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
Vol.
189, No. 1, 1992
BIOCHEMICAL
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