The Mechanism of Plasmid Curing in Bacteria

2006, 7, 000-000 1

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

The Mechanism of Plasmid Curing in Bacteria

Gabriella Spengler1,*, Annamria Molnr1, Zsuzsanna Schelz1, Leonard Amaral2, Derek Sharples3,Joseph Molnr1

1Institute of Medical Microbiology and Immunobiology, Albert Szent-Gyrgyi Medical Centre, University of Szeged,Szeged, Hungary; 2Unit of Mycobacteriology, UPMM, Institute of Hygiene and Tropical Medicine, Universidade Novade 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 Btransporter 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 extentof this effect was significantly less than might have been expected based on a complex interaction with plasmid DNA. Theeffects of heterocyclic compounds on the plasmids responsible for the virulence of Yersinia and A. tumefaciens, or onnodulation, 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 thepresence 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 planarring system with substitution in the L-molecular region. A symmetrical p-electron conjugation at the highest occupiedmolecular orbitals favours the antiplasmid effect.

The antiplasmid effect of heterocyclic compounds is expressed differentially in accordance with the structural form of theDNA to which they bind. In this manner extrachromosomal plasmid DNA that exists in a superhelical state binds morecompound than its linear or open-circular form; and least to the chromosomal DNA of the bacterium, that carries theplasmid. 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 anew perspective in rational drug design against bacterial plasmids. The inhibition of conjugational transfer of antibioticresistance plasmid can be exploited to reduce the spread of antibiotic resistance plasmid in the ecosystem. Inhibition ofplasmid replication at various stages, as shown in the rolling circle model (replication, partition, conjugal transfer) mayalso be the theoretical basis for the elimination of bacterial virulence in the case of plasmid mediated pathogenicity andantibiotic resistance.

The large number of compounds tested for antiplasmid effects provides opportunities for QSAR studies in order to find acorrelation between the antiplasmid effect and the supramolecular chemistry of these plasmid curing compounds. Plasmidelimination in vitro provides a method of isolating plasmid free bacteria for biotechnology without any risk of inducingmutations.

INTRODUCTION

This report reviews the mechanisms by which the an-tiplasmid activity of heterocyclic compounds is expressed inplasmid carrying bacteria and the reversal of drug resistanceby these bacteria by elimination of plasmids containing anti-biotic resistant genes and the relationship of these mecha-nisms to the inhibition of antibiotic efflux produced by thesesame compounds. The complete inhibition of plasmid en-coded hemolysin B transporter activity and poor eliminationof plasmid DNA encouraged the study of the role of the

*Address correspondence to this author at the Institute of Medical Microbi-ology and Immunobiology, Albert Szent-Gyrgyi Medical Centre, Univer-sity of Szeged, Szeged, Hungary; E-mail: [email protected]

membrane efflux pumps in bacteria and the recognition ofthe importance of ABC transporters in general.

The study of the potential release and acquisition of re-sistance genes presents an interesting research challenge.The phenomenon has resulted in part from the misuse ofantibiotics and from horizontal gene transfer in the ecosys-tem. This horizontal gene transfer contributes to evolutionaryprocesses in a wide variety of organisms. In this process theauto-transmissible plasmids have a key role in the expansionof gene pools involved in gene transfer between various or-ganisms [1]. Other types of gene transfer can occur at theintracellular level and between bacterial chromosomes,plasmids and transposons. The third level of antibiotic resis-tance transmission occurs between microorganisms and ani-mals, humans included.

SAIF

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

RELATIONSHIP BETWEEN THE CHEMICALSTRUCTURE AND THE ANTIBACTERIAL EFFECT

Heterocyclic compounds such as phenothiazines havebeen shown to possess activity against a large number ofbacterial species [2-5]. E. coli however is relatively resistantto phenothiazines having MIC values that range from 100 to150 m g/ml [6, 7]

Despite this innate resistance, phenothiazines are quiteeffective in the elimination of plasmids from this bacterium[8]. The use of E. coli in studies that attempt to relate thestructure of the heterocyclic phenothiazine to the degree ofantimicrobial activity and to its antiplasmid activity maytherefore provide essential insights that may in turn lead tothe rational synthesis of new antimicrobial agents. This hasbeen achieved by studies that test both the antibacterial andantiplasmid activities of heterocyclic compounds groupedinto two major categories, namely, substituted phenothiazi-nes and non-phenothiazine heterocyclic compounds.

The activities of substituted phenothiazine compoundsagainst the plasmid model strain of E. coli K12LE140 arepresented in Table 1. The results for non-phenothiazine het-erocyclic compounds in Table 2. As shown in Table 1 all ofthe compounds listed possess activity against E. coliK12LE140. However, the phenothiazine derivatives ofGroup A are in general, more effective than those of GroupsB, D, E and F. Group A consists of chloro substituted phe-nothiazines all of which have been derived from the parentchlorpromazine.

The antibacterial activity of promazine, which has equalneuroleptic activity to chlorpromazine but with milder sideeffects, is similar to that of chlorpromazine as has 2-chloro-10-(2-dimethylaminoethyl)phenothiazine. The antibacterialactivity significantly increases in the derivatives, des-dime-thylchlorpromazine (MIC 0.73m g/ml) and des-methylchlor-promazine (MIC of 0.47m g/ml), primary and secondaryamines being more effective as antibacterial agents. Thisfinding demonstrates the importance of amine order in theantibacterial effect of phenothiazines. In contrast to this, thederivative 2-chloro-7-hydroxyphenothiazine is far less active(MIC 16 m g/ml). The differential antibacterial activity ofthese derivatives suggests that substitution in the ring systemand the conformation of the side chain can modify the an-timicrobial effect.

The antibacterial activity of chlorpromazine can be sig-nificantly decreased by oxidation at the S-5 atom as shownby Table 1 Group B. Position 5 seems to be an important siteon the ring in determining antibacterial activity (CPZ-5-oxide MIC 24.3 m g/ml) whereas oxidation at the phenothiaz-ine ring nitrogen has no effect on the antibacterial activity(CPZ-N-oxide MIC 2.1 m g/ml). The irrelevance of the phe-nothiazine ring nitrogen is also evidenced by the decreasedeffect noted with CPZ-5, N-dioxide (MIC 27.0 m g/ml), aneffect that is essentially the result of oxidation at the S-5position. Trihydroxylation of carbons 3, 7, and 8 reduces theantibacterial activity of the CPZ derivative whereas single ordihydroxylation at these carbon atoms does not significantlyaffect the activity. One can therefore conclude that reductionof CPZ antibacterial activity is focused on the Sulfur atom atposition 5 and on substitution at Carbons 3, 7 and 8, 6, 9.

The antibacterial activity of thioridazine, a derivative ofCPZ that is substituted by sulfur at carbon 2, is equal to thatof CPZ [9-11]. Phenothiazine enantiomers show similar ac-tivities to CPZ (Table 1 Group D). The phenothiazine dyesmethylene blue and toluidine blue have MICs that are con-siderably lower than those shown by CPZ or thioridazine.The importance of substitution at C-2 is thus exemplified bythe presence of chloride and sulfur, respectively. The anti-bacterial activity of non-phenothiazine tricyclic compoundsis presented in Table 2. Other than the demonstration thatmany tricyclic compounds do indeed have antibacterial ac-tivity, and that this activity can be modified by selective sub-stitution at important sites of the rings, it can be noted thatthe tricyclic psychopharmacons imipramine and desipraminethat have non-planar tricyclic skeletons have similar anti-bacterial activities and become relatively inactive on satura-tion of the ring system (Table 2 Group A). It can be con-cluded that one of the aromatic rings is necessary for theantibacterial activity although which of the three rings isprimarily responsible is yet to be determined.

ANTIPLASMID EFFECTS IN VITRO

Bacteria have developed the ability to resist the toxicaction of antibiotics by a variety of mechanisms such as:enzymatic inactivation, as is frequently the case with resis-tance to b -lactams and aminoglycosides [12-15], alterationof the permeability of the cell envelope to given antibiotics[16-20] and modification of the relevant antibiotic target[21]. To date all bacteria studied have been shown to haveefflux pumps that are capable of extruding antibiotics whenthe concentration of the antibiotic is of itself, not sufficientlytoxic to the organism [22].

Phenothiazines, which have been shown to have antimi-crobial activity against bacteria (see Table 1), have also beenshown to inhibit the efflux pumps of bacteria whenever thishas been studied [20, 23-27]. These phenothiazines have alsobeen shown to cause the elimination of plasmids from bacte-ria [28-30, 33]. A relationship between antimicrobial activ-ity, elimination of bacterial plasmids and the inhibition ofefflux pumps that extrude antibiotics is strongly suggestedby a number of studies that have investigated agents thatexhibit all three effects [34-38].

The relationship between agents that have antibacterialand antiplasmid activities and also act as inhibitors of bacte-rial efflux pumps has been further studied. In addition,agents such as promethazine and acridine orange, which alsohave these activities, have also been shown to reverse antibi-otic resistance in various bacterial species, when applied ascontrols [39-41]. Such reversal of resistance is associatedwith the ability of the agent to inhibit the efflux pump. Thisconfers on the bacteria the ability to grow in concentrationsof an antibiotic that would normally be toxic to the bacteria.

The compounds listed in Tables 1 and 2, and whose mo-lecular structures are described by Fig. (2), have been inves-tigated for their ability to eliminate bacterial plasmids. It isimportant to note that plasmid elimination in some com-pounds only occurs at sub-inhibitory concentrations of therespective agent. The use of sub-inhibitory concentrationsallows the bacterium to grow. This is a necessary pre-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

CompoundsMIC values

M(x10-4)Plasmid curing % at

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-g -propyl-phenothiazine-a ,g -glutamylamid 0.9 2.0

F) Dyes

Methylene blue 16.3 0.01>

Toluidine blue 17.0 0.01>


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

CompoundsMIC values

M(x10-4)Plasmid curing % at

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



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

D 8-tetrahydrocannabinol 3.1 0.01>

D 9-tetrahydrocannabinol 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


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

Inoculum/mL at the beginningof the experiments

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

2x102 3.0 12.0 1050

2x103 8.0 57.0 840

2x104 12.0 6.0 520

2x105 16.0 1.0 642

2x106 20.0 0.1 811

2x108 22.0 0.1 876

plasmid replication is dependent upon bacterial replicationand the number of subsequent generations (Table 3).

After incubation of the bacterium carrying the lac plas-mid with sub-inhibitory concentrations of the agents, ali-quots of the culture are streaked onto drug-free mediumcontaining eosin-methylene blue (EMB). The EMB reagentaids in the identification of colonies that either contain or aredeficient in the lac plasmid, showing deep violet (lac+) orpink (lac -), respectively. The percentage of plasmid elimina-tion (curing) is determined by the number of pink coloniesdivided by the total number of colonies (pink and deep vio-let) present on the surface of the agar plate. The majority ofthe substituted phenothiazines of Groups A and C have highplasmid curing activities below the MIC at sub-inhibitoryconcentrations. The most active agent of these groups is 2-chloro-10-(2-dimethylaminoaethyl)-phenothiazine which hasa plasmid curing activity of 90% and an MIC of 3.1m g/ml.The important contribution of electronic configuration toplasmid curing activity is further shown by compounds suchas thioridazine, thiethylperazine and promazine derivatives,all of which have symmetrical p -electron distributions in theL-molecular region (Fig. (1), Fig. (2) structures 22-27). An-tidepressants possessing a non planar tricyclic structure witha secondary amine side chain are more effective in eliminat-ing plasmid than are compounds with tertiary amine sidechains, whereas the plasmid eliminating potency of quater-nary amines is much weaker probably because they are un-able to cross the bacterial cell membrane. The 7-hydroxy, 7,8-dihydroxy and 7, 8-dioxo substituted CPZ derivatives alsoappear to have lower plasmid curing effects than CPZ. Nei-ther the 6, 9-dihydroxy, 6, 9-dioxo, 5-oxide nor the 7, 8- and6, 9-siloxy derivatives exert any significant antiplasmid ac-tivity. These derivatives are considerably more polar thanchlorpromazine as indicated by the comparative ClogPvalues (ClogP: CPZ = +5.50, 7-OHCPZ = + 4.58, 7, 8-DiOHCPZ = 2.06, 3, 7, 8-TriOHCPZ = 0.77). This wouldimply that cell penetration is an important factor to be con-sidered in assessing plasmid curing activity.

It can be concluded from these results that substitution inthe aromatic rings might prevent an interaction between thep -electrons of the phenothiazine ring and target moleculesessential to plasmid replication. Structure 29 for example hassome marginal plasmid curing activity that might be relatedto the additional p electron cloud provided by the second

chlorpromazine ring. The plasmid curing activity of non-phenothiazine tricyclic compounds is similar to that of thesubstituted phenothiazines. The MIC values and antiplasmideffects do not directly correlate but some of the compoundswere effective in sub-inhibitory concentrations. A largenumber of compounds only have antibacterial activity with-out any antiplasmid activity however some degree of anti-bacterial activity is essential for antiplasmid activity. Thecannabinol derivatives are rather interesting with respect toplasmid curing activity which is present in tetrahydocan-nabidiolic acid, and to a lesser extent in cannabidiolic acidand absent in cannabinol, tetrahydrocannabinols and can-nabidiol.

Fig. (1). Electronic Structure of the phenothiazine skeleton.

The substituted secondary amines, desipramine and pro-triptyline, eliminated F lac plasmid at lower concentrationsthan did the substituted tertiary amines alimemazine andnoxiptyline (Table 4). The majority of phenothiazines elimi-nated plasmid with a frequency similar to the antidepressants(Table 4). Compounds such as chlorpromazine and imi-pramine may become attached through their cationic aminegroup to phospholipids in the cell membrane, while the hy-drophobic part of the molecule intercalates between the fattyacid chains of the membrane. This would indicate that theplasmid eliminating ability of a given compound may be afunction of the membrane-lipid: water distribution coeffi-cient. This can be substantiated by the fact that the distribu-tion coefficient for chlorpromazine is 1700 and for imi-pramine is 2395. Conversely the distribution coefficient forlidocaine is only 17.


Phenothiazine derivative

1. 2-chloro-7-hydroxyphenothiazine

S

NH

HO

Cl

16. CPZ-5-oxide

S

N

NH3C CH3

Cl

O

2. 2-chloro-10-(2-dimethylaminoaethyl)-phenothiazine

H3C

S

N

Cl

N

CH3

17. CPZ-5N-oxide

S

N

N+ Cl

O

CH3

O

3. Promazine

S

N

NH3C CH3

18. CPZ-N-oxide

S

N

N+

CH3

O

4. Desdimethyl-CPZ

S

N

NH2 Cl

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

S

N

N+ Cl

CH3

O

OSi

CH3

H3CCH3

H3C

H3C

5. Desmethyl-CPZ

S

N

NH ClH3C

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

S

N

NH3C

Cl

OSi

CH3

H3CCH3

H3C

H3C

OSi

H3C CH3

CH3H3C

H3C

CH3


(Fig. 2) contd.


6. Chlorpromazine (CPZ)

S

N

N ClH3C CH3

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

S

N

NH3C

ClCH3

O

O

Si

SiH3C

CH3

CH3

CH3

CH3

H3C CH3

CH3

CH3

CH3

7. CPZ methylammonium iodide

S

N

N+ ClH3C CH3

H3C I-

22. Thioridazine

S

N

N

N

CH3

S

CH3

8. 7-hydroxyCPZ

S

N

N ClH3C CH3

OH

23. Thiethylperazine

S

N

SCH3

N

CH3

9. 3,7-hydroxyCPZ

S

N

N ClH3C CH3

OH

OH

24. Levomepromazine

S

N

OCH3

N

CH3

CH3H3C


(Fig. 2) contd.


10. 7,8-hydroxyCPZ

S

N

N ClH3C CH3

OH

HO

25. Dextromepromazine

S

N

OCH3

NCH3H3C

H3C

11. 7,8-dioxoCPZ

S

N

N ClH3C CH3

O

O

26. Diethazine

S

N

N

H3C

H3C

12. 6,9-dihydroxyCPZ

S

N

NH3C CH3

OH

HO

27. Promethazine

S

N

N

CH3H3C

CH3

13. 6,9-dioxoCPZ

S

N

NH3C CH3

O

O

Cl

28. Tiazinamium

S

N

N+CH3H3C

H3CCH3

14. 8-hydroxyCPZ

S

N

NH3C CH3

Cl

HO

29. Bis-(2-chloro-10-g -propyl)-phenothiazine- a ,g -glutamylamide

S

N

NH

O

H2N

Cl

HN

O

N

SCl


(Fig. 2) contd.


15. 3,7,8-trihydroxyCPZ

S

N

NH3C CH3

Cl

HO

OH

OH

30. Methylene blue

S+

N

NH3C

CH3

NCH3

CH3

31. Toluidine blue

S

N

NH3C

CH3

NH2

Cl

CH3

Non-phenothiazine derivatives

32. Desipramine (DMI)

NNH

CH3

46. 1-dimethylamino-3-(9-anthryl)-3-propanol

NCH3

CH3

HO

33. DMIH

NNH

CH3

47. Maprotiline

NCH3H3C

34. DMIH2

NNH

CH3

48. 4,4-dimethylaminodiphenylmethane

N NH3C

CH3

CH3

CH3


(Fig. 2) contd.


35. Imipramine

NN

CH3

CH3

49. Chlorprothixene

S

N

H3C

CH3

Cl

36. Imipramine (saturated)

NN

CH3

CH3

50. Cis-chlopenthixol

S

N

N

OH

37. Imipramine methylammonium iodide

NN+

CH3

H3CCH3

I-

51. Trans-chlopenthixol

S

N

N

OH

38. 2-hydroxyimipramine

NN

CH3

CH3

OH

52. D 8-tetrahydrocannabinol

O

CH3

CH3CH3

H3C OH

39. 3-chloro-8-hydroxyimipramine

NN

CH3

CH3

Cl

OH

53. D 9-tetrahydrocannabinol

O

CH3

CH3CH3

H3C OH


(Fig. 2) contd.


40. 3-chloro-8-dimethyl-tertbutyl-siloxy-imipramine

NN

CH3

CH3

Cl

O

Si

CH3

H3C

H3C

CH3H3C

54. Cannabinol

O

CH3

CH3CH3

H3C OH

41. Protriptyline

NH

CH3

55. Cannabidiol

OH

H2C CH3

HO

CH3

CH3

42. Amitriptyline

NCH3

CH3

56. Cannabidiolic acid

OH

H2C CH3

CH3

CH3

O

OH

HO

43. Amitriptyline-methylammonium iodide

N+CH3

H3CCH3

I-

57. Morphine

O

N

CH3

44. 1-dimethylamino-3(1-anthryl)-3-propanol

HO

N

CH3

CH3

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

NCH3

CH3

OH


(Fig. 2) contd.


45. 1-dimethylamino-3(2-anthryl)-3-propanole

OH

N CH3

H3C

59. Ethidium bromide

CH3

NH2H2N

60. Acridine orange

CH3

NN NH3C

CH3

CH3

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

Table 4. Plasmid Elimination Action of Various Psychotherapeutics

CompoundsConcentrationm g/mL

Plasmid eliminationpercent

ColoniesExamined

MICm g/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-fluoperazine was investigated by replica plating [32] onplasmid-mediated doxycycline resistant bacterial strains iso-lated from clinical specimens. The frequency of plasmidelimination was low for the majority of the tested strains,despite the formation of complexes between antiplasmidcompounds (promethazine, 9-aminoacridine) and plasmidDNA isolated from the plasmid containing clinical strains. Itmay be suggested that inefficient penetration of the an-

tiplasmid compounds was responsible for the weak plasmidcuring effect in these clinical isolates. It is probable that thecell wall/cell membrane served as a barrier resulting in weakplasmid elimination [20]. Indirect evidence also supportsthis, since the co-administration of verapamil (Ca2+-anta-gonist) and trifluoperazine (intercalating plasmid curingcompound) resulted in a remarkable increase in plasmidelimination [unpublished results].


The inhibition of plasmid replication resulted from a sin-gle nick, outside of the replication origo of the superhelicalstructure. The process leads to further relaxation of plasmidDNA. 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 andits promethazine complex were analysed by agarose-gelelectrophoresis, the superhelical form was missing from thepromethazine treated plasmid DNA. The open circular andlinear 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 plasmidDNS. (In: Waring, M.J. (1966) Symp. Soc. Gen. Microb. 16, 235-265).

Plasmid replication can also be inhibited by blocking theactivity of the enzyme DNA gyrase. This time the introduc-tion of superhelical turns into plasmid DNA is prevented.The activity of DNA gyrase isolated from M. luteus was in-hibited in the presence of promethazine and imipramine [45],both compounds having plasmid curing effects. Apart fromgeneral intercalation into dsDNA, a specific binding site forphenothiazine intercalation was found in the GC-rich region.[46, 47]. This means that the expression of the GC rich pro-moter of the MDR-1 gene responsible for drug resistance ofcancer cells can be blocked simultaneously.

All of the tested plasmids were curable from E. colistrains, except the E1 colicin [43, 45]. The simultaneouselimination of Flac and pBR322 plasmid from E. coli alsoshowed relatively high frequency. It turns out that the curingefficiency of phenothiazines depends also on the host cells,since the same plasmid was curable with different frequen-cies from various E. coli strains [44, 48]. The Ca++ bindingprotein encoding the plasmid responsible for the virulence ofY. enterocolitica and Y. pseudotuberculosis isolates wascured with rather low frequency [49].

The antiplasmid compounds were also able to inhibitplasmid transfer [8, 50]. In these experiments E. coli r144drdKmr strain with depressed pilus synthesis was used as a do-nor, and a chromosomally sodium azide resistant E. colistrain was used as a recipient. A dose dependent inhibition ofconjugal plasmid transfer was observed [42] where both

transconjugal DNA synthesis and mating pair formationswere inhibited. The inactivation of sex pili was shown by

Fig. (4). The effect of the binding of ethidium bromide to SV40DNA I and II. The upper part of the diagram presents three stagesin the reversible binding of dye to SV40 DNA I.

a) represents the dye-free molecule with 14 superhelical turns; theaddition of 420 molecules of ethidium bromide completely unwindsthe superhelical turns to form the relaxed molecule (b). The addi-tion of a further 720 dye molecules leads to the formation of a posi-tive superhelical molecule, shown in (c). The lower part of the dia-gram shows three stages in the reversible binding of dye to SV40DNA II, which remains relaxed throughout. (e) represents the re-laxed, nicked molecule with the same number of dye moleculesbound as to the relaxed, intact molecules in (b). The arrows joining(b) and (e) indicate that the nick may be introduced or repairedwithout change of the 420 molecules of ethidium bromide bound.The nicked molecule is nearly saturated, as shown in (f), with 1860molecules of ethidium bromide bound. Introduction of a single-strand scisson into I in the presence of a high dye concentrationresults an irreversible unwinding of the superhelix. (In: Waring, M(1970) J. Mol. Biol. 51, 247-279).

the inhibition of absorption of pilus specific phage [39, 40](Tables 5, 6).

ANTIPLASMID EFFECTS IN VIVO

The adhesion of urinary pathogen E. coli strains was in-hibited in HEP2 tissue culture cells by inhibition of the adhe-sive type I and p-fimbriae [51]. Based on these results thesynergistic effect of promethazine with gentamycin wasstudied in children with frequently recurring pyelonephritis[52] and in adults [53, 54]. In these experiments 10 children


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

No. of cells/mLCompounds

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-dioxoCPZ 0.05 0.5 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-g -propyl)phenothiazine-a ,g -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 37C. The data are the results of two parallel experiments.


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

No. of cells/mLCompounds

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



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

D 8-tetrahydrocannabinol 3.0 1.5 1.9

D 9-tetrahydrocannabinol 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 37C. The data are the results of two parallel experiments.


were given a combination of gentamycin and promethazinefor 7 days (Group l.). In the second group 11 children re-ceived gentamycin alone and in the 3rd group 19 childrenwere on long-term oral antibiotic prophylaxis with episodesof intensive treatment. Over a three year follow up period,the number of pyelonephritis recurrences were significantlylower in Group1 than in Groups 2 and 3. Plasmid eliminationwas also studied by in vivo or ex vivo experiments, when theplasmid profile of freshly isolated colonies from the urine ofpromethazine and imipramine treated adult patients werecompared. A heterogenicity of antibiotic sensitivities and theplasmid profile of bacteria isolated from promethazine orimipramine treated patients was found after only five daystreatment with tricyclic psychopharmacons. These changeswere found in only a few percent of the E. coli colonies iso-lated from the urine of treated patients, therefore it has to beconsidered that plasmid elimination has no clinical impor-tance on its own [43]. Among 50 treated patients, 12 hadsignificant bacteriuria, however 21 of the control 50 patientsalso had significant bacteriuria. The resistance patterns of tendifferent isolates from the urine of maprotiline treated pa-tients changed after 5 days of treatment, when carbenicillinand 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 serumconcentrations in patients, consequently the frequency of invivo plasmid curing was low. Synergism between antibioticsand antiplasmid effect can be produced by complex mecha-nisms.

STRUCTURE ACTIVITY RELATIONSHIPS

It has been proposed that the conjugated p -electron sys-tem of the tricyclic skeleton has a special importance wherethe conjugation of the p -electrons of the two aromatic ringsis symmetrical to the L-molecular region (Fig. (1)). It is rea-sonable to conclude that the antibacterial and antiplasmideffect noted with active phenothiazines or tricyclic non-phenothiazines is dependent on the available p -electrons andtheir distribution.

Thus knowing the mechanism of plasmid curing, an idealplasmid curing or resistance modifier compound can be pro-posed. The correlation between antiplasmid effects andchemical structure of the curing compounds was studied. Inquantitative structure activity relationship (QSAR) studies itwas shown that the HOMO orbital energy, the symmetry ofthe p electron distribution to the L-molecular region and thesuperdelocalizability of the p electron system of tricyclicskeleton on atoms 10, 12 and 13 were responsible for effec-tive binding through stacking interaction to the biologicaltarget eg. DNA or DNA gyrase. Based on the results of theQSAR correlation [56-61] novel substituted anthranyl- com-pounds were synthesized and found to possess remarkableplasmid curing effects [62-64]. The antiplasmid action oftricyclics on the other hand was found to be rather specificand depended upon the chemical structures of the com-pounds [14, 65].

Drug treatment of bacterial cells resulted in the inhibitionof plasmid replication and finally in the formation of plas-mid-free cells. In order to analyze the mechanism of action at

the molecular level the effects of these drugs at severalpoints in the course of transformation, in plasmid DNA rep-lication and the topological state of plasmid DNA was ex-amined (Fig. (5)).

Fig. (5). Electrophoretic analysis of antiplasmid effects of pro-methazine.

Samples:

1. E. coli HB101/pBR322 culture was treated with 250 m g/mL pro-methazine; 2. E. coli HB101/pBR322 culture was treated with 1000m g/mL promethazine; 3. E. coli HB101/pBR322 culture was treatedwith 2000 m g/mL promethazine; 4. E. coli HB101/pBR322 cellswere lysed with lysosyme and 250 m g/mL promethazine was addedto the lysed cells; 5. E. coli HB101/pBR322 cells were lysed withlysosyme and 1000 m g/mL promethazine was added to the lysedcells; 6. E. coli HB101/pBR322 cells were lysed with lysosyme and2000 m g/mL promethazine was added to the lysed cells; 7. pBR322plasmid DNA; 8. pBR322 DNA was treated with Hind III at 37Cfor 2 hours, which made the DNA linear; 9. pBR322 plasmid DNA.

a: linear; b: relaxed circular; c: covalently closed circular. (In: Mol-nr J, Fldek S, Nakamura MJ, Rausch H, Domonkos K, Szab M.(1992) APMIS Suppl. 30/100, 24-31).

Two possible target sites were identified in plasmid DNAreplication. One of them involved membrane binding sites,the other one is in DNA replication. The other effect ob-served in vivo and in vitro was the influence on the topologi-cal state of plasmid DNA (Fig. 4). The presence of tricyclicdrugs promoted the relaxation of plasmid DNA by interfer-ing with the supercoiling activity of DNA gyrase causing acessation in plasmid replication.

Therefore in order to improve drug design, some quan-tum chemical parameters, including: p electron superdelo-calizibility, HOMO, LUMO orbital energies, size of the Vander Waals surface in a watery environment were calculatedby several computer programs (MM2, CNDO) for phenothi-


azines, acridines and naphthyridines. Based on these resultsnew anthracene derivatives were synthesized [66-70]. In thisway the antiplasmid and carcinogenic molecular orbitalswere clearly differentiated [71, 72]. Based on the QSARstudies, new compounds with well-defined and predictableelectronic structures in the molecular orbitals [31] were pre-pared. The compounds possessed antiplasmid activity butdisplayed no mutagenic or carcinogenic effects [59, 60]. Thebinding affinity changes caused by promethazine and imi-pramine indicated that the resulting plasmid elimination wasconnected at least partly to membrane proteins and plasmidDNA complexes (Fig. (5)).

Drugs may affect the binding affinity of replicating plas-mid DNA to membrane proteins producing more stablecomplexes that negatively interfere with the processing ofthe replication fork and the expression of plasmid encodedgenes. This was shown for the F lac plasmid.

EVALUATION OF PLASMID CURING

Although bacterial resistance is different from eukaryoticresistance in many respects there are common sensitivepoints, such as transporter protein mediated efflux pumpsystems. In this respect the mechanism of resistance in bacte-ria, protozoa and tumour cells is similar and therefore it maybe possible to overcome it in a similar way.

In preliminary in vivo experiments it was shown that theefficiency of plasmid curing was rather low and apparentlyof little practical importance. However considering the exis-tence of efflux pump inhibitors it was decided to check theresults of the interaction of plasmid curing compounds andantibiotics in vitro . Among various plasmids, the hemolysinand tetracyline transporter encoding plasmid was eliminatedfrom the bacteria. Detailed analysis of the plasmidless bacte-ria showed that the MIC value for tetracyline was reduced inthe plasmidless bacterial cells. In addition the antibacterialeffect of tetracycline is synergized by plasmid curing com-pounds. This was independent from the elimination of plas-mid DNA [52]. This resistance modifying effect of selectedphenothiazines and structurally related compounds wassimilar for all individual cells of the culture, showing that theinhibition of drug efflux was able to increase the intracellularconcentration of chemotherapeutics in bacteria and cancercells. Plasmid elimination was shown to exist in microbialeco-systems, however a number of clinical isolates wereresistant to many antibiotics and simultaneously had verylow sensitivity to high concentrations of the plasmid curingcompounds. Using mixed cultures, including plasmid bear-ing bacterial cells (E. coli K12 LE 140), the conditions of apolimicrobial flora were simulated. Plasmid elimination wasstudied under various conditions, including co-inhabitingbacteria, at various temperatures and in the presence of theplasmid curing promethazine. It was established that plasmidelimination from E. coli K12 LE 140 promoted by sub-inhibitory concentrations of promethazine was significantlyexalted by elevation of temperature either in the monocultureor when incubated together with either B. cereus or S. epi-dermidis. The efficiency of plasmid elimination of phenothi-azine was markedly enhanced by the presence of a secondspecies of bacteria [73].

Doxycycline resistant bacterial isolates from clinicalspecimens were found to have a cell membrane that was im-permeable to resistance modifying antiplasmid drugs. Thefrequency of the elimination of tetracycline resistance waslow for the majority of strains investigated, despite the factthat it has been shown that complexes are formed betweenantiplasmid compounds and plasmid DNA. The results sug-gest that the curing effects were dependent on the perme-ability barrier of the clinical isolates studied. Promethazineand trifluoperazine as well 9-aminoacridine have pronouncedplasmid curing activity in E. coli K12 LE 140 and these ef-fects were substantially enhanced by administering the pro-ton pump inhibitor trifluoromethyl ketone at concentrationsranging from 0.05 to 1.0 mg/L. Thus it can be shown thatvarious type of resistance modifying drugs, such as calcium-channel blockers or proton pump inhibitors, can enhance theactivity of plasmid curing drugs. The results suggest thatinhibitors of membrane ABC transporters and proton pumpsmay be combined to produce plasmid curing in some antibi-otic-resistant bacterial strains [20].

There is evidence for that the activity of plasmid medi-ated haemolysin transporter in bacteria [74] and other bacte-rial transport proteins have a close homology to mammalianmultidrug resistance transporters, the so called efflux pumps[75]. This multidrug resistance mechanism was modified inboth bacteria and in cancer cells by antiplasmid compounds[76]. The intracellular accumulation of antibiotics or che-motherapeutics increases as a consequence of decreased an-tibiotic efflux in both bacterial and tumour cell systems. Theinhibition of the efflux pump is the same for all individualmembers of the population of bacterial and cancer cells,however the antiplasmid effects occurred in only a smallfraction of the growing bacterial cell populations [76] but theinhibition of exporter proteins can also be exploited to in-crease plasmid curing.

IMPORTANCE OF PLASMIDS AND ABC TRANS-PORTERS

Membrane transporters can be encoded by genes local-ised on the chromosomes and on plasmids such as haemo-lysin and tetracycline transporters [77, 78]. The multidrugmembrane transporters are classified into two main groupssuch as ABC transporters and proton pump systems based onenergetic requirements and the second class is sub-dividedinto a large number of subclasses.

Some transporters such as the tetracycline efflux proteinmediate the extrusion of the particular antibiotic. This activeefflux is important in ensuring a significant level of resis-tance to tetracycline or other antibiotics. In contrast the tettransporters are multidrug-transporters, which confer resis-tance against a wide variety of structurally unrelated com-pounds [79-82]. The mdr transporters can be inhibited a widevariety of compounds such as uncouplers and calcium an-tagonists .The proton motive force utilizing efflux pumps issensitive to compounds that dissipate the proton gradient inthe membrane. These pumps mediate the efflux of xenobiot-ics in a coupled exchange with protons.

The majority of multidrug transporters use ATP as theenergy to pump the antibiotics out of the cells, while the sec-ond largest groups of transporters utilize the transmembrane-


proton gradient to drive the antibiotics or other xenobioticsout of the cells. Several subclasses belong into this group[83-85, 89, 90]. Experiments suggested that drug resistanceby bacteria and cancer cells can be achieved in various ways,however the inhibition of efflux pump systems is the mostpromising mechanism in this respect because the intracellu-lar concentration of antibiotics is enhanced in all individualcells of the population simultaneously and at much lowerconcentration of resistance modifier compounds than isneeded for plasmid elimination. This resistance modifyingeffect is apparently independent from the antibacterial orcytotoxic effects.

CONCLUSIONS

The aim of the study was to clarify the mechanisms ofaction of the drugs on plasmid replication and to improve thecuring activity of existing compounds by computer aideddrug design in order to obtain effective plasmid curing com-pounds. The ordered replication, partition and segregation ofplasmid DNA is essential, if they are not to be lost from theirhost cells [42, 86].

From the mechanisms described above it is evident thatthe spread of antibiotic resistance can be reduced at differentlevels in a rather complex approach. Apparently one of thesimplest ways to reduce antibiotic resistance is to halt plas-mid replication, partition and transfer by plasmid elimina-tion. Consequently the co-administration of antibiotics andresistance modifiers can be recommended in serious poly ormultidrug resistant infections as well as in multidrug resis-tant cancer. The inhibition of the efflux-pump systems in themembrane of bacteria or in cancer cells occurs simultane-ously in all the individual cells of the resistant population,therefore blockade of the efflux pumps as targets would ap-pear to be an effective approach to combinational chemo-therapy. The comparison of minimal inhibitory concentra-tions of drug candidates measured on the haemolysin trans-porter plasmid containing E. coli cells and on its plasmidlessderivative can be exploited for pre-screening particular re-sistance modifiers (Fig. (6)) [23]. In addition, since plasmidcuring compounds destabilize the maintenance of extrachromosomal elements in a fraction of bacterial population

Fig. (6). Schematic structure of the HlyA translocon complex.(http://bmec1.igmors.u-psud.fr).

without mutagenic effect, the results can be exploited in or-der to isolate plasmid free bacteria for biotechnology withoutany risk of mutations.

The main goal of this study is to investigate ways ofcombating antibiotic resistance by the selective inhibition ofplasmid replication and transfer and by blocking the bacterialefflux pumps. The results of the model experiments on thesynergistic interactions between antibiotics and resistancemodifiers found in vitro can be exploited in the rational drugdesign of drugs to counteract antibiotic resistant pathogens.

ACKNOWLEDGEMENTS

These studies were supported by the Szeged Foundationfor Cancer Research and Cooperation in Science and Tech-nology, COST Action B16 "Reversal of Antibiotic Resis-tance" at the European Commission.

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