Jan 17, 1989 - 13. Meyers, J. D., E. C. Reed, D. H. Shepp, M. Thornquist, P. S.. Dandliker, C. A. Vicary, N. Flournoy, L. E. Kirk, J. H. Kersey,. E. D. Thomas, and ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1989, p. 1606-1608 0066-4804/89/091606-03$02.00/0 Copyright © 1989, American Society for Microbiology
Vol. 33, No. 9
NOTES
Antagonism of the Cytotoxic but Not Antiviral Effects of Ara-Sangivamycin by Adenosine GARY M. BIRCH,1 STEVEN H. KRAWCZYK,2 LEROY B. TOWNSEND,2 AND JOHN C. DRACHl* Department o Biologic and Materials Sciences, School of Dentistry,' and Department of Medicinal Chemistry,
College of Pharmacy,2 The University of Michigan, Ann Arbor, Michigan 48109-1078 Received 17 January 1989/Accepted 30 May 1989
Inhibition of DNA synthesis by ara-sangivamycin was antagonized by adenosine. The 50% inhibitory concentrations increased 1.6- to 32-fold in the presence of 1.0 to 50 ,uM adenosine, respectively. In contrast, the inhibition of human cytomegalovirus replication by ara-sangivamycin was not antagonized by as much as 50 ,uM adenosine. This suggests that different enzymes were responsible for the phosphorylation of ara-sangivamycin in uninfected and infected cells.
Human cytomegalovirus (HCMV) infections are serious, debilitating, and often life-threatening opportunistic diseases in immunocompromised individuals (8, 10). Moreover, HCMV may interact at the molecular level with human immunodeficiency virus and enhance the consequences of human immunodeficiency virus infection (3). Although a number of compounds are active against HCMV in vitro (2, 5, 11), only gancyclovir, acyclovir (ACV), and foscarnet are sufficiently promising for clinical use against certain HCMV infections (8, 13, 15, 16, 19). We have found that certain deoxyribosyl and arabinosyl pyrrolo[2,3-d]pyrimidines have selective antiviral activity against HCMV in vitro (21). Of these fraudulent nucleosides, ara-sangivamycin (ara-S), ara-toyocamycin, and 2'-deoxysangivamycin show the most selective activity against HCMV; but these same compounds show little selectivity against herpes simplex virus type 1. Analogs such as ara-S or deoxysangivamycin may be selectively phosphorylated by HCMV in infected cells, based on evidence that deoxyguanosine, deoxyadenosine, and gancyclovir are phosphorylated to a greater extent in infected cells than in uninfected cells (6). These observations suggest the possibility that a HCMV-encoded kinase or HCMV-induced cellular kinase is responsible for activating the compounds. To better understand this selective antiviral activity, we examined the antagonism of the antiviral and anticellular activities of ara-S by natural nucleosides. The capacity of adenosine (Ado), deoxyadenosine (dAdo), and deoxycytidine (dCyd) to antagonize the biological effects of fraudulent nucleosides was determined by measuring the amount of incorporation of [3H]thymidine ([3H]dThd) into acid-precipitable material and by an HCMV plaque reduction assay. The former assay, which is indicative of cellular DNA synthesis, was essentially a modification of the cytotoxicity assay described previously (21). In brief, KB cells or human foreskin fibroblasts (HFF cells) were planted in 96-well cluster dishes at a concentration of 12,000 cells per well in 200 ,ul of medium. After incubation of the dishes for 20 to 24 h in a humidified atmosphere at 37°C, 150 pul of spent medium was removed from each well. Combinations of 50 ,ul *
of drug and 50 ,ul of antagonist at four times their final concentrations were added. [3H]dThd (50 IlI; ICN Pharmaceuticals, Irvine, Calif.) was diluted with unlabeled dThd to give a final concentration of 3 p.M and was then added to each well to give a final concentration of 2 ,uCiIml. In experiments with Ado or dAdo as the antagonist, 2'-deoxycoformycin (dCof; a gift from Warner-Lambert/Parke-Davis Ann Arbor, Mich.), an adenosine deaminase inhibitor (22), was added to the medium at a final concentration of 1.0 p.g/ml (3.7 ,uM) as described previously (7, 20). After incubation of the plates for 24 to 26 h, cells were harvested and the label incorporated into acid-precipitable material was determined as described previously (21). All combinations were assayed in triplicate. Ara-S and sangivamycin were synthesized in the laboratory of L. B. Townsend (21). Cytarabine (ara-C) was a gift from The Upjohn Co. (Kalamazoo, Mich.). In uninfected KB cells, Ado antagonized the inhibition of DNA synthesis by ara-S. Figure 1 shows that in the absence of Ado, ara-S inhibited DNA synthesis with a 50% inhibitory concentration (IC50) of 5.3 p.M. When Ado was added in the presence of dCof, the dose-response curves shifted to the right, illustrating the antagonism of the cytotoxic effects of ara-S. This antagonism was observed with as little as 1.0 p.M Ado (the IC50 increased to 8.5 ,IM). With 50 p.M Ado, the antagonism was so profound that 100 p.M ara-S inhibited DNA synthesis by only 20%. Thus, the IC50 increased from 5.3 to 170 p.M in the presence of 50 p.M Ado. Comparable increases in IC50s were obtained when the HFF cell line was used under similar experimental conditions (Table 1). Visual inspection of the cells also revealed that KB and HFF cells were spared from toxicity at the higher Ado concentrations (data not shown). A more extensive comparison of the effects of drugantagonist combinations on DNA synthesis in uninfected KB and HFF cells is presented in Table 1. In KB cells, at increased concentrations of Ado, a large increase in the IC50 was observed for ara-S and sangivamycin. There was a slight increase for ara-C. These results are consistent with the fact that sangivamycin, a known inhibitor of DNA synthesis (18, 21), is antagonized by Ado in sarcoma 180 cells (17) and is phosphorylated by adenosine kinase (14). When dAdo was used as the antagonist, smaller increases in the IC50 of
Corresponding author. 1606
NOTES
VOL. 33, 1989 0
1607
responsible for phosphorylation of ara-S to its monophosphate in uninfected cells was adenosine kinase or deoxy-
120
adenosine kinase.
0 100
0
St0
80
0
ir
60
oh 40 8 00 c
0.
20
-c
00 -.1
1 0
1
Ara-S Conc-entration
100
1 000
(gM)
FIG. 1. Dose-response relationshi tions of ara-S and Ado on DNA synithesis in uninfected KB cells. DNA synthesis was measured by the iincorporation of [3H]dThd into acid-insoluble material. Data are from 7, 3, 3, and 3 separate experiments in the presence of 0 (l) 1 (A), 5 (0), and 50 (A) ,uM Ado, respectively. sangivamycin were observed coImpared with the increases observed when Ado was used as the antagonist. In the case of ara-S, dAdo produced a larger increase in the IC50 (Table 1). To verify further the antagoni sm with this system, ara-C was used in combination with ciCyd. Ara-C was selected based on its known potent inhibiltion of DNA synthesis and phosphorylation to the monophorsphate by 2'-deoxycytidine kinase (9). The IC50 of ara-C incrreased from 0.10 puM in the absence of dCyd to 1.8 p.M in thie presence of 10 ,uM dCyd (Table 1). In contrast, the IC50s of ara-S and sangivamycin did not change significantly up to 32 uM dCyd. In HFF cells, similar large increases in the IC5,Os of both ara-S and sangivamycin occurred at increased Pkdo concentrations. Taken together, these results strongly indicate that the enzyme
Based on these results in uninfected cells and the known selective activity of ara-S against HCMV, similar antagonism experiments were performed in HCMV-infected HFF cells. HCMV replication was measured by using a modification of the plaque reduction assay described previously (21). In brief, HFF cells were planted in 24-well cluster dishes at a concentration of 50,000 cells per well in 1.0 ml of medium. After 44 to 48 h, when cells were -80% confluent, monolayers were inoculated with 0.20 ml of a suspension containing 100 PFU of HCMV (Towne strain) and incubated for 1 h. Following adsorption, the virus suspension was aspirated and 1.0 ml of overlay medium was added which contained drug-antagonist combinations and 1.0 ,ug of dCof per ml. All combinations were assayed in triplicate. The plates were incubated for 6 to 9 days, cells were fixed and stained with 0.1% crystal violet in 20% methanol, and microscopic
plaques were enumerated at 20-fold magnification with a profile projector (Nikon). Dose-response relationships were
constructed as shown in Fig. 1, and IC50s were interpolated and tabulated as shown in Table 1. IC50s for the inhibition of HCMV plaque reduction at selected antagonist-drug combinations were compared with those for the inhibition of [3H]dThd. Figure 2 presents these data for ara-S as the amount of change in the IC50 in the presence of antagonist divided by the IC50 in the absence of antagonist. In marked contrast to the effects in uninfected KB and HFF cells, in which 50 ,uM Ado increased the IC50s more than 30-fold, concentrations of Ado up to 50 ,uM did not antagonize the antiviral activity of ara-S (IC50s in the absence and presence of 50 ,uM Ado were 0.84 and 1.2 ,uM, respectively). Similarly, 10 puM dAdo effected an increase in the IC50 for inhibition of DNA synthesis in KB cells, whereas 10 ,uM dAdo caused a 6.5-fold decrease in the IC50 for activity against HCMV in infected HFF cells. Comparable to ara-S, the cytotoxic effect of sangivamycin was reversed by
TABLE 1. Comparison of combinations of drug and antagonists on DNA synthesis on uninfected cellsa IC50 (,uM) produced by combination at the following antagonist concn (p.M)b:
Cell type,
antagonist, and drug
0
0.50
1.0
3.2
5.0
10
23 ± 4.7 0.66 NDc
35 ± 14 4.3 0.34
20
32
50
100
179 ± 2 5.9 ND
>100 ND 0.50
KB cells
Ado Ara-S Sangivamycin Ara-C
5.3 + 1.6 0.12 ± 0.03 0.10
7.0 ± 3.7 0.18 0.15
dAdo Ara-S Sangivamycin
5.3 ± 1.6 0.12 ± 0.03
15 ± 13 ND
18 ± 1.3 0.17
dCyd Ara-S Sangivamycin Ara-C
5.3 ± 1.6 0.12 ± 0.03 0.10
7.3 ND 0.31
8.1 0.13 ND
HFF cells Ado Ara-S
Sangivamycin
0.59 ± 0.52 0.022
ND ND 0.18
56 ± 22 0.28
ND >0.32
ND ND 0.59
8.0 0.15 1.8
6.8 0.15 ND
1.6 ± 1.6 0.25
2.6 ± 2.0 0.52
6.0 ND
56 1.6
DNA synthesis was measured by determining the amount of incorporation of [3H]dThd into acid-insoluble material. b IC50s were interoplated by using dose-response curves such as those shown in Fig. 1. All assays were performed in triplicate. Results are means + standard deviations for data from two or more experiments. c ND, No determination at the indicated antagonist concentration. a
1608
NOTES
ANTIMICROB. AGENTS CHEMOTHER. 3. Davis, M. G., S. C. Kenney, J. Kamine, J. S. Pagano, and E.-S.
°
Unlntected Cell
10
,c
4. 5.
10qs
6.
7.
Concentration Ado or dAdo (SM) FIG. 2. Change in IC50s caused by combinations of ara-S and Ado or dAdo in uninfected HFF (A) and KB (El, *) cells and in HCMV-infected HFF cells (O, *). IC'50s for the inhibition of DNA synthesis in uninfected cells were obtained from the data in Table 1. IC50s for HCMV plaque reduction in infected HFF cells were determined in one to seven separate experiments, as described in the text. Solid symbols denote the use of Ado as the antagonist; open symbols denote the use of dAdo as the antagonist.
50 Ado, but the antiviral activity was not affected by up toanM Ado (data not shown). If these compounds were phosphory-
lated infectediHfected cells by adenosine kinase, they should have been antagonized by Ado at 50 ,uM, considering that the Km, of Ado for adenosine kinase is -0.4 ,uM (14). The differences in antagonism between uninfected KB and HFF cells and HCMV-infected HFF cells may be indicative that different enzymes are responsible for phosphorylating ara-S and other pyrrolo[2,3-dfpyrimidines to their monodwhich phosphorylates phosphates. The nucleosidfthee gancyclovir to its monophosphate in HCMV-infected cells has not been identified, although a cellular deoxyguanosine kinase has been suggested (12). In herpes simplex virus type 1-infected cells, viral thymidine kinase phosphorylates gancyclovir and acyclovir to their monophosphates (15, 16). Unlike herpes simplex virus type 1, however, HCMV does not encode
a
vir-resistant
thymidine kinase (4). Studies with a gancycloof HCMV show that HCMV may en-
mutant
code another nucleoside kinase (1). Our results support this possibility and suggest that different enzymes are involved in the activity of ara-S in uninfected and infected cells. Thus, in uninfected cells, antagonism may have occurred because Ado and ara-S competed for adenosine kinase or a closely related enzyme. In HCMV-infected cells, antagonism was not apparent because an enzyme with little or no specificity toward Ado or dAdo was involved. This project
was
supported with federal funds from the DepartServices under contracts AI-42554 and
ment of Health and Human
AI-72641.
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