Inhibition of Drug Efflux in Mycobacteria with ...

Recent Patents on Anti-Infective Drug Discovery, 2011, 6, 000-000

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Inhibition of Drug Efflux in Mycobacteria with Phenothiazines and Other Putative Efflux Inhibitors Liliana Rodrigues1,2, José A. Aínsa3,4, Leonard Amaral1,2,4 and Miguel Viveiros1,4,* 1

Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 2UPMM, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, 3Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 4COST Action BM0701 (ATENS) Received: March 25, 2011; Accepted: March 30, 2011; Revised: April 7, 2011

Abstract: Mycobacteria are responsible for some of the oldest diseases known to Man, usually associated with high morbility and mortality rates. An example is tuberculosis (TB), a major public health problem that accounts for eight million new cases each year. Furthermore, the increase of multidrug and extremely-drug resistance seriously threatens the success of the TB control programmes. Resistance to anti-mycobacterial drugs is often due to spontaneous mutations in target genes, followed by selection of resistant mutants during treatment. However, this does not explain all cases of drug resistance and other mechanism(s) may be involved, namely efflux pumps that extrude the drug to the exterior of the cell. Efflux pumps are becoming attractive drug targets for the development of new anti-mycobacterial compounds and several efflux inhibitors have been developed and published in patent applications (i.e., WO2004062674, US2004204378, US2003118541, WO2008141012, WO2009110002, WO2010054102). However, none of these inhibitors is used in clinical practice. This review will focus on the potential use of efflux inhibitors as adjuvants of the anti-mycobacterial therapy, an approach that may restore the activity of antibiotics that are subject to efflux and render the mycobacteria more susceptible to drugs transported by these pumps.

Key words: Efflux inhibitors, efflux pumps, mycobacteria, phenothiazines, thioridazine, tuberculosis. INTRODUCTION The Mycobacterium genus comprises several clinically relevant pathogens, of which the most known is Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) one of the oldest diseases known to Man. In fact, TB is still a major threat for public health worldwide and the World Health Organization (WHO) estimates that approximately two billion people are infected with Mycobacterium tuberculosis, with eight million new cases and two million deaths each year [1]. Furthermore, the increase of multidrugresistant TB (MDRTB; TB caused by M. tuberculosis resistant to at least isoniazid and rifampicin, the two first-line drugs that constitute the backbone of TB treatment) and extensively-drug resistant TB (XDRTB; MDRTB with additional resistance to fluoroquinolones and one of the three injectable second-line drugs kanamycin, amikacin and capreomycin) has reduced the options available for treatment of these patients and seriously threaten the control of TB worldwide [1].

Mycobacterium fortuitum complex, frequently responsible for abscess formation in surgical wounds and pulmonary disease in the case of imunossupression of the host [2]. Only a few antibiotics are active against mycobacterial pathogens, which frequently acquire resistance leaving very limited therapeutic options. For these reasons, treatment of mycobacterial infections generally consists of a combination of three or more drugs that must be given for a long period of time for a successful outcome and for preventing the selection of resistant strains.

In addition to M. tuberculosis, the Mycobacterium genus also comprises other clinically important mycobacteria such as Mycobacterium leprae and the non-tuberculous or atypical mycobacteria like Mycobacterium avium complex, highly associated with infections in AIDS patients, and

Acquired drug resistance in M. tuberculosis is mainly attributed to chromosomal mutations that alter the antibiotic target or the activator of the pro-drug [3]. The selection of drug resistant mutants has to do with several causes: first, the long generation time of M. tuberculosis, its low metabolic activity and capacity for dormancy [4]; second, M. tuberculosis may be located inside pulmonary cavities, with difficult antibiotic access [5]. Such compartmentalization of the infection increases the probability of exposure to monotherapy or lower antibiotic doses, which may result in higher probability of acquiring resistance. This effect is enhanced in the presence of an inadequate dosage of anti-TB drugs, due to inadequate prescription by the physician or non-adherence by the patient.

*Address correspondence to this author at the Grupo de Micobactérias, Unidade de Microbiologia Médica, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Rua da Junqueira 100, 1349-008 Lisboa, Portugal; Tel: + 351 21 365 26 52; Fax: + 351 21 363 21 05; E-mail: [email protected]

In addition to the mutation-mediated acquisition of drug resistance, M. tuberculosis and other mycobacteria are provided with a diversity of intrinsic drug resistance mechanisms, mainly the impermeability of the cell-wall and the activity of drug efflux pumps. These mechanisms also

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2 Recent Patents on Anti-Infective Drug Discovery, 2011, Vol. 6, No. 2

contribute to overall bacterial resistance and act in synergy with drug resistance mutations [6]. In fact, mycobacteria are naturally resistant to several antimicrobial drugs, mainly due to the permeability barrier provided by the mycobacterial cell-wall that causes a slow uptake of compounds. This diminished membrane permeability in synergy with the extrusion of drugs through efflux pumps can decrease the antibiotic concentration in the cell [7, 8]. Altogether, this allows the bacteria to survive for a longer period of time in the presence of that antibiotic, conferring a low-level resistance phenotype. It is possible that this prolonged survival under antibiotic pressure may increase the probability of selection of spontaneous mutants that present a high-level resistance phenotype, from this subpopulation of bacteria [911]. By this manner, the efflux mechanisms would occur prior to the acquisition of target gene mutations and overproduction of efflux pumps could result in an increase in antibiotic resistance [11, 12]. This may be particularly relevant in the case of long-term therapies such as that used in TB treatment, where a sustained pressure of sub-inhibitory concentrations of an antibiotic can result in an increased efflux activity and allow the selection of spontaneous mutants, thus rendering the organism resistant to that antibiotic. A way to prevent these events from occurring would be the inhibition of efflux pumps by using efflux inhibitors. This strategy would increase the activity of antibiotics that are subject to efflux and decrease the frequency of selection of drug resistant mutants. What would be the impact of such strategy in the treatment of mycobacterial infections? The use of efflux inhibitors would be of particular importance in the case of highly prevalent pathogenic mycobacteria, namely M. tuberculosis. The need for new antimycobacterial therapeutic strategies is now urgent for the control of the TB epidemic [1] and if efflux inhibitors may represent an interesting therapeutic approach, certainly, it is worth to explore it. This review will focus on the usefulness of non-antibiotic agents that can act as efflux inhibitors in mycobacteria, which being used as adjuvants of the conventional antimycobacterial therapy would restore the activity of antibiotics that are subject to efflux, prevent the selection of drug resistant strains and help in the control of the infections caused by these pathogens. INTRINSIC RESISTANCE IN MYCOBACTERIA: MEMBRANE IMPERMEABILITY AND EFFLUX PUMPS In many Gram-negative bacterial pathogens, such as Pseudomonas aeruginosa, intrinsic drug resistance mainly relies on the impermeability of the cell-wall and the activity of efflux pumps [13]. Mycobacteria phylogenetically cluster among the Gram-positive bacteria, although their cell-wall structurally resembles that of Gram-negative bacteria. This lipid-rich mycobacterial cell-wall establishes a double barrier to the entry of antimicrobials protecting the cell from toxic compounds [14, 15]. The peptidoglycan and arabinogalactan layers limit the entry of hydrophobic molecules, whereas the mycolic acid layer limits the access of both hydrophobic and hydrophilic molecules [14]. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may enter

Rodrigues et al.

the cell by diffusion through the hydrophobic bilayer. However, hydrophilic antibiotics and nutrients could cross the cell-wall through porin-like channels that have been characterised in some mycobacterial species such as M. smegmatis [16-18]. However, in these species, mycobacterial porin-like proteins are much less abundant than in the Gramnegative outer membrane and only allow low rates of uptake for small hydrophilic nutrients and antibiotics [17, 19]. Intriguingly, the genome of M. tuberculosis lacks genes homologues to those encoding porin-like proteins in other mycobacteria. Many mycobacterial-specific drugs, such as isoniazid or ethambutol, target the biosynthesis of cell-wall mycolic acids and arabinogalactan respectively, so their action also will weaken the permeability of the cell-wall, hence facilitating the uptake of other drugs used simultaneously in the combination therapy. Along with cell-wall impermeability, active efflux systems can also provide resistance by extruding drug molecules that enter the cell. Although bacterial efflux pumps are known for their association with antimicrobial resistance, their natural physiological role consists in the extrusion of noxious agents (i.e. intracellular metabolites) from the cell, allowing the bacteria to survive in a hostile environment [10]. Efflux pump systems can be organized into different families according to their energetic and structural characteristics: the ATP-binding cassette (ABC) superfamily; the major facilitator superfamily (MFS); the multidrug and toxic compound extrusion (MATE) family; the small multidrug resistance (SMR) family; and the resistance nodulation division (RND) family. Efflux pumps that are included in the ABC superfamily are considered primary transporters because they hydrolyze ATP as a source of energy, whereas the other families of efflux pumps use the proton (or sodium in the case of MATE family) gradient as an energy source and are thus called secondary transporters [10]. Antimicrobial resistance due to an increased efflux activity can be caused by the genetic overexpression of the efflux pump, or by aminoacid substitutions in the protein itself that can render the pump more efficient. Both mechanisms cause the reduction of the intracellular concentration of the antimicrobial and, consequently, the organism becomes less susceptible to that agent. Several mycobacterial drug efflux pumps have been identified and characterized to the present day [20-22]. In laboratory conditions, many of these efflux pumps have been associated with low-level resistance to multiple drugs, including the main drugs in use against mycobacterial infections. One example of this is the P55 efflux pump from M. bovis and M. tuberculosis which has been associated with low-level resistance to rifampicin among other drugs [23]. The genome of M. tuberculosis contains several genes encoding putative efflux pumps. Other mycobacterial species such as M. smegmatis have additional efflux pumps, like the LfrA efflux pump responsible for low-level resistance to fluoroquinolones and other compounds [24-25]. Some of the genes encoding complete mycobacterial efflux pumps or components of them have been shown to be induced upon diverse drug treatments: for example, the operon Rv0341Rv0342-Rv0343 is induced by treatment with isoniazid [26].

Phenothiazines and Other Efflux Inhibitors in Mycobacteria

In mycobacteria, efflux pumps also contribute to the transport of other molecules. This is the case of the DrrC protein from M. tuberculosis, an ATP-binding cassette (ABC) transporter that is involved in the transport of phthiocerol dimycocerosate (PDIM), an important lipid for the virulence of this pathogen [27]. This lipid is also transported by MmpL7, a protein of the resistance nodulation division (RND) family of efflux pumps; remarkably, this family of efflux transporters was thought to be restricted only to Gram-negative bacteria. Like in many other bacterial species, most of the studies on mycobacterial efflux pumps rely on the overexpression of a particular efflux pump, which is often associated with moderate increases in the MICs to several drugs [23, 24, 28]. This phenotype needs to be corroborated by using compounds known to be efflux inhibitors (EIs), which results in a decrease of the MICs values, reaching again those of the wild-type or even lower values. This phenomenon has led to consider the usefulness of potential helper compounds that would inhibit efflux pumps, rendering the bacteria more susceptible to antibiotics. EFFLUX PUMP INHIBITORS AND THE “HELPER COMPOUND” CONCEPT The concept of “helper compound” in antimicrobial chemotherapy is not new. The highly successful combination of -lactam antibiotics with -lactamase inhibitors was a milestone to overcome resistance to -lactam antibiotics. This has fuelled research in similar strategies, such as the use of protein kinase inhibitors to inhibit aminoglycoside resistance mediated by aminoglycoside phosphotransferase enzymes [29]. In these two cases, the helper compound only affects resistance to one single class of antibiotics. However, in the case of efflux pumps, potential helper compounds would have a much greater impact. The contribution of efflux pumps to resistance to multiple drugs, to the acquisition of chromosomal mutations and higher resistance levels, and to other processes such as virulence, has made these pumps attractive drug targets for the development of new compounds that would inhibit the efflux activity and can be used as adjuvants (also called ‘helper compounds’) in combination with existing antibiotics [9, 30]. An ideal inhibitor of a particular efflux pump should satisfy several criteria: 1) it must enhance the activity of multiple substrates of the pump; 2) it must not potentiate antibiotics that are not efflux substrates; 3) it must not change MICs in strains lacking efflux pumps; 4) it must increase accumulation and decrease extrusion of efflux pump substrates; and 5) it must not affect the proton gradient across the inner membrane [31]. Research is being done in this direction, targeting specific efflux pumps of important Gram-negative and fungal pathogens [32, 33]. Several compounds shown to be efflux inhibitors have been developed and some of them published in patent applications: WO2004062674 (substituted polyamines as inhibitors of bacterial efflux pumps) [34]; WO2008141012 (quaternary alkyl ammonium bacterial efflux pump inhibitors and therapeutic uses thereof) [35]; WO2009110002 (novel efflux pump inhibitors) [36]; WO2010054102 (polybasic bacterial efflux pump inhibitors and therapeutic uses thereof) [37].

Recent Patents on Anti-Infective Drug Discovery, 2011, Vol. 6, No. 2

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Other compounds have been used in laboratory conditions to inhibit efflux activity in vitro indirectly, by depleting the energy source used by the efflux pump. A classic example is carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which affects the energy level of the bacterial membrane and is used to dissipate the proton motive force and inhibit the efflux of several drugs. However, this compound also reduces the viability of the bacterium and cause cell death and, therefore, the observed effect on the efflux activity may be due to causes other than efflux inhibition. Moreover, this compound is described as highly noxious and cytotoxic and is also a substrate of efflux pumps [9, 38]. Table 1 presents some representative compounds that have been used as efflux inhibitors in mycobacteria. Several of these compounds have been or are used in clinical practice for purposes other than bacterial efflux inhibition. For example, the phenothiazines have been used for several years in the treatment of psychoses and verapamil in the treatment of arrhythmia. The potential use of some of these compounds as efflux inhibitors that may restore the activity of antibiotics subject to efflux has lead the scientific community to consider these compounds with renewed interest. Reserpine is a plant alkaloid known to inhibit P-glycoprotein in eukaryotic cells and to decrease the emergence of resistance in Gram-positive bacteria [39-41]. In mycobacteria, reserpine has been used to reduce resistance to isoniazid in M. tuberculosis strains with induced resistance to this drug by the activity of efflux pumps [42]. The calcium channel antagonist verapamil, another known inhibitor of Pglycoprotein, also inhibits several bacterial ABC efflux pumps, including DrrAB of M. tuberculosis [43]. Verapamil also showed activity in the inhibition of ethidium bromide efflux by M. smegmatis and M. avium complex, as well as in the reduction of MICs of several antibiotics [44, 45]. Plant derivative compounds such as luteolin, biochanin A, piperine and farnesol have also shown promising results as efflux pump inhibitors in M. smegmatis and M. tuberculosis [46-48]. In summary, various compounds have been used to inhibit efflux activity in vitro, but none of them can be used in clinical practice for this purpose. There is a concern regarding the adverse effects and selectivity of these compounds, since some of them have been shown to inhibit both eukaryotic and bacterial efflux systems [30, 39]. In conclusion, the search for safe and effective efflux inhibitors that could be used as ‘helper compounds’ administered in combination with conventional antibiotics to which the organism was initially resistant has proven to be a challenge. THE PHENOTHIAZINES: SAFE AND EFFECTIVE EFFLUX INHIBITORS Phenothiazines are heterotricyclic compounds derived from methylene blue, the dye discovered in the 19th Century by Paul Ehrlich, and reported by him to render mobile bacteria immobile [49]. The potential neuroleptic properties of methylene blue resulted in the synthesis of the phenothiazine chlorpromazine in 1953, which was used for the therapy of psychosis for the next 30 years. Later studies


Table 1.

Rodrigues et al.

List of Representative Compounds Used as Efflux Inhibitors in Mycobacteria

Efflux inhibitor

Chlorpromazine

Chemical Structure

Description/ Clinical use

Availability (brand names)

Phenothiazine. Antipsychotic indicated for the treatment of schizophrenia.

Largactil, NovoChlorpromazine,

Adverse Effects

Reference

Sedation, slurred speech constipation cardiac arrhythmias

[42, 44, 45, 53]

Drowsiness dizziness, fatigue Prolongs the QT interval on the Electrocardiograph

[42, 44, 45, 51, 54, 55]

Intensol,

Thorazine.

Thioridazine

Phenothiazine. Antipsychotic used in the management of psychoses.

Mellaril, Novoridazine, Thioril

Verapamil

Calcium channel blocker. Used in the treatment of hypertension and arrhythmia.

Isoptin, Verelan, Verelan PM, Calan, Bosoptin, Covera-HS

Reserpine

Plant alkaloid. Inhibits the uptake of norepinephrine and dopamine. Used as an antihypertensive and antipsychotic.

Novoreserpine, Diupres, Regroton, Serpex, Serathide, Hydroserpine, Hydropres, Salutensin, Diutensen-R, ReneseR, Metatensin.

depression, asthma, nausea vomiting, gastric intolerance and ulceration diarrhea

Luteolin

Plant flavonoid. Antioxidant, antiinflammatory, and anti-tumour properties

N.A.

N.A.

[46]

Biochanin A

O-methylated isoflavone. Has putative benefits in dietary cancer prophylaxis.

N.A.

N.A.

[46]

Piperine

Plant alkaloid found in black pepper and long pepper.

N.A.

N.A.

[47]

Farnesol

Natural plant metabolite. Isoprenoid component.

N.A.

N.A.

[48]

N.A. – not available.

Headaches dizziness swelling increased urination fatigue, nausea ecchymosis

[43-45]

Hypotension bradycardia fatigue [39-41]


showed that chlorpromazine directly inhibited the in vitro growth of M. tuberculosis [50, 51]. However, the side effects produced by this phenothiazine were severe and thus it was not considered as an anti-TB compound [52]. Other phenothiazines have derived from chlorpromazine, namely thioridazine, promazine, promethazine, trifluoperazine, triflupromazine, acetopromazine and fluphenazine. In particular, thioridazine (trade names Mellaril, Novoridazine, Thioril) was introduced into clinical practice and is currently a FDAapproved drug for treatment of psychiatric disorders. This phenothiazine caused much milder side effects than chlorpromazine, thus making them potential candidates as effective anti-TB drugs. However, the in vitro effects noted still took place at concentrations that were clinically unmanageable [52]. In 1992, Crowle et al. showed that chlorpromazine had activity against intracellular mycobacteria at a concentration that was far lower than that needed to inhibit their growth in vitro [53]. The in vitro activity of thioridazine against antibiotic susceptible and resistant strains of M. tuberculosis was demonstrated by Amaral et al. [51]. Later studies showed that thioridazine promoted the killing of intracellular MDRTB and the cure of M. tuberculosis or MDRTB infected mice [54, 55]. The mechanism of action of phenothiazines remains to be completely clarified. Several suggestions have been made, namely interference with cell-wall integrity, binding to calcium transport membranes and inhibition of adherence of calcium [56, 57]. These compounds are mainly described to inhibit the transport of calcium (Ca2+) in eukaryotic cells by preventing its binding to Ca2+-binding proteins, such as calmodulin [30, 58]. As a consequence, enzyme systems dependent of Ca2+, such as those involved in generating cellular energy from hydrolysis of ATP, are inhibited [59]. In bacterial cells, it has been demonstrated that phenothiazines reduce or reverse resistance to multiple drugs [52, 60]. The reason for this is that phenotiazines inhibit bacterial Ca2+-dependent enzyme systems, which are needed for generating the proton-motive force, which is the energy source used by many efflux pumps in order to confer resistance to multiple antibiotics [30, 61]. Furthermore, it has been shown that phenothiazines also enhance the killing of intracellular bacteria, such as M. tuberculosis. This can be due to a concentration effect, as the agent reaches lethal concentrations inside the macrophage, possibly due to the inhibition of K+ and Ca2+ transport processes [62]. Although the concentration effect cannot be ruled out, recent experiments have shown that inhibitors of K+ and Ca2+ transport enhance the killing of intracellular bacteria, further suggesting that phenothiazines enhance killing by the same mechanism [30, 63]. Thioridazine and chlorpromazine have been used to demonstrate the presence of efflux activity in mycobacteria like M. smegmatis, M. avium complex and M. tuberculosis [42, 44, 45]. Also, thioridazine or chlorpromazine treatment produced an increase of expression levels of the efflux pump P55 in M. tuberculosis, although none of these compounds was found to be a substrate of that particular efflux pump [23, 64].


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THIORIDAZINE, CHLORPROMAZINE AND VERAPAMIL INHIBIT THE EFFLUX ACTIVITY AND DECREASE THE INTRINSIC RESISTANCE OF MYCOBACTERIUM SMEGMATIS AND M. AVIUM The phenothiazines thioridazine and chlorpromazine and also the calcium-channel inhibitor verapamil were tested for activity against efflux pumps in the reference strains M. smegmatis mc2155 and M. avium ATCC25291T [44]. As shown in Figs. (1A) for M. smegmatis mc2155 and (1B) for M. avium ATCC25291, the tested compounds inhibited the efflux of ethidium bromide (a broad efflux pump substrate) during the period of the assay. The same study also showed that these efflux inhibitors had an effect on the MIC of the macrolides erythromycin and clarithromycin. It is important to note that the efflux inhibitors were used at the MIC in order to prevent an effect on the viability of the organisms, a condition that must be satisfied if the effect of the efflux inhibitor on the MIC of an antibiotic is to be exclusively that against the efflux system itself [44, 65]. Table 2 summarizes the effect of thioridazine, chlorpromazine and verapamil on the MICs of erythromycin and clarithromycin for M. smegmatis mc2155 and M. avium ATCC25291. The efflux inhibitors significantly increased the susceptibility of the M. avium ATCC25291 strain to erythromycin and in a lesser extent to clarithromycin. Relatively to M. smegmatis mc2155, verapamil showed the highest effect, whereas

Fig. (1). Effect of chlorpromazine (CPZ), thioridazine (TZ) and verapamil (VP) on the efflux of ethidium bromide in M. smegmatis mc2155 (A) and M. avium ATCC25291T (B) (adapted from reference #44).

thioridazine and chlorpromazine only produced a slight increase of the susceptibility to erythromycin. This work


Table 2.

Rodrigues et al.

Effect of Thioridazine, Chlorpromazine and Verapamil on the MICs (mg/L) of Several Antibiotics for M. Smegmatis mc2155 and M. Avium ATCC25291T (Adapted from Reference #44) MIC

MIC + TZ

MIC + CPZ

MIC + VP

M. smegmatis

M. avium

M. smegmatis

M. avium

M. smegmatis

M. avium

M. smegmatis

m. avium

200

16

100

0.5

100

0.5

50

4

12.5

4

12.5

2

12.5

2

12.5

2

Erythromycin Clarithromycin

TZ, thioridazine; CPZ, chlorpromazine; VP, verapamil. Data in bold type represent significant (at least 4-fold) reduction of resistance produced by the presence of the EPIs.

suggested the presence of an intrinsic efflux system for M. avium ATCC25291, responsible for the intrinsic resistance of this organism to erythromycin. Thioridazine, chlorpromazine and verapamil were shown to be effective inhibitors of this intrinsic efflux system. Another study, evaluated the effect of chlorpromazine and other efflux inhibitors was tested in M. smegmatis mc2155 cells that produced the Tap efflux pump from Mycobacterium fortuitum, which confers low-level resistance to tetracycline and aminoglycosides [66, 67]. In this study, chlorpromazine used at the MIC was the most potent efflux inhibitor for reducing tetracycline MICs Table 3 since it reduced the MIC of tetracycline 128 times, from 16 to 0.12 in M. smegmatis cells expressing the Tap efflux pump from M. fortuitum. Other efflux inhibitors, namely CCCP and reserpine, only decreased 16 times the MIC of tetracycline in these cells. In control cells not expressing such efflux pump, chlorpromazine was again the most potent inhibitor, since it decreased 16 times the MIC of tetracycline, while CCCP and reserpine reduced 4 times such resistance values. Tetracycline and chlorpromazine were determined to act synergistically in these conditions. In the same report, chlorpromazine was not able to produce an increase of tetracycline accumulation levels, in contrast with CCCP and reserpine that clearly increased five-fold the accumulation levels for this antibiotic. Table 3.

Effect of CCCP, Reserpine and Chlorpromazine (CPZ) on the MICs (mg/L) of Tetracycline for M. smegmatis mc2155 Cells Containing Plasmid pSUM36 (Empty Vector) or Plasmid pAC48 (pSUM36 Derivative Containing the Gene encoding Tap Efflux Pump from M. fortuitum) (Adapted from Reference #67). pSUM36 (vector)

pAC48 (Tap)

Efflux Inhibitor

MIC

Relative MIC

MIC

Relative MIC

-

2

1

16

1

CCCP

0.5

-4x

1

-16x

Reserpine

0.5

-4x

1

-16x

CPZ

0.12

-16x

0.12

-128x

THIORIDAZINE, CHLORPROMAZINE AND VERAPAMIL AFFECT EFFLUX AND MACROLIDE RESISTANCE IN CLINICAL ISOLATES OF M. AVIUM COMPLEX Following the promising results obtained above, a later study tested the same efflux inhibitors for activity against

efflux in reference and clinical M. avium complex strains, in particular the effect of these compounds in the reduction of resistance to macrolides [45]. The MICs for clarithromycin and erythromycin against a collection of M. avium and M. intracellular reference and clinical strains are summarized in Table 4. Among the clinical strains tested, four isolates presented clinically significant resistance to clarithromycin and a high MIC for erythromycin (see Table 4 for breakpoints according to CLSI guidelines). This high-level clarithromycin/erythromycin resistance was due to the presence of mutations at position 2058 (AG/C) in domain V of the 23S rRNA gene in three of these strains. The other clarithromycin resistant isolate, M. avium 386/08, presented a wild-type sequence in this region of the 23S rRNA gene, which suggests that this phenotype could result from mutation(s) located outside this region, or be due to other resistance mechanism(s), including efflux. In the same report, the MIC for clarithromycin and erythromycin was determined in the absence and presence of of the MICs of the EIs. As shown in Table 4, chlorpromazine, thioridazine and verapamil reduced the MIC of clarithromycin for most of the strains tested. However, in the particular case of the strains with high-level resistance to clarithromycin, the reduction of the MICs promoted by the efflux inhibitors did not reach a level of susceptibility, i.e. an MIC 16mg/L. The same EIs also decreased efflux of EtBr in both reference and clinical strains Fig. (2). Altogether, these results confirmed that at least one active efflux system is involved in the extrusion of macrolides and contributes to the resistance of M. avium complex to these drugs, which may be decreased in the presence of efflux inhibitors. CURRENT & FUTURE DEVELOPMENTS The increase of drug resistance in TB, especially those forms associated with higher mortality rates such as MDRTB and XDRTB, along with the scarcity of drugs for treating TB has promoted a worldwide effort in discovering and developing novel anti-TB agents. In this context, efflux pumps play an important role, not only for their contribution to resistance to drugs currently in use but more importantly for two facts: the activity of novel drugs against TB could be greatly affected by efflux pumps; and efflux pumps themselves can be drug targets for designing compounds that would inhibit their activity, thus, when used as adjuvants in anti-TB therapy, they would render the bacillus more susceptible to drugs transported by the pump. In fact, targeting membrane functions has become an attractive novel approach for persister pathogens such as M. tuberculosis [68].


Table 4.


7

Effect of Thioridazine, Chlorpromazine and Verapamil on the MICs of Clarithromycin and Erythromycin Against M. avium Complex Reference Strains and Clinical Isolates (Adapted from Reference # 45) MICs (mg/L) Strain

Clarithromycin

Erythromycin

No EI

TZ

CPZ

VP

No EI

TZ

CPZ

VP

M. avium ATCC25291T

4

2

2

2

16

0.5

0.5

4

M. avium 104

8

8

8

8

256

256

256

256

M. avium isolate 1

8

2

2

4

256

256

256

256

M. avium isolate 2

8

2

2

8

256

256

256

256

M. avium isolate 3

8

2

2

8

256

64

16

128

M. avium isolate 4

8

1

1

8

256

128

128

256

M. avium isolate 5

8

2

2

2

256

256

256

256

M. avium isolate 6

8

2

2

8

256

256

256

256

M. avium isolate 7

8

2

1

1

128

64

16

16

M. avium isolate 8

1024

1024

1024

512

512

256

512

128

M. avium isolate 9

512

512

128

512

1024

256

256

512

M. intracellulare ATCC13950T

0.25

0.25

0.25

0.25

0.06

0.06

0.06

0.06

M. intracellulare isolate 1

8

8

4

8

256

256

256

64


8

8

8

8

256

16

16

256


1

0.5

0.5

1

256

256

256

16


1

0.5

0.5

1

256

256

256

256


1

0.25

0.5

0.25

256

256

256

64


2

0.5

1

0.125

256

256

128

256


1024

1024

256

1024

2048

1094

512

256


2048

256

2048

1024

2048

256

1094

256

EI, efflux inhibitor; TZ, thioridazine; CPZ, chlorpromazine; VP, verapamil. EIs were used at the MIC. A 4-fold reduction was considered to denote significant synergistic effect between the antibiotic and the efflux inhibitor and is identified in bold. Breakpoints for clarithromycin MIC determination by microdilution in 7H9/pH 6.8: S 16mg/L; I = 32mg/L; R 64mg/L [71].

Efflux inhibitors like the phenothiazines represent interesting alternatives in the search for effective compounds for treatment of MDR/XDRTB. An interesting example is thioridazine, a neuroleptic used for the therapy of psychosis for decades, has passed beyond patent protection and is of little interest to the pharmaceutical industry for future development [69]. The fact that it inhibits in vitro growth of M. tuberculosis, including MDR/XDRTB isolates, has a low price, good availability and a generally good tolerability in humans, makes thioridazine a compound worthy of consideration in the area of research and development of new anti-TB therapeutic strategies. In fact, thioridazine has been repeatedly recommended for the management of the MDRTB infected patient due to its ability to enhance the killing of intracellular MDRTB and cure the mouse of TB infection [55]. Therapy of XDRTB with thioridazine has been used for the therapy of patients who did not respond to any antibiotic therapy and whose prognosis was poor and hence were selected for thioridazine therapy on the basis of

compassionate reasons [69, 70]. All these evidences established the basis for an international call to start clinical trials with thioridazine [71]. Thioridazine is ready for clinical trials because it has been widely used in clinical practice, albeit for other purposes, and may prolong the life of or cure thousands of patients that are infected with a terminal XDRTB infection. Nevertheless, this is a unique circumstance and further investigation is required to develop other efflux inhibitors, either more effective thioridazine derivatives or other compounds, in order to prevent the continuous increase of multidrug resistant mycobacteria with efflux-mediated resistance. ACKNOWLEDGEMENTS This work was supported by grants EU-FSE/FEDERPTDC/BIA-MIC/71280/2006, EU-FSE/FEDER-PTDC/BIAMIC/105509/2008 and EU-FSE/FEDER-PTDC/SAU-


Rodrigues et al.

Fig. (2). Efflux of ethidium bromide by clinical strains of M. avium (A) and M. intracellulare (B). Relative fluorescence was obtained by normalization of data against the conditions of no efflux (presence of efflux inhibitor and no glucose) (adapted from reference #45).

FCF/102807/2008 provided by Fundação para a Ciência e a Tecnologia (FCT) of Portugal. L. Rodrigues was supported by grant SFRH/BD/24931/2005 (FCT, Portugal). CONFLICT OF INTERESTS

[8] [9]

None to declare. [10]

REFERENCES [1] [2] [3] [4] [5]

[6] [7]

World Health Organization (WHO). Global tuberculosis control report. WHO: Geneva 2010. Griffith DE. Therapy of nontuberculous mycobacterial disease. Curr Opin Infect Dis 2007; 20: 198-203. Somoskovi A, Parsons LM, Salfinger M. The molecular basis of resistance to isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir Res 2001; 2: 164-8. Wayne LG. Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 1994; 13: 908-14. Elliott AM, Berning SE, Iseman MD, Peloquin CA. Failure of drug penetration and acquisition of drug resistance in chronic tuberculous empyema. Tuber Lung Dis 1995; 76: 463-7. Nikaido H. Multidrug resistance in bacteria. Annu Rev Biochem 2009; 78: 119-46. Davin-Regli A, Bolla JM, James CE, Lavigne JP, Chevalier J, Garnotel E, et al. Membrane permeability and regulation of drug

[11]

[12]

[13]

[14] [15]

"influx and efflux" in enterobacterial pathogens. Curr Drug Targets 2008; 9: 750-9. Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Pagès JM, Amaral L. Antibiotic stress, genetic response and altered permeability of E. coli. PLoS ONE 2007; 2: e365. Pagès JM, Amaral L. Mechanisms of drug efflux and strategies to combat them: challenging the efflux pump of Gram-negative bacteria. Biochim Biophys Acta 2009; 1794: 826-33. Piddock LJ. Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin Microbiol Rev 2006; 19: 382-402. Quinn T, O'Mahony R, Baird AW, Drudy D, Whyte P, Fanning S. Multi-drug resistance in Salmonella enterica: efflux mechanisms and their relationships with the development of chromosomal resistance gene clusters. Curr Drug Targets 2006; 7: 849-60. Baucheron S, Tyler S, Boyd D, Mulvey MR, Chaslus-Dancla E, Cloeckaert A. AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrob Agents Chemother 2004; 48: 3729-35. Hancock RE, Speert DP. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat. 2000; 3: 247-55. Brennan PJ, Nikaido H. The envelope of mycobacteria. Annu Rev Biochem 1995; 64: 29-63. Draper P. The outer parts of the mycobacterial envelope as permeability barriers. Front Biosci 1998; 3: 1253-61.

Phenothiazines and Other Efflux Inhibitors in Mycobacteria [16]

[17] [18] [19] [20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34] [35]

[36]

[37]

Lambert PA. Cellular impermeability and uptake of biocides and antibiotics in gram-positive bacteria and mycobacteria. Symp Ser Soc Appl Microbiol 2002; 31: 46S–54S. Niederweis M. Nutrient acquisition by mycobacteria. Microbiology 2008; 154: 679-92. Niederweis M, Danilchanka O, Huff J, Hoffmann C, Engelhardt H. Mycobacterial outer membranes: In search of proteins. Trends Microbiol 2010; 18: 109-16. Trias J, Jarlier V, Benz R. Porins in the cell wall of mycobacteria. Science 1992; 258: 1479-81. De Rossi E, Aínsa JA, Riccardi G. Role of mycobacterial efflux transporters in drug resistance: an unresolved question. FEMS Microbiol Rev 2006; 30: 36-52. Louw GE, Warren RM, van Pittius NC, McEvoy CR, Van Helden PD, Victor TC. A balancing act: efflux/influx in mycobacterial drug resistance. Antimicrob Agents Chemother 2009; 53: 3181-9. Viveiros M, Leandro C, Amaral L. Mycobacterial efflux pumps and chemotherapeutic implications. Int J Antimicrob Agents 2003; 22: 274-8. Ramon-Garcia S, Martín C, Thompson CJ, Aínsa JA. Role of the Mycobacterium tuberculosis P55 efflux pump in intrinsic drug resistance, oxidative stress responses, and growth. Antimicrob Agents Chemother 2009; 53: 3675-82. Takiff HE, Cimino M, Musso MC, Weisbrod T, Martinez R, Delgado MB, et al. Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis. Proc Natl Acad Sci USA 1996; 93: 362-6. Sander P, De Rossi E, Böddinghaus B, Cantoni R, Branzoni M, Böttger EC, et al. Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance. FEMS Microbiol. Lett 2000; 193: 19-23. Alland D, Steyn AJ, Weisbrod T, Aldrich K, Jacobs WR Jr. Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition. J Bacteriol 2000; 182: 1802-11. Camacho LR, Constant P, Raynaud C, Laneelle MA, Triccas JA, Gicquel B, et al. Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J Biol Chem 2001; 276: 19845-54. Gupta AK, Reddy VP, Lavania M, Chauhan DS, Venkatesan K, Sharma VD, Tyagi AK, Katoch VM. jefA (Rv2459), a drug efflux gene in Mycobacterium tuberculosis confers resistance to isoniazid & ethambutol. Indian J Med Res. 2010; 132: 176-88. Daigle DM, McKay GA, Wright GD. Inhibition of aminoglycoside antibiotic resistance enzymes by protein kinase inhibitors. J Biol Chem 1997; 272: 24755-8. Martins M, Dastidar SG, Fanning S, Kristiansen JE, Molnar J, Pagès JM, et al. Potential role of non-antibiotics (helper compounds) in the treatment of multidrug-resistant Gram-negative infections: Mechanisms for their direct and indirect activities. Int J Antimicrob Agents 2008; 31: 198-208. Lomovskaya O, Watkins W. Inhibition of efflux pumps as a novel approach to combat drug resistance in bacteria. J Mol Microbiol Biotechnol 2001; 3: 225-36. Lomovskaya O, Zgurskaya HI, Totrov M, Watkins WJ. Waltzing transporters and 'the dance macabre' between humans and bacteria. Nat Rev Drug Discov 2007; 6: 56-65. Watkins WJ, Chong L, Cho A, Hilgenkamp R, Ludwikow M, Garizi N, et al. Quinazolinone fungal efflux pump inhibitors. Part 3: (N-methyl)piperazine variants and pharmacokinetic optimization. Bioorg Med Chem Lett 2007; 17: 2802-6. Nelson, M.L., Alekshun, M.N. Substituted polyamines as inhibitors of bacterial efflux pumps. WO2004062674 (2004). Glinka, T., Rodny, O., Bostian, K., Wallace, D.M. Quaternary alkyl ammonium bacterial efflux pump inhibitors and therapeutic uses thereof. WO2008141012 (2008). Koul, S., Thota, N., Mallepally, V.R., Sangwan, P.L., Taneja, S.C., Khan, I.A., Kumar, A., Raja, A.F.,Saxena, A.K., Agrawal, S.K., Johri, R.K., Abdullah, S.T., Singh, G., Bachu, L.N.R., Dhar, A.K., Purnima, B., Qazi, G.N. Novel efflux pump inhibitors. WO2009110002 (2009). Glinka, T., Rodny, O., Bostian, K.A., Wallace, D.M., Higuchi, R., Chow, C., Mak, C.C., Hirst, G., Eastman, B. Polybasic bacterial efflux pump inhibitors and therapeutic uses thereof. WO2010054102 (2010).

Recent Patents on Anti-Infective Drug Discovery, 2011, Vol. 6, No. 2 [38] [39] [40] [41]

[42]

[43]

[44]

[45]

[46]

[47]

[48] [49] [50]

[51]

[52] [53]

[54]

[55] [56]

[57]

[58] [59]

[60]

9

Krulwich TA, Quirk PG, Guffanti AA. Uncoupler-resistant mutants of bacteria. Microbiol Rev 1990; 54: 52-65. Marquez B. Bacterial efflux systems and efflux pumps inhibitors. Biochimie 2005; 87: 1137-47. Stavri M, Piddock LJ, Gibbons S. Bacterial efflux pump inhibitors from natural sources. J Antimicrob Chemother 2007; 59: 1247-60. Markham PN, Neyfakh A. Inhibition of the multidrug transporter NorA prevents emergence of norfloxacin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 1996; 40: 2673-4. Viveiros M, Portugal I, Bettencourt R, Victor TC, Jordaan AM, Leandro C, Ordway D, Amaral L. Isoniazid-induced transient highlevel resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2002; 46: 2804-10. Pasca MR, Guglierame P, Arcesi F, Bellinzoni M, De Rossi E, Riccardi G. Rv2686c-Rv2687c-Rv2688c, an ABC fluoroquinolone efflux pump in Mycobacterium tuberculosis. Antimicrob Agents Chemother 2004; 48: 3175-8. Rodrigues L, Wagner D, Viveiros M, Sampaio D, Couto I, Vavra M, et al. Thioridazine and chlorpromazine inhibition of ethidium bromide efflux in Mycobacterium avium and Mycobacterium smegmatis. J Antimicrob Chemother 2008; 61: 1076-82. Rodrigues L, Sampaio D, Couto I, Machado D, Kern WV, Amaral L, et al. The role of efflux pumps in macrolide resistance in Mycobacterium avium complex. Int J Antimicrob Agents 2009; 34: 529-33. Lechner D, Gibbons S, Bucar F. Plant phenolic compounds as ethidium bromide efflux inhibitors in Mycobacterium smegmatis. J Antimicrob Chemother 2008; 62: 345-8. Sharma S, Kumar M, Sharma S, Nargotra A, Koul S, Khan IA. Piperine as an inhibitor of Rv1258c, a putative multidrug efflux pump of Mycobacterium tuberculosis. J Antimicrob Chemother 2010; 65: 1694-701. Jin J, Zhang JY, Guo N, Sheng H, Li L, Liang JC, et al. Farnesol, a potential efflux pump inhibitor in Mycobacterium smegmatis. Molecules 2010; 15: 7750-62. Ehrlich P. The Collected Papers of Paul Ehrlich. Himmelweit F, Marquardi M, Dale H (Eds). Pergammon Press: London 1956. Viveiros M, Amaral L. Enhancement of antibiotic activity against poly-drug resistant Mycobacterium tuberculosis by phenothiazines. Int J Antimicrob Agents 2001; 17: 225-8. Amaral L, Kristiansen JE, Abebe LS, Millett W. Inhibition of the respiration of multi-drug resistant clinical isolates of Mycobacterium tuberculosis by thioridazine: potential use for initial therapy of freshly diagnosed tuberculosis. J Antimicrob Chemother 1996; 38: 1049-53. Amaral L, Viveiros M, Molnar J. Antimicrobial activity of phenothiazines. In Vivo 2004; 18: 725-32. Crowle AJ, Douvas GS, May MH. Chlorpromazine: A drug potentially useful for treating mycobacterial infections. Chemotherapy 1992; 38: 410-19. Ordway D, Viveiros M, Leandro C, Amaral L. Clinical concentrations of thioridazine kill intracellular multi-drug resistant Mycobacterium tuberculosis. Antimicrob. Agents Chemother 2003; 47: 917-22 Martins M, Viveiros M, Kristiansen JE, Molnar J, Amaral L. The curative activity of thioridazine on mice infected with Mycobacterium tuberculosis. In Vivo 2007; 21: 771-5. Amaral L, Martins M, Viveiros M. Phenothiazines as anti-multidrug resistant tubercular agents. Infect. Disord. Drug Targets 2007; 7: 257-65. Dutta NK, Mehra S, Kaushal D. A Mycobacterium tuberculosis sigma factor network responds to cell-envelope damage by the promising anti-mycobacterial thioridazine. PLoS One 2010; 5(4): e10069. doi:10.1371/journal.pone.0010069. Weiss B, Prozialeck W, Cimino M, Barnette MS, Wallace TL. Pharmacological regulation of calmodulin. Ann NY Acad Sci 1980; 356: 319-45. Garcia JJ, Tuena de Gomez-Puyou M, Gomez-Puyou A. Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO4. J Bioenerg Biomembr 1995; 27: 127-36. Kristiansen MM, Leandro C, Ordway D, Martins M, Viveiros M, Pacheco T, et al. Phenothiazines alter resistance of methicillin resistant strains of Staphylococcus aureus (MRSA) to oxacillin in vitro. Int J Antimicrob Agents 2003; 22: 250-3.

10 Recent Patents on Anti-Infective Drug Discovery, 2011, Vol. 6, No. 2 [61]

[62]

[63] [64]

[65]

[66]

Bhatnagar K, Singh VP. Ca2+-dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris. Curr Microbiol 2003; 46: 265-9. Wittekindt OH, Schmitz A, Lehmann-Horn F, Hansel W, Grissmer S. The human Ca2+-activated K+ channel, IK, can be blocked by the tricyclic antihistamine promethazine. Neuropharmacology 2006; 50: 458-67. Ahluwalia J, Tinker A, Clapp LH, Duchen MR, Abramov AY, Pope S, et al. The large-conductance Ca2+-activated K+ channel is essential for innate immunity. Nature 2004; 427: 853-8. Boshoff, H. I., T. G. Myers, B. R. Copp, M. R. McNeil, M. A. Wilson, C. E. Barry III. The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 2004: 279: 40174–84. Viveiros M, Martins A, Paixão L, Rodrigues L, Martins M, Couto I, et al. Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. Int J Antimicrob Agents 2008; 31: 458-62. Aínsa JA, Blokpoel MC, Otal I, Young DB, De Smet KA, Martín C. Molecular cloning and characterization of Tap, a putative

Rodrigues et al.

[67]

[68]

[69]

[70]

[71]

multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis. J Bacteriol 1998; 180: 5836-43. Ramon-Garcia S, Martín C, Aínsa JA, De Rossi E. Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum. J Antimicrob Chemother 2006; 57: 2529. Hurdle JG, O'Neill AJ, Chopra I, Lee RE. Targeting bacterial membrane function: an underxploited mechanism for treating persistent infections. Nat Rev Microbiol 2011; 9: 62-75. Abbate E, Vescoso M, Natiello M, Cufre M, Garcia A, Ambroggi M, et al. Tuberculosis extensamente resistente (XDR-TB) en Argentina: aspectos destacbles epidemiologicos, bacteriologicos, terapeuticos y evolutivos. Revista Argentina de Medicina Respiratoria 2007; 1: 19-25. Amaral L, Boeree MJ, Gillespie SH, Udwadiad ZF, van Soolingen D. Thioridazine cures extensively drug-resistant tuberculosis (XDR-TB) and the need for global trials is now! Int J Antimicrob Agents 2010; 35: 524-6. Clinical and Laboratory Standards Institute (CLSI). Susceptibility testing of mycobacteria, nocardia and other aerobic actinomycetes; Approved standard; M24-A. Vol 23, Nº18. CLSI: Wayne, Pa, 2003.