The most important group of inhibitors of the hydroxylases so far discovered is comprised of certain substituted derivatives of phenyl
alanine, tyrosine, and tryptophan. Table VII (143-147) lists most of the compounds of this class that have been found to show considerable inhibitory activity toward any one of the hydroxylases, while Table VIII (148-151) indicates the percentage of inhibition found for a few
aromatic amino acids in various in vitro assay systems. It is evident that the reported results are quite discordant in some cases as are, for example, the data for diiodotyrosine. More detailed studies with
3,5-T A B L E V I I
PERCENT INHIBITION OF AROMATIC AMINO ACID HYDROXYLASES FOUND in Vitro WITH SOME AMINO ACID A N A L O G S
0
Tyrosine hydroxylase Tryptophan Phenylalanine Adrenal Brain hydroxylase, brain hydroxylase, liver
Inhibitor 0.1
3,5-Diiodo-L- < 1 5 81 33 2-Fluoro-<*-methyl-DL 95
2-Chloro-a-methyl-DL- 84 3-Bromo-a-methyl-DL- 73
2a-Dimethyl-DL- 69 < 1 5 < 1 5
3-Amino-L- 65 < 1 5
3-Nitro-L- 64
a-Ethyl-DL- 60
3-Chloro-a-methyl-DL- 60 3-Fluoro-L- 50 ryptophan derivatives
a-methyl-5-hydroxy-DL- 80
5-Iodo-DL- 100 42
5-Bromo-DL- 98 45
5-Chloro-2-methyl-DL- 96 23
5-Chloro-DL- 90 44
5-Methyl-DL- 75 30
2-Methyl-DL- 68 28
5-Hydroxy-DL- 30 59 < 2 0
L-> 20 58
a-Methyl-DL- 17 52 27
6-Methyl-DL- 45 53
5-Fluoro-DL- 42 50
6-Chloro-DL- < 1 5 75
6-Fluoro-DL- < 1 5 80
° Value above each column indicates millimolar concentration of inhibitor; values within table indicate percent inhibition b y inhibitor. b
Data from 1^0.
c
Data from 143.
d
Data from 144, 145.
e
Data from 9, 146.
f D a t a from 83, 147.
0 D a t a from 126.
h
D a t a from 145.
l
' D a t a from 131.
1 Unsubstituted C o m p o u n d .
67
T A B L E V I I I
PERCENT INHIBITION OF AROMATIC A M I N O A C I D H Y D R O X Y L A S E S B Y SOME A M I N O A C I D A N A L O G S IN V A R I O U S in Vitro A S S A Y SYSTEMS
Concentration (mM) of Phenylalanines (%) Tyrosines (%) Tryptophans (%)
Enzyme Source Sub
strate DMPH4
Noncompetitive with substrate.
b
Concentration 5mAf in tetrahydrofolate.
c
Competitive with substrate.
* With tryptophan as substrate.
2. AMINO ACID HYDROXYLASE INHIBITORS 69
3-Iodo-a-methyl-DL-tyrosine 1.8 X 10~
7
p-Chloro-DL-phenylalanine 1.6-2 X
10-{
6-Fluoro-DL-tryptophan 9-12 X 10"'
° Approximate Ki expressed as molar value. 6
Data from 39.
c
Data from 146.
d
Data from 147.
diiodotyrosine suggest that it is as potent an in vitro inhibitor of tyrosine hydroxylase as a-methyl-p-tyrosine (Table I X ) .
The extensive screening of amino acid analogs as inhibitors of tyrosine and tryptophan hydroxylases allows some conclusions as to the relation
ship between structure and activity (9, 39, 83, lift, 144-147). The posi
tion and nature of the substituents appear to be critical for activity and for selectivity toward each of the hydroxylases.
Electrophilic substitution in the para position of phenylalanine or in the 6 position of tryptophan by a nitro group or halogen seems to increase the activity toward tryptophan hydroxylase while decreasing the activity toward tyrosine hydroxylase. Substitution of a halogen in the meta position of phenylalanine or tyrosine or in the 5 position of tryptophan causes a strong enhancement of inhibitory action toward tyrosine hydroxylase without much effect on the activity toward trypto
phan hydroxylase. Thus, 3-iodotyrosine and 5-iodotryptophan are effec
tive tyrosine hydroxylase inhibitors in vitro but are relatively weak against tryptophan hydroxylase. p-Chlorophenylalanine and 6-fluoro-tryptophan, on the other hand, are potent inhibitors of tryptophan hy
droxylase but have little action on tyrosine hydroxylase. In both series
70 E. G. MCGEER AND P. L. MCGEER
of inhibitors, the nature of the halogen appears to be much more critical for tyrosine hydroxylase inhibition than for tryptophan hydroxylase in
hibition. Introduction of a methyl group on the a position of the side chain seems to decrease inhibitory potency toward tryptophan hydrox
ylase with possible enhancement of inhibitory potency toward tyrosine hydroxylase. Among the phenylalanine and tyrosine derivatives, esters appear to be approximately equal to the parent amino acid as inhibitors of either hydroxylase. However, other alterations of the side chain, such as N substitution, hydroxyl substitution as in phenylserine, decarboxyla
tion, rearrangement as in /?-aminophenylpropionic acid, or lengthening or shortening as in 3-iodophenylglycine or 4-(3-iodophenyl)-2-methyl-2-aminobutanoic acid, appear to destroy inhibitory activity.
No amino acid inhibitor of phenylalanine hydroxylase has yet been found with an in vitro activity comparable to those of the most potent amino acid inhibitors of tyrosine and tryptophan hydroxylases. Halo-genated phenylalanines are the only type that have shown promise in the limited screening so far done. The weight of evidence suggests that 2-halogenated phenylalanines have little or no action against phenyl
alanine hydroxylase {131, H5), although there is one report (126) of 2-fluorophenylalanine showing promise. The distinction in activity be
tween 3- and 4-substituted derivatives is not as clear-cut as for inhibition of the other hydroxylases, and the nature of the halogen appears critical.
If the substitution is in the 3 position, the order of activity is I > Br > CI > F, but in the 4 position the reverse order holds. Thus, the most active inhibitors in these series are 3-iodophenylalanine and 4-fluorophenylalanine (145). Introduction of an a-methyl group into the halogenated phenylalanines does not enhance inhibition of phenylalanine hydroxylase (14$)- This is one of a number of examples in which struc
tural requirements for phenylalanine hydroxylase inhibition are closer to those for tryptophan than for tyrosine hydroxylase inhibition. As has been found for tyrosine and tryptophan hydroxylases, reduction or lengthening of the alanine side chain destroys inhibitory potency.
Some amino acids have shown stimulatory effects on the hydroxylases.
Phenylalanine, for example, is said to enhance hydroxylation of trypto
phan by brain (54) and liver (149) at low concentrations and to inhibit it at high concentrations. The inhibition of phenylalanine hydroxylase by excess substrate has already been mentioned. Thyroxine has been reported to stimulate tyrosine hydroxylase from adrenals (148) and brain (23) in vitro but the in vivo effects may be more complex (see Section V,C).
Competition with substrate has been demonstrated to be the
mech-2. AMINO ACID HYDROXYLASE INHIBITORS 71 anism of action for the in vitro inhibition of tyrosine hydroxylase by phenylalanine (144) , 3-iodophenylalanine (144, H5), L-3-iodotyrosine
(148) j 3,5-diiodo-L-tyrosine (148), 5-halotryptophans (146), and a-methyl-p-tyrosine (4, 148) despite one report (140) that the last-named compound is competitive with cofactor and not with substrate.
It has also been demonstrated for tryptophan hydroxylase inhibition by p-chlorophenylalanine and 6-fluorotryptophan (147). a-Methyl-5-hydroxytryptophan, on the other hand, has been reported to be com
petitive toward pteridine cofactor (148) for tyrosine hydroxylase inhibi
tion as have p-halogenated phenylalanines for phenylalanine hydroxylase (182).
IV. IN VIVO INHIBITORS OF THE HYDROXYLASES Hydroxylase inhibition in vivo presents more complex challenges than inhibition in vitro. A compound active in vitro may be rapidly metabol
ized to an inactive derivative or, conversely, to a more active derivative.
Its effect on the rate of hydroxylation in vivo may be on the control mechanisms that normally regulate the synthesis rather than on. the hydroxylation enzyme per se. The compounds most easily classified are those that have been demonstrated to inhibit the hydroxylases in vitro.
A. Catechols
Most of the catechols have only minimal effects in vivo. Very large doses of a variety of catechol derivatives such as 3,4-dihydroxy-6-methyl-o-methylaminoacetophenone (6-methylarterenone) (128, 180), ethyl 3,4-dihydroxyphenylpropionate, or 3,4-dihydroxybutyrophenone (88) have been used in rats without significant changes in brain 5-HT, brain tryptophan hydroxylase, or brain tyrosine hydroxylase, a-n-Propyldopacetamide (H 22/54) has been reported to reduce mouse or rat brain 5-HT (152-156), NE, and dopamine levels (155, 157) after single IP doses of 300-500 mg/kg. The extent of depletion was dependent on the environmental temperature (155). Pineal 5-HT was particularly affected (158). Ross and Haljasmaa (156) also found brain 5-HT de
creases after the administration of a variety of related catechols includ
ing 2,3-dihydroxy isomers of compounds such as H 22/54. Both H 22/54 and a,/?,/?-trimethyl-DOPA have been reported to decrease levels of NE
72 E. G. MCGEER AND P. L. MCGEER
in rat heart, spleen, liver, and adrenals with the extent of depletion in each organ being affected by the environmental temperature {157, 159).
Others (83, 128) have used similar doses of H 22/54 in mice, rats, and guinea pigs without finding significant effects on brain 5-HT levels.
There were questionable effects on NE concentrations. a,/?,/?-Trimethyl-DOPA does not affect endogenous brain levels of 5-HT or NE (126).
Replenishment of 5-HT by 5-HTP in rats treated with H 22/54 has been taken as evidence that the drug is acting in vivo on tryptophan hydroxylation (154). Assays in vitro on the livers of rats sacrificed after injection of H 22/54 or a,/?,/Mrimethyl-DOPA in vivo show sig
nificant decreases in the ability to hydroxylate phenylalanine (160, 161) or tryptophan (126, 132, i62), although the inhibition can be largely overcome by addition of pteridine cofactor to the in vitro assay system (132). Similar results have been obtained with a variety of 2,3- and 3,4-dopacetamides related to H 22/54 (156).
Rats treated with large doses of H 22/54 show a slight decrease in locomotor activity but no change in estrous behavior such as obtained with more potent inhibitors of tryptophan hydroxylase (153).
Rats treated with apomorphine (25 mg/kg sc) show a 50% decrease in the synthesis in telencephalon and brain stem of [
1 4
C ] catecholamines from intraventricularly injected [
1 4
C]tyrosine (134)- The interactions of apomorphine with the catecholinergic system (163) are complex, how
ever, and direct inhibition of tyrosine hydroxylase probably plays a very minor role in its physiological actions. Apomorphine is thought to have a more prominent action on dopaminergic receptors.
The catechol, «-methyl-DOPA, has been shown to lower slightly brain 5-HT (156) and dopamine levels and, more profoundly, brain and heart NE levels (46, 164). This effect is, however, primarily due to displace
ment of normal amines by the a-methyldopamine and a-methyl-NE formed in vivo (46, 164-167) rather than to any inhibitory effect on either the hydroxylases or the decarboxylase (168-170). Equivalent degrees of decarboxylase inhibition can be reached in vivo by compounds such as «-methyl-2,3-dihydroxyphenylalanine with little or no effect on tissue amine levels (46). Gal et al. (171) showed that administration of a-methyl-DOPA in amounts calculated to produce a concentration of 1-5 X 10"
4
M in brain did not impair the ability of brain tissue to hydroxylate tryptophan in vivo.
Another catechol, 6-hydroxydopamine, has been found to deplete catecholamines from heart and sympathetic nervous system (172-173a) on peripheral administration and from brain on intraventricular admin
istration, but this is also due, not to any inhibitory effect on tyrosine
2. AMINO ACID HYDROXYLASE INHIBITORS 73 hydroxylase, but to an immediate displacement effect (166) coupled with a destructive action on the terminal binding sites (174) and neurons themselves (173, 175-178e). The damage to adrenergic nerve terminals by 6-hydroxydopamine is at least partially reversible in the sympathetic nervous system (179).
In summary, the catechols have not yet yielded a drug of great practi
cal utility as a hydroxylase inhibitor in vivo. It may be expected that any active catechol will interfere to some extent with any enzyme that requires a pteridine cofactor and possibly with other oxidative systems;
H 22/54 (158), related dopacetamides (180), and 3',4'-dihydroxy-2-methylpropiophenone (181) have, for example, been shown to inhibit catechol O-methyltransferase (COMT) in vivo.