Vol. 189, No. 1, 1992 November 30, 1992

BIOCHEMICAL

RESEARCH COMMUNICATIONS Pages 33-39

AND BIOPHYSICAL

ACTIVATION OF AN ADRENERGIC PRO-DRUG THROUGH SEQUENTIAL STEREOSELECTIVE ACTION OF TANDEM TARGET ENZYMES Jayanta School

Debnath,

of

Philip

A.

Husain

Sheldon

and

Chemistry and Biochemistry, Technology, Atlanta,

Georgia GA 30332

W. May*

Institute

of

Received October 12, 1992 SUMMARY: The synthetic amino acid, 3,4-dihydroxyphenylserine (DOPS) has been of great interest for many years as an adrenerqic pro-drug, since the L-threo diastereomer of DOPS can be a precursor of R-(-)-norepinephrine, the natural form of this neurotransmitter. We now report bioactivation of DOPS to the potent pharmacological agent, noradrenalone (arterenone), via sequential stereoselective action by two target enzymes -dopamine P-monooxyqenase (DBM) and L-aromatic amino acid decarboxylase (AADC) -- acting in tandem. Enzymatic activation is stereospecific, with only the L-erythro DOPS diastereomer producing noradrenalone; this is consistent with the known stereospecificities of AADC and DBM. These results provide a heretofore unrecognized rationale for the bioactivity of L-erythro DOPS and provide a basis for the design of new adrenerqic prodrugs. 0 1992Academic PE55, Inc.

The synthetic amino has been of great interest adrenerqic pro-drug (l-9). (+)-L-erythro, to

the

rine

(-)-D-erythro,

chirality

(P-carbon) DOPS (as,

of both

which

neurotransmitter. has been

proposed

(8) r and successful

for

deficiency

(-)-L-threo, the

amino

and

acid

(+)-D-threo

(a-carbon)

--

due

and carbinol

moieties. It has long been recognized that L-threoPR) can be converted in many tissues to (-)-norepineph-

(NE),

administered

3,4-dihydroxyphenylserine (DOPS) acid, for many years as a potential DOPS exists as four stereoisomers --

is

the

natural

Thus,

the

for

as

use

of

hypotensive

Alzheimer's clinical

form

of

this

disorders

has been

a therapeutic

bypass

adrenerqic

DOPS as an adrenergic

and Parkinson's

study

primary (7),

for

diseases

carried for

out

depression

(8), in

which

a catecholamine

pro-drug and a DOPS was enzyme

(9).

* To whom correspondence

should

be addressed.

33

0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form rewrved.

Vol.

189,

No.

In

1, 1992

BIOCHEMICAL

contrast

to

decarboxylation

of

"unnatural" configuration of

the

L-threo

that readily

phenylethanolamines

such

(S)-norepinephrine,

stereospecific Thus,

of

the

we reasoned amino

L-erythro-DOPS (arterenone) highly potent represent active

the target

the

--

that acid the

catalyzes

produce

from

dopamine

in

the

sequential result

ketone

in

P-monooxygenase of

P-carbon

(S)-p-

of

agent

production

of

(10-12).

of

--

Such

a desired

precursor

via

of and DBM on

noradrenalone

norepinephrine (13-15).

via by

stereoselective action (AADC; EC 4.1.1.38)

analog

an inactive

followed

gem-diols

production

all

and We have

ketonization

at

decarboxylase

of

the

as (S)-octopamine, norpseudoephedrine, producing the corresponding ketones

pharmacological

an example

adrenergic

enzyme

enzymatically-produced

should

compound

stereoselective In this

enzymatic

(CG, PS) would

(R)-hydroxylation

L-aromatic

COMMUNICATIONS

phenylalkanolamine neurotransmitters NE, epinephrine and their cognates).

(i.e., demonstrated

dehydration

RESEARCH

of norepinephrine, whose @i to the PR configuration present

(DBM, EC 1.14.17.1) and

BIOPHYSICAL

diastereomer,

L-erythro-DOPS

(+)-enantiomer is opposite

functional

hormones previously

the

AND

which

a process

is

a

would

pharmacologicallysequential

action by two target enzymes acting in tandem. report we demonstrate stereoselective activation DOPS, through tandem action of these pro-drug,

of two

enzymes. EXPERIMENTAL

Dopamine p monooxygenase was isolated and purified from bovine adrenal glands as described previously (10,ll). L-aromatic amino acid decarboxylase was obtained from hog kidney by ammonium sulfate fractionation using the method of Voltattorni et. a1.(16). Noradrenalcne was synthesized as described by Remizov (17) and recrystallized from ethanol/ether as the hydrochloride salt; mp. 133-137(d); Mass spec (CI): m/e 138.1 (M+l); lH nmr (8,D20): 7.456.85 (m,3H), 4.43 (s,'2H). The four diastereomers of 3,4dihyroxyphenylserine were generously supplied by Sumitomo Pharmaceuticals CO., Ltd. (Osaka, Japan), and purity was confirmed by hplc analysis. L-threo: mp 230(d); [cY.]~ 20, -39.2. D-Threo: mp 230(d); [a]n 20, +42.4. L-erythro: mp 162(d); [ti]D 20, +49.2. D-erythro: mp 153-156(d); [a]D 20, -59.5. All other chemicals and solvents were obtained from standard chemical sources and were of highest purity. L-aromatic amino acid decarboxylase reactions were carried containing 100 mM phosphate buffer (pH out at 37 OC in a medium 10 w pyridoxal phosphate, and 15 mM of the 6.8) I 20 w pargyline, particular DOPS isomer. After a 5 minute preincubation of the medium (0.45 ml), reaction was initiated with 0.15 ml of enzyme solution (ca. 2.5 mg protein). Aliquots for HPLC analyses were 34

Vol.

189, No. 1, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

taken at various times and quenched with an equal volume of icecold 0.4 N perchloric acid containing 2.5 mg/ml sodium metabisulfate and 50 mg/ml EDTA, and then centrifuged at 5000 g for 10 minutes. For coupled assays with dopamine P-monooxygenase, 0.5 ml of the decarboxylase assay mixture from above was combined with 0.45 ml of a solution of 20 mM ascorbic acid, 20 mM sodium fumarate, 10 w cusoq, 500 pg/ml catalase in 0.1 M sodium acetate buffer (pH 5.25). After preincubated for five minutes, the reaction was initiated with 0.1 ml of a DBM solution (ca. 1 pg protein). Aliquots for HPLC analysis were quenched as described above. HPLC analyses were performed using a dual electrode electrochemical detection system equipped to oxidize catecholamines (ESA SlOOA, Analytical Cell 5011 with cell 1 set at -100 mV, cell 2 set at +4OOmV, and the conditioning cell 5021 set at -25OmV), equipped with a dual piston pump (ESA Model 4201, and strip recorder (Fisher 500). Products were separated utilizing an ESA Catecholamine HR-80 column (8cm X 4.6mmID, 3 micron ODS) and a mobile phase of 6.9 g/L sodium phosphate monobasic, 250 mg/L 1-heptanesulfonic acid (Na salt), 80 mg/L EDTA, and 5% methanol, adjusted to pH 3.6 with NaOH. RESULTS AND DISCUSSION In

separate

experiments,

3,4-dihydroxyphenylserine decarboxylase The results threo-

of

(AADC) and then with obtained are presented

and L-erythro-

configuration

Table

each

at

1.

Isomer

the

the

was incubated

DOPS, both a-carbon,

Reactivities

of DOPS

Stereochemistry

four

with

dopamine in Table

of which are active

diastereomers L-aromatic

@-monooxygenase 1. As expected,

Activity with AADP

+

L-threo

+

in Coupled

Activity with DBMb

+

D-erythro as determined the production of norepinephrine, with AADC for 60 minutes. the production of noradrenalone, as determined incubation with AADC for 90 minutes, followed

35

(DBM). L-

Assays

D-threo

a Measured by after incubation b Measured by after sequential minutes.

acid

possess the (S) substrates for AADC.

Diastereomers

L-erythro

of

amino

by

HPLC

analysis,

by HPLC analysis, by DBM for 60

In

Vol.

189, No. 1, 1992

BIOCHEMICAL

A

01

B

T

0

7

I

AND BIOPHYSICAL

C

RESEARCH COMMUNICATIONS

A

B

C

: 7

‘0

Retention

time

Retention

(minutes)

time

(minutes)

Figure 1. Identlflcatlon of proacts bv HPJ,C of T,-ervthro-DOPS A) Chromatrogram of assay medium prior to from. incubation with enzymes. The large peak consists primarily of Lerythro-DOPS. B) Chromatogram after incubation with AADC for 60 minutes. The new peak with an elution time of 2.5 minutes was identified as norepinephrine by comparison with an authenic C) Chromatogram of assay medium after incubation with standard. DBM for 60 minutes, preceded by AADC incubation for 90 minutes. The third peak at 4 minutes co-elutes with an authentic standard of noradrenalone. ,. Figure 2. &&ntrflcatlon of uroducts by HPLC of L-threo-DOPS from A) Chromatrogram of assay medium prior to couDled. incubation with enzymes. The large peak consists primarily of LB) Chromatogram after incubation with AADC for 60 threo-DOPS. The new peak with an elution time of 2.5 minutes is minutes. norepinephrine. C) Chromatogram of assay medium after incubation with DBM for 60 minutes, preceded by AADC incubation for 90 minutes. A noradrenalone peak does not appear. The reduction in height of the norepinephrine peak is due to dilution of concentrations upon addition of medium for the DBM reaction.

contrast,

both

configuration It is also diastereomers L-threo-DOPS a subsequent Figures reaction figures,

results expected

D-threo-

and D-erythro-

at their a-carbon atoms, evident from the Table that are AADC substrates, s&.y by AADC to -- is converted reaction

with

1 and 2 illustrate

DOPS, possessing

the

are inactive toward although both "L"

(R) AADC.

L-erythro-DOPS -- and a a product which undergoes

DBM. HPLC chromatograms

obtained

after

of DOPS diastereomers with AADC and DBM. As shown in the reaction of AADC with either L-erythroor L-threo-DOPS in time-dependent formation of norepinephrine (NE), the product of enzymatic decarboxylation. Control

36

Vol.

189,

No.

experiments D-threo-

BIOCHEMICAL

1, 1992

confirmed

that

or D-erythro-

enzymatic

both L-erythrotwo substrates R-(-)-NE previously

does

under the AADC-catalyzed

of It

indeed the case. L-erythro-DOPS with noradrenalone adrenalone

in

AADC, and that

not

with

any of

occur

the with

clearly

DOPS

produces

NE from

products arising from L-threo-DOPS producing

producing S-(+)-NE. DBM stereospecifically

DOPS would

the

non-

undergo

these

Since we have catalyzes (lo-12), our by AADC from

the

DBM-catalyzed

from Figures 1C and 2C that this in Figure lC, sequential reaction AADC and DBM results in formation of

a time

enzyme

when either

is clear As shown

formation

when either

occur

with

(S)-@-phenylethanolamines only the product produced

L-erythro-diastereomer ketonization.

not

COMMUNICATIONS

incubated

and L-threo-DOPS, are enantiomeric,

of only was that

RESEARCH

does

reaction conditions. decarboxylation

and L-erythro-DOPS established that

ketonization expectation

BIOPHYSICAL

NE formation

DOPS is

decarboxylation

diastereomers While

AND

dependent

from is

the

omitted

process.

L-erythro from

the

As expected, substrate does

experiment.

Figure 2C shows that noradrenalone contrast, occur when L-threo-DOPS is reacted sequentially

In

formation with

is of

not

occur

sharp does

m

AADC and DBM.

Taken together, these results establish that activation DOPS through sequential action by AADC and DBH indeed occurs the stereochemistry stereospecificities

expected on the basis of the respective of AADC (3) and DBM (10). Thus, of the

stereoisomers of DOPS, u the of being enzymatically converted agent,

noradrenalone,

target

enzymes.

through

L-erythro to the the

are

similar,

the

tandem

6.4 x 10m5 mM/s obtained DOPS and L-threo-DOPS,

Vm,, values under our respectively,

of

four

diastereomer is capable potent pharmacological action

of

Kinetic experiments were carried out with the Km values of 2.81 mM and 2.39 DOPS diastereomers. for L-erythro-DOPS and L-threo-DOPS, respectively. values

of with

6.6

these

two

AADC-reactive mM were obtained While

these

assay conditions for L-erythrodiffer markedly. Thus, it is that L-erythro-DOPS -- the only bioactivation to noradrenalone

evident from these kinetic results diastereomer which can undergo the which we report here -- is indeed the more reactive diastereomer. The possibility of developing specific, enzyme-targeted molecules of potential clinical interest is the primary goal of much current effort directed toward rational activatable "pro-drugs". The usual design specific target enzyme capable of converting 37

K,

x 10m4 mM/s and

design of enzymestrategy focuses on a the pro-drug to a

Vol.

189, No. 1, 1992

BIOCHEMICAL

pharmacologically-active illustrate

species.

an example

desired

AND BIOPHYSICAL

of

The results

"pre-pro

drug"

pharmacologically-active

inactive

precursor

target

enzymes

Over unexpected

via

acting

the years, bioactivity

(18) . These decarboxylation

reported

activation,

compound

sequential in

RESEARCH COMMUNICATIONS

is

produced

stereoselective

an by two

tandem.

many investigators for

the

(+)-norepinephrine,

which

that

undergoes

have

L-erythro

reported

is

diastereomer

receptor-inactive. the

finding of

DOPS

metabolic to "unnatural" Our demonstration

sequential

enzymatic

biactivation provides

process an attractive

reported here to produce noradrenalone rationale which may account for the

obtained

in

studies,

design

of

previous

future

adrenergic

the

from

action

observations have been puzzling since of L-erythro-DOPS should give rise

L-erythro-DOPS

here

whereby

and may provide

a basis

for

results the

pro-drugs. ACKNOWLEDGMENT

The financial support of the National (HL 28167) is gratefully acknowledged.

Institutes

of

Health

REFERENCES 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Beyer, H., Blascko, H., Burn, J. H., and Langemann, H. (1950) Nature. 165, 926; Blaschko, H., Burn, J.H., and Langemann, H. (1950) Br. J. Pharmacol. 5, 431-437; Schmiterlow, C.G. (1951) ibid, 6, 127-134; Creveling, C.R., Daly, J., Tokuyama, T., Witkop, B. (1968) Biochem. Pharmacol. 17, 65-70. Bartholini, G., Constantinidis, J., Tissot, R., and Pletscher, Pharmacol. 20, 1243-1247. A. (1971) Biochem. Bartholini, G., Constantinidis, J., Tissot, R., Puig, M., and Pletcher, A. (1975) J Pharmacol. Exp Ther. 193, 523-532. Porter, C.C., Torchiana, M.L., and Stone, C.A. (1972) Life Sci. 11, 787-795. Bioorg. Chem. 14, 429-443 (1986). Jung, M. L. Inagaki, C., and Tanaka, C. (1978) Biochem. Pharmac. 27, 10811086. Araki, H., Tanaka, C., Fujiwara, H., Nakamura, M., and Ohmura, (1981) J. Pharm. Pharmacol. 33, 772-777. I. Life Sci. 42,95-102. Gibson, C. (1988) Biaggioi, I., and Robertson, D. (1987) Lancet. 1170-1172. May, S.W., Phillips, R.S., Mueller, P.W., and Herman, H.H. (1981) J. Biol. Chem. 256, 2258-2261. May, S.W., Phillips, R.S., Mueller, P.W., and Herman, H.H. (1981) J. Biol. Chem. 256, 8470-8475. May, S-W., Phillips, R.S., Herman, H.H., and Mueller, P.W. (1982) Biochem. Biophys. Res Comm. 104, 38-44. Daly, J.W., Creveling, C.R., and Witkop, B. (1966) J. Med. Chem. 9, 273-280. McGreer E.G., and McGreer, P.L. (1973) in Metabolic Inhibitors (R.M. Hochster, M. Kates, and J.H. Quastrel, Eds.), Vol 4, pp. 45-105, Academic Press, New York. 38

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189, No. 1, 1992

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AND BIOPHYSICAL

15.

RESEARCH COMMUNICATIONS

Goldstein, M. (1966) in The Biochemistry of Copper (J. Peisach, P. Aisen, and W.E. Blumberg, Eds.), pp 443-454, Academic Pres, New York. 16. Voltattorni, C.B., Minelli, A., Vecchini, P., Fiori, A., and Turano, C. (1979) Eur. J. Biochem. 93, 181-188. 17. Remizov, A.L. (1958) J. Gen. Chem USSR. 28, 2530. 18. See, for example, references 3 and 4, and earlier studies cited therein.

39

Activation of an adrenergic pro-drug through sequential stereoselective action of tandem target enzymes.

The synthetic amino acid, 3,4-dihydroxyphenylserine (DOPS) has been of great interest for many years as an adrenergic pro-drug, since the L-threo dias...
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