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Inhibition of tyrosine hydroxylase by pyrroloisoxazoles
LIFE SCIENCES V o l . 6, p p . Printed in Great Britain.
1463-1466,
1967.
Pergamon P r e s s L t d
INHIBITION OF TYROSINE HYDROXYIASE
BY 'PYRROLO ISOXAZOLE S R. J. Taylor, Jr. and Leon Ellenbogen Department of Endocrine Research, Experimental Therapeutics Research Section Lederle Laboratories, American Cyanamid Co., Pearl River, New York
( R e c e i v e d 9 F e b r u a r y 1967;
in final
f o r m 22 March 1967)
The enzyme tyrosine hydroxylase which catalyzes the initial step in the biosynthesis of norepinephrine has been found to occur in brain, adrenal medulla, and other sympathetically innervated tissues (1) , and has been shown to require a structurally specific pteridine cofactor (1'2)
Two classes of
compounds, aromatic amino acids and catechols, have thus far been reported to inhibit the purified beef adrenal enzym e(3).
The present paper reports the
inhibition of beef adrenal tyrosine hydroxylase in vitro by a novel class of compounds, pyrroloisoxazoles. Materials and Methods Tyrosine hydroxylase was prepared from beef adrenal medulla by the procedure of Na~atsu et al. (1).
The enzyme was precipitated from the
105,000 x g supernatant by addition of ammonium sulfate to 40~io saturation, and the precipitate was resuspended in 1 x lO-3M aqueous mercaptoethanol (pH 6.5) and stored at -20°C until used.
Tyrosine hydroxylase activity was
assayed by a previously reported modification (2) of the procedure developed by Nagatsu et al. (4).
The standard incubation mixture consisted of O.1 umole
of L-tyrosine containing 3 to 4 x lO 4 c.p.m, of L-tyrosine-3,5-3H,
200 ~moles
of acetate buffer (pH 6.0), 1.O umole of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine in O.1 ml of O.1 M phosphate (pH 7.1~) containing 1 M mercaptoethanol,
O.1 ~mole of m-hydroxy-p-bromobenzyloxyamine
0.25 ml of enzyme preparation.
(NSD-1055), and
The pyrroloisoxazoles were added to the
1463
1464
Vol. 6, No. 14
PYRROLOISOXAZOLES
reaction mixture in O.i ml dlmethylformam~de. 1.Omlwlthwater.
The final volume was made to
All incubations were for 30 minutes at 37°C.
Controls
were included to correct for the slight inhibition due to dimethylformam~de. Ferrous ~ n i u m
sulfate solutions were prepared immediately before use.
S-Methyl ~-tyrosine was obtained from Merck, Sharp and Dohme Research Laboratory, m-hydroxy-p-bromobenzyloxyamlne (NSD-1055) from Smith and Nephew Research, Ltd., and 3-4-dihydroxyphenyl-propylacetamide (H-22/54, Hassle Laboratories) from Dr. Sidney Udenfriend, N.I.H.
The procedure for the
synthesis of the pyrroloisoxazoles will be reported elsewhere (5). Results and Discussion The effects of five pyrroloisoxazoles on tyrosine hydroxylase activity are shown in Table I.
All five pyrroloisoxazoles inhibited the enzyme at TABLE 1
Pyrroloisoxazoles as Inhibitors of Tyrosine Hydroxylase Concentration Required For 50% Inhibition% Compound
(M)
3-Amino-4H-pyrrolo[3,4c]isoxazole-5(6H)carboxyllc acid, ethyl ester (CL-65263)
1 x lO -h
3-Amino-4H-pyrrolo[3,4c]isoxazole-5(6H)carboxylic acid, benzyl ester (CL-62219)
4 x 10 -4
3-Form-mido-4H-pyrrolo[3,4c]isoxazole-5 (6H)-carboxyllc acid, ethyl ester (CL-67908)
2 x 10 -3
5-Acetyl-3-amino-5,6-dihydro-4H-pyrrolo [3,4c]isoxazole (CL-62664)
1 x 10 -3
3-Propionamido-4H-pyrrolo[3,4c]-isoxazole-5 (6H)-carboxyllc acid, ethyl ester (CL-62726)
h x 10 -3
3,4 Dihydroxyphenylpropylacetamide (H-22i54 , Hassle Laboratories)
1 x 10 -4
DL-~-Methyl-p-tyrosine 3-Iodo-L-tyrosine
6.5 x 10 -5 2 x lO -6
All compounds were added to the incubation mixtures in 0.i ml dimethylformamlde. @Data represent a range of 45-55~ inhibition.
Vol. 6, No. 14
PYRROLOISOXAZOLES
concentrations ranging from i x 10-4}4 to 4 x IO-3M.
1465
One of the compounds
(CL-65263) inhibited 50% at 1 x 10-4M and was equal in potency to 3,4dihydroxyphenylpropylacetamide (H°22/54). in Fig. 1.
The structure of CL-65263 is shown
The tyrosine analogues, G-methyl-~-tyrosine and 3-iodotyrosine,
were found to inhibit at 6.5 x lO -5 and 2 x lO-6M, respectively, in our assay system.
""h
o
-c-
I
NHz
Fig. t 5h, ucfure oF 5-Amino-4-H-pyrro]o [3,4cl isoxazole -5 (6H)-ca rboxylic acid, efhyl esfer (CL 652.65) The addition of 0.2 ~nole of Fe +2 completely eliminated the inhibition when the CL-65263 concentration was 2 x lO'4M (Table 2).
The inhibition was
eliminated by less Fe +2 when the CL-65263 concentration was lowered to 5 x lO-5M.
Variation of the pteridine cofactor concentration from 5 x lO-4M
to 5 x lO-3M did not significantly affect the inhibition by CL-65263. Similarly, variation of the tyrosine concentration from 5 x lO-5M to 2 x lO-4M had no effect on the inhibition. The catechol, 3,4-dihydroxyphenylpropylacetamide (H-22/54), was reported to be the most potent of a series of catechols which appear to inhibit by competing with the pteridine cofactor (3).
The other reported class of
inhibitors, tyrosine analogues, is represented by G-methyl-p-tyrosine and 3-iodotyrosine. substrate (3).
These compounds are known to be competitive with the Pyrroloisoxazoles are not structural analogues of tyrosine,
nor do they possess a catechol moiety, and thus represent a novel chemical. structure which inhibits tyrosine hydroxylase.
1466
PYRROLOISOXAZOLES
Vol.
6,
No.
TABLE 2 Effect of Varied Concentrations of Tyrosine, Pteridine Cofactor, and Ferrous Iron on Inhibition of Tyrosine Hydroxylase by 3-Amino-4Hpyrrolo[B,4c]isoxazole-5(6H)-carboxylic acid, ethyl ester (CL-65263) Concentration
of CL-65263 (M)
Additions
Per Cent Inhibition $
2 x i0 -4
no added Fe+2^ i x IO-4M Fe+~ 2 x IO-4M Fe ~
68 56 0
5 X 10 -5
no adde~ Fe++2^ i x IO-TM Fe+ 2_ 2 x 10-4M Fe ~
44 16 0
2 x 10 -4
5 x Io-SM tyrosine i x IO-/M tyrosine 2 x IO-4M tyrosine
~8 65 60
2 x 10 -4
5 x IO-4M pteridine i x IO-~M pteridine 5 x IO-/M pteridine
46 45 43
SRelative to non-inhibited control.
The inhibition by pyrroloisoxazoles appears to be dependent on the concentration of ferrous iron, but not on tyrosine or pteridine cofactor.
This
would suggest the inhibition by pyrroloisoxazoles may involve the chelation of Fe +2.
We have previously reported inhibition of the enzyme by o-phenan-
throline, and inhibition by (~,~-dipyridyl was observed by Nagatsu et al. (1). Further studies on the inhibition of tyrosine hyc~roxylase and other Fe +2enzymes are in progress. References i.
T. NAGATSU, M. LEVITT and S. UDE~FRIE~[D, J. Biol. Chem. 239, 2910 (1964).
2.
L. E ~ O G E N , R. J. TAYLOR, JR., and G. B. BRUNDAGE, Biochem. and Biophys. Res. Commun. 19, 708 (1965).
3.
S. UDENFRIEND t P. ZALTZ~.~N-NIRENBERG and T. NAGATSU~ Biochem. Pharmacol. i_~4, 837 (1965).
4.
T. NAGATSU, M. LEVITT and S. UDENFRIEND, Anal. Biochem. 9--,122 (1964).
5.
S. M. GADEEAR and E. COHEN, to be published.
14
1463-1466,
1967.
Pergamon P r e s s L t d
INHIBITION OF TYROSINE HYDROXYIASE
BY 'PYRROLO ISOXAZOLE S R. J. Taylor, Jr. and Leon Ellenbogen Department of Endocrine Research, Experimental Therapeutics Research Section Lederle Laboratories, American Cyanamid Co., Pearl River, New York
( R e c e i v e d 9 F e b r u a r y 1967;
in final
f o r m 22 March 1967)
The enzyme tyrosine hydroxylase which catalyzes the initial step in the biosynthesis of norepinephrine has been found to occur in brain, adrenal medulla, and other sympathetically innervated tissues (1) , and has been shown to require a structurally specific pteridine cofactor (1'2)
Two classes of
compounds, aromatic amino acids and catechols, have thus far been reported to inhibit the purified beef adrenal enzym e(3).
The present paper reports the
inhibition of beef adrenal tyrosine hydroxylase in vitro by a novel class of compounds, pyrroloisoxazoles. Materials and Methods Tyrosine hydroxylase was prepared from beef adrenal medulla by the procedure of Na~atsu et al. (1).
The enzyme was precipitated from the
105,000 x g supernatant by addition of ammonium sulfate to 40~io saturation, and the precipitate was resuspended in 1 x lO-3M aqueous mercaptoethanol (pH 6.5) and stored at -20°C until used.
Tyrosine hydroxylase activity was
assayed by a previously reported modification (2) of the procedure developed by Nagatsu et al. (4).
The standard incubation mixture consisted of O.1 umole
of L-tyrosine containing 3 to 4 x lO 4 c.p.m, of L-tyrosine-3,5-3H,
200 ~moles
of acetate buffer (pH 6.0), 1.O umole of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine in O.1 ml of O.1 M phosphate (pH 7.1~) containing 1 M mercaptoethanol,
O.1 ~mole of m-hydroxy-p-bromobenzyloxyamine
0.25 ml of enzyme preparation.
(NSD-1055), and
The pyrroloisoxazoles were added to the
1463
1464
Vol. 6, No. 14
PYRROLOISOXAZOLES
reaction mixture in O.i ml dlmethylformam~de. 1.Omlwlthwater.
The final volume was made to
All incubations were for 30 minutes at 37°C.
Controls
were included to correct for the slight inhibition due to dimethylformam~de. Ferrous ~ n i u m
sulfate solutions were prepared immediately before use.
S-Methyl ~-tyrosine was obtained from Merck, Sharp and Dohme Research Laboratory, m-hydroxy-p-bromobenzyloxyamlne (NSD-1055) from Smith and Nephew Research, Ltd., and 3-4-dihydroxyphenyl-propylacetamide (H-22/54, Hassle Laboratories) from Dr. Sidney Udenfriend, N.I.H.
The procedure for the
synthesis of the pyrroloisoxazoles will be reported elsewhere (5). Results and Discussion The effects of five pyrroloisoxazoles on tyrosine hydroxylase activity are shown in Table I.
All five pyrroloisoxazoles inhibited the enzyme at TABLE 1
Pyrroloisoxazoles as Inhibitors of Tyrosine Hydroxylase Concentration Required For 50% Inhibition% Compound
(M)
3-Amino-4H-pyrrolo[3,4c]isoxazole-5(6H)carboxyllc acid, ethyl ester (CL-65263)
1 x lO -h
3-Amino-4H-pyrrolo[3,4c]isoxazole-5(6H)carboxylic acid, benzyl ester (CL-62219)
4 x 10 -4
3-Form-mido-4H-pyrrolo[3,4c]isoxazole-5 (6H)-carboxyllc acid, ethyl ester (CL-67908)
2 x 10 -3
5-Acetyl-3-amino-5,6-dihydro-4H-pyrrolo [3,4c]isoxazole (CL-62664)
1 x 10 -3
3-Propionamido-4H-pyrrolo[3,4c]-isoxazole-5 (6H)-carboxyllc acid, ethyl ester (CL-62726)
h x 10 -3
3,4 Dihydroxyphenylpropylacetamide (H-22i54 , Hassle Laboratories)
1 x 10 -4
DL-~-Methyl-p-tyrosine 3-Iodo-L-tyrosine
6.5 x 10 -5 2 x lO -6
All compounds were added to the incubation mixtures in 0.i ml dimethylformamlde. @Data represent a range of 45-55~ inhibition.
Vol. 6, No. 14
PYRROLOISOXAZOLES
concentrations ranging from i x 10-4}4 to 4 x IO-3M.
1465
One of the compounds
(CL-65263) inhibited 50% at 1 x 10-4M and was equal in potency to 3,4dihydroxyphenylpropylacetamide (H°22/54). in Fig. 1.
The structure of CL-65263 is shown
The tyrosine analogues, G-methyl-~-tyrosine and 3-iodotyrosine,
were found to inhibit at 6.5 x lO -5 and 2 x lO-6M, respectively, in our assay system.
""h
o
-c-
I
NHz
Fig. t 5h, ucfure oF 5-Amino-4-H-pyrro]o [3,4cl isoxazole -5 (6H)-ca rboxylic acid, efhyl esfer (CL 652.65) The addition of 0.2 ~nole of Fe +2 completely eliminated the inhibition when the CL-65263 concentration was 2 x lO'4M (Table 2).
The inhibition was
eliminated by less Fe +2 when the CL-65263 concentration was lowered to 5 x lO-5M.
Variation of the pteridine cofactor concentration from 5 x lO-4M
to 5 x lO-3M did not significantly affect the inhibition by CL-65263. Similarly, variation of the tyrosine concentration from 5 x lO-5M to 2 x lO-4M had no effect on the inhibition. The catechol, 3,4-dihydroxyphenylpropylacetamide (H-22/54), was reported to be the most potent of a series of catechols which appear to inhibit by competing with the pteridine cofactor (3).
The other reported class of
inhibitors, tyrosine analogues, is represented by G-methyl-p-tyrosine and 3-iodotyrosine. substrate (3).
These compounds are known to be competitive with the Pyrroloisoxazoles are not structural analogues of tyrosine,
nor do they possess a catechol moiety, and thus represent a novel chemical. structure which inhibits tyrosine hydroxylase.
1466
PYRROLOISOXAZOLES
Vol.
6,
No.
TABLE 2 Effect of Varied Concentrations of Tyrosine, Pteridine Cofactor, and Ferrous Iron on Inhibition of Tyrosine Hydroxylase by 3-Amino-4Hpyrrolo[B,4c]isoxazole-5(6H)-carboxylic acid, ethyl ester (CL-65263) Concentration
of CL-65263 (M)
Additions
Per Cent Inhibition $
2 x i0 -4
no added Fe+2^ i x IO-4M Fe+~ 2 x IO-4M Fe ~
68 56 0
5 X 10 -5
no adde~ Fe++2^ i x IO-TM Fe+ 2_ 2 x 10-4M Fe ~
44 16 0
2 x 10 -4
5 x Io-SM tyrosine i x IO-/M tyrosine 2 x IO-4M tyrosine
~8 65 60
2 x 10 -4
5 x IO-4M pteridine i x IO-~M pteridine 5 x IO-/M pteridine
46 45 43
SRelative to non-inhibited control.
The inhibition by pyrroloisoxazoles appears to be dependent on the concentration of ferrous iron, but not on tyrosine or pteridine cofactor.
This
would suggest the inhibition by pyrroloisoxazoles may involve the chelation of Fe +2.
We have previously reported inhibition of the enzyme by o-phenan-
throline, and inhibition by (~,~-dipyridyl was observed by Nagatsu et al. (1). Further studies on the inhibition of tyrosine hyc~roxylase and other Fe +2enzymes are in progress. References i.
T. NAGATSU, M. LEVITT and S. UDE~FRIE~[D, J. Biol. Chem. 239, 2910 (1964).
2.
L. E ~ O G E N , R. J. TAYLOR, JR., and G. B. BRUNDAGE, Biochem. and Biophys. Res. Commun. 19, 708 (1965).
3.
S. UDENFRIEND t P. ZALTZ~.~N-NIRENBERG and T. NAGATSU~ Biochem. Pharmacol. i_~4, 837 (1965).
4.
T. NAGATSU, M. LEVITT and S. UDENFRIEND, Anal. Biochem. 9--,122 (1964).
5.
S. M. GADEEAR and E. COHEN, to be published.
14