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The Mechanism of Plasmid Curing in Bacteria

Article  in  Current Drug Targets · August 2006


DOI: 10.2174/138945006777709601 · Source: PubMed

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Current Drug Targets, 2006, 7, 000-000 1

The Mechanism of Plasmid Curing in Bacteria

Gabriella Spengler1,*, Annamária Molnár1, Zsuzsanna Schelz1, Leonard Amaral2, Derek Sharples3,
Joseph Molnár1

1
Institute of Medical Microbiology and Immunobiology, Albert Szent-Györgyi Medical Centre, University of Szeged,
Szeged, Hungary; 2Unit of Mycobacteriology, UPMM, Institute of Hygiene and Tropical Medicine, Universidade Nova
de Lisboa, Lisbon, Portugal and 3School of Pharmacy and Pharmaceutical Sciences, University of Manchester,
Manchester, UK

Abstract: Bacterial plasmids have a major impact on metabolic function. Lactose fermentation of E. coli or hemolysin B
transporter expressed by the plasmids that carry these respective genes could be readily obviated by heterocyclic com-
pounds that readily bind to plasmid DNA. These compounds could also reverse the resistance to antibiotics of E. coli ,
Enterobacter, Proteus, Staphylococcus and Yersinia strains by eliminating plasmids. However, the frequency and extent
of this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. The
effects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or on
nodulation, nitrogen fixation of Rhizobia accounted for the elimination of 0.1 to 1.0 % of plasmids present in the popula-
tions studied. Bacterial plasmids can be eliminated from bacterial species grown as pure or mixed bacterial cultures in the
presence of sub-inhibitory concentrations of non-mutagenic heterocyclic compounds.
The antiplasmid action of the compounds depends on the chemical structure of amphiphillic compounds having a planar
ring system with substitution in the L-molecular region. A symmetrical π-electron conjugation at the highest occupied
molecular orbitals favours the antiplasmid effect.
The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of the
DNA to which they bind. In this manner “extrachromosomal” plasmid DNA that exists in a superhelical state binds more
compound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries the
plasmid. It can also be noted that these compounds are not mutagenic and their antiplasmid effects correlate with the en-
ergy of HOMO-orbitals.
Plasmid elimination is considered also to take place in ecosystems containing numerous bacterial species. This opens up a
new perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibiotic
resistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition of
plasmid replication at various stages, as shown in the “rolling circle” model (replication, partition, conjugal transfer) may
also be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity and
antibiotic resistance.
The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find a
correlation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmid
elimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducing
mutations.

INTRODUCTION membrane efflux pumps in bacteria and the recognition of


the importance of ABC transporters in general.
This report reviews the mechanisms by which the an-
tiplasmid activity of heterocyclic compounds is expressed in The study of the potential release and acquisition of re-
plasmid carrying bacteria and the reversal of drug resistance sistance genes presents an interesting research challenge.
by these bacteria by elimination of plasmids containing anti- The phenomenon has resulted in part from the misuse of
biotic resistant genes and the relationship of these mecha- antibiotics and from horizontal gene transfer in the ecosys-
nisms to the inhibition of antibiotic efflux produced by these tem. This horizontal gene transfer contributes to evolutionary
same compounds. The complete inhibition of plasmid en- processes in a wide variety of organisms. In this process the
coded hemolysin B transporter activity and poor elimination auto-transmissible plasmids have a key role in the expansion
of plasmid DNA encouraged the study of the role of the of gene pools involved in gene transfer between various or-
ganisms [1]. Other types of gene transfer can occur at the
intracellular level and between bacterial chromosomes,
*Address correspondence to this author at the Institute of Medical Microbi- plasmids and transposons. The third level of antibiotic resis-
ology and Immunobiology, Albert Szent-Györgyi Medical Centre, Univer- tance transmission occurs between microorganisms and ani-
sity of Szeged, Szeged, Hungary; E-mail: spenglerg@freemail.hu mals, humans included.

1389-4501/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.


2 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

RELATIONSHIP BETWEEN THE CHEMICAL The antibacterial activity of thioridazine, a derivative of


STRUCTURE AND THE ANTIBACTERIAL EFFECT CPZ that is substituted by sulfur at carbon 2, is equal to that
of CPZ [9-11]. Phenothiazine enantiomers show similar ac-
Heterocyclic compounds such as phenothiazines have tivities to CPZ (Table 1 Group D). The phenothiazine dyes
been shown to possess activity against a large number of methylene blue and toluidine blue have MICs that are con-
bacterial species [2-5]. E. coli however is relatively resistant
siderably lower than those shown by CPZ or thioridazine.
to phenothiazines having MIC values that range from 100 to The importance of substitution at C-2 is thus exemplified by
150 µg/ml [6, 7]
the presence of chloride and sulfur, respectively. The anti-
Despite this innate resistance, phenothiazines are quite bacterial activity of non-phenothiazine tricyclic compounds
effective in the elimination of plasmids from this bacterium is presented in Table 2. Other than the demonstration that
[8]. The use of E. coli in studies that attempt to relate the many tricyclic compounds do indeed have antibacterial ac-
structure of the heterocyclic phenothiazine to the degree of tivity, and that this activity can be modified by selective sub-
antimicrobial activity and to its antiplasmid activity may stitution at important sites of the rings, it can be noted that
therefore provide essential insights that may in turn lead to the tricyclic psychopharmacons imipramine and desipramine
the rational synthesis of new antimicrobial agents. This has that have non-planar tricyclic skeletons have similar anti-
been achieved by studies that test both the antibacterial and bacterial activities and become relatively inactive on satura-
antiplasmid activities of heterocyclic compounds grouped tion of the ring system (Table 2 Group A). It can be con-
into two major categories, namely, substituted phenothiazi- cluded that one of the aromatic rings is necessary for the
nes and non-phenothiazine heterocyclic compounds. antibacterial activity although which of the three rings is
primarily responsible is yet to be determined.
The activities of substituted phenothiazine compounds
against the plasmid model strain of E. coli K12LE140 are ANTIPLASMID EFFECTS IN VITRO
presented in Table 1. The results for non-phenothiazine het-
erocyclic compounds in Table 2. As shown in Table 1 all of Bacteria have developed the ability to resist the toxic
the compounds listed possess activity against E. coli action of antibiotics by a variety of mechanisms such as:
K12LE140. However, the phenothiazine derivatives of enzymatic inactivation, as is frequently the case with resis-
Group A are in general, more effective than those of Groups tance to β-lactams and aminoglycosides [12-15], alteration
B, D, E and F. Group A consists of chloro substituted phe- of the permeability of the cell envelope to given antibiotics
nothiazines all of which have been derived from the parent [16-20] and modification of the relevant antibiotic target
chlorpromazine. [21]. To date all bacteria studied have been shown to have
efflux pumps that are capable of extruding antibiotics when
The antibacterial activity of promazine, which has equal the concentration of the antibiotic is of itself, not sufficiently
neuroleptic activity to chlorpromazine but with milder side toxic to the organism [22].
effects, is similar to that of chlorpromazine as has 2-chloro-
10-(2-dimethylaminoethyl)phenothiazine. The antibacterial Phenothiazines, which have been shown to have antimi-
activity significantly increases in the derivatives, des-dime- crobial activity against bacteria (see Table 1), have also been
thylchlorpromazine (MIC 0.73µg/ml) and des-methylchlor- shown to inhibit the efflux pumps of bacteria whenever this
promazine (MIC of 0.47µg/ml), primary and secondary has been studied [20, 23-27]. These phenothiazines have also
amines being more effective as antibacterial agents. This been shown to cause the elimination of plasmids from bacte-
finding demonstrates the importance of amine order in the ria [28-30, 33]. A relationship between antimicrobial activ-
antibacterial effect of phenothiazines. In contrast to this, the ity, elimination of bacterial plasmids and the inhibition of
derivative 2-chloro-7-hydroxyphenothiazine is far less active efflux pumps that extrude antibiotics is strongly suggested
(MIC 16µg/ml). The differential antibacterial activity of by a number of studies that have investigated agents that
these derivatives suggests that substitution in the ring system exhibit all three effects [34-38].
and the conformation of the side chain can modify the an- The relationship between agents that have antibacterial
timicrobial effect. and antiplasmid activities and also act as inhibitors of bacte-
The antibacterial activity of chlorpromazine can be sig- rial efflux pumps has been further studied. In addition,
nificantly decreased by oxidation at the S-5 atom as shown agents such as promethazine and acridine orange, which also
by Table 1 Group B. Position 5 seems to be an important site have these activities, have also been shown to reverse antibi-
on the ring in determining antibacterial activity (CPZ-5- otic resistance in various bacterial species, when applied as
oxide MIC 24.3µg/ml) whereas oxidation at the phenothiaz- controls [39-41]. Such reversal of resistance is associated
ine ring nitrogen has no effect on the antibacterial activity with the ability of the agent to inhibit the efflux pump. This
(CPZ-N-oxide MIC 2.1µg/ml). The irrelevance of the phe- confers on the bacteria the ability to grow in concentrations
nothiazine ring nitrogen is also evidenced by the decreased of an antibiotic that would normally be toxic to the bacteria.
effect noted with CPZ-5, N-dioxide (MIC 27.0µg/ml), an The compounds listed in Tables 1 and 2, and whose mo-
effect that is essentially the result of oxidation at the S-5 lecular structures are described by Fig. (2), have been inves-
position. Trihydroxylation of carbons 3, 7, and 8 reduces the tigated for their ability to eliminate bacterial plasmids. It is
antibacterial activity of the CPZ derivative whereas single or important to note that plasmid elimination in some com-
dihydroxylation at these carbon atoms does not significantly pounds only occurs at sub-inhibitory concentrations of the
affect the activity. One can therefore conclude that reduction respective agent. The use of sub-inhibitory concentrations
of CPZ antibacterial activity is focused on the Sulfur atom at allows the bacterium to grow. This is a necessary pre-
position 5 and on substitution at Carbons 3, 7 and 8, 6, 9. condition for the demonstration of plasmid elimination as
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 3

Table 1. The MIC Values and the Plasmid Curing Effect of Phenothiazine Tricyclic Compounds on E. coli K12LE140 Strain

MIC values Plasmid curing % at


Compounds
M(x10-4) subinhibitory concentration

A) Phenothiazine derivatives

Chlorpromazine (CPZ) 2.0 29.0

Promazine 3.09 26.0

2-Chloro-10-(2-dimethylaminoaethyl)phenothiazine 3.1 90.0

Desdimethylchlorpromazine 0.73 8.0

Desmethylchlorpromazine 0.47 10.0

Chlorpromazine-methylammonium-iodide 4.7 38.0

2-chloro-7-hydroxyphenothiazine 16.0 0.1

B) Hydroxy-, silcoxy- and oxo-chlorpromazine (CPZ) derivatives

7-hydroxyCPZ 2.42 2.0

3,7-dihydroxyCPZ 5.7 0.8

7,8-dihydroxyCPZ 3.6 0.3

7,8-dioxoCPZ 3.87 0.1

6,9-dihydroxyCPZ 5.6 0.01>

6,9-dioxoCPZ 6.4 0.01>

8-hydroxyCPZ 0.9 1.0

3,7,8-trihydroxyCPZ 22.5 0.01>

CPZ-5-oxide 24.3 0.01>

CPZ-5,N-dioxide 27.0 0.01>

CPZ-N-oxide 2.1 2.0

7-(dimethyl-tert-butyl-siloxy)CPZ 2.2 10.0

7,8-bis-( dimethyl-tert-butyl-siloxyCPZ 3.5 0.01>

6,9-bis-( dimethyl-tert-butyl-siloxy)CPZ 3.5 0.01>

C) Thioalkyl-substituted phenothiazines

Thioridazine 1.9 34.0

Thiethylperazine 2.5 31.0

D) Phenothiazine enantiomers

Levomepromazine 3.5 20.0

Dextromepromazine 3.5 18.0

E) Phenothiazines with different side chains

diethazine 4.3 18.0

promethazine 3.0 25.0

thiasinamide 8.0 0.01>

Bis-(2-chloro-10-γ-propyl-phenothiazine-α,γ-glutamylamid 0.9 2.0

F) Dyes

Methylene blue 16.3 0.01>

Toluidine blue 17.0 0.01>


4 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

Table 2. The MIC Values and the Plasmid Curing Effect of Non-Phenothiazine Tricyclic Compounds on E. Coli K12LE140

MIC values Plasmid curing % at


Compounds
M(x10-4) subinhibitory concentration

A) Hydroxy- and siloxy derivatives of dibenzoazepines (DMI)

desipramine (DMI) 3.7 23.0

DMIH (partially saturated DMI) 4.3 16.0

DMIH2 (saturated DMI) 30.0 0.5

Imipramine 5.6 40.0

Imipramine (saturated) 23.9 0.01>

Imipramine-methylammonium iodide 10.0 7.0

2-hydroxyimipramine 5.4 4.0

3-chloro-8-hydroxyimipramine 1.8 3.9

3-chloro-8-dimethyl-tert-butyl-siloxyimipramine 3.5 6.0

B) Dibenzocycloheptane derivatives

Protriptyline 2.5 22.0

Amitriptyline 5.1 50.0

Amitriptyline-methylammonium iodide 7.1 0.5

C) Anthracene derivatives

1-dimethylamino-3-(1-anthryl)-3-propanol 7.1 5.0

1-dimethylamino-3-(2-anthryl)-3-propanol 10.7 0.2

1-dimethylamino-3-(9-anthryl)-3-propanol 32.2 24.0

Maprotiline 1.7 90.0

4,4’-bis-dimethylaminodiphenylmethane 30.0 0.01>

D) Thioxanthene derivatives

Chlorprothixene 1.7 32.0

Cis-clopenthixol 2.3 26.0

Trans-clopenthixol 1.2 33.0

E) Cannabis and phenanthryl derivatives

∆8-tetrahydrocannabinol 3.1 0.01>

∆ -tetrahydrocannabinol
9
3.3 0.01>

Cannabinol 3.2 0.01>

Cannabidiol 9.6 0.3

Cannabidiolic acid 13.9 2.5

Tetrahydrocannabidiolic acid 14.0 30.0

Morphine 20.0 0.01>

1-dimethylamino-3-(9-phenanthryl)-3-propanol 4.1 40.0

F) Fluorescent Dyes

Ethidium bromide 3.8 0.1

Acridine orange 4.1 20.0


The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 5

Table 3. The effect of inoculum size (number of generations during the growth of bacteria in the presence of compounds) on the
plasmid elimination of imipramine (4.68x10-4 M) on E. coli K12 F’lac strain

Inoculum/mL at the beginning


Number of colony formers x107 Plasmidless (lac-) colonies % Number of colonies tested
of the experiments

2x102 3.0 12.0 1050


3
2x10 8.0 57.0 840
4
2x10 12.0 6.0 520
5
2x10 16.0 1.0 642

2x106 20.0 0.1 811

2x108 22.0 0.1 876

plasmid replication is dependent upon bacterial replication chlorpromazine ring. The plasmid curing activity of non-
and the number of subsequent generations (Table 3). phenothiazine tricyclic compounds is similar to that of the
substituted phenothiazines. The MIC values and antiplasmid
After incubation of the bacterium carrying the lac plas-
effects do not directly correlate but some of the compounds
mid with sub-inhibitory concentrations of the agents, ali-
were effective in sub-inhibitory concentrations. A large
quots of the culture are streaked onto drug-free medium
containing eosin-methylene blue (EMB). The EMB reagent number of compounds only have antibacterial activity with-
out any antiplasmid activity however some degree of anti-
aids in the identification of colonies that either contain or are
bacterial activity is essential for antiplasmid activity. The
deficient in the lac plasmid, showing deep violet (lac+) or
cannabinol derivatives are rather interesting with respect to
pink (lac -), respectively. The percentage of plasmid elimina-
plasmid curing activity which is present in tetrahydocan-
tion (curing) is determined by the number of pink colonies
nabidiolic acid, and to a lesser extent in cannabidiolic acid
divided by the total number of colonies (pink and deep vio-
and absent in cannabinol, tetrahydrocannabinols and can-
let) present on the surface of the agar plate. The majority of
nabidiol.
the substituted phenothiazines of Groups A and C have high
plasmid curing activities below the MIC at sub-inhibitory
concentrations. The most active agent of these groups is 2-
chloro-10-(2-dimethylaminoaethyl)-phenothiazine which has
a plasmid curing activity of 90% and an MIC of 3.1µg/ml.
The important contribution of electronic configuration to
plasmid curing activity is further shown by compounds such
as thioridazine, thiethylperazine and promazine derivatives,
all of which have symmetrical π-electron distributions in the
L-molecular region (Fig. (1), Fig. (2) structures 22-27). An-
tidepressants possessing a non planar tricyclic structure with
a secondary amine side chain are more effective in eliminat-
ing plasmid than are compounds with tertiary amine side
chains, whereas the plasmid eliminating potency of quater-
nary amines is much weaker probably because they are un- Fig. (1). Electronic Structure of the phenothiazine skeleton.
able to cross the bacterial cell membrane. The 7-hydroxy, 7,
8-dihydroxy and 7, 8-dioxo substituted CPZ derivatives also The substituted secondary amines, desipramine and pro-
appear to have lower plasmid curing effects than CPZ. Nei- triptyline, eliminated F’ lac plasmid at lower concentrations
ther the 6, 9-dihydroxy, 6, 9-dioxo, 5-oxide nor the 7, 8- and than did the substituted tertiary amines alimemazine and
6, 9-siloxy derivatives exert any significant antiplasmid ac- noxiptyline (Table 4). The majority of phenothiazines elimi-
tivity. These derivatives are considerably more polar than nated plasmid with a frequency similar to the antidepressants
chlorpromazine as indicated by the comparative ClogP (Table 4). Compounds such as chlorpromazine and imi-
values (ClogP: CPZ = +5.50, 7-OHCPZ = + 4.58, 7, 8- pramine may become attached through their cationic amine
DiOHCPZ = 2.06, 3, 7, 8-TriOHCPZ = 0.77). This would group to phospholipids in the cell membrane, while the hy-
imply that cell penetration is an important factor to be con- drophobic part of the molecule intercalates between the fatty
sidered in assessing plasmid curing activity. acid chains of the membrane. This would indicate that the
It can be concluded from these results that substitution in plasmid eliminating ability of a given compound may be a
the aromatic rings might prevent an interaction between the function of the membrane-lipid: water distribution coeffi-
π-electrons of the phenothiazine ring and target molecules cient. This can be substantiated by the fact that the distribu-
essential to plasmid replication. Structure 29 for example has tion coefficient for chlorpromazine is 1700 and for imi-
some marginal plasmid curing activity that might be related pramine is 2395. Conversely the distribution coefficient for
to the additional π electron cloud provided by the second lidocaine is only 17.
6 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

Phenothiazine derivative
1. 2-chloro-7-hydroxyphenothiazine 16. CPZ-5-oxide
HO S
O
S
N Cl
H N

N Cl
H3C CH3

2. 2-chloro-10-(2-dimethylaminoaethyl)-phenothiazine 17. CPZ-5N-oxide


Cl
O
S
S
N
N

H3C
N N+ Cl
O
CH3
CH3

3. Promazine 18. CPZ-N-oxide

S
S
N
N

N+
N O
H3C CH3 CH3

4. Desdimethyl-CPZ 19. 7-(dimethyl-tertbutyl-siloxy)CPZ


CH3
H3C
H3C CH3
Si
S H3C
O

S
N
NH2 Cl

N+ Cl
O
CH3

5. Desmethyl-CPZ 20. 7,8-bis-(dimethyl-tert-butyl-siloxy)CPZ


CH3
H3C
H3C CH3
H3C Si
CH3
S H3C O
H3C O
N Si
H3C
CH3
S
N
NH Cl
H3C

N Cl
H3C CH3
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 7

(Fig. 2) contd….

Phenothiazine derivative

6. Chlorpromazine (CPZ) 21. 6,9-bis-(dimethyl-tert-butyl-siloxy)CPZ

CH3 CH3
H3C Si CH3
S CH3
O
N

CH3 O S
H3C Si CH3 N
N Cl
H3C CH3 CH3 CH3

N Cl
H3C CH3

7. CPZ methylammonium iodide 22. Thioridazine

S S

N N

N S
N+ Cl
H3C CH3
H3C CH3
I- N
CH3

8. 7-hydroxyCPZ 23. Thiethylperazine

OH

S
S N
CH3
N
N

S
N Cl CH3
H3C CH3

9. 3,7-hydroxyCPZ 24. Levomepromazine

OH

S
S N
N

CH3
OH N O
N Cl H3C CH3 CH3
H3C CH3
8 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

(Fig. 2) contd….
Phenothiazine derivative
10. 7,8-hydroxyCPZ 25. Dextromepromazine
OH
HO
S
S N
N

H3C
N O
N Cl H3C CH3 CH3
H3C CH3

11. 7,8-dioxoCPZ 26. Diethazine


O
O S

N
S
N

N
H3C
N Cl H3C
H3C CH3

12. 6,9-dihydroxyCPZ 27. Promethazine


OH
S

HO S
N
N

CH3
H3C N
N CH3
H3C CH3

13. 6,9-dioxoCPZ 28. Tiazinamium


O
S
O S
N N

CH3
H3C
N+
N Cl CH3
H3C CH3 H3C

14. 8-hydroxyCPZ 29. Bis-(2-chloro-10-γ-propyl)-phenothiazine-α,γ-glutamylamide


HO
S

S N
N
Cl

NH
N Cl O
H3C CH3 O
H2N
HN

Cl S
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 9

(Fig. 2) contd….
Phenothiazine derivative

15. 3,7,8-trihydroxyCPZ 30. Methylene blue

OH N
HO
H3C CH3
N S+ N
S CH3 CH3
N

OH
N Cl
H3C CH3

31. Toluidine blue

N CH3

H3C
N S NH2
CH3 Cl

Non-phenothiazine derivatives
32. Desipramine (DMI) 46. 1-dimethylamino-3-(9-anthryl)-3-propanol

CH3
N
H
N

HO
CH3
N
CH3

33. DMIH 47. Maprotiline

CH3
N
H
N

N
H3C CH3

34. DMIH2 48. 4,4’-dimethylaminodiphenylmethane

CH3
N
H H3C CH3
N N N
CH3 CH3
10 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

(Fig. 2) contd….
Non-phenothiazine derivatives
35. Imipramine 49. Chlorprothixene

CH3
N
N CH3
S

N CH3
H3C
Cl

36. Imipramine (saturated) 50. Cis-chlopenthixol

CH3
N
N CH3
S

OH

37. Imipramine methylammonium iodide 51. Trans-chlopenthixol

CH3 OH
N+
CH3
N H3C N
I-

38. 2-hydroxyimipramine 52. ∆8-tetrahydrocannabinol

CH3 CH3
CH3
N O
N CH3

OH
H3C OH CH3

39. 3-chloro-8-hydroxyimipramine 53. ∆9-tetrahydrocannabinol

Cl CH3
CH3 CH3
N O
N CH3

H3C OH CH3
OH
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 11

(Fig. 2) contd….
Non-phenothiazine derivatives
40. 3-chloro-8-dimethyl-tertbutyl-siloxy-imipramine 54. Cannabinol

Cl CH3
CH3 CH3
N O
N CH3

H3C OH CH3
H3C CH3
O
H3C Si
H3C CH3

41. Protriptyline 55. Cannabidiol

CH3
CH3
N
H OH

H2C CH3
HO

CH3

42. Amitriptyline 56. Cannabidiolic acid

CH3
CH3
N
OH O
CH3
OH

H2C CH3
HO

CH3

43. Amitriptyline-methylammonium iodide 57. Morphine

CH3
N+ O
CH3
H3C -
I
N
CH3

44. 1-dimethylamino-3(1-anthryl)-3-propanol 58. 1-dimethylamino-3(9-phenanthryl)-3-propanol

CH3
CH3
N
N CH3
CH3 OH
HO
12 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

(Fig. 2) contd….
Non-phenothiazine derivatives
45. 1-dimethylamino-3(2-anthryl)-3-propanole 59. Ethidium bromide

H3C
N CH3 CH3

OH
H2N NH2

60. Acridine orange

CH3 CH3
N N N
H3C CH3

Fig. (2). Molecular structures of agents examined for antimicrobial and plasmid curing activity.

Table 4. Plasmid Elimination Action of Various Psychotherapeutics

Concentration Plasmid elimination Colonies MIC


Compounds
µg/mL percent Examined µg/mL

Desipramine 50 23,4 3600 70

Trimipramine 180 25,8 5700 200

Protriptyline 50 22,9 4400 80

Noxiptyline 260 41,0 3300 300

Promazine 60 40,0 6950 90

Trimeprazine 130 22,0 4700 150

Trifluopromazine 60 25,0 3500 80

Chlorprothixine 40 87,0 3450 50

Profenamine 220 54,0 7100 230

Thiazinamium 190 0,0 5000 230

Chlorpromazine methoioiodide 160 24,0 2900 180

Toluidine blue 240 0,0 3500 280

Lidocaine 2950 0,3 4360 3500

Procaine 18000 0,0 4020 >20.000

The plasmid curing activity of promethazine and tri- tiplasmid compounds was responsible for the weak plasmid
fluoperazine was investigated by replica plating [32] on curing effect in these clinical isolates. It is probable that the
plasmid-mediated doxycycline resistant bacterial strains iso- cell wall/cell membrane served as a barrier resulting in weak
lated from clinical specimens. The frequency of plasmid plasmid elimination [20]. Indirect evidence also supports
elimination was low for the majority of the tested strains, this, since the co-administration of verapamil (Ca2+-anta-
despite the formation of complexes between antiplasmid gonist) and trifluoperazine (intercalating plasmid curing
compounds (promethazine, 9-aminoacridine) and plasmid compound) resulted in a remarkable increase in plasmid
DNA isolated from the plasmid containing clinical strains. It elimination [unpublished results].
may be suggested that inefficient penetration of the an-
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 13

The inhibition of plasmid replication resulted from a sin- transconjugal DNA synthesis and mating pair formations
gle nick, outside of the replication origo of the superhelical were inhibited. The inactivation of sex pili was shown by
structure. The process leads to further relaxation of plasmid
DNA. Intercalation of the compounds was proved by an in-
crease in the melting point of the DNA and by circular di-
chroism (Fig. (3) [14]). When the native plasmid DNA and
its promethazine complex were analysed by agarose-gel
electrophoresis, the superhelical form was missing from the
promethazine treated plasmid DNA. The open circular and
linear forms of plasmid DNA were present in the pro-
methazine treated samples in increased proportions [42-44]
(Fig. (4)).

Fig. (3). Intercalation of the antiplasmid compound into the plasmid


DNS. (In: Waring, M.J. (1966) Symp. Soc. Gen. Microb. 16, 235- Fig. (4). The effect of the binding of ethidium bromide to SV40
265). DNA I and II. The upper part of the diagram presents three stages
in the reversible binding of dye to SV40 DNA I.
Plasmid replication can also be inhibited by blocking the
a) represents the dye-free molecule with 14 superhelical turns; the
activity of the enzyme DNA gyrase. This time the introduc-
addition of 420 molecules of ethidium bromide completely unwinds
tion of superhelical turns into plasmid DNA is prevented.
the superhelical turns to form the relaxed molecule (b). The addi-
The activity of DNA gyrase isolated from M. luteus was in- tion of a further 720 dye molecules leads to the formation of a posi-
hibited in the presence of promethazine and imipramine [45], tive superhelical molecule, shown in (c). The lower part of the dia-
both compounds having plasmid curing effects. Apart from
gram shows three stages in the reversible binding of dye to SV40
general intercalation into dsDNA, a specific binding site for
DNA II, which remains relaxed throughout. (e) represents the re-
phenothiazine intercalation was found in the GC-rich region.
laxed, nicked molecule with the same number of dye molecules
[46, 47]. This means that the expression of the GC rich pro-
bound as to the relaxed, intact molecules in (b). The arrows joining
moter of the MDR-1 gene responsible for drug resistance of (b) and (e) indicate that the nick may be introduced or repaired
cancer cells can be blocked simultaneously.
without change of the 420 molecules of ethidium bromide bound.
All of the tested plasmids were curable from E. coli The nicked molecule is nearly saturated, as shown in (f), with 1860
strains, except the E1 colicin [43, 45]. The simultaneous molecules of ethidium bromide bound. Introduction of a single-
elimination of F’lac and pBR322 plasmid from E. coli also strand scisson into I in the presence of a high dye concentration
showed relatively high frequency. It turns out that the curing results an irreversible unwinding of the superhelix. (In: Waring, M
efficiency of phenothiazines depends also on the host cells, (1970) J. Mol. Biol. 51, 247-279).
since the same plasmid was curable with different frequen-
cies from various E. coli strains [44, 48]. The Ca++ binding the inhibition of absorption of pilus specific phage [39, 40]
protein encoding the plasmid responsible for the virulence of (Tables 5, 6).
Y. enterocolitica and Y. pseudotuberculosis isolates was
cured with rather low frequency [49]. ANTIPLASMID EFFECTS IN VIVO
The antiplasmid compounds were also able to inhibit The adhesion of urinary pathogen E. coli strains was in-
plasmid transfer [8, 50]. In these experiments E. coli r144drd hibited in HEP2 tissue culture cells by inhibition of the adhe-
Kmr strain with depressed pilus synthesis was used as a do- sive type I and p-fimbriae [51]. Based on these results the
nor, and a chromosomally sodium azide resistant E. coli synergistic effect of promethazine with gentamycin was
strain was used as a recipient. A dose dependent inhibition of studied in children with frequently recurring pyelonephritis
conjugal plasmid transfer was observed [42] where both [52] and in adults [53, 54]. In these experiments 10 children
14 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

Table 5. The Effect of Substituted Phenothiazines on R-Plasmid Transfer in E. coli Strains

No. of cells/mL
Compounds
Transconjugant x 104 Donor x 108 Recipient x 108

A) Phenothiazine derivatives
2-chloro-7-hydroxyphenothiazine 0.2 1.6 2.6
2-chloro-10-(2-dimethylaminoaethyl)phenothiazine 0.12 1.4 1.8
Promazine 0.30 1.0 1.9
DesdimethylCPZ 0.1 1.1 1.9
DesmethylCPZ 0.15 0.9 1.6
Chlorpromazine (CPZ) 0.2 0.8 1.4
CPZ-methylammonium Iodide 0.4 1.9 2.1
B) Hydroxy-, siloxy- and oxo-CPZ derivatives
7-hydorxyCPZ 0.15 0.85 1.2
3,7-dihydroxyCPZ 0.08 0.9 0.5
7,8-dihydroxyCPZ 0.07 0.8 0.77
7,8-dioxoCPZ 0.05 0.5 0.9
6,9-dihydroxyCPZ 0.07 1.5 0.9
6,9-dioxoCPZ 0.45 1.5 2.2
8-hydroxyCPZ 0.08 1.2 2.1
3,7,8-trihydroxyCPZ 1.9 1.7 1.8
CPZ-5-oxide 1.5 2.6 1.0
CPZ-5, N-dioxide 2.0 1.6 1.4
CPZ-N-oxide 0.05 1.3 1.1
7-(dimethyl-tertbutyl-siloxy)-CPZ 3.1 2.4 2.2
7,8-bis-(dimethyl-tertbutyl-siloxy)CPZ 3.5 2.8 1.9
6,9-bis-(dimethyl-tertbutyl-siloxy)CPZ 2.3 2.6 1.5
C) Thioalkyl-substituated phenothiazines
Thioridazine 0.8 0.8 2.2
Thiethylperazine 0.63 0.6 1.1
D) Phenothiazine enantiomers
Levomepromazine 0.09 1.2 2.2
Dextromepromazine 0.09 1.4 2.4
E) Phenothiazines with different side chains
Diethazine 0.25 1.9 2.1
Promethazine 0.18 1.3 1.6
Thiazinamine 0.95 1.5 2.1
Bis-(2-chlor-10-γ-propyl)phenothiazine-α,γ-glutamyl-amide 1.8 1.6 1.8
F) Dyes
Methylene blue 1.2 1.4 2.4
Tholuidine blue 1.9 1.7 2.9
Control 3.4 2.1 2.8
Concentration of the compounds: 50% MIC. The samples were incubated for 120 min. at 37°C. The data are the results of two parallel experiments.
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 15

Table 6. The Effect of Non-Phenothiazine Tricyclic Compounds on R-Plasmid Transfer of E. coli Strains

No. of cells/mL
Compounds
Transconjugant x104 Donor x108 Recipient x108

A) Hydroxy- and siloxy derivatives of dibenzoazepines

Dezipramine (DMI) 0.02 0.75 1.9

DMIH (partially saturated DMI) 0.04 1.3 1.6

DMIH2 (saturated DMI) 2.5 1.8 2.1

Imipramine 0.23 0.8 1.4

Imipramine (saturated) 3.0 1.7 2.3

Imipramine methylammonium iodide 0.7 1.5 2.4

2-hydroxyimipramine 0.1 1.0 1.7

3-chloro-8-hydroxyimipramine 0.05 1.05 1.5

3-chloro-8-dimethyl-tertbutyl-siloxyimipramine 0.38 1.5 2.4

B) Dibenzocycloheptane derivatives

Protriptyline 0.34 1.3 1.15

Amitriptyline 0.23 0.9 0.87

Amitriptyline methylammonium iodide 0.7 1.4 2.3

C) Anthracene derivatives

1-dimethylamino-3-(1-anthryl)-3-propanol 0.3 0.4 0.9

1-dimethylamino-3-(2-anthryl)-3-propanol 0.06 0.9 0.65

1-dimethylamino-3-(9-anthryl)-3-propanol 0.08 0.8 1.7

Maprotiline 0.16 1.5 1.9

4,4’-bis-dimethylaminodiphenylmethane 2.5 2.0 2.3

D) Thioxanthene derivatives

Chlorprothixene 0.6 1.3 1.8

Cis-clopenthixol 0.4 1.1 1.5

Trans-clopenthixol 0.1 0.9 1.5

E) Cannabis and phenanthryl derivatives


∆8-tetrahydrocannabinol 3.0 1.5 1.9

∆ -tetrahydrocannabinol
9
2.2 2.2 2.5

Cannabinol 1.7 1.9 2.0

Cannabidiol 1.5 1.8 1.5

Cannabidiolic acid 2.1 1.9 2.0

Morphine 3.0 1.9 1.8

1-dimethylamino-3-(9-phenanthryl)-3-propanol 1.2 2.0 1.9

F) Dyes

Ethidium bromide 0.34 1.2 1.9

Acridine orange 0.12 0.6 0.9

Control 3.1 1.9 2.1


Concentration of the compounds: 50% MIC. The samples were incubated for 120 min. at 37°C. The data are the results of two parallel experiments.
16 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

were given a combination of gentamycin and promethazine the molecular level the effects of these drugs at several
for 7 days (Group l.). In the second group 11 children re- points in the course of transformation, in plasmid DNA rep-
ceived gentamycin alone and in the 3rd group 19 children lication and the topological state of plasmid DNA was ex-
were on long-term oral antibiotic prophylaxis with episodes amined (Fig. (5)).
of intensive treatment. Over a three year follow up period,
the number of pyelonephritis recurrences were significantly
lower in Group1 than in Groups 2 and 3. Plasmid elimination
was also studied by in vivo or ex vivo experiments, when the
plasmid profile of freshly isolated colonies from the urine of
promethazine and imipramine treated adult patients were
compared. A heterogenicity of antibiotic sensitivities and the
plasmid profile of bacteria isolated from promethazine or
imipramine treated patients was found after only five days
treatment with tricyclic psychopharmacons. These changes
were found in only a few percent of the E. coli colonies iso-
lated from the urine of treated patients, therefore it has to be
considered that plasmid elimination has no clinical impor-
tance on its own [43]. Among 50 treated patients, 12 had
significant bacteriuria, however 21 of the control 50 patients
also had significant bacteriuria. The resistance patterns of ten
different isolates from the urine of maprotiline treated pa-
tients changed after 5 days of treatment, when carbenicillin
and cefuroxime resistance was eliminated from three isolates
[52]. A similar in vivo curing was also found by Mehtar [55].
The in vitro effective concentrations of plasmid curing com-
pounds were found to be higher than the achievable serum
concentrations in patients, consequently the frequency of in
vivo plasmid curing was low. Synergism between antibiotics
and antiplasmid effect can be produced by complex mecha-
nisms. Fig. (5). Electrophoretic analysis of antiplasmid effects of pro-
methazine.
STRUCTURE ACTIVITY RELATIONSHIPS
Samples:
It has been proposed that the conjugated π-electron sys-
1. E. coli HB101/pBR322 culture was treated with 250 µg/mL pro-
tem of the tricyclic skeleton has a special importance where
methazine; 2. E. coli HB101/pBR322 culture was treated with 1000
the conjugation of the π-electrons of the two aromatic rings
µg/mL promethazine; 3. E. coli HB101/pBR322 culture was treated
is symmetrical to the L-molecular region (Fig. (1)). It is rea-
with 2000 µg/mL promethazine; 4. E. coli HB101/pBR322 cells
sonable to conclude that the antibacterial and antiplasmid
were lysed with lysosyme and 250 µg/mL promethazine was added
effect noted with active phenothiazines or tricyclic non-
to the lysed cells; 5. E. coli HB101/pBR322 cells were lysed with
phenothiazines is dependent on the available π-electrons and
lysosyme and 1000 µg/mL promethazine was added to the lysed
their distribution.
cells; 6. E. coli HB101/pBR322 cells were lysed with lysosyme and
Thus knowing the mechanism of plasmid curing, an ideal 2000 µg/mL promethazine was added to the lysed cells; 7. pBR322
plasmid curing or resistance modifier compound can be pro- plasmid DNA; 8. pBR322 DNA was treated with Hind III at 37°C
posed. The correlation between antiplasmid effects and for 2 hours, which made the DNA linear; 9. pBR322 plasmid DNA.
chemical structure of the curing compounds was studied. In
a: linear; b: relaxed circular; c: covalently closed circular. (In: Mol-
quantitative structure activity relationship (QSAR) studies it
nár J, Földeák S, Nakamura MJ, Rausch H, Domonkos K, Szabó M.
was shown that the HOMO orbital energy, the symmetry of
(1992) APMIS Suppl. 30/100, 24-31).
the π electron distribution to the L-molecular region and the
superdelocalizability of the π electron system of tricyclic Two possible target sites were identified in plasmid DNA
skeleton on atoms 10, 12 and 13 were responsible for effec- replication. One of them involved membrane binding sites,
tive binding through stacking interaction to the biological the other one is in DNA replication. The other effect ob-
target eg. DNA or DNA gyrase. Based on the results of the served in vivo and in vitro was the influence on the topologi-
QSAR correlation [56-61] novel substituted anthranyl- com- cal state of plasmid DNA (Fig. 4). The presence of tricyclic
pounds were synthesized and found to possess remarkable drugs promoted the relaxation of plasmid DNA by interfer-
plasmid curing effects [62-64]. The antiplasmid action of ing with the supercoiling activity of DNA gyrase causing a
tricyclics on the other hand was found to be rather specific cessation in plasmid replication.
and depended upon the chemical structures of the com-
Therefore in order to improve drug design, some quan-
pounds [14, 65].
tum chemical parameters, including: π electron superdelo-
Drug treatment of bacterial cells resulted in the inhibition calizibility, HOMO, LUMO orbital energies, size of the Van
of plasmid replication and finally in the formation of plas- der Waals’ surface in a watery environment were calculated
mid-free cells. In order to analyze the mechanism of action at by several computer programs (MM2, CNDO) for phenothi-
The Mechanism of Plasmid Curing in Bacteria Current Drug Targets, 2006, Vol. 7, No. 7 17

azines, acridines and naphthyridines. Based on these results Doxycycline resistant bacterial isolates from clinical
new anthracene derivatives were synthesized [66-70]. In this specimens were found to have a cell membrane that was im-
way the antiplasmid and carcinogenic molecular orbitals permeable to resistance modifying antiplasmid drugs. The
were clearly differentiated [71, 72]. Based on the QSAR frequency of the elimination of tetracycline resistance was
studies, new compounds with well-defined and predictable low for the majority of strains investigated, despite the fact
electronic structures in the molecular orbitals [31] were pre- that it has been shown that complexes are formed between
pared. The compounds possessed antiplasmid activity but antiplasmid compounds and plasmid DNA. The results sug-
displayed no mutagenic or carcinogenic effects [59, 60]. The gest that the curing effects were dependent on the perme-
binding affinity changes caused by promethazine and imi- ability barrier of the clinical isolates studied. Promethazine
pramine indicated that the resulting plasmid elimination was and trifluoperazine as well 9-aminoacridine have pronounced
connected at least partly to membrane proteins and plasmid plasmid curing activity in E. coli K12 LE 140 and these ef-
DNA complexes (Fig. (5)). fects were substantially enhanced by administering the pro-
ton pump inhibitor trifluoromethyl ketone at concentrations
Drugs may affect the binding affinity of replicating plas-
ranging from 0.05 to 1.0 mg/L. Thus it can be shown that
mid DNA to membrane proteins producing more stable
complexes that negatively interfere with the processing of various type of resistance modifying drugs, such as calcium-
the replication fork and the expression of plasmid encoded channel blockers or proton pump inhibitors, can enhance the
genes. This was shown for the F’ lac plasmid. activity of plasmid curing drugs. The results suggest that
inhibitors of membrane ABC transporters and proton pumps
EVALUATION OF PLASMID CURING may be combined to produce plasmid curing in some antibi-
otic-resistant bacterial strains [20].
Although bacterial resistance is different from eukaryotic
There is evidence for that the activity of plasmid medi-
resistance in many respects there are common sensitive
ated haemolysin transporter in bacteria [74] and other bacte-
points, such as transporter protein mediated efflux pump
rial transport proteins have a close homology to mammalian
systems. In this respect the mechanism of resistance in bacte-
multidrug resistance transporters, the so called efflux pumps
ria, protozoa and tumour cells is similar and therefore it may [75]. This multidrug resistance mechanism was modified in
be possible to overcome it in a similar way.
both bacteria and in cancer cells by antiplasmid compounds
In preliminary in vivo experiments it was shown that the [76]. The intracellular accumulation of antibiotics or che-
efficiency of plasmid curing was rather low and apparently motherapeutics increases as a consequence of decreased an-
of little practical importance. However considering the exis- tibiotic efflux in both bacterial and tumour cell systems. The
tence of efflux pump inhibitors it was decided to check the inhibition of the efflux pump is the same for all individual
results of the interaction of plasmid curing compounds and members of the population of bacterial and cancer cells,
antibiotics in vitro. Among various plasmids, the hemolysin however the antiplasmid effects occurred in only a small
and tetracyline transporter encoding plasmid was eliminated fraction of the growing bacterial cell populations [76] but the
from the bacteria. Detailed analysis of the plasmidless bacte- inhibition of exporter proteins can also be exploited to in-
ria showed that the MIC value for tetracyline was reduced in crease plasmid curing.
the plasmidless bacterial cells. In addition the antibacterial
effect of tetracycline is synergized by plasmid curing com- IMPORTANCE OF PLASMIDS AND ABC TRANS-
pounds. This was independent from the elimination of plas- PORTERS
mid DNA [52]. This resistance modifying effect of selected
Membrane transporters can be encoded by genes local-
phenothiazines and structurally related compounds was
similar for all individual cells of the culture, showing that the ised on the chromosomes and on plasmids such as haemo-
inhibition of drug efflux was able to increase the intracellular lysin and tetracycline transporters [77, 78]. The multidrug
membrane transporters are classified into two main groups
concentration of chemotherapeutics in bacteria and cancer
such as ABC transporters and proton pump systems based on
cells. Plasmid elimination was shown to exist in microbial
energetic requirements and the second class is sub-divided
eco-systems, however a number of clinical isolates were
into a large number of subclasses.
resistant to many antibiotics and simultaneously had very
low sensitivity to high concentrations of the plasmid curing Some transporters such as the tetracycline efflux protein
compounds. Using mixed cultures, including plasmid bear- mediate the extrusion of the particular antibiotic. This active
ing bacterial cells (E. coli K12 LE 140), the conditions of a efflux is important in ensuring a significant level of resis-
polimicrobial flora were simulated. Plasmid elimination was tance to tetracycline or other antibiotics. In contrast the tet
studied under various conditions, including co-inhabiting transporters are multidrug-transporters, which confer resis-
bacteria, at various temperatures and in the presence of the tance against a wide variety of structurally unrelated com-
plasmid curing promethazine. It was established that plasmid pounds [79-82]. The mdr transporters can be inhibited a wide
elimination from E. coli K12 LE 140 promoted by sub- variety of compounds such as uncouplers and calcium an-
inhibitory concentrations of promethazine was significantly tagonists .The proton motive force utilizing efflux pumps is
exalted by elevation of temperature either in the monoculture sensitive to compounds that dissipate the proton gradient in
or when incubated together with either B. cereus or S. epi- the membrane. These pumps mediate the efflux of xenobiot-
dermidis. The efficiency of plasmid elimination of phenothi- ics in a coupled exchange with protons.
azine was markedly enhanced by the presence of a second
The majority of multidrug transporters use ATP as the
species of bacteria [73].
energy to pump the antibiotics out of the cells, while the sec-
ond largest groups of transporters utilize the transmembrane-
18 Current Drug Targets, 2006, Vol. 7, No. 7 Spengler et al.

proton gradient to drive the antibiotics or other xenobiotics without mutagenic effect, the results can be exploited in or-
out of the cells. Several subclasses belong into this group der to isolate plasmid free bacteria for biotechnology without
[83-85, 89, 90]. Experiments suggested that drug resistance any risk of mutations.
by bacteria and cancer cells can be achieved in various ways,
The main goal of this study is to investigate ways of
however the inhibition of efflux pump systems is the most
combating antibiotic resistance by the selective inhibition of
promising mechanism in this respect because the intracellu- plasmid replication and transfer and by blocking the bacterial
lar concentration of antibiotics is enhanced in all individual efflux pumps. The results of the model experiments on the
cells of the population simultaneously and at much lower synergistic interactions between antibiotics and resistance
concentration of resistance modifier compounds than is modifiers found in vitro can be exploited in the rational drug
needed for plasmid elimination. This resistance modifying
design of drugs to counteract antibiotic resistant pathogens.
effect is apparently independent from the antibacterial or
cytotoxic effects.
ACKNOWLEDGEMENTS
CONCLUSIONS These studies were supported by the Szeged Foundation
for Cancer Research and Cooperation in Science and Tech-
The aim of the study was to clarify the mechanisms of
nology, COST Action B16 "Reversal of Antibiotic Resis-
action of the drugs on plasmid replication and to improve the
tance" at the European Commission.
curing activity of existing compounds by computer aided
drug design in order to obtain effective plasmid curing com-
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