Archa oral
B~al.Vol. 20. pp. 581 to 585. Pergamon Press 1975. Printed in Great Britain.
IN
VITRO ANTIBACTERIAL ACTIVITY OF
ACTINOBOLIN ANALOGUES AGAINST CARIOGENIC STREPTOCOCCI AND OTHER ORAL BACTERIA D. E.
HUNT
and K. E.
EDWARDS,
III
Department of Oral Biology, Emory University, School of Dentistry, Atlanta. Georgia 30322, U.S.A.
Summary-Thirty-one synthetic analogues of the antibiotic actinobolin were evaluated for in uitro inhibitory activity against 6 strains of Streptococcus mutans, six mixed cultures obtained from humac. dental plaque and three Lactobacillus strains obtained from deep carious lesions of adult patients. The analogues were of two general types in which: (A) additions were made to the free amino group of the parent compound or (B) the L-alanine moiety was replaced with different groups. None of the analogues tested were found to have antibacterial activity superior to the natural antibiotic; however, the data suggest some of the structural and molecular features of the actinobolin molecule that appear to be essential for antibacterial activity.
INTRODUCTION
MATERIALS
AND METHODS
Many studies have established the potential value of Bacteria antimicrobial agents in the control of dental caries The cariogenic streptococci used in this study were (McClure and Hewitt, 1946; Shaw, 1959; Keyes, 1969; Streptococcus mutuns strains 6715, AHT, LM-7, Bibby, 1970). Additional reports which have shown NCTC 10449, PK-1, and SL-1. They were selected strong correlations between cariostatic effect and the because they represent a spectrum of serotypes of spectrum of antibiotic activity of individual antibioStrep. mutans. These strains were maintained in stab tics, have helped establish that dental caries is probcultures of brain-heart infusion (BHI) agar (Difco, ably associated with a small spectrum of Gram-posiDetroit); cultures were incubated for 24 hr at 37°C tive oral bacteria (Stephan et al., 1952; Fitzgerald, in an atmosphere of 95 per cent nitrogen and 5 per 1955). These types of investigations, combined with cent carbon dioxide. The lactobacilli used were the availability of a vast literature covering more than obtained as follows: plates of modified (Hunt and 25 yr of medical applications of antibiotics, have Kohn, 1974) Tomato Juice Agar, Special, (Difco) were allowed the formulation of a list of chemical and bioinoculated with debris from deep carious lesions of logical properties .that should be found in an ideal adult patients and incubated for 48 hr under the antimicrobial cariostatic agent (Fitzgerald, 1972). above indicated conditions. Microscopic examination Actinobolin is an antibiotic that has most of the of selected isolated colonies growing on these plates properties desired in an ideal cariostatic antibiotic revealed short, Gram-positive, nonsporing rods show(Hunt. Sandham and Caldwell, 1970; Hunt, Navia ing palisade arrangements. These isolates were further and Lopex, 1971; Hunt et al., 1972; Armstrong, Feacharacterized according to criteria described by Burgin and Hunt, 1973). Additionally, this antibiotic has nett and Scherp (1968) and three were identified as been reported to be strongly inhibitory for bacteria strains of Lactobacillus acidophilus, L. casei, and L. associated with periodontal disease (Armstrong and fermenti. The lactobacilli were maintained on BHI Hunt, 1972) to effectively control the periodontal synslants which were incubated as described. Six mixed drome in rice rats (Shaw and Ivimey, 1973). In microbial cultures were also tested for sensitivity to vitro data suggest that actinobolin may be synergistic the actinobolin analogues. The mixed cultures were with fluoride in preventing dental caries and perioobtained by inoculating BHI broth with dental dontal disease (Armstrong and Feagin, 1973). These plaque obtained from approximal tooth surfaces of observations encouraged a study to evaluate in vitro adults. In all primary in vitro evaluations of the analoa variety of chemical analogues of actinobolin for ingues, 2 sets of plates were prepared: one set was incuhibitory activity against cariogenic streptococci and bated aerobically and the second set was incubated other oral bacteria to: (a) determine if any of the anain an atmosphere of 95 per cent nitrogen and 5 per logues have antibacterial activity superior to the parcent carbon dioxide. All plates were incubated at ent compound; (bl identify those chemical modilica37°C for 24 hr. tions to the parent compound that appear to alter Analogues antibiotic activity; and (c) acquire information about The actinobolin analogues tested are shown in Fig. structure-activity relationships which may be corre1. On the basis of the chemical modifications to the lated with what is currently known about the subcelnatural compound, the analogues were divided into lular mechanism by which actinobolin inhibits bac2 general types arbitrarily designated as types A and terial growth. 581
582
D. E. Hunt
and K. E. Edwards,
13
Omlnrclldln~
L
13..
I-AC*tYl
lo
Fig. 1. Comparative
structural
features
alamlm. .eeily yield
of actinobolin
B, and for reasons of brevity, assigned numbers l-30. Type A analogues were modified by making various chemical additions to the free amino group of the actinobolin molecule. Type B were those in which the L-alanine moeity of actinobolin was replaced with a variety of chemical groups. All analogues were readily soluble in water except analogue number 8 which was insoluble in water and was tested as an aqueous suspension. As no data are available concerning the toxicity of the analogues, and because one of the main objectives of this study were to compare the antibacterial activity of the analogues with the parent compound, the analogue concentration selected for testing reflects therapeutic levels of natural actinobolin which can be safely applied topically in the oral cavity. Antibacterial activity For primary in vitro evaluations of the analogues, a paper-disc agar diffusion method (Hunt and Pittillo, 1967) was used in which BHI agar plates, uniformly
111
l..logu.s
r.,,ao,d ritb
..I
and synthetic
0
AHT PK-1 SL-1 6715 LM-7 NCI-C 10449
O$ 0 0 0 0 0
Acb*
lt
2
3.4 2.3 3.2 3.1 3.0 3.3
1.6 1.0 1.4 1.4 l-5 1.3
1.4 1.0 1.3 1.1 l-l 1.2
I
yr.,,s
analogues.
inoculated with test bacteria, were overlaid with paper discs (740-E, Schleicher and Schuell, Keene, N. H.) which had been charged with aqueous solutions of the individual analogues or actinobolin. The relative antibacterial activity of the analogues was determined by measuring the diameters of zones of growth inhibition which developed around the discs. Additional in vitro evaluations were conducted in which the analogues showing the strongest activity in the primary evaluations were tested for inhibitory activity against proliferating broth cultures of strains of Strep. mutans. For these studies, test tubes containing IOml of sterile BHI broth were uniformly inoculated with a 0.1 ml of a BHI broth culture of each strain of Strep. mutans. Tubes were incubated aerobically at 37°C and growth was monitored turbidimetrically at 540 nm in a Bausch and Lomb Spectronic 20 Colorimeter. When the turbidity in these tubes indicated an early phase of rapid proliferation, actinobolin or the selected analogues were added. Appropriate control tubes were untreated.
Table 1. In vitro inhibitory activity of synthetic analogues of actinobolin Streptococcus mutans strains
ap,ro,ri.t.
14-30.
against cariogenic streptococci
Test compounds (I 00 &disc) 5 6 7 9 4 2.4 1.4 2.2 2.1 2.1 2.3
2.5 2.6 2.6 2.6 26 2.5
1.0 1.0 1.1 1.2 1.2 1.1
0% 0.8 0.8 1.0 I.0 I.1
I.7 1.4 2.6 1.6 I,4 1.7
11
13
25
30
1.5 1.0 I.3 1.4 1.2 1.3
2.1 1.7 2.0 2.0 1% 1.9
2.0 2.2 1.6 2.4 2.3 2.4
I.8 1.2 1.0 1.5 1.5 1.6
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. $ Numbers in Table refer to diameters (cm) of zones of growth inhibition that developed (under aerobic conditions) around paper discs containing the indicated concentration of the respective test compounds. Discs were 0.63 cm in diameter. Analogues 3, 8, 10, 12, 13a-24, and 2629 were non-inhibitory for the streptococci tested.
583
Antibacterial activity of actinobolin analogues Table 2. III vitro inhibitory activity of synthetic analogues of actinobolin obtained from dental plaque Plaque (subjects)
0
AG DH JO PB LH BP
01 0 0 0 0 0
Acb*
It
2
2.4 2.4 2.0 2.4 2.5 2.3
1.4 1.0 0.7 0.8 1.0 I -0
1.2 0.7 0.7 0 0.7 1-I
against mixed microbial cultures
Test compounds (100 pg/disc) 4 5 6 7 9 1.4 1.5 1.1 1.6 1.6 1.4
2.0 1.9 2.1 2.1 2.4 1.5
1.1 1.1 1.0 0.9 1.3 l-4
1.0 0.9 1.0 0.9 1.3 1.0
1.5 1.3 1.7 1.5 1.7 1.7
11
13
25
30
1.3 1.1 1.4 1.5 1.3 1.1
2.0 1.6 2.2 2.1 2.0 2.1
1.5 2.1 2.0 2.4 2.5 2.5
1.1 1.2 1.7 1.5 1.7 1.5
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. TNumbers in Table refer to diameters (cm) of zones of growth inhibition that developed (under aerobic conditions) around paper discs containing the indicated concentration of the respective test compounds. The diameter of the discs was 0.63 cm. Analogues 3, 8, 10, 12, 13a-24 and 26-29 were non-inhibitory for the plaque cultures tested. RESULTS The in-vitro inhibitory activity of the analogues against a spectrum of cariogenic streptococci is shown in Table 1. Under aerobic conditions and at a concentration of lOOpg/disc, analogues 1, 2, 4-7, 9, 11, 13, 25 and 30 reve.aled various levels of antibacterial activity against the streptococcal strains tested as indicated by the zones of bacterial growth inhibition. Analogues 3, 8, IO, 12, 13a-24 and 2629 were non-inhibitory at the concentrations tested. Data presented in Table 2 show similar inhibitory effects when the analogues were tested for antibacterial activity against mixed microbial populations prepared from human dental plaque. Nearly identical results were obtained when the analogues were tested against the plaque cultures and the streptococcal strains under anaerobic conditions. The analogues were generally less inhibitory for lactobacilli since only analogues 4, 5, 9 and 13 produced measurable zones of growth inhibition against the Lactobacillus strains tested (Table 3). A dose-related effect was also noted since lower concentrations of the respective analogues (50 and 25 pg/ disc-not shown in Tables l-3) produced smaller zones of growth rnhibition. As a basis for selecting those compounds with the strongest inhibitory activity, all analogues that produced a zone of growth inhibition 12.0cm dia were selected for secondary
evaluations. Consequently, analogues 4, 5, 9, 13, 25 and actinobolin were added to rapidly dividing broth cultures of Strep. mutans strains 6715, AHT, LM-7, NCTC 10449, PK-1 and SL-1. Representative data in Fig. 2 show that analogue 5 was less inhibitory than actinobolin for proliferating broth cultures of Strep mutans 6715, and nearly identical results were obtained when analogues 5, 9, 13 and 25 were added to growing broth cultures of the six above indicated strains of Strep. mutans.
DISCUSSION
Under the described conditions, none of the analogues were found to be as strongly antibacterial as the parent compound. The lactobacilli were generally less sensitive to inhibition by the analogues than were strains of Strep. mutans tested, an observation that might be useful in identifying agents that have highly specific anticaries activity. Type A analogues were generally more inhibitory than those of type B, which suggests some of the structural features of the actinobolin molecule that appear to be required for antibacterial activity. The L-alanine moeity appears to be critical for inhibitory activity since the removal of, substitution of, or additions to this portion of the actinobolin molecule caused a reduction or total loss
Table 3. In-uirro inhibitory activity of synthetic analogues of actinobolin 0 Lactobacillus strains
AnI
Ae$
L. acidophilus
O/I
0
L. casei L. ,jhzenti
0 0
0 0
Acb* An Ae 2.0 1.6 2.3
2.8 2.0 2-4
Test compounds (100pg/disc) 5 4t An Ae An Ae 1.3 1.2 0
0 1.2 0
I.2 1.3 I-3
1.4 I.3 i.5
against oral lactobacilli 9
13
An
Ae
An
Ae
0 1.3 I.2
1.3 1.3 1.4
0 1.2 1.2
1.2 1.2 1.3
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. $ Zones of growth inhibition that developed under anaerobic conditions. 4 Zones of growth inhibition that developed under aerobic conditions. 11Numbers in Table refer to diameters (cm) of zones of growth inhibition that developed around paper discs containing the indicated concentration of the respective test compounds. The diameter of the discs was 0.63 cm. Analogues 3, 8, 10, 12, 13a-24 and 2629 were non-inhibitory for the lactobacilli tested.
584
D. E. Hunt and K. E. Edwards, III Control A 5Opg Acb/ml
l
n
A 5Opg Anologue 5/ml . IOOpg Anologue 5/ml
IOOpg Acb/ml
Time,
Fig. 2. Comparative analogue
hr
Time,
hr
in-citro inhibitory activity of actinobolin (Acb) and a representative (5) against exponentially dividing broth cultures of Strep. ~nutc~r~s6715.
of activity. Since studies have shown that actinobolin inhibits protein synthesis (Hunt et al., 1966; Hunt and Narkates, 1971) and because of the key role of free amino groups in protein synthesis, it seemed logical to assume that the free amino group on the actinobolin molecule might be the specific binding site. Support for this assumption was found in the observation that the N-acetyl derivative of actinobolin (analogue 13a), in which the free amino group is blocked, lost all antibacterial activity for strains of Strep. mutuns, mixed oral cultures, and lactobacilli. Additionally, this assumed role of the free amino group in the inhibition of protein synthesis is consistent with the report (Smithers, Bennett and Struck, 1969) that the most probable site of action of actinobolin is interference with the binding of phenylalanyl -tRNA to the polyuridylic acid-ribosome complex. It is of interest that a free amino group has been shown to be the specific binding site on puromycin, an antibiotic that is also an inhibitor of bacterial protein synthesis. Its inhibitory action is due to the formation of a peptide bond between the terminal carboxyl group of the growing peptide chain and the free amino group 01 puromycin (Nathans, 1967). At this time, no definite conclusions can be drawn about the exact way that the free amino group of the actinobolin molecule might act to inhibit bacterial protein synthesis since certain analogues, i.e. analogue 13 which also has a blocked amino group, retained substantial antibacterial activity. Additionally, some of the peptide derivatives retained partial inhibitory activity. These seeming inconsistencies could be attributed to differences in the permeability of the bacterial cells to the various analogues, to the hydrolysis of the peptides to yield actinobolin, or to steric effects. The latter are suggested by the delay in growth inhibition of the analogues in broth cultures (Fig. 2) of Strep. mutans. Also of interest is the diminished inhibitory activity of analogue 25. This analogue differs from the parent compound only by the presence of the beta hydroxy group which further emphasizes that the slightest change in the structure of an antibiotic molecule often has a profound effect on its antibacterial activity. Ackrlow(edyements~This work was partially supported by Grant RR-05308 and by McCandless Research
USPHS
Control
l
synthetic
Funds from Emory University. Our thanks are extended to Fran Hardy for her assistance in the preparation of this paper. Actinoholin and the analogttcs t&e; wcrc supolied bv Parke-Davis & Co.. Detroit. The six strains of Strep. t&tans were supplied by Richard Facklam. Portions of this paper were presented at the 52nd General Session of the International Association for Dental Research, Atlanta, Georgia, 1974.
REFERENCES Armstrong P. J. Jr. and Feagin F. F. 1973. Effect of fluoride ions and actinobolin on apatite growth at enamel surfaces. J. dent. Rex 52, 1346. Armstrong P. J. Jr., Feagin F. F. and Hunt D. E. 1973. 1,~ uitro antimicrobial activity of actinobolin applied to tooth surfaces. J. prriodont. kes. 8. 404405. _. Armstrong P. J. Jr. and Hunt D. E. 1972. 1n vitro evaluation ofactinobolin as an antibiotic for the treatment of periodontal disease. Appl. Micro/M 23, 88-90. Bibbv B. G. 1970. Antibiotics and dental caries. In: Dietarw Chemicals us Detztal Curies (Edited by Harris R. S.), pp’. 4&54. American Chemical Society Publication. Washington, DC. Burnett G. W. and Scherp H. W. 1968. In: Oral Microbiology and It~j~tious Disease, p. 285. Williams & Wilkins, Baltimore. Fitzgerald R. J. 1955. Influence of antibiotics on experimental rat caries. In: Advances in Eqwrimental Caries Research (Edited by Sognnaes R. F.), pp. 187-196. American Association for the Advancement of Sciences, Washington, D.C. Fitzgerald R. J. 1972.‘ Inhibition of experimental dental caries. Atitirnicroh. Ag. Chemother. 1, 296302. Hunt D. E., Armstrong P. J. Jr.. Black C. III and Narkates A. J. 1972. III vitro comparison of actinobolin and other antibiotics used in the treatment of periodontal disease and dental caries. Proc. Sot. rrp. EioL Mrd. 140, 14291433. Hunt D. E. and Kohn D. W. 1974. The use of amphotericin B to increase the selectivity of media used for isolating oral lactobacilli. Archs oral Biol. 19. 1073-m1076. Hunt D. E. and Narkates A. J. 1971. Etrect of actinobolin on nucleic acid and protein synthesis in Streptococcus faecalis. J. dent. Res. 50, 161@1615. Hunt D. E., Navia J. M. and Lopez H. 1971. III uiuo cariostatic activity of actinobolin. J. dent. Res. SO, 371-373. Hunt D. E. and Pittillo R. F. 1967. Antimicrobial activitv of 1,2,5-selenadiazoles. In: Antimicrobial Agents a& Chemotherapy (Edited by Hobby G. L.), pp. 551-554. American Society for Microbiology, Ann Arbor.
Antibacterial activity of actinobolin analogues Hunt D. E., Pittillo R. F., Johnson E. P. and Moncrief F. C. 1966. The effect of actinobolin on nucleic acid and protein synthesis in Escherichia coli. Cm. J. Microbio/. 12, 515-520. Hunt D. E., Sandham 1-I. J. and Caldwell R. C. 1970. In vitro antibiotic sensitivity of oral microorganisms to actinobolin. J. dent. Rex 50, 371-373. Keyes P. H. 1969. Present and future measures for dental caries control. J. Am. dent. Ass. 79, 1395-1404. McClure F. J. and Hewitt W. L. 1946. The relation of penicillin to induced rat dental caries and oral L. acidophilus. J. dent. Res. 25, 441-443. Nathans D. 1967. Puromycin. In: Antibiotics I Mechanism of Action (Edited by Gottlieb D. and Shaw P. D.), pp. 259-277. Springer-Verlag. New York.
585
J. H. 1959. Caries inhibiting agents. Pharmac. Rev. 705-716. J. H. and Ivimey J. K. 1973. Actinobolin as an inhibiof the periodontal syndrome in rice rats. Archs oral Biol. 18, 357-360. Smithers D., Bennett L. L. and Struck R. F. 1969. Inhibition of protein synthesis in mammalian cells by actinobolin. MO&. Pharm. 5, 433-445. Stephan R. M., Fitzgerald R. J., McClure F. J., Harris M. R. and Jordan H. V. 1952. The comparative effects of penicillin, bacitracin, chloromycetin, aureomycin, and streptomycin on experimental dental caries and on certain oral bacteria in the rat. J. dent. Res. 31, 421427. Shaw 11, Shaw tor
B~al.Vol. 20. pp. 581 to 585. Pergamon Press 1975. Printed in Great Britain.
IN
VITRO ANTIBACTERIAL ACTIVITY OF
ACTINOBOLIN ANALOGUES AGAINST CARIOGENIC STREPTOCOCCI AND OTHER ORAL BACTERIA D. E.
HUNT
and K. E.
EDWARDS,
III
Department of Oral Biology, Emory University, School of Dentistry, Atlanta. Georgia 30322, U.S.A.
Summary-Thirty-one synthetic analogues of the antibiotic actinobolin were evaluated for in uitro inhibitory activity against 6 strains of Streptococcus mutans, six mixed cultures obtained from humac. dental plaque and three Lactobacillus strains obtained from deep carious lesions of adult patients. The analogues were of two general types in which: (A) additions were made to the free amino group of the parent compound or (B) the L-alanine moiety was replaced with different groups. None of the analogues tested were found to have antibacterial activity superior to the natural antibiotic; however, the data suggest some of the structural and molecular features of the actinobolin molecule that appear to be essential for antibacterial activity.
INTRODUCTION
MATERIALS
AND METHODS
Many studies have established the potential value of Bacteria antimicrobial agents in the control of dental caries The cariogenic streptococci used in this study were (McClure and Hewitt, 1946; Shaw, 1959; Keyes, 1969; Streptococcus mutuns strains 6715, AHT, LM-7, Bibby, 1970). Additional reports which have shown NCTC 10449, PK-1, and SL-1. They were selected strong correlations between cariostatic effect and the because they represent a spectrum of serotypes of spectrum of antibiotic activity of individual antibioStrep. mutans. These strains were maintained in stab tics, have helped establish that dental caries is probcultures of brain-heart infusion (BHI) agar (Difco, ably associated with a small spectrum of Gram-posiDetroit); cultures were incubated for 24 hr at 37°C tive oral bacteria (Stephan et al., 1952; Fitzgerald, in an atmosphere of 95 per cent nitrogen and 5 per 1955). These types of investigations, combined with cent carbon dioxide. The lactobacilli used were the availability of a vast literature covering more than obtained as follows: plates of modified (Hunt and 25 yr of medical applications of antibiotics, have Kohn, 1974) Tomato Juice Agar, Special, (Difco) were allowed the formulation of a list of chemical and bioinoculated with debris from deep carious lesions of logical properties .that should be found in an ideal adult patients and incubated for 48 hr under the antimicrobial cariostatic agent (Fitzgerald, 1972). above indicated conditions. Microscopic examination Actinobolin is an antibiotic that has most of the of selected isolated colonies growing on these plates properties desired in an ideal cariostatic antibiotic revealed short, Gram-positive, nonsporing rods show(Hunt. Sandham and Caldwell, 1970; Hunt, Navia ing palisade arrangements. These isolates were further and Lopex, 1971; Hunt et al., 1972; Armstrong, Feacharacterized according to criteria described by Burgin and Hunt, 1973). Additionally, this antibiotic has nett and Scherp (1968) and three were identified as been reported to be strongly inhibitory for bacteria strains of Lactobacillus acidophilus, L. casei, and L. associated with periodontal disease (Armstrong and fermenti. The lactobacilli were maintained on BHI Hunt, 1972) to effectively control the periodontal synslants which were incubated as described. Six mixed drome in rice rats (Shaw and Ivimey, 1973). In microbial cultures were also tested for sensitivity to vitro data suggest that actinobolin may be synergistic the actinobolin analogues. The mixed cultures were with fluoride in preventing dental caries and perioobtained by inoculating BHI broth with dental dontal disease (Armstrong and Feagin, 1973). These plaque obtained from approximal tooth surfaces of observations encouraged a study to evaluate in vitro adults. In all primary in vitro evaluations of the analoa variety of chemical analogues of actinobolin for ingues, 2 sets of plates were prepared: one set was incuhibitory activity against cariogenic streptococci and bated aerobically and the second set was incubated other oral bacteria to: (a) determine if any of the anain an atmosphere of 95 per cent nitrogen and 5 per logues have antibacterial activity superior to the parcent carbon dioxide. All plates were incubated at ent compound; (bl identify those chemical modilica37°C for 24 hr. tions to the parent compound that appear to alter Analogues antibiotic activity; and (c) acquire information about The actinobolin analogues tested are shown in Fig. structure-activity relationships which may be corre1. On the basis of the chemical modifications to the lated with what is currently known about the subcelnatural compound, the analogues were divided into lular mechanism by which actinobolin inhibits bac2 general types arbitrarily designated as types A and terial growth. 581
582
D. E. Hunt
and K. E. Edwards,
13
Omlnrclldln~
L
13..
I-AC*tYl
lo
Fig. 1. Comparative
structural
features
alamlm. .eeily yield
of actinobolin
B, and for reasons of brevity, assigned numbers l-30. Type A analogues were modified by making various chemical additions to the free amino group of the actinobolin molecule. Type B were those in which the L-alanine moeity of actinobolin was replaced with a variety of chemical groups. All analogues were readily soluble in water except analogue number 8 which was insoluble in water and was tested as an aqueous suspension. As no data are available concerning the toxicity of the analogues, and because one of the main objectives of this study were to compare the antibacterial activity of the analogues with the parent compound, the analogue concentration selected for testing reflects therapeutic levels of natural actinobolin which can be safely applied topically in the oral cavity. Antibacterial activity For primary in vitro evaluations of the analogues, a paper-disc agar diffusion method (Hunt and Pittillo, 1967) was used in which BHI agar plates, uniformly
111
l..logu.s
r.,,ao,d ritb
..I
and synthetic
0
AHT PK-1 SL-1 6715 LM-7 NCI-C 10449
O$ 0 0 0 0 0
Acb*
lt
2
3.4 2.3 3.2 3.1 3.0 3.3
1.6 1.0 1.4 1.4 l-5 1.3
1.4 1.0 1.3 1.1 l-l 1.2
I
yr.,,s
analogues.
inoculated with test bacteria, were overlaid with paper discs (740-E, Schleicher and Schuell, Keene, N. H.) which had been charged with aqueous solutions of the individual analogues or actinobolin. The relative antibacterial activity of the analogues was determined by measuring the diameters of zones of growth inhibition which developed around the discs. Additional in vitro evaluations were conducted in which the analogues showing the strongest activity in the primary evaluations were tested for inhibitory activity against proliferating broth cultures of strains of Strep. mutans. For these studies, test tubes containing IOml of sterile BHI broth were uniformly inoculated with a 0.1 ml of a BHI broth culture of each strain of Strep. mutans. Tubes were incubated aerobically at 37°C and growth was monitored turbidimetrically at 540 nm in a Bausch and Lomb Spectronic 20 Colorimeter. When the turbidity in these tubes indicated an early phase of rapid proliferation, actinobolin or the selected analogues were added. Appropriate control tubes were untreated.
Table 1. In vitro inhibitory activity of synthetic analogues of actinobolin Streptococcus mutans strains
ap,ro,ri.t.
14-30.
against cariogenic streptococci
Test compounds (I 00 &disc) 5 6 7 9 4 2.4 1.4 2.2 2.1 2.1 2.3
2.5 2.6 2.6 2.6 26 2.5
1.0 1.0 1.1 1.2 1.2 1.1
0% 0.8 0.8 1.0 I.0 I.1
I.7 1.4 2.6 1.6 I,4 1.7
11
13
25
30
1.5 1.0 I.3 1.4 1.2 1.3
2.1 1.7 2.0 2.0 1% 1.9
2.0 2.2 1.6 2.4 2.3 2.4
I.8 1.2 1.0 1.5 1.5 1.6
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. $ Numbers in Table refer to diameters (cm) of zones of growth inhibition that developed (under aerobic conditions) around paper discs containing the indicated concentration of the respective test compounds. Discs were 0.63 cm in diameter. Analogues 3, 8, 10, 12, 13a-24, and 2629 were non-inhibitory for the streptococci tested.
583
Antibacterial activity of actinobolin analogues Table 2. III vitro inhibitory activity of synthetic analogues of actinobolin obtained from dental plaque Plaque (subjects)
0
AG DH JO PB LH BP
01 0 0 0 0 0
Acb*
It
2
2.4 2.4 2.0 2.4 2.5 2.3
1.4 1.0 0.7 0.8 1.0 I -0
1.2 0.7 0.7 0 0.7 1-I
against mixed microbial cultures
Test compounds (100 pg/disc) 4 5 6 7 9 1.4 1.5 1.1 1.6 1.6 1.4
2.0 1.9 2.1 2.1 2.4 1.5
1.1 1.1 1.0 0.9 1.3 l-4
1.0 0.9 1.0 0.9 1.3 1.0
1.5 1.3 1.7 1.5 1.7 1.7
11
13
25
30
1.3 1.1 1.4 1.5 1.3 1.1
2.0 1.6 2.2 2.1 2.0 2.1
1.5 2.1 2.0 2.4 2.5 2.5
1.1 1.2 1.7 1.5 1.7 1.5
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. TNumbers in Table refer to diameters (cm) of zones of growth inhibition that developed (under aerobic conditions) around paper discs containing the indicated concentration of the respective test compounds. The diameter of the discs was 0.63 cm. Analogues 3, 8, 10, 12, 13a-24 and 26-29 were non-inhibitory for the plaque cultures tested. RESULTS The in-vitro inhibitory activity of the analogues against a spectrum of cariogenic streptococci is shown in Table 1. Under aerobic conditions and at a concentration of lOOpg/disc, analogues 1, 2, 4-7, 9, 11, 13, 25 and 30 reve.aled various levels of antibacterial activity against the streptococcal strains tested as indicated by the zones of bacterial growth inhibition. Analogues 3, 8, IO, 12, 13a-24 and 2629 were non-inhibitory at the concentrations tested. Data presented in Table 2 show similar inhibitory effects when the analogues were tested for antibacterial activity against mixed microbial populations prepared from human dental plaque. Nearly identical results were obtained when the analogues were tested against the plaque cultures and the streptococcal strains under anaerobic conditions. The analogues were generally less inhibitory for lactobacilli since only analogues 4, 5, 9 and 13 produced measurable zones of growth inhibition against the Lactobacillus strains tested (Table 3). A dose-related effect was also noted since lower concentrations of the respective analogues (50 and 25 pg/ disc-not shown in Tables l-3) produced smaller zones of growth rnhibition. As a basis for selecting those compounds with the strongest inhibitory activity, all analogues that produced a zone of growth inhibition 12.0cm dia were selected for secondary
evaluations. Consequently, analogues 4, 5, 9, 13, 25 and actinobolin were added to rapidly dividing broth cultures of Strep. mutans strains 6715, AHT, LM-7, NCTC 10449, PK-1 and SL-1. Representative data in Fig. 2 show that analogue 5 was less inhibitory than actinobolin for proliferating broth cultures of Strep mutans 6715, and nearly identical results were obtained when analogues 5, 9, 13 and 25 were added to growing broth cultures of the six above indicated strains of Strep. mutans.
DISCUSSION
Under the described conditions, none of the analogues were found to be as strongly antibacterial as the parent compound. The lactobacilli were generally less sensitive to inhibition by the analogues than were strains of Strep. mutans tested, an observation that might be useful in identifying agents that have highly specific anticaries activity. Type A analogues were generally more inhibitory than those of type B, which suggests some of the structural features of the actinobolin molecule that appear to be required for antibacterial activity. The L-alanine moeity appears to be critical for inhibitory activity since the removal of, substitution of, or additions to this portion of the actinobolin molecule caused a reduction or total loss
Table 3. In-uirro inhibitory activity of synthetic analogues of actinobolin 0 Lactobacillus strains
AnI
Ae$
L. acidophilus
O/I
0
L. casei L. ,jhzenti
0 0
0 0
Acb* An Ae 2.0 1.6 2.3
2.8 2.0 2-4
Test compounds (100pg/disc) 5 4t An Ae An Ae 1.3 1.2 0
0 1.2 0
I.2 1.3 I-3
1.4 I.3 i.5
against oral lactobacilli 9
13
An
Ae
An
Ae
0 1.3 I.2
1.3 1.3 1.4
0 1.2 1.2
1.2 1.2 1.3
* Actinobolin. t See Fig. 1 for names and chemical features of the respective analogues. $ Zones of growth inhibition that developed under anaerobic conditions. 4 Zones of growth inhibition that developed under aerobic conditions. 11Numbers in Table refer to diameters (cm) of zones of growth inhibition that developed around paper discs containing the indicated concentration of the respective test compounds. The diameter of the discs was 0.63 cm. Analogues 3, 8, 10, 12, 13a-24 and 2629 were non-inhibitory for the lactobacilli tested.
584
D. E. Hunt and K. E. Edwards, III Control A 5Opg Acb/ml
l
n
A 5Opg Anologue 5/ml . IOOpg Anologue 5/ml
IOOpg Acb/ml
Time,
Fig. 2. Comparative analogue
hr
Time,
hr
in-citro inhibitory activity of actinobolin (Acb) and a representative (5) against exponentially dividing broth cultures of Strep. ~nutc~r~s6715.
of activity. Since studies have shown that actinobolin inhibits protein synthesis (Hunt et al., 1966; Hunt and Narkates, 1971) and because of the key role of free amino groups in protein synthesis, it seemed logical to assume that the free amino group on the actinobolin molecule might be the specific binding site. Support for this assumption was found in the observation that the N-acetyl derivative of actinobolin (analogue 13a), in which the free amino group is blocked, lost all antibacterial activity for strains of Strep. mutuns, mixed oral cultures, and lactobacilli. Additionally, this assumed role of the free amino group in the inhibition of protein synthesis is consistent with the report (Smithers, Bennett and Struck, 1969) that the most probable site of action of actinobolin is interference with the binding of phenylalanyl -tRNA to the polyuridylic acid-ribosome complex. It is of interest that a free amino group has been shown to be the specific binding site on puromycin, an antibiotic that is also an inhibitor of bacterial protein synthesis. Its inhibitory action is due to the formation of a peptide bond between the terminal carboxyl group of the growing peptide chain and the free amino group 01 puromycin (Nathans, 1967). At this time, no definite conclusions can be drawn about the exact way that the free amino group of the actinobolin molecule might act to inhibit bacterial protein synthesis since certain analogues, i.e. analogue 13 which also has a blocked amino group, retained substantial antibacterial activity. Additionally, some of the peptide derivatives retained partial inhibitory activity. These seeming inconsistencies could be attributed to differences in the permeability of the bacterial cells to the various analogues, to the hydrolysis of the peptides to yield actinobolin, or to steric effects. The latter are suggested by the delay in growth inhibition of the analogues in broth cultures (Fig. 2) of Strep. mutans. Also of interest is the diminished inhibitory activity of analogue 25. This analogue differs from the parent compound only by the presence of the beta hydroxy group which further emphasizes that the slightest change in the structure of an antibiotic molecule often has a profound effect on its antibacterial activity. Ackrlow(edyements~This work was partially supported by Grant RR-05308 and by McCandless Research
USPHS
Control
l
synthetic
Funds from Emory University. Our thanks are extended to Fran Hardy for her assistance in the preparation of this paper. Actinoholin and the analogttcs t&e; wcrc supolied bv Parke-Davis & Co.. Detroit. The six strains of Strep. t&tans were supplied by Richard Facklam. Portions of this paper were presented at the 52nd General Session of the International Association for Dental Research, Atlanta, Georgia, 1974.
REFERENCES Armstrong P. J. Jr. and Feagin F. F. 1973. Effect of fluoride ions and actinobolin on apatite growth at enamel surfaces. J. dent. Rex 52, 1346. Armstrong P. J. Jr., Feagin F. F. and Hunt D. E. 1973. 1,~ uitro antimicrobial activity of actinobolin applied to tooth surfaces. J. prriodont. kes. 8. 404405. _. Armstrong P. J. Jr. and Hunt D. E. 1972. 1n vitro evaluation ofactinobolin as an antibiotic for the treatment of periodontal disease. Appl. Micro/M 23, 88-90. Bibbv B. G. 1970. Antibiotics and dental caries. In: Dietarw Chemicals us Detztal Curies (Edited by Harris R. S.), pp’. 4&54. American Chemical Society Publication. Washington, DC. Burnett G. W. and Scherp H. W. 1968. In: Oral Microbiology and It~j~tious Disease, p. 285. Williams & Wilkins, Baltimore. Fitzgerald R. J. 1955. Influence of antibiotics on experimental rat caries. In: Advances in Eqwrimental Caries Research (Edited by Sognnaes R. F.), pp. 187-196. American Association for the Advancement of Sciences, Washington, D.C. Fitzgerald R. J. 1972.‘ Inhibition of experimental dental caries. Atitirnicroh. Ag. Chemother. 1, 296302. Hunt D. E., Armstrong P. J. Jr.. Black C. III and Narkates A. J. 1972. III vitro comparison of actinobolin and other antibiotics used in the treatment of periodontal disease and dental caries. Proc. Sot. rrp. EioL Mrd. 140, 14291433. Hunt D. E. and Kohn D. W. 1974. The use of amphotericin B to increase the selectivity of media used for isolating oral lactobacilli. Archs oral Biol. 19. 1073-m1076. Hunt D. E. and Narkates A. J. 1971. Etrect of actinobolin on nucleic acid and protein synthesis in Streptococcus faecalis. J. dent. Res. 50, 161@1615. Hunt D. E., Navia J. M. and Lopez H. 1971. III uiuo cariostatic activity of actinobolin. J. dent. Res. SO, 371-373. Hunt D. E. and Pittillo R. F. 1967. Antimicrobial activitv of 1,2,5-selenadiazoles. In: Antimicrobial Agents a& Chemotherapy (Edited by Hobby G. L.), pp. 551-554. American Society for Microbiology, Ann Arbor.
Antibacterial activity of actinobolin analogues Hunt D. E., Pittillo R. F., Johnson E. P. and Moncrief F. C. 1966. The effect of actinobolin on nucleic acid and protein synthesis in Escherichia coli. Cm. J. Microbio/. 12, 515-520. Hunt D. E., Sandham 1-I. J. and Caldwell R. C. 1970. In vitro antibiotic sensitivity of oral microorganisms to actinobolin. J. dent. Rex 50, 371-373. Keyes P. H. 1969. Present and future measures for dental caries control. J. Am. dent. Ass. 79, 1395-1404. McClure F. J. and Hewitt W. L. 1946. The relation of penicillin to induced rat dental caries and oral L. acidophilus. J. dent. Res. 25, 441-443. Nathans D. 1967. Puromycin. In: Antibiotics I Mechanism of Action (Edited by Gottlieb D. and Shaw P. D.), pp. 259-277. Springer-Verlag. New York.
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J. H. 1959. Caries inhibiting agents. Pharmac. Rev. 705-716. J. H. and Ivimey J. K. 1973. Actinobolin as an inhibiof the periodontal syndrome in rice rats. Archs oral Biol. 18, 357-360. Smithers D., Bennett L. L. and Struck R. F. 1969. Inhibition of protein synthesis in mammalian cells by actinobolin. MO&. Pharm. 5, 433-445. Stephan R. M., Fitzgerald R. J., McClure F. J., Harris M. R. and Jordan H. V. 1952. The comparative effects of penicillin, bacitracin, chloromycetin, aureomycin, and streptomycin on experimental dental caries and on certain oral bacteria in the rat. J. dent. Res. 31, 421427. Shaw 11, Shaw tor
