`
`BIOCHEMICAL EFFECTS AND METABOLIC TRANSFORMATIONS
`OF 5-AZACYTIDINE IN Escherichia coli
`
`Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague
`
`A. CraAxK and F. Sorm
`
`Received November 4th, 1964
`
`5-Azacytidine (1-6-p-ribofuranosyl 5-azacytosine) is a new antimetabolite
`with distinctly antibacterial effects. Complete inhibition of growth of Escheri-
`chia coli brought about by 1:2.10~°m 5-azacytidine can be removed by
`simultaneous addition of uridine, cytidine or thymidine, but not of uracil or
`cytosine. At increased temperature 5-azacytidine is decomposed and loses
`its biological activity. In a bacterial medium andin an E.coli cell-free extract
`5-azacytidine isreadily metabolized whereby ribosyl N-formylbiuret and ribo-
`syl biuret are formed as decomposition products of the unstable 5-azauridine,
`accompanied by 5-azauracil, N-formylbiuret, biuret and other unidentified
`substances. After pre-incubation 5-azacytidine with the cell-free extract of
`E. coli brings about a slight
`inhibition of uridine phosphorolysis and
`a slight inhibition of cytosine deaminase activity. The mechanism ofthe strik-
`ing inhibitory effect of 5-azacytidine is discussed in view of the incorporation
`of 5-azacytidine into nucleic acids.
`
`Attemps to elucidate the mechanism of biological action of synthetic analogues
`of nucleic acid precursors have provided a number of fundamental data. At this
`Institute the synthesis and biological effects of the aza-analogues of nucleic acid
`pyrimidine precursors have been investigated for a numberof years! ’?. 5-Azacytidine,
`a new antimetabolite of nucleic acid biosynthesis was synthesized here recently*
`and was found to possess marked antibacterial and cancerostatic properties*. The
`study of cancerostaticeffects of 5-azacytidine revealed that it has a specific effect
`on the lymphatic and hematopoietic systems causing a pronounced decrease in the
`number ofcirculating lymphocytes and of mature myeloid cells of bone marrow”.
`The present communication is intended to contribute to the understanding of the
`inhibitory effect of 5-azacytidine using a microbial system. The growth of E. coli
`in a simple mineral medium and the possibility to study the antagonism between
`the inhibitor and the natural precursors of nucleic acids should yield fundamental
`information about the nature of the inhibitory effect of the substance in question.
`CELGENE 2012
`CELGENE 2012
`APOTEX v. CELGENE
`APOTEX v. CELGENE
`IPR2023-00512
`IPR2023-00512
`
`Vol. 30 (1965)
`
`2091
`
`

`

`Cihdk, Sorm:
`
`Experimental
`
`Reagents. 5-Azauracil, 5-azacytosine, N-formylbiuret, 5-azauridine? (this designation is used
`here in spite of the fact that the substance does not possess the usual nucleosidic structure),
`5-azacytidine® and 5-deoxyazacytidine® were prepared at this Institute. «-p-Ribose 1-phosphate
`and 2-deoxy-«-D-ribose 1-phosphate were obtained from Calbiochem. Ribosyl N-formylbiuret
`and ribosyl biuret were prepared enzymatically from 5-azauracil and ribose 1-phosphate as de-
`scribed earlier’. The pyrimidine precursors of nucleic acids were of analytical purity. 5-Aza-
`cytidine-4-1*C (1 mC/mmole) was prepared by Dr J. Moravek, uracil-2-!*C (9-9 mC/mmole),
`uridine-G-!*C (6-3 mC/mmole), cytidine-G-1*C (1-4mC/mmole) and cytosine-2-'*C (3 mC/
`mmole) were obtained from the Institute for Research, Production and Application of Radioactive
`Isotopes in Prague, thymine-2-!4+C (9-5 mC/mmole) was from Amersham.
`Cultivation of E. coli and preparation of the cell-free extract. As a test of growth inhibition the
`growthof a bacterial culture was estimated after 16 h of cultivation at 37°C. The growth tookplace
`in 10 ml synthetic medium containing glucose® in test-tubes provided with Kapsenberg stoppers;
`inoculation was carried out with a single drop of a 24-h-old £. coli culture. When the reversal of
`inhibition was estimated the inhibitors and precursors of nucleic acids were added to the medium
`immediately before inoculation. The cell-free extract was prepared from a culture aerated for 8 h
`at 37°C in 51 synthetic medium. After centrifugation and washing the bacteria were crushed in-
`a cooled (—40°C) bacterial press?. The crushed cells were suspended in 50 ml 0-1m Tris-HCl
`buffer of pH 7-4 and the cell debris was centrifuged (12000 g, 20 min, 3°C). The supernatant was
`divided in small lots and kept at —15°C, the protein content was determined according to Lowry
`and co-workers?°,
`
`Determination of uridine phosphorylase, thymidine phosphorylase and cytosine deaminase activ-
`ities. Incubation was carried out at 37°C in 2. 107 7m Tris-HCI buffer of pH7-4 in a total volume
`of 1 ml. The cell-free extract of E. coli was added in 0-1—0-3 ml amounts. Uracil, uridine, thymine
`or thymidine concentrations were 2. 107 *m, that of cytosine 2-4. 107 4m, those of magnesium
`chloride, ribose 1-phosphate and deoxyribose 1-phosphate 4.10 ~*m. The antimetabolites were
`addedin the solution to a final concentration of 1-4.10~°m after adjusting the pH to 7-0. After
`definitive time intervals 0-1-0-2 ml samples of the incubation mixture were placed on Whatman
`No 1 paper without deproteinizing. Chromatography took place in mixtures of n-butanol—acetic
`acid—water (10:1: 3),
`isobutyric acid~water-ammonia (66: 33:1:5) or ethyl acetate-water—
`formic acid (60 : 35: 5).
`Assay of metabolic transformations of 5-azacytidine. Incubation took place in 2. 107 *u Tris-HCl
`buffer or in 107 ‘m phosphate buffer of pH 7-4 at 37°C; 5-azacytidine concentration was selected
`within the range of 10~*-10~7m. For the investigation of metabolic changes of 5-azacytidine
`a cell-free extract of E. coli was used and incubation lasted 5—60 min. For the identification of
`substances formed during incubation of 5-azacytidine their instability in alkaline solution was
`madeuse of. In this case substances were formed that had been identified earlier during the in
`vestigation of metabolic transformation of 5-azauridine’, For this reason the chromatographically
`isolated compounds were heated for 5-30 min in a boiling-water bath in 1IN-NH,OH. In order
`to determine the effects of substances that are formed by spontaneous decomposition of 5-aza-
`cytidine 5-azacytidine was aseptically incubated 24 h in Tris-HCl buffer and subsequently treated
`with a cell-free extract from E. coli.
`Determination of radioactivity. The position of radioactive compounds on chromatograms was
`determined from an automatic record (Friesecke-Hoepfner) of samples analyzed in parallel.
`Radioactive zones were eluted with water onto planchets and their radioactivity measured after
`drying in a methane-flow proportional 27 scaler in an infinitely thin layer. Radioactivity of whole
`cells was measured on membranefilters. Bacteria from 2 ml medium were washed with 5 ml 5%
`trichloroacetic acid, twice with 10 ml distilled water and thefilters dried for 10 min at 100°C.
`
`2092
`
`Collection Czechoslov. Chem. Commun.
`
`

`

`
`
`Biochemical Effects and Metabolic Transformations of 5-Azacytidine
`
`Results
`
`CHARACTER OF INHIBITION OF E. coli GROWTH BY 5-AZACYTIDINE
`
`In order to obtain somebasic information abouttheefficiency of the new synthetic
`analogues of nucleic acid pyrimidine precursors the inhibition of E. coli growth was
`measured. The inhititory effect of new and some previously tested''* aza-analogues
`of uracil and cytosine is shown in Table I. It appears that with 6-azapyrimidines the
`bases are more effective than the corresponding ribonucleosides. Similarly, 5-aza-
`uracil is a more potent inhibitor of growth than the unstable 5-azauridine’. A sur-
`prising exception is formed here by 5-azacytidine which shows the highest bacterio-
`static effects of all the substances tested and for E. coli it thus represents the most
`powerful synthetic anomalous pyrimidine ribonucleoside inhibitor. The nature of
`E. coli growth inhibition by 5-azacytidine and 5-deoxyazacytidine may be seen in
`Fig. 1. 5-Azacytidine is about 20 times more powerful in inhibiting growth than is
`5-deoxyazacytidine. Full inhibition of growth ensues in the presence of 1:2. 107°M |
`5-azacytidine or 2:5. 10~°m 5-deoxyazacytidine.
`In view of the instability of 5-azauridine’ it appeared useful first to determine the
`stability of 5-azacytidine. The stability of 5-azacytidine was evaluated on the basis
`of inhibition of E. coli growth. The results shown in Fig. 2 reveal how the biological
`effect of 5-azacytidine depends on temperature and on the length of previous in-
`cubation in a bacterial medium. It follows from the character of the curves that
`5-azacytidine is readily decomposed. The decomposition proceeds readily at the
`boiling temperature but at low temperature a solution of 5-azauracil is relatively
`stable. It appears likely that in common with the situation with 5-azauridine’ the
`5-azapyrimidine ring of 5-azacytidine is spontaneously opened and the biological
`activity lost.
`E. coli growth inhibition due to 5-azacytidine can be suppressed by simultaneous
`addition of uridine, cytidine or deoxyuridine and deoxycytidine and by higher
`concentrations of thymidine. Uracil and cytosine do not remove the inhibition due
`
`Table I
`
`Antibacterial Effects of some Azapyrimidines in E. coli
`
`
`50% Growth
`|
`50% Growth
`
`Antimetabolite
`
`Inhibition 7 Antimetabolite
`
`Inhibition
`
`
`
`
`
`
`
`|
`M
`|
`ug/ml
`M —t
`|
`ug/ml
`_
`_
`|
`7 i
`
`
`
`
`
`
`5-Azauracil |=2503 26.107° || 5-Azauridine 1.0.1073 |
`
`5-Azacytosine 0-06|24.1077280 26.1077 || 5-Azacytidine
`
`
`
`6-Azauracil
`7
`62.107>
`6-Azauridine
`1300
`53.10 3
`
`520
`
`2:1.1073
`
`12.1074nes6-Azacytidine
`
`||
`
`14
`
`6-Azacytosine
`
`Vol. 30 (1965)
`
`2093
`
`

`

`Cihak, Sorm:
`
`
`
`0-01
`
`005
`
`04
`
`05
`
`1
`
`dg /mi
`
`Fig. 1
`Inhibition of Z. coli Growth by 5-Azacytidine
`(1) and 5-Deoxyazacytidine (2)
`Growthis expressed as % of uninhibited cul-
`ture.
`
`
`
`Fig. 2
`Decrease in Biological Activity of 5-Azacyti-
`dine after Pre-Incubation at Higher Tempera-
`ture
`100 zg S-azacytidine/ml heated aseptically in
`the synthetic medium at pH 7:2. In 60 min
`intervals samples were withdrawn and after
`dilution to 1 ug/ml growth inhibition assayed.
`
`Table II
`
`Antagonism between 5-Azacytidine and the Pyrimidine Precursors of Nucleic Acids
`Cultivation of E. coli for 16h at 37°C. Concentration of 5-azacytidine 1 vg/ml.
`
`Culture Growth, %
`Pyrimidine ———_
`
`5-azacytidine : pyrimidine precursor
`Precursor
`1:100 |
`1:10
`1:1
`| 1:01
`|
`|
`
`|
`
`
`
`
`
`
`Uracil
`Uridine
`Deoxyuridine
`Cytosine
`Cytidine
`
`Deoxycytidine
`Thymidine
`
`|
`
`|
`|
`
`i
`
`is
`100
`100
`11
`100
`
`100
`91
`
`|
`|
`
`18
`100
`73
`8
`100
`
`55
`49
`
`10
`55
`44
`5
`55
`
`22
`3
`
`0
`6
`0
`0
`4
`
`0
`0
`
`to 5-azacytidine even at high concentrations (Table II). This finding is somewhat
`surprising since inhibition of E. coli growth brought about by any of the azapyrimi-
`dines shown in Table I with the exception of 5-azacytosine’’ can be suppressed by
`an addition of uracil and cytosine. Thymine and purine precursors of nucleic acids
`were always without effect. The inhibitory effects of 5-deoxyazacytidine can be
`removed by adding both uridine and cytidine, or uracil and cytosine.
`In the presence of 4.10~ 7M 5-azacytidine E. coli cells display pronounced morphological
`changes manifested by the formation of filamentous forms. At lower concentrations of the anti-
`metabolite (1-6. 107 7M)the filamentous forms ofcells do not occur although growthis inhibited.
`
`2094
`
`Collection Czechoslov. Chem. Commun,
`
`

`

`
`
`ce
`
`Biochemical Effects and Metabolic Transformations of 5-Azacytidine
`
`On simultaneously adding uridine or cytidine (final concentration 4. 107°) to a culture heavily
`inhibited by 5-azacytidine (4. 107 ©m) no formation of filamentous cell forms was observed. An
`addition of uracil or cytosine did not prevent the formation of the filamentous forms. 5-Deoxy-
`azacytidine did not cause any filamentous forms to be produced even at the concentration of
`4.107 °.
`
`Possibilities to enhance the inhibitory effects of 5-azacytidine were also considered.
`On applying simultaneously 6-azauracil or 6-azauridine with N-formylbiuret which
`has an inhibitory effect during the first stages of formation of nucleic acid pyrimidine
`precursors’? a marked potentiation of the biological activity of both antimetabolites
`was observed'*. According to our unpublished findings biological activity is also
`increased on combining N-formylbiuret with 6-azacytosine and 6-azacytidine. By
`measuring the inhibition of E. coli growth it was found that the combination of
`N-formylbiuret with 5-azacytidine (and similarly with 5-azauracil or 5-azacytosine)
`does not result in any potentiation of inhibitory effects. On applying simultaneously
`5-azacytidine and 6-azauracil no increase in the inhibitory activity of either of the
`antimetabolites could be observed.
`
`In addition to 5-azacytidine and 5-deoxyazacytidine 1-G-p-glucopyranosyl 5-azacytosine and
`1-8-p-ribopyranosyl 5-azacytosine were synthesized?. Neither of the substances inhibits FE. coli
`growth at a concentration of | mg/ml.
`
`TRANSFORMATIONS OF 5-AZACYTIDINE IN THE COURSE OF CULTIVATION
`
`When the pronounced antibacterial activity of 5-azacytidine was established it
`appeared to be of interest to find out by what mechanism the growth of E.coli is
`inhibited. During the first phase the changes were investigated which 5-azacytidine
`undergoes during cultivation. The medium after inhibition with 5-azacytidine was
`analyzed and it was found that even during the first minutes after addition 5-aza-
`cytidine-4-!*C undergoes metabolic changes. The medium was found to contain new
`radioactive substances which were analyzed in analogy with the degradation products
`of 5-azauridine’, and ribosyl biuret, 5-azauracil and biuret were found among the
`products. After 30 min ofcultivation no 5-azacytidine (10~*m) can be detected in the
`medium. Fig. 3 showsthe level of 5-azauracil found in the medium of E. coli during
`cultivation in the presence of 5-azacytidine.
`It was further analyzed what changes 5-azacytidine undergoes in E. coli cells.
`To this end 5-azacytidine-4-1*C was added to a final concentration of 10~*m to an
`aerated culture at the beginning of the logaritmic phase of growth and samples were
`withdrawn after definite time intervals to be filtered through membranefilters and
`their radioactivity determined. In order to remove the nucleotide pool cells on the
`filters were washed with ice-cold 5° trichloroacetic acid and distilled water. It
`follows from Fig. 4 that within a short interval after adding 5-azacytidine-4-1*C
`a markedrise ofintracellular radioactivity can be observed. A simultaneous addition
`ofcytidine and 5-azacytidine-4-'*C (10 : 1) resulted in a removalof growth inhibition
`and in a 4-7-fold decrease of intracellular radioactivity.
`
`Vol. 30 (1965)
`
`2095
`
`

`

`6 000
`
`c.p.m.
`
`4000
`
`2000
`
`Cihdk, Sorm:
`
`1500
`
`c.p.m.
`
`1 000
`
`500
`
`0:75
`
`Asas
`
`0-45
`
`Fig. 3
`Level of 5-Azauracil in E. coli Medium Du-
`ring Cultivation with 5-Azacytidine
`Cultivation of £. coli under aeration at 37°C,
`5-azacytidine (10~*m, 0-2 mC/ml) addedat
`the beginning of the logarithmic phase of
`growth. 0-J ml medium samples were analy-
`zed.
`
`Fig. 4
`Radioactivity of E. coli Cells during Cultiva-
`tion with 5-Azacytidine-4-!4C
`5-Azacytidine-4-!*C (6-2 . 10* counts/min/ml)
`added after 6h of growth to an aerated cul-
`ture at 37°C.
`1 Radioactivity of cells in 2ml
`medium in the presence of 10~ *m-5-azacyti-
`dine, 2 in the presence of 10~*m 5-azacyti-
`dine + 107 7m cytidine, 3 growth curve in
`the presence of 10~*m 5-azacytidine.
`
`It appeareduseful to determine what part of total radioactivity contained in the
`low-molecular fraction of inhibited cells is due to 5-azacytidine. Cells were extracted
`after different periods of growth in the presence of 5-azacytidine with 0-2N-HCIO,
`under cooling with ice. Chromatographic analysis of the extracts obtained failed
`to detect 5-azacytidine after 1 h after beginning of growthofE. coli with 5-azacytidine.
`It did reveal a greater amount of ribosyl biuret, traces of ribosyl N-formylbiuret and
`of a substance which appears to be chromatographically identical with 5-azacytidine
`5’-phosphate prepared enzymatically in a cell-free extract from mouse liver using
`5-azacytidine and adenosine 5’-triphosphate'*.
`
`METABOLIC CHANGES OF 5-AZACYTIDINE IN A CELL-FREE EXTRACT OF E. coli
`
`In order further to elucidate the transformations of 5-azacytidine taking place
`in E. coli cells during cultivation a cell-free extract was used. In view of the fact
`that 5-azacytidine undergoes spontaneous decomposition it was necessary to
`consider this factor together with metabolic transformations of 5-azacytidine. It was
`found in orientation experiments that substances formed by spontaneous decomposi-
`tion of 5-azacytidine do not undergo any further metabolic changes and do not
`possess antibacterial effects toward E.coli.
`
`2096
`
`Collection Czechoslov. Chem. Commun-
`
`

`

`Sed
`
`Biochemical Effects and Metabolic Transformations of 5-Azacytidine
`
`Nucleic acids Qa Decomposition of the 5-azapyrimidine ring
`
`/oN
`
`5-Azacytidine 5“-phosphate
`
`|
`
`
`
`5-Azauridine 5*-phosphate
`4
`;
`
`5-Azacytidine —__————_ ||»_5-Azauridine
`
`Uridine
`
`
`
`Spontaneous
`decomposition
`
`_5-Azauracil
`
`
`
`Cytosine
`
`Uracil
`
`In investigating the anabolic changes of 5-azacytidine it was established at first
`whether 5-azacytidine is phosphorylated. 5-Azacytidine 5’-phosphate could not be
`detected in a cell-free extract from E. coli with certainty. It was found that under
`the incubation conditions 5-azacytidine is rapidly metabolized. The incubation
`mixture was found to contain ribosyl N-formylbiuret and ribosyl biuret formed
`through decomposition of unstable 5-azauridine’, and 5-azauracil. Changes in the
`amountsofthese substances in the course of incubation are apparent from TableIII.
`
`Relationships between the individual metabolites formed in the course of incubation
`from 5S-azacytidine are rather complex. The transiently formed S-azauridine can
`
`Table Ill
`
`Time Dependence of Metabolic Transformations of 5-Azacytidine in a Cell-Free Extract
`of E. coli
`Incubated in 2. 107m Tris-HCI buffer of pH 7-4, 1:3 mg protein, 10~>m 5-azacytidine-4-!*C
`(6-65. 10° counts/min/ml). Values obtained after chromatographic separation of 0-1 ml sample
`of incubation mixture.
`
`Duration
`5-Azacytidine + |
`Ribosyl
`;
`;
`;
`of Incubation
`é-Avauridine
`N-Formylbiuret
`Ribosyl Biuret
`5-Azaaracil
`min
`counts/min
`counts/min
`counts/min
`counts/min
`
`
`
`
`
`
`58 120
`49 960
`41 750
`34 640
`28 260
`20 750
`15 000
`
`4 876
`5 464
`7010
`10 115
`12 300
`14 500
`17 515
`
`4 380
`8 020
`11 600
`13 125
`18 600
`21 720
`22 700
`
`1 100
`2 380
`3 860
`4 845
`7 660
`8 520
`9 805
`
`5
`10
`15
`20
`30
`45
`60
`
`
`
`Vol. 30 £1965}
`
`2097
`
`

`

`Cihak, Sorm:
`
`exist during the first phase both as an usual ribonucleoside andin the cyclized form’?.
`It is not known whetheroneof the 5-azauridine formsis preferentially decomposed’
`while the other more readily undergoes enzymatic deribosidization.
`
`INHIBITION OF SOME ENZYMATIC SYSTEMS IN THE PRESENCE OF 5-AZACYTIDINE
`
`Due to metabolic transformations of 5-azacytidine both in E. coli cells in vivo and
`in the cell-free extract in vitro new biologically active substances are formed (e.g.
`5-azauracil’’’!°, N-formylbiuret'*). For this reason an application of 5-azacytidine
`can be expected to result
`in a polyvalent inhibitory effect. In view of structural
`similarities between 5-azacytidine and cytidine we took upa study of cytidine dea-
`minase activity. However, in the cell-free extract of E. coli the metabolic changes
`of cytidine are not affected by the presence of 5-azacytidine.
`Significant changes can be observed in the activity of uridine phosphorylase.
`While the synthesis of uridine from uracil and ribose 1=phosphate is not affected a
`phosphorolytic cleavage of uridine is diminished in the presence of 5-azacytidine
`{Table IV). Similarly the phosphorolysis of thymidine is slightly diminished in the
`presence of 5-azacytidine (by 16%).
`In analogy to the synthesis of uridine from
`uracil and ribose 1-phosphate an increased level of thymidine (and of B-thymine
`riboside) formed by the reaction of thymine with deoxyribose 1-phosphate can be
`found in the presence of 5-azacytidine.
`In view of the metabolic changes of 5-azacytidine it appeared to be of interest
`in what way the inhibition of uridine phosphorylase will change with proceeding
`incubation. It follows from Fig. 5A that during short-term incubation (up to 5 min)
`no inhibition of uridine phosphorylase is apparent in the presence of 5-azacytidine.
`
`Table IV
`
`Inhibition of Uridine Phosphorolysis in the Presence of 5-Azacytidine
`Incubation for 20 min at 37°C in 2. 107m Tris-HCI buffer of pH 7-4, 1:8 mg protein. 2. 10° +m
`uracil-2-'4C (1-1. 10° counts/min/ml) or uridine-G-'*C (5-6. 10* counts/min/ml); 4.10~4m
`ribose 1-phosphate and MgCl,. Analysis of 0-2 ml incubation mixture.
`
`
`
`
`
`
`
`5-Azacytidine Reacted Substrate|InhibitionUracil Uridine
`
`
`
`M
`counts/min
`counts/min
`pf
`
`% S
`
`ynthesis of uridine
`10 470
`9 210
`Phosphorolysis of uridine
`5 180
`4090
`
`
`
`11 560
`12 300
`
`5 920
`7 310
`
`52:3
`57-24
`
`46-6
`35-9
`
`_
`0
`
`—
`23
`
` |
`
`_
`10-3
`
`ws
`1073
`
`“ Uridine level is by 9% higher than in the incubation mixture without 5-azacytidine.
`
`2098
`
`Collection Czechoslov. Chem. Commun.
`
`

`

`
`
`Biochemical Effects and Metabolic Transformations of 5-Azacytidine
`
`For this reason it was assumed that the actual inhibitor is not 5-azacytidine but
`rather the transiently formed S-azauridine. In order to verify this view the cell-free
`extract was pre-incubated with 5-azacytidine before estimating the uridine phosphoryl-
`ase inhibition. It follows from the results obtained (Fig. 6) that the inhibition of
`
`3 000
`
`c.p.m.
`
`2 000
`
`1000
`
`FP
`
`6 000
`
`4000
`
`2 000
`
`Fig. 5
`Inhibition of some Enzymatic Systems in a Cell-Free Extract of EF. coli during Incubation with
`5-Azacytidine
`A Activity of uridine phosphorylase: 2. 10~*m uridine-G-!*C (4-3 . 10* counts/min/ml) 2:0 mg
`protein. B Activity of cytosine deaminase: 2.10~*m uracil-2-'*C (3-2. 10* counts/min/ml),
`1-8 mg protein. Incubation in 2. 107 7m Tris-HCl buffer of pH7-4 at 37°C.
`1 Without inhibitor,
`2 with 107 3m 5-azacytidine.
`
`- 60
`Inhibition
`°/o
`
`20
`
`
`
`20
`
`60
`
`‘
`min
`
`100
`
`Fig. 6
`Dependence of the Inhibition of E. coli Uridine Phosphorylase on the Length of Pre-Incubation
`of a Cell-free Extract with 5-Azacytidine
`Incubated for 20 min at 37°C in 2.1072Tris-HCl buffer of pH 7-4, 2.10~* uridine-G-!4C
`(2:8 . 10+ counts/min/ml). 1 Activity of uridine phosphorylase after pre-incubation of the cell-free
`extract without 5-azacytidine (52% phosphorolysis of uridine to uracil), 2 after pre-incubation
`with 107 3m 5-azacytidine.
`
`Vol. 30 (1965)
`
`2099
`
`

`

`Cihdk, Sorm:
`
`uridine phosphorylase rises somewhat for a certain period of 5-azacytidine pre-
`incubation. This situation persists probably until the deamination of 5-azacytidine
`is overcompensated by spontaneous cleavage of the 5-azauridine formed which
`results in a decreased inhibition of uridine phosphorylase activity. Pre-incubation
`of the cell-free extract without 5-azacytidine does not affect the activity of uridine
`phosphorylase.
`
`During phosphorolysis of uridine in the presence of 5-azacytidine in a phosphate buffer no
`inhibition of uridine phosphorylase activity takes place, in contrast with the application of Tris-
`HCIbuffer. This phenomenonis probably connected with a different type of metabolic changes of
`5-azacytidine in the two buffers.
`
`=
`
`Table V
`
`Metabolic Transformations of Cytosine in the Presence of 5-Azacytidine
`Incubated for 20 min at 37°C in 2. 10~ 2m Tris-HCl buffer of pH 7-4, 2-0 mg protein, 4-107 4m
`cytosine-2-!*C (9-5 . 10* counts/min/ml). Analysis of 0:2 ml incubation mixture.
`
`
`5-Azacytidine|Cytosine|—Uracil SEN|CETREAIGE:Uridine |
`
`
`
`
`
`M counts/min|counts/min|counts/min 7 Tota [| SPyiesins |
`
`
`|
`Radioactivity |
`%
`|
`_t a
`_
`
`
`
`'
`
`
`
`
`
`
`
`
`
`—
`1.10°3
`2. 10-3
`4.10°% ~
`
`:
`
`518
`1 425
`1 885
`2 220
`
`17 850
`14370)
`12 220
`10 690
`
`|
`
`1 045
`|
`3 540
`5550
`6 460
`
`5*3
`18-3
`28-2
`33-4
`
`97-3
`92:7
`90-4
`88-6
`
`In the presence of 5-azacytidine a decreased formation of uracil from cytosine
`was observed. It follows from Fig. 5B that cytosine deaminase activity is lowered
`in the presence of 5-azacytidine similarly as with uridine phosphorylase only after
`a longer period of incubation with S-azacytidine (10 min). It was observed before
`that cytosine deaminase is markedly inhibited by 5-azauracil and 5-azacytosine!!.
`It is assumed that the inhibition of cytosine deaminase observed in the presence
`of 5-azacytidine is due to 5-azauracil formed from 5-azacytidine during incubation.
`Metabolic changes of cytosine in the presence of increasing concentrations of 5-aza-
`cytidine are apparent from data in Table V.
`
`Discussion
`
`5-Azacytidine is a new antimetabolite with pronounced antibacterial and cancero-
`static effects**>. The mechanism of the antibacterial action of 5-azacytidine is poly-
`valent due to spontaneous and enzymatic transformations of the compound. 5-Aza-
`uridine formed by deamination of 5-azacytidine is readily further transformed to the
`biologically active 5-azauracil'>+>. The main inhibitory effect of 5-azacytidine does
`not consist in its metabolic transformation to 5-azauridine or 5-azauracil even if a
`
`2100
`
`-_
`
`Collection Czechoslov. Chem. Commun.
`
`

`

`
`
`Biochemical Effects and Metabolic Transformations of 5-Azacytidine
`
`certain analogy exists between the mentioned 5-azapyrimidines. A substantial part
`of the overall biological activity of 5-azacytidine may be due to higher anabolites
`of 5-azacytidine or its incorporation into nucleic acids.
`With regard to the site of inhibition by 5-azacytidine it is of interest that the
`inhibition of E. coli growth brought about by 5-azacytidine can be removed by
`simultaneous application of the antimetabolite with uridine or cytidine but not with
`uracil or cytosine. Analogously,
`the formation of filamentous cell forms in the
`presence of 5-azacytidine can be prevented by simultaneous addition of uridine or
`cytidine but not of uracil or cytosine. It appearslikely that the differences in the ability
`to remove the inhibitory effects of 5-azacytidine depend on the rate with which the
`individual pyrimidine bases or nucleosides can be utilized for nucleic acid synthesis.
`The inhibitory effect that can be expected in E. coli on applying 5-azacytidine
`can be schematically represented as follows. 5-Azauridine and S-azauracil formed
`from 5-azacytidine slight
`inhibit uridine phosphorylase, 5-azauracil blocks the
`activity of cytosine deaminase. For the sake of explaining the pronouncedinhi-
`bitory effect of 5-azacytidine it
`is assumed that during the first minutes after
`addition of 5-azacytidine E. coli cells effect a deamination as well as a phos-
`phorylation of 5-azacytidine to 5-azacytidine 5’-triphosphate which is incommon
`with animal
`systems'* incorporated into nucleic acids. The main inhibitory
`effect of 5-azacytidine apparently consists in the spontaneous decomposition of the
`S-azapyrimidine ring incorporated in a nucleic acid molecule. Spontaneous de-
`composition of the 5-azapyrimidine ring was demonstrated for the first time with
`5-azauracil'? and later at the nucleoside or nucleotide level with 5-azauridine or
`5-azauridine 5'-phosphate’.
`
`We are indebted to Mrs R. Lerochova and Miss I. Fialova for technical assistance.
`
`Reférences
`
`1. Gut J., Moravek J., Parkanyi C., PrystaS’ M., Skoda J., Sorm F.: This Journal 24, 3154 (1959).
`2. Skoda J., Cihak A., Gut J., Prysta’ M., Piskala A., Parkanyi C., Sorm F.: This Journal 27,
`1736 (1962).
`3. Piskala A., Sorm F.: This Journal 29, 2060 (1964).
`4. Sorm F., Piskala A., Cihdk A., Vesely J.: Experientia 20, 202 (1964).
`5. Sorm F., Vesely J.: Neoplasma //, 123 (1964).
`6. Pliml J., Sorm F.: This Journal 29, 2576 (1964).
`7. Cihak A., Skoda J., Sorm F.: This Journal 29, 300 (1964).
`8. Skoda J., Hess V. F., Sorm F.: This Journal 22, 1130 (1957).
`9. Hughes D. E.: Brit. J. Exptl. Pathol. 32, 97 (1951).
`10. Lowry O. H., Rosebrough N. I., Farr A. L., Randall R. L.: J. Biol. Chem. 193, 265 (1951).
`11. Cihak A., Sorm F.: This Journal 30, 2137 (1965).
`12. Cihak A., Skoda J., Sorm F.: Biochim. Biophys. Acta 72, 125 (1963).
`13. Cihak A., Skoda J., Sorm F.: This Journal 29, 3297 (1963).
`14. Sorm F., Sormova Z., Jurovéik M., RaSka K.: This Journal, in the press.
`15. Cihak A., Sorm F.: This Journal 30, 324 (1965).
`
`Translated by A. Kotyk.
`
`Vol. 30 (1965)
`
`210L
`
`

`

`Cihak, Sorm
`
`Pesrome
`
`A. Unrax u ®. Dlopm: Buoxumuueckoe delicmeue u smemadoauveckue npeepaiyenus 5-azayumu-
`ouna y Escherichia coli. 5-A3auaTaqun (1-6-p-puOodypano3usi-5-a3aluTO3HH) — HOBbI aHTH-
`MeTaOOsJIMT, OONaaarollai BLIPAa3HTeJIbHbIM AHTHOaKTepHasIbHbIM Jelictpvem. IlonHoe warnOupo-
`BaHue pocta Escherichia coli, BbizbIBaeMoe 5-a3al{MTHQHHOM B KOHWeHTpayHH 1,2. 10— ©m, BO3MO%K-
`HO YCTpaHHTb OJHOBPeMeHHOL DOOaBKON ypuanHa, UMTHAMHA, WIM TOrda Kak ypallM WIM WATO3HH
`He OKa3bIBaloT HHKaKOro Baas. Tipu noBbImeHHOM TemMepatype 5-a3alMTUQUH pa3ziaraerca HM Te-
`paer Ouvosormyeckyio akTHuBHOCTs. B nutratenbHOl cpene Gaxtepuii u B GecKneTOUHOM akKCTpakTe
`E. coli 5-a3auuTaquH Nerko e3amMHUpyetca, IpH4em oOO6pa3yroTca puGo3n-N-dbopmunGuyper
`u puGo3muiOuypeT B KayecTBe MpOAYKTOB pa3oxKeHHA HeycToiunBoro 5-a3aypHoMNa; Walee nosy-
`yarotca 5-a3aypauus, N-popmMunOnuypet, GuypeT HW MalbHeltinime HeMAeHTUpUUMpOBAHHBIe BelLe-
`cTBa. [locue npeaqBapHTesbHOrO HHKYOHPOBaHHA C 5-a3alMTHOMHOM B Oe€CKIIETOYHOM 3KCTpaKkTe M3
`E. coli uMeror Mecro HHTHONpOBanHe PochoporH3a ypuauHAa WM THUMMouHa wu CHaGoe MHrHOMpOBaHHe
`WMTO3HHeaMnua3HOW aKTHBHOCTH. OOcy2xHeH MeXaHH3M BbIPaSHTeCIbHOrO TOPMO3AIUerO DelicTBHA
`5-a3auWTHAnHa C TOUKH 3peCHHA BOSMOXKHOLO BKJIIOURHHA 5-a3alMTHAMHa B HYKJICMHOBbIe KHCJIOTHI.
`
`
`
`2102
`
`Collection Czechoslov. Chem. Commun.
`
`