Download Biochemical Screening of Pyrimidine

Biochemical Screening of Pyrimidine Antimetabolites
I. Systems with Oxidative Energy Source*
JOSEPHE. STONEANDVANR. POTTER
(McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wis.}
Orotic acid (uracil-4-carboxylic acid) has been the livers were quickly excised and placed in a chilled bath of
isotonic saline solution. A 20 per cent homogenate in chilled
shown under both in vivo and in vitro conditions, 0.25
M sucrose was made with the use of an all-glass Potterin both microorganisms and mammals, to be a pre
Elvehjem homogenizer, and Ihis homogenate was centrifuged
cursor of the mono-, di-, and triphosphate pyrim- at approximately 600 g for 10 minutes to remove nuclei and
idine nucleotides, the pyrimidine coenzymes, and whole cells.
Portions of 0.8 ml. of the cytoplasmic liver fraction were
also of the pyrimidine moieties in both ribo- and
placed in 25-ml. Erlenmeyer flasks, each of which contained
deoxyribonucleic acid (5, 6, 11, 12, 13, 15-17).
2.20 ml. of a reaction mixture which had the following com
This study was prompted by the current position:
progress in the study of nucleic acid metabolism
15.0 Amóles
Potassium glutamate
and its relationship to tumor growth and metabo
6.0 /«moles
Potassium fumarate
15.0 /iiiiole-s
lism. The purposes of this research are the Potassium pyruvate
15.0 /¿moles
selection of agents as possible components of KHjPO,
9.0 /uñóles
MgCl2
sequential (9) or concurrent blocks (4), the clari
Ribose-5-phosphate (RSP)
6.0 /¿moles
fication of biochemical pathways, and the develop
Uridine-5-monophosphate (UMP-5')
1.0 /imoles
ment of concepts and technics in a biochemical Orotic acid-6-C"
0.3 /tmoles
approach to pharmacological research.
Adenosine triphosphate (ATP)
3.0 /uñóles
The data presented here represent the results of A sufficient quantity of sucrose to make an isotonicity equiva
lent to 0.25 Msucrose.
a screening program with a biochemical system
A sufficient quantity of water to make a volume of 2.20 ml.
which converts orotic acid to the uridine nucleo
The pH was adjusted to 7.2-7.4 with 0.2 N KOH. All flasks
tides. Normal rat liver cytoplasm furnished the
were
in duplicate. Drugs were added to the system in a drug:
enzyme source, and oxidative substrates furnished
orotic acid ratio of 10:1 (3.0 /IMof the drug to 0.3 /IMof the
the source of energy for the reactions. A primary
orotic acid).2 Whenever possible, the drugs were added in
objective of the study was to ascertain which aqueous solution; however, when necessary, acetate solutions
drugs could be shown to act upon one or more or propylene glycol solutions were utilized. In these cases, ap
steps in the conversion of orotic acid to the uridine propriate control flasks were added.
The reaction mixtures were incubated with constant agita
nucleotides.
tion for 20 minutes at 30°C. The reactions were terminated by
MATERIALS AND METHODS
It has been demonstrated that under in vitro
conditions orotic acid is converted to uridine
nucleotides by preparations of normal rat liver
(6, 10). The latter observation is the basis of the
test system used in this study. The drugs were
added to a standardized system, and the con
version of orotic acid to the uridine nucleotides
was measured and compared with the conversion
in appropriate controls.
White rats of either sex,1 weighing 150-450 gm., were used
in this study. The animals were sacrificed by decapitation, and
* This work was supported in part by a grant (No. C2409)
from the National Cancer Institute, National Institutes of
Health, United States Public Health Service.
Received for publication July 10, 1956.
the addition of 1.50 ml. of 1.5 N perchloric acid to each flask,
which resulted in a final perchloric acid concentration of 0.5 N.
The resulting mixture was centrifuged at 600 g for 10 minutes
and the pellet discarded. The acid-soluble supernatant liquid
was neutralized with 2.0 N KOH with phenol red as an internal
indicator, and the neutral solution was then allowed to stand
for 10 minutes at —¿10°
C. for the precipitation of residual
potassium perchlorate. After recentrifugation, the supernatant
fluid was placed upon 5-cni. Dowex l (X10) aniónexchange
resin columns (14).
The Chromatographie columns were submitted to the elution scheme which is summarized in Table 1.
Fractions 1, 2, and 3 were not examined and were discarded
(see below). Fractions 4, 5, 6, and 7 were read at 260 and 275
1Obtained from the Holtzman-Rolfsmeyer Rat Company,
Madison, Wis.
2The orotic acid used in this study was labeled in carbon 6.
Its specific activity was 4.1 X 10* counts/min/mg (5.75
/imoles). Approximately 213,000 counts/min were added in
0.3 /imoles added to each flask.
1033
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1034
Cancer Research
nan in a Beckman model DU spectrophotometer. The radio
activity of the samples was determined by plating a 0.2-ml.
aliquot from each tube upon weighed aluminum planchets
which were inscribed with a circle 3.8 sq. cm. in area. Approxi
mately 0.05 ml. of a 0.1 per cent solution of gelatin was used to
spread the solution upon the inscribed area.8 The samples were
then dried under a heat lamp, and the determination of radio
activity was done in gas-flow proportional counters.4
It should be emphasized that to insure activity of the system
all glassware should be washed in sulfurie-nitric acid cleaning
solution and glass-distilled water. All reagents should also be
prepared with glass-distilled water.
Under control conditions, the standard sys
tem5 converts an average of 7 per cent (range, 3-12
per cent among all preparations used) of the orotic
acid to the uridine nucleotides. The UTP-UTPX6
complex accounts for 80-90 per cent of the orotic
acid converted.
4) UMP-5' + ATP ^ UDP + ADP (3).
4a) UDP + ATP ^±UTP + ADP (3).
4b) UTP-> UDPX (UDP conjugates) and
UTPXS (3).
5) The ATP renewal is accomplished by oxidative phosphorylation with the Krebs cycle
fortified at three points.
In testing drugs against the standard system,
the following points were taken as criteria of the
activity of the system:
1. The total radioactivity in the UTP-UTPX
complex is the most reliable index to the effect of
the drug upon the conversion of orotic acid to the
uridine nucleotides. In this study it has been taken
as the major criterion.
2. The quantity of nucleotide in the UTP-
TABLE1*
SCHEDULE
OFELUENTS
ANDFRACTIONS
COLLECTED
Fraction
no.
1
2
3
4
5
6
7
Eluent
Ml/total
fraction
No. samples/
fraction
Compounds eluted
HjO
30
Nucleosides, bases
Adenosine monophosphate (AMP), UMP-5', orotic acid
3.0 N formic acid t
150
0.3 N ammonium formate!
Orotic acid and ADP
30
Mixed uridine coenzymes (UDPX's)
0.4 N ammonium formate
30
0.7 N ammonium formate
Uridine diphosphate (UDP)
30
l. 0 N ammonium formate
30
Uridine triphosphate (UTP) and UTPX
1.25 N ammonium formate
ATP
30
* AHsubstances eluted in thé
Chromatographie scheme presented in Table 1 were characterized by either chromatography of
known samples or reference to previous research done in this laboratory (3).
t All formic acid solutions were at pH 2.O.
ÃŽ
All ammonium formate solutions were at pH 5.O.
The known reactions included in the test sys
tem may be summarized as follows:
1) R5P + ATP -> PRPP (phosphoribosylpyrophosphate) + AMP (7).
2) Orotic acid + PRPP —¿Â»
Orotidylic acid (orotic
acid ribotide) + PP (pyrophosphate) (8).
3) Orotidylic acid -> UMP-5' + C02 (8).
3The gelatin solution facilitates spreading of the plated
solutions; it also equalizes the crystallization of the ammonium
formate present (M. J. Johnson, personal communication).
4After being counted, the plates were again weighed, and
correction was made for self-absorption. The assistance of Dr.
Charles Heidelberger and his staff in radioactivity determina
tions is gratefully acknowledged.
6The first system investigated consisted essentially of the
same ox¡dativesubstrates employed in the standard system,
including 3.0 jumólesof ATP, a relatively large amount of
UMP-5' (6 /¿moles),and a pool of 3.0 /¿molesof orotic acid
which contained 0.3 /¿molesof the labeled compound. These
substances were incubated with 0.8 ml. of 20 per cent rat liver
homogenate for 20 minutes at 30°C. Under these conditions,
the conversion of orotic acid to the uridine nucleotides could
not be demonstrated; however, under these conditions the
UMP-5' pool was almost entirely converted to uridine.
The addition of 6.0 /¿moleseach of fructose and ribose-5phosphate to the system outlined above and the substitution of
the cytoplasmic fraction of rat liver in the place of the whole
homogenate resulted in the production of UTP which had a
UTPX fraction is an indication of the transphosphorylase ability of the system in the presence
specific activity of 1200 counts/min//*mole. However, this sys
tem was unsatisfactory as a test object because of the extreme
ly low percentage of orotic acid converted to the uridine
nucleotides and because of excessive variation between dupli
cate flasks (20-40 per cent). It was found that, with this and all
subsequent systems investigated, the major uridine derivative
produced was uridine triphosphate (UTP). This fraction con
tains small amounts of a uridine nucleotide (Dr. L. Hecht,
unpublished data) whose specific activity is about 60 per cent
of that of the UTP. This compound has not been completely
characterized and is designated as UTPX because it follows
UTP on the chromatogram. The radioactivity of the UTPUTPX fraction has been adopted as the major criterion of
activity.
The second system was further modified by the deletion of
the fructose and the reduction of the UMP-5' pool to 1.0
/¿mole.
This small amount of UMP-5' serves as a marking pool,
and its phosphorylation to UTP-UTPX serves as a measure
ment of the transphosphorylative ability of the preparation.
Amounts of UMP-5' greater than 1 /¿moledepress the con
version of orotic acid into the uridine nucleotides by actual
inhibition, aside from the effects due to competition for high
energy phosphate and the dilution of the radioactivity. This
aspect will be treated fully in a later paper. Ribose-5-phosphate
was shown to be present at optimal amounts when used at a
level of 2.0 /xmoles/ml. Fructose may be used in place of ribose5-phosphate; however, its inclusion adds a number of addi
tional reactions to an already complex system.
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STONEANDPOTTER—Screeningof Pyrimidine Antimetabolites
of the drug. Theoretically, the specific activity of
the UTP-UTPX (counts/min/Mmole) can be taken
as an indication of the effect of the drug upon
oxidative phosphorylation. However, it must be
noted that the large ATP pool used in the standard
system is sufficient for the transphosphorylation
of the UMP-5' pool to UTP.6 Under these condi
tions, the UMP-5' pool is converted to UTP re
gardless of the effect a drug may have on the
1035
UDP, and UTP-UTPX are good indices of a pri
mary effect upon oxidative phosphorylation rather
than upon orotic acid metabolism per se.
The total radioactivity found in UDP, UDPX's,
UMP-5', or in orotic acid itself can be used as an
additional criterion of the conversion of orotic
acid. However, these fractions (with the exception
of the orotic acid fraction) contain relatively little
radioactivity and are not so reliable as the UTP
TABLE2*
DRUGS
WHICHDEMONSTRATED
No SIGNIFICANT
ACTION
AGAINST
THESTANDARD
SYSTEM
Compound
no.
Name
1.
2-Hydroxy-4-amino-S-carbethoxypyrimidine
2.
2-Ammo-4-hydroxy-5-carbethoxypyrimidine
8.
2-Mercapto-4-amino-S-carbethoxypyrimidine
4.
2-Ethoxy-4-hydroxy-5-carbethoxypyrimidine
5.
2-Ethylmercapto-4-chloro-5-carbethoxy pyrimidine
6.
l-(D-glucopyranosyl)-carbamylcytosine
7.
2-EthyImercapto-4-ammo-5-4^1orophenoxymethylpyrimidine
8.
2-Ethylsulfonyl-4-amino-S-(2,4-dichlorophenoxymethyl)-pyrimidine
9.
2-Hydroxy-4-amino-5-aminomethylpyrimidine
10.
2,4-Diethoxy-5-carbethoxypyrimidine
11.
Na-carbethoxy-L-asparagine
12.
Na-carbethoxy-D-asparagine
13.
4-Amino-5-hydroxymethylpyrimidine
14.
5-Methyl-orotic acid
15.
5-Iodo-orotic acid
16.
4-Methyl-uraciI
17.
4-Methyl-2-sulfo-uracil
18.
4-Trifluoromethyl-2-sulfo-uracil
19.
2-Mercapto-orotic acid
20.
5-Amino-orotic acid
21.
Thiothymine
22.
Bromouracil
23.
4-Oxy-5-(o,p-dichlorophenoxy)-pyrimidine
24.
Thiocytosine
25.
Thiouracil
26.
Azathymine
27.
Cytosamine
* Drugs nos. 1—15,
28-35, 40, and 46-51 were supplied by Sharpe and Dohme,
Inc. Nos. 19, 20, 36-38, and 41 were supplied by Lederle Laboratories. Nos. 21-26,
44, and 45 were supplied by Welcome Research Laboratories. Nos. 27 and 52 were
supplied by Eli Lilly and Co. Nos. 16-18, 39, 42, and 43 were supplied by Dr.
Charles Heidelberger of this department.
energy source of the system. However, in cases
of an extreme inhibitory effect of an agent upon
the energy source, the absolute amounts of ATP,
6During the course of a later investigation, an experiment
was performed in which the following changes were made in
the test system employed in this study: The ATP pool was
reduced from 3.0 ¿imolesto 0.5 /linole, and the nuclei were
washed with chilled 0.25 M sucrose solution and the washings
added to the cytoplasmic fraction. The time of incubation was
increased to 45 minutes. These modifications resulted in the
conversion of 47 per cent (45.6-50.0 per cent) of the orotic
acid. Moreover, under these conditions the specific activity of
the UTP complex is a valid measurement of the oxidative
capacity of the system. It should be noted that this modifica
tion allows for a finer differentiation between drug action upon
oxidative phosphorylation and upon orotic acid metabolism
per se. Such a system, because of its ability to convert a high
proportion of orotic acid at low molari! ics to uridine nucleotides, could be used for production of the latter in radioactive
form.
complex. The UMP-5' fraction is extremely
small and is separated only with difficulty from
the orotic acid. The orotic acid fraction itself is
extremely radioactive, and its net conversion is
low. For these reasons, the UMP-5' peak and the
orotic acid peak are eluted from the column with
3.0 N formic acid and discarded.
RESULTS AND DISCUSSION
The drugs which have been tested in this study
have been divided into three groups on the basis
of their effects upon the test system. Those com
pounds which depressed the conversion of orotic
acid 0-20 per cent were regarded as demonstrating
no significant effect upon orotic acid metabolism
(Table 2).
Those agents which gave an inhibition of 20-40
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1036
Cancer Research
per cent were placed in an intermediate group
(Table 3).
The drugs which depressed orotic acid conversion 40-100 per cent were selected for further
study (Table 4).
In general, oxidative phosphorylation is perhaps not the energy source of choice in a biochemical-pharmacological screening system. This
source of energy has certain disadvantages,
Among these are the rigorous standards of cleanliness which must be observed and the instability of
the enzyme system. However, the most serious
disadvantage lies in the fact that the energy
source may be the most sensitive component of the
system. Agents which act upon one or more steps
in the oxidative cycle could appear to be inhibitors
of reactions in which they actually have no effect.
However, in this study oxidative phosphorylation was deliberately chosen as an energy source
because of its extreme sensitivity. Under these
conditions, it allows for the screening not only of
orotic acid antimetabolites but also of agents
which may have an effect upon some of the
respiratory enzymes. The agents found active
against this system were then tested against a
more specific system with a glycolytic energy
source. The differential screening systems show
clearly which agents are active against orotic acid
metabolism per se and which are active against
oxidative phosphorylation alone,
Differential screening with multiple systems
can also give information on agents with multiple
points of action and could be an aid in the correlation of biochemical and pharmacological phenomena. Programs of this type should also give
rise to a dictionary of drugs whose point or points
of action are more or less known. It would seem
that such a body of knowledge would be invaluable in integrative biochemistry,
The thirteen agents which demonstrated some
inhibitory capacity can be divided into four
chemical and to some extent functional groups,
Group 1 consists of the 5-substituted analogs
of orotic acid and 5-diazo-uracil. The chloro,
bromo, and diazo analogs of orotic acid demonstrated moderate inhibition (average, 45 per cent
TABLE 8
DRUGSWHICHDEPRESSEDTHE CONVERSION
OF
OHOTICACIDBY20-40 PER CENT
Compound
no.
Name
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
2-Ethylmercapto-4-hydroxy-5-carbethoxypyrimidine
2-Ethylrnercapto-4-amino-5-aminomethylpyrimidine
2,4-Diamino-5-carbethoxypyrimidine
2-Mercapto-4-amino-5-hydroxymethylpyrimidine
2-Ethylmercapto-4-amino-5-hydroxymethylpyrimidine
D-dihydro-orotic acid
5-Hydroxymethylcytosine
Fumarylurea
5-Carboxy-orptic acid
2-Amjno-orotic acid
6-Acetic-erotic acid
4-Trifluoromethyl-uracil
TABLE 4*
DRUGSWHICHINHIBITEDTHECONVERSION
OFOROTICACIDBY40-100 PER CENT
UTP-UTPXUTP-UTPX ATP
Compound
no.
Name
radioactivity
a«per cent
of control
/¿moles
as per cent
of control
pmoles
ns per cent
of control
40.
5-Chloro-orotic acid
55 (2)
139
138
41.
5-Bromo-orotic acid
33 (3)
129
131
42.
5-Diazo-orotic acid
47(3)
95
83
43.
5-Diazo-uracil
27(3)
64
57
44.
2,4-Diammo-5-(3',4'-dichlorophenyl)-6-methylpyrimidine
25 (2)
38
27
45.
Daraprim
9(2)
14
17
46.
2-Benzylmercapto-4-amino-5-carbethoxypyrimidine
29 (2)
90
93t
47.
2-Ethylmercapto-4-amino-5-chlorornethylpyrimidine
35 (2)
118
84
48.
2-Benzylmercapto-4-araino-5-hydroxymethylpyrimidine
9 (2)
48
60
49.
2-Ethoxy-4-amino-5-carbethoxypyrimidine
17 (2)
73
63
50.
2-Ethylmercapto-4-amino-5-carbethoxypyrimidine
11 (2)
47
43
51.
N"-carbethoxy-DL-asparagine
50 (l)
91
52.
Amicetin
41 (2)
63
50
* Figures in parentheses give number of experiments.
t In one experiment a duplicate flask was extremely aberrant and demonstrated a total count which was 180 per
cent of the control levels. This drug is extremely insoluble.
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STONEANDPOTTER—Screeningof Pyrimidine Antimetabolites
of control). This inhibition is accompanied by an
actual increase in the amount of UTP-UTPX and
in ATP. From these data it would appear that
these agents have no inhibitory effect upon oxidative phosphorylation and that the inhibition oc
curs in the steps between orotic acid and UMP-5'.
The occurrence of abnormal metabolites or the
"piling up" of other metabolites could not be
demonstrated because of the low net conversion of
orotic acid. The inhibition produced by 5-diazouracil would seem to be of a different type be
cause of the depression of the amounts of triphosphate which accompany the inhibition. It should
be noted that the 5-carboxy, amino, methyl, and
iodo analogs of orotic acid were ineffective in this
system. The 2-amino and 2-mercapto analogs
were likewise ineffective.
Group 2 includes Daraprim (2,4-diamino-5-parachlorophenyl-6-ethylpyrimidine)
and its analog
2,4-diamino-5- (3',4'-dichlorophenyl) -6-methylpyrimidine. In this system these drugs are marked
inhibitors of orotic acid conversion to the uridine
nucleotides (average, 17 per cent of control).
However, this phenomenon is accompanied by
marked depression of the amounts of triphosphate.
Thus, it would seem that these drugs have a
marked effect upon the energy source of the sys
tem. The effect of these drugs upon orotic acid
metabolism cannot be ascertained until they are
tested against a system whose energy source is
unaffected by them. Daraprim has been shown by
other workers to be a folie acid antagonist (1). The
action shown here may be a corollary of this or
may be an additional point of action.
Group 3 is composed of the 2,5-substituted
4-amino pyrimidines. The inhibition was accom
panied by moderate depression of the amounts of
both uridine triphosphate and ATP. In this case,
the data derived from a system of this type cannot
differentiate between action upon orotic acid
metabolism and the energy source. This group of
drugs demonstrates the variability between dupli
cate flasks and experiments. This phenomenon
may be due to variation in solubility with slight
variation in pH. These compounds all have an
amino group in position 4, and it would seem that
a large variety of groups can be used in positions
2 and 5. It is difficult to draw conclusions from these
data, because a potentially active compound may
be almost completely insoluble.
The antibiotic Amicetin (2) produced an aver
age inhibition of 41 per cent of the control levels;
there was moderate diminution of the amounts of
triphosphate.
N" carbethoxy-DL-asparagine diminished orotic
acid conversion to 50 per cent of the controls.
The drugs discussed above were reinvestigated
1037
in a semispecific system which essentially tested
orotic acid conversion only. These data will be
presented in another publication.
SUMMARY
Fifty-two pyrimidine derivatives and related
compounds have been tested for orotic acid antimetabolite activity against a biochemical system
which converts orotic acid to the uridine nucleo
tides. Thirteen of these drugs have been demon
strated to be active against one or more of the
components of the system.
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Biochemical Screening of Pyrimidine Antimetabolites: I.
Systems with Oxidative Energy Source
Joseph E. Stone and Van R. Potter
Cancer Res 1956;16:1033-1037.
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