Chem., 246, 1725-1732. Amine Oxidases and Methods for Their Study, 9. Kapeller-Adler, R., (1970), Wiley-Interscience, New York, 191-216. 10. Janne, J., Williams-Ashman, H.G. and Schenone, A., (1971), Biochem. Biophys. Res. Comm., 43, 1362-1368. 11. Janne, J. and Williams-Ashman, H.G., (1971), Biochem. Biophys. Res. Comm., 42, 222-229. 12. Harik, S.I., Pastemak, G.W. and Snyder, S.H., (1973), Biochem. Biophys. Acta, 304, 753-764. 13. Weber, G. and Singhal, R.L., (1964), J. Biol. Chem., 239, 521-526. 14. Weber, G.,.Queener, S.F. and Morris, H.P., (1972), Can. Res., 32, 1933-1940. 15. Peraino, C. and Pitot, H.C., (1963), Biochem. Biophys. Acta, 73, 222-231. 16. Rando, R.R., (1974), Biochemistry, 13, 3859-3863. 17. Karlen, B., and Lindeke, B., (1969), Acta Phann. Suecica, 6, 613-616. 18. Russell, D.H. and Snyder, S.H., (1968), Proc. Nat. Acad. Sci., 60, 1420-1427. 19. Atkins, F.L. and Beaven, M.A., (1975), Biochem. Pharm., 24, 763-768. 20. Jones, M.E., Anderson, A.D., Anderson, C. and Hodes, S., (1961), Arch. Biochem. Biophys., 95, 499-507.

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