DOI: 10.1002/cmdc.201100404

Quaternary Ammonium Salts and Their AntimicrobialPotential: Targets or Nonspecific Interactions?Maximilian Tischer,[a] Gabriele Pradel,[b] Knut Ohlsen,[b] and Ulrike Holzgrabe*[a]

Dedicated to Prof. Dr. Dr. h.c. G. Bringmann on the occasion of his 60th birthday.

22 � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2012, 7, 22 – 31

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Introduction

Since the discovery in 1876 by Robert Koch that microorgan-isms cause infectious diseases, physicians, microbiologists, andmedicinal chemists have been searching for drugs to preventinfections. As early as the 1930s quaternary ammonium com-pounds (QACs) were found to have antimicrobial activities ;patents were immediately filed for this purpose.[1] QACs arethe most useful antiseptics and disinfectants, and have beenused for a variety of clinical purposes such as preoperative dis-infection of unbroken skin, application to mucous membranes,and disinfection of noncritical surfaces.[2]

A systematic variation of bisisoquinolinium and bisquinolini-um salts led to the first-in-class bisquaternary ammonium com-pound (BQAC) dequalinium chloride, i.e. , 1,1’-(1,10-decanediyl)-bis(4-amino-2-methyl)quinolinium dichloride (CAS 6707-58-0,Figure 1).[3] For the past 55 years, this biscation has beenwidely used as an antiseptic and disinfectant. It has also beeninvestigated for manifold activities (Table 1), although its mech-anism of action is not yet clear.

With this review we demonstrate the anti-infective power ofQACs and BQACs toward bacteria and protozoa, especiallyagainst plasmodia. The structure–activity relationships (SAR) ofthe various series of QACs and BQACs toward the different mi-croorganisms as well as their modes of action are also dis-cussed.

Dequalinium

Today dequalinium is licensed only as a topical medication.This includes pharyngeal sprays, throat lozenges, mouthwashes and decongestant sprays, topical creams, gels, andointments, as well as tablets or suppositories for vaginal appli-cation. Beside its antibacterial activity, dequalinium has beenreported to exhibit an array of other biological effects such aspotent antitumor activity through toxicity against mitochon-dria,[8–12] inhibition of protein kinase C,[13, 14] antimalarial,[15–17]

antitrypanosomal,[3, 6] and antifungal[7] activities, inhibition ofmycothiol ligase in Mycobacterium tuberculosis,[5] selectiveblockade of small-conductance Ca2+-activated K+ chan-

nels,[18–24] and disintegration of amyloid fibrils.[25] In addition tothese effects, dequalinium’s toxicity has also been reported; itmay cause skin necrosis if administered on intertriginous skinareas under occlusive conditions.[26, 27] Despite this, none ofthese effects are relevant for its current medical indications.

Possible mode of action of dequalinium chloride and relatedcompounds

In contrast to some other QACs, the mode of action of dequali-nium seems to be more specific. For example, the low dose of10 mg per vaginal tablet, 0.25 mg per lozenge, or 10 mg per100 mL gurgling solution points to a specific mode of action.Moreover, SARs derived by Babbs et al.[3] indicate that dequali-nium has one or more specific targets; otherwise, distinct dif-ferences in activity between closely related compounds wouldbe difficult to explain. Figure 2 clearly shows the influence of

[a] M. Tischer, Prof. Dr. U. HolzgrabeInstitute of Pharmacy and Food ChemistryUniversity of W�rzburg, Am Hubland, 97074 W�rzburg (Germany)E-mail : u.holzgrabe@pharmazie.uni-wuerzburg.de

[b] Dr. G. Pradel, Dr. K. OhlsenResearch Center for Infectious Diseases, University of W�rzburgJoseph-Schneider-Str. 2, Building D15, 97080 W�rzburg (Germany)

For more than 50 years dequalinium chloride has been usedsuccessfully as an antiseptic drug and disinfectant, particularlyfor clinical purposes. Given the success of dequalinium chlo-ride, several series of mono- and bisquaternary ammoniumcompounds have been designed and reported to have im-

proved antimicrobial activity. Furthermore, many of them ex-hibit high activity against mycobacteria and protozoa, especial-ly against plasmodia. This review discusses the structure–activi-ty relationships and the modes of action of the various seriesof (bis)quaternary ammonium compounds.

Figure 1. Structure of dequalinium chloride.

Table 1. Activities of dequalinium toward various microorganisms.

Bacteria MIC [mg mL�1]

Staphylococcus aureus 1.28[4]

Streptococcus agalactiae 3[4]

Streptococcus pyogenes 1[4]

Enterococcus faecalis 24[4]

Listeria monocytogenes 8[4]

Escherichia coli 64[4]

Gardnerella vaginalis 128[4]

Proteus mirabilis >1024[4]

Proteus vulgaris 63.0[3]

Mycobacterium phlei 1.66[3]

Mycobacterium tuberculosis 1.2 (aerobic)0.3 (anaerobic)[5]

Parasites Activity

Trypanosoma brucei ED50 = 0.043 mm[6]

Trichomonas vaginalis MIC = 57.6 mg mL�1[4]

Leishmania major IC50 = 0.8 mm[a]

Plasmodium falciparum IC50 = 0.055 mm[b]

Fungi IC50 [mm]

Candida albicans 5.5[7]

Cryptococcus neoformans 11[7]

[a] T. �lschl�ger, unpublished results. [b] L. Sologub, G. Pradel, unpublish-ed results.

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varying the length of the central linker on minimal inhibitoryconcentration (MIC) values against Staphylococcus aureus. Re-placing the quinoline moiety with an isoquinoline group in-creases the activity threefold (10 methylene groups). With fur-ther modification of the structure toward dequalinium, the ac-tivity increases another 50-fold. Substances without a specifictarget would not show such subtle differences in activity dueto slight alterations in the chemical structure. Similar findingswere reported by Babbs et al. for other series of correspondingcompounds (see Figure 3 and Figure 4).[3]

Maximilian Tischer completed his gradu-

ate studies in pharmacy at the University

of W�rzburg in 2006 and received his

pharmaceutical license in 2007. He is

now working toward his PhD in medici-

nal chemistry at the University of W�rz-

burg under the supervision of Professor

Holzgrabe. His fields of research are or-

ganic synthesis, SAR studies, and struc-

tural development of anti-infective qua-

ternary ammonium compounds.

Gabriele Pradel studied biology at the

University of Gießen and received her

PhD from the University of Frankfurt am

Main in 1999. She then worked as a

postdoctoral fellow and instructor at the

New York University Medical Center and

the Weill Cornell Medical Center of New

York, respectively. In 2005 she was

awarded by the Emmy Noether Program

of the Deutsche Forschungsgemein-

schaft, and since then has headed a re-

search group at the W�rzburg Research Center for Infectious Dis-

eases. Her research focus lies in the gametocyte stages of malaria

parasites as targets for transmission-blocking strategies.

Knut Ohlsen studied biology at the Uni-

versity of Leipzig. In 1998 he received

his PhD in microbiology from the Uni-

versity of W�rzburg. After a postdoctoral

fellowship in the research group of Jçrg

Hacker at the University of W�rzburg,

he became a group leader at the Univer-

sity of W�rzburg’s Institute for Molecular

Infection Biology. His research areas are

host–pathogen interactions of Staphylo-

coccus aureus, antibacterial drug devel-

opment, and in vivo imaging tech-

niques.

Ulrike Holzgrabe studied chemistry and

pharmacy at the Universities of Marburg

and Kiel, respectively, receiving her PhD

under the supervision of Prof. Dr. R.

Haller. In 1989 she finished her habilita-

tion, and in 1990 she accepted an asso-

ciate professorship at the University of

Bonn, where she served as a vice rector

from 1997 to 1999. Since 1999 she has

held a full professorship at the Universi-

ty of W�rzburg. She was president of

the German Pharmaceutical Society from 2003 to 2007. Her main

research interests include drug discovery in the fields of muscarinic

ligands and anti-infectives, as well as quality analysis of active

pharmaceutical ingredients.

Figure 2. Dependence of anti-S. aureus activity on the length of the centrallinker of dequalinium derivatives. The negative logarithm of the MIC values(pMIC) is plotted as a function of the number of methylene groups (n) ;hence, an increase in pMIC value reflects an increase in anti-infective poten-cy.

Figure 3. Dependence of anti-S. aureus activity on the length of the centrallinker of isoquinolinium derivatives.

Figure 4. Dependence of anti-S. aureus activity on the length of the centrallinker of quinolinium derivatives.

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MED U. Holzgrabe et al.

In 1961, Caldwell et al. investigated the antibacterial activityof a series of dequalinium derivatives differing in the alkyllinker chain length. It was demonstrated that the mode ofaction of dequalinium may be closely related to its interactionwith proteins such as pepsin, casein, gelatin, and egg albu-min.[28] Investigations into the site of action revealed a dequali-nium’s high affinity for membranes, no dissolution of the cellwall in S. aureus, and almost no lytic effect on B. megateriumprotoplasts. Additionally, the substance is taken up by thecells, and the morphological abnormalities are mainly confinedto intracellular changes.[29] A few years later, Hugo and Frierfound that dequalinium is taken up by the cells and that thecell wall is not the primary site of absorption. Although Gram-negative E. coli were observed to be much less susceptible todequalinium than S. aureus, the uptake under inhibitory condi-tions was similar, indicating a difference in affinity rather thanin mode of action. The outer cell membrane of Gram-negativeorganisms seems to constitute a permeability barrier to dequa-linium. Additional experiments with the aerobic and anaerobicmetabolism of glucose indicated no specific attack on the cy-tochrome system. Taken together, it is rather likely that dequa-linium exhibits its bactericidal action by access to the cyto-plasm followed by precipitation of cytoplasmic material. Nucle-ic acids are particularly susceptible to precipitation by thiscompound.[30] Using fluorescence microscopy, Weiss et al. dem-onstrated that dequalinium accumulates in the mitochondriaof several human and murine tumor cell lines, resulting inpotent anticarcinoma activity.[9] Furthermore, it binds DNA andinhibits calmodulin as well as ATP production in isolated ratliver mitochondria. Through delocalization of the positivecharge over the quinaldine moiety, it is a lipophilic biscationthat can readily penetrate the lipid bilayers of mitochondriaand bacteria, for example.[9–12, 31, 32] This observation lends sup-port to the endosymbiotic theory, which proposes free-livingbacteria as the evolutionary origin of mitochondria.

Targets of QACs in Bacteria: More Than Non-specific Interactions with Membranes

Many surface-active compounds are bactericidal, and some ex-planations have been proposed for this action. In 1951, Saltoninvestigated the adsorption of cetyltrimethylammonium bro-mide (CTAB), a monoquaternary ammonium compound(MQAC), in S. aureus and some other bacteria.[33] It was ob-served in electron microscopy studies that the cytoplasmicmembrane contracts away from the cell wall, indicating cyto-lytic damage and cell leakage.[34] CTAB-treated bacteria re-leased their cell contents into their suspending media. Thissupports the hypothesis, previously raised by Baker et al. , ofcell membrane disorganization by detergent-like activity as themain mode of action of QACs.[35] More than 50 years later,Denyer also found that the main effects of QACs in bacteriaare structural and functional changes in the cell wall, release ofwall components and initiation of autolysis, inhibition of mem-brane ATPase, and electrostatic interactions with negativelycharged polar head groups of phospholipids.[36, 37]

In another study by Locher et al. ,[38] the antimicrobial prop-erties of nostocarbolines (see Figure 7 below) were investigat-ed; these compounds were observed to exhibit activities onlyas dimers. There was a strong correlation between the lengthof the linker chain and antimicrobial activity. The most activecompounds had linkers of 10 and 12 methylene units andwere bactericidal against methicillin-resistant S. aureus (MRSA),vancomycin-resistant enterococci (VRE), and Escherichia coli.Killing activity correlated with membrane damage, as indicatedby ATP leakage. Moreover, a SOS response following DNAdamage was observed in E. coli. Clearly, these substances inter-act with bacterial membranes, given their chemical structureswith positive charges and hydrophobic regions. However, therole of intracellular targets remains vague. The observed effecton the SOS response may suggest some specific interactionwith DNA; however, this has never been proven. It is possiblethat the most potent agents exerted membrane damage, lead-ing to disruption of the cell envelope, disturbing protonmotive force, and arresting intracellular activity by binding tar-gets in the cytoplasm.[38]

Interestingly, Gram-positive bacteria are generally more sen-sitive than Gram-negative bacteria. This has been attributed tothe outer membrane of Gram-negative bacteria, which isabsent in Gram-positive strains (cf. dequalinium findings). As aconsequence, QACs must first pass the outer membrane bylysis of this layer, for example, and then interact with the innermembrane and later with intracellular components. The Gram-negative bacterium Pseudomonas aeruginosa and bacteria thatare embedded in biofilm matrices are less susceptible to QACsand other antiseptics.[39, 40] These observations indicate a crucialrole played by particular surface structures of various bacterialspecies in sensitivity to QACs. Likewise, mycobacteria, whichhave a unique cell wall consisting of a hydrophobic mycolatelayer and a peptidoglycan layer linked by the polysaccharidearabinogalactan, show high resistance to QACs.[41] Importantly,many antibiotic-resistant staphylococci (such as MRSA or me-thicillin-resistant S. epidermidis) have acquired plasmids such asqacA, qacB, qacC or qacD, which encode efflux pumps andthus confer resistance determinants to QACs.[42–44] This sug-gests that intracellular accumulation of QACs is important forfull activity and underscores the importance of the interactionbetween QACs and intracellular targets for antibacterial activi-ty.

Several bisquaternary bisnaphthalimides have been evaluat-ed against a variety of human pathogens, leading to the dis-covery of compounds that show inhibitory effects againstGram-positive bacteria, including multi-resistance staphylococ-ci. Originally, bistertiary bisnaphthalimides were described asputative anticancer drugs, but clinical trials were unsuccess-ful.[45, 46] In a reevaluation of bisquaternary bisnaphthalimidesagainst bacteria, a highly active substance was identified,which has been designated as MT02 (Figure 5). This compoundexerts high antimicrobial activity, with a MIC value of0.31 mg mL�1 against community-acquired MRSA lineageUSA300. It is also active against other Gram-positive bacteriasuch as Streptococcus pneumonia, Listeria monocytogenes, andBacillus subtilis. Gram-negative bacteria such as Citrobacter

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Quaternary Ammonium Salts

koseri, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis,Pseudomonas aeruginosa, Salmonella typhimurium, Serratiamarcescens, and Shigella dysenteriae were not susceptible, pos-sibly owing to their outer membrane, which acts as a barrierthat prevents access of the compound into the cytoplasm.[47]

Different lengths of the methylene linker showed no correla-tion with antimicrobial activity (Figure 6). However, derivativesof MT02 without nitro groups and with shorter or longer con-necting chains also exerted activity against S. aureus, but onlyat higher concentrations.

Importantly, studies into the mode of action based on DNAmicroarray global expression analysis and radiolabeling experi-ments generated evidence that MT02 is a DNA binding com-pound that leads to the inhibition of DNA replication. The tran-scriptional signature was characterized by a strong expressionof genes involved in DNA metabolism, DNA replication, SOS re-sponse, and transport of positively charged compounds.[47] Thedirect binding of MT02 to DNA was demonstrated by surfaceplasmon resonance and gel retardation experiments. Impor-tantly, the substance did not show cytotoxic activity in cell cul-ture systems using four different eukaryotic cell lines. Further-more, expression analysis suggested that MT02 indirectly im-pacts cell wall metabolism and cell propagation. In contrast tobistertiary bisnaphthalimides, which exhibit antitumor activity(see above),[46, 48] the two permanent positive charges of MT02prevent penetration of the compound into eukaryotic cells andmay therefore be the cause of its low cytotoxicity despite itsbinding to eukaryotic DNA. Binding of MT02 to double-strand-ed DNA is reversible, and is probably not restricted to a specif-

ic base sequence as reported for the bisnaphthalimide elina-fide.[49] The direct binding of MT02 to DNA as a mode of actionrepresents an example in which the molecular target of aBQAC has been evaluated in detail.

So far, no specific target has been identified for most QACs;it is assumed that the effect is rather generalized than specificto one target. However, as already pointed out, there shouldbe some target specificities, as shown for the bisquaternarybisnaphthalimide MT02, because the activity of QACs towarddifferent bacterial species varies substantially and cannot beexplained simply by the structure of cationic charge and hy-drophobic portions.

Interestingly, similar to QACs, membrane damage has longbeen assumed as the major mechanism of action of cationicantimicrobial peptides.[50] These peptides share chemical fea-tures with QACs, such as a cationic portion and a hydrophobicregion. It became clear more recently that other target sites,including interaction with lipid II, an important precursor ofcell wall synthesis, are major targets for some cationic antibac-terial peptides.[51] Whether QACs also interact with lipid II hasnot yet been investigated, and remains an attractive hypothe-sis for further evaluation.

With all these findings taken into account, the generalmechanism of QAC action can be summarized as follows:

* adsorption and penetration through the cell wall (the cellwall has no penetration barrier) ;

* binding to components of the cell membrane, probablywith both lipids and proteins;

* disorganization of cell membranes and, at higher concen-trations, causing leakage with subsequent loss of low-mo-lecular-weight material ;

* intracellular degradation of proteins and nucleic acids;* lysis of cell wall components by release of autolytic en-

zymes;* complete loss of structural organization of the cell.

QACs in the Treatment of Malaria

Malaria is the most prevalent tropical disease in theworld, with 225 million new cases reported eachyear and an annual death toll of 0.8 millionpeople.[52] Many of these fatal cases are instances ofdrug-resistant malaria tropica, caused by the proto-zoan parasite Plasmodium falciparum.

In the search for new drugs for chemotherapeuticintervention, dequalinium was tested for its effect onmalaria parasites. In vitro parasite growth testsshowed that dequalinium is active against the P. fal-ciparum blood stages, with an IC50 value of 55 nm

(see Table 1; references [16] , [17] , and [60]; L. Solo-gub and G. Pradel, unpublished observations). Themode of action included compound accumulation inmitochondria combined with an inhibition of calmo-dulin and of the membrane-bound ATPase of theparasitophorous vacuole.[17, 53] The in vitro results

Figure 5. Structure of MT02.

Figure 6. Chain length–activity correlation of bisquaternary naphthalimides againstS. aureus.

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MED U. Holzgrabe et al.

were confirmed in vivo by using Plasmodium berghei-infectedmice. Dequalinium was able to cure 40 % of the infected ani-mals, monitored 30 days post infection.[16]

While dequalinium was tested in malaria parasites due to itsoverall antimicrobial effect, other QACs were investigated fortheir potential as antimalarials by a target-oriented approachthat is linked to the de novo membrane biosynthesis of blood-stage parasites. The rapid, manifold, and recurrent replicationof malaria parasites in red blood cells (RBCs) requires highturnover rates in membrane components, making parasitelipid metabolism an attractive weak spot for targeting drugs.[54]

Phospholipid synthesis in malaria parasites

After infection by Plasmodium, the total amount of phospholi-pids increases sixfold in parasitized human RBCs.[55] The mem-brane composition of malaria parasites differs significantlyfrom that of uninfected erythrocytes, and is primarily com-posed of phospholipids such as phosphatidylcholine (PtdCho,40–50 %; see Figure 7) and phosphatidylethanolamine (PtdEtn,35–45 %).

Malaria parasites synthesize phospholipids from polar headgroups such as choline (Figure 7), ethanolamine, or serine.[54, 55]

PtdEtn is synthesized by the parasite via a pathway followingphosphorylation of ethanolamine. Ethanolamine is obtained inlimited amounts from plasma and in larger quantities throughdecarboxylation of serine, which, in turn, is either imported orderived from hemoglobin degradation.[56] PtdCho is synthe-

sized from choline by two routes in Plasmodium. During thefirst route, which is highly conserved in eukaryotes, PtdCho isderived from choline via an enzymatic cascade of the de novocytidine diphosphate (CDP)–choline pathway. This route in-volves three enzymes: choline kinase, CTP phosphocholine cy-tidylyltransferase, and choline/ethanolamine phosphotransfer-ase (PfCEPT). The second route of PtdCho synthesis was onlyrecently identified in P. falciparum and is typical for plants andnematodes, but is absent in mammals.[57] Through this route,which is termed the serine decarboxylase–PtdEtn methyltrans-ferase (SDPM) pathway, phosphoethanolamine is convertedinto phosphocholine, a rate-limiting step mediated by theenzyme phophoethanolamine methyltranserase PfPMT.[57] Bothroutes of PtdCho synthesis represent excellent targets fornovel chemotherapeutics. Only the CDP–choline pathway,however, was hitherto reported to be sensitive to QACs(Figure 8).

The effect of QACs on the plasmodial choline carrier

The CDP–choline pathway requires the transport of host chol-ine from plasma into the infected erythrocyte, a process thatinvolves the remnant erythrocytic choline carrier as well as anunknown transporter of the parasite-induced new permeationpathway (NPP).[58] Subsequently choline crosses the barriers ofthe parasitophorous vacuole membrane and the parasiteplasma membrane by an as-yet unidentified organic cationtransporter of Plasmodium,[54, 55, 59, 60] which is proposed to be lo-

cated in the plasmalemma of the parasite (H. Vial,personal communication). The transport of cholinefrom plasma is a rate-limiting step in membrane bio-synthesis by malaria parasites, and thus the cholinecarriers of infected erythrocytes represent excellenttarget structures for the design of phospholipid polarhead analogues as competitors of choline.

In 1985, Vial and colleagues reported for the firsttime that compounds which mimic the structures oflipid precursors such as ethanolamine, serine, or chol-ine are able to block blood-stage replication of themalaria parasite in the low micromolar range. Thefirst three compounds tested as structural analoguesof choline were the mono-QAC (MQAC) dodecyltri-methylammonium and the bis-QACs (BQACs) deca-methonium and hemicholinium 3 (Figure 7), whichexhibited antimalarial activities toward P. falciparumwith half-maximal inhibitory concentration (IC50)values of 0.7–10 mm.[61] Treatment of parasites withthese compounds resulted in a specific decrease inthe biosynthesis of PtdCho, paving the way for amore rational approach in designing choline ana-logues.[62]

In follow-up studies, Vial and co-workers designedextensive sets of primary, secondary, and tertiaryamines as well as MQACs and BQACs as potentialcholine analogues, which were tested in various P. fal-ciparum strains.[63, 64] SAR analyses indicated that theshape, electronegativity, and lipophilicity of the com-

Figure 7. Structures of choline, phosphatidylcholine (PtdCho), hemicholinium 3, G25, 3m,M34, M38, M40, M60, T3/T4, T16, TE3, MAP-412, and nostocarbolines.

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Quaternary Ammonium Salts

pounds are related to in vitro antiplasmodial activity. ForMQACs, increasing lipophilicity around the nitrogen atom wasshown to be beneficial for activity, as was an increase in alkylchain length up to 12 methylene groups. Beyond 12 methyl-ene units, the antimalarial activity decreased slightly, probablydue to curling up of the chain. In contrast, increasing alkylchain length (n) between the two nitrogen atoms constantlyand dramatically increased the antimalarial properties ofBQACs (e.g. , compound G19: IC50 = 3 pm at n = 21; seeFigure 9).[64] Furthermore, a moderate increase in lipophilicityaround the nitrogen atom is beneficial for the BQACs. While itremains to be determined which of the three above-men-tioned choline carriers acts as the main target for the QACs,these new data point toward targeting the organic cationtransporter within the parasite plasmalemma (H. Vial, personalcommunication).

Rational drug design of antiplasmodial BQACs

SAR analyses pointed to a lead compound, G25 (Figure 7),which has an alkyl chain length of n = 16 and IC50 values of0.6–5.3 nm toward various drug-sensitive and drug-resistantP. falciparum strains.[64, 67] A radioactive G25 analogue accumu-lated in infected RBCs, with a 180-fold increase of substance inthe membrane fraction of infected relative to uninfectedRBCs.[67] G25 had no cytotoxic effects in cell culture, with a se-lectivity index (SI) �1000. During a 4 day suppression test inmice, which were infected with P. chabaudi, G25 successfullycleared parasites from the blood, but with a rather low thera-peutic index (TI) of 18.[67, 68] At high doses, G25 provoked rapidand transient hypoxia in the rodents. G25 was subsequently

tested in Aotus monkeys, which were infected with P. falcipa-rum, as well as in Rhesus monkeys infected with P. cynomolgi,and cured the infected monkeys at doses of 0.03 mg kg�1 anda TI>30. These promising results, however, experienced adrawback in the discovery that subcutaneously administeredradioactive compounds had a short elimination half-life inmice (3.3 h) with low bioavailability (17.3 %). The effective doseof orally administered G25 was 100-fold higher than the intra-peritoneally or subcutaneously administered compound.[68]

G25 was thus limited as a lead compound owing to its weakoral absorption, which was assigned to the permanent positivecharge of the quaternary ammonium moiety, which preventedthe compound from crossing the gastrointestinal barrier.

To remedy the problems of low bioavailability and toxicity,in follow-up studies the pyrrolidinium moiety of G25 was sub-stituted by a thiazolium group that is present in vitamin B1.[69]

Two highly active compounds were identified, T3 and T4 (n =

12 for both compounds; see Figure 7), which exhibited in vitroactivities against P. falciparum with IC50 values of 2.6 and0.7 nm and in vivo activities against P. vinckei in the mousemodel with half-maximal effective dose (ED50) values of 0.2 and0.14 mg kg�1, respectively.[69] T3 and its neutral bio-precursorTE3 (Figure 7) were subsequently investigated for their phar-macological properties. Low doses administered intraperito-neally offer protection against malaria in the P. vinckei mousemodel, with ED50 values of 0.2 mg kg�1 for T3 and 0.25 mg kg�1

for TE3, while oral administration led to protection, with ED50

values of 13 and 5 mg kg�1, respectively.[70] The bioavailabilityof T3 was 72 % after intraperitoneal administration and 15 %after oral administration in rats. Furthermore, the T3 plasmaconcentrations of 8 nm at 24 h following oral administration ofTE3 were higher than the IC50 values for most chloroquine-re-sistant strains of P. falciparum.[70] Based on these promising re-sults, compound T3/SAR97276 is now being clinically investi-gated by Sanofi–Aventis and has meanwhile entered phase IIclinical trials.[54]

Two recent studies by the Vial research group were aimed atdesigning more effective neutral prodrugs of T3 to enhance its

Figure 8. SDPM and CDP-choline pathways for PtdCho synthesis in P. falcipa-rum.[54, 55] Cho, choline; CS, cytostome; NPP, new permeation pathway; E, er-ythrocyte; eChoT, erythrocyte choline transporter ; EM, erythrocyte mem-brane; Etn, ethanolamine; FV, food vacuole; Hb, hemoglobin; OCT, organic-cation transporter; P-Cho, phosphocholine; PfCCT, CTP-phosphocholine cyti-dylyltransferase; PfCEPT, choline/ethanolamine cytidylyltransferase; PfCK,choline kinase; PfEK, ethanolamine kinase, PfPMT, phosphoethanolaminemethyltransferase; PLMT, phospholipid methyltransferase; PPM, parasiteplasma membrane; PtdCho, phosphatidylcholine; PtdEtn, phosphatidyletha-nolamine; PtdEtnMT, phosphatidylethanolamine methyltransferase; PV, para-sitophorous vacuole; PVM, parasitophorous vacuole membrane; Ser, serine;SD, serine decarboxylase.

Figure 9. Chain length–activity correlation of BQACs reported by Vial andcolleagues.[64–66]

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MED U. Holzgrabe et al.

bioavailability.[71, 72] To circumvent the poor oral absorption ofprevious BQACs, the Vial group further replaced the quaternaryammonium heads by bioisosteric groups that are highly basic,charged at physiological pH, and capable of creating bondswith the target similar or stronger than those of conventionalBQACs.[65] Because of the equilibrium between protonated andunprotonated forms, these compounds are expected to diffusemore easily through cell barriers. Out of 60 new compounds,2-aminopyridium salts, amidines, and guanidines showed thebest antiplasmodial activities in the low nanomolar range.Again, dimerization of the cationic head produced effectorswith enhanced antimalarial activities, and the optimal alkylchain length between the cationic heads was n = 12. Threelead compounds, the amidines M34, M38, and M40 (Figure 7),were identified, but the high flexibility of the linker was consid-ered a drawback for membrane permeation.[65] In a subsequentstudy, hybrid biscationic salts were designed from efficientsymmetrical derivatives (T3 or T4 and M34, 38, or M40).[66]

However, the generation of hybrids did not result in any syner-gy between amidine and thiazolium cationic heads, and theoral absorption was still weak. Another lead compound of thestudy by Calas et al. was M64, a reversed N-alkylamidine, inwhich the alkyl chain is attached to the functional nitrogenatom (Figure 7).[65, 73] Because M64 turned out to be unstablein vivo, reverse benzamidine derivatives were designed. Severalpromising candidates were identified, among them compound3m (Figure 7), which exhibited an IC50 value of 6.6 nm in P. falci-parum in vitro and an ED50 value of 5 mg kg�1 in the P. vinckeimouse model.[73]

Further advances in the design of antiplasmodial QACs

Other research groups joined the search for antimalarialBQACs. Sasaki and co-workers synthesized antimalarial com-pounds from isonicotinic acid as a starting material. The firstlead compounds, PAM-86 (Figure 7), exhibited promising anti-plasmodial activities in vitro (IC50 = 10 nm), but not in vivo.[74]

The subsequently designed MAP series showed increased ac-tivities with increased alkyl chain linker length, whereas the cy-totoxicity of the compounds increased with the number ofside methylene groups (m).[75] A lead compound, MAP-412 (n =

12, m = 4; see Figure 7), exhibited an IC50 value of 100 nm withno cytotoxicity and an ED50 value of 8.2 mg kg�1 in mice infect-ed with P. berghei.[75]

The inhibitory effect of the cyanobacterium-derived alkaloidnostocarboline (Figure 7) against protozoan parasites was de-scribed.[76] Biscationic dimeric derivatives exhibited activitiesagainst Leishmania donovani in the sub-micromolar range,against Trypanosoma brucei in the low-micromolar range, andagainst P. falciparum in the low-nanomolar range with weak cy-totoxicity. When tested in the P. berghei mouse model, howev-er, the dimers did not show significant activity, while nostocar-boline exhibited an ED50 value of 50 mg kg�1.[77]

Furthermore, the above-mentioned bisquaternary naphthali-mides were tested for their antimalarial properties.[78] SAR anal-yses showed that an alkyl chain length of n = 8 between thepositively charged nitrogen atoms exhibited optimal antiplas-

modial activities, with IC50 values of 60 nm in P. falciparum in vi-tro (Figure 10), while n>8 decreased the effect on the parasite.A dysfunction in membrane biosynthesis was shown on the ul-trastructural level for two of the compounds.[78]

Additional modes of action of antiplasmodial QACs

Because PtdCho can be synthesized by the parasite via theSDPM pathway and thus plasma-derived choline is not essen-tial for parasite blood-stage survival, it was postulated that theinhibition of choline uptake alone cannot account for the anti-malarial activity of BQACs. In this context, Biagini et al. studiedthe uptake of the antimalarially active BQAC T16 (IC50 = 25 nm)and its radioactive analogue (Figure 7).[79] The compound wasreported to enter infected erythrocytes by NPP-induced chan-nels, as shown by using the NPP inhibitor furosemid. Approxi-mately 40 % of total T16 accumulated in the food vacuole,where it bound to the hemoglobin-derived ferriprotoporphyrinIX as well as to hemozoin.[79, 80] The authors proposed thatBQACs act on hemozoin, which would thus strongly contributeto their lethal effect on malaria parasites. In contrast to theseassumptions, two new studies indicate that BQACs affect enzy-matic activities downstream of choline entry. A recent proteo-mics analysis on T4-treated P. falciparum showed that T4 treat-ment decreases the expression of the PfCEPT in these para-sites.[81] The enzyme is responsible for the final steps of PtdChoand PtdEtn synthesis (Figure 8). A follow-up study revealedthat the BQACs hemicholinium 3 and T3 inhibit the phosphory-lation activities of recombinantly expressed choline kinase(PfCK) and ethanolamine kinase (PfEK, Figure 8).[82] The lattertwo studies indicate that BQACs exhibit multiple modes ofaction during phospholipid biosynthesis in Plasmodium by tar-geting the choline carrier as well as enzymes of both theSDPM and the CDP–choline pathways, which would conclu-sively explain the lethal effect of BQACs on malaria parasites.

Summary and Outlook

Since the advent of dequalinium chloride as a potent disinfec-tant and antiseptic drug that is also active against mycobacte-ria and protozoa, a variety of new BQACs were found to havehigher activity especially against Gram-positive bacteria and

Figure 10. Chain length–activity correlation of bisquaternary naphthalimidesreported by Tischer et al.[78]

ChemMedChem 2012, 7, 22 – 31 � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.chemmedchem.org 29

Quaternary Ammonium Salts

plasmodia. On one hand, the antimicrobial activity seems todepend on the length of the chain between the two positivecharges, indicating that a specific target, for example, relatedto the structure and biosynthesis of the cell wall components,is addressed. On the other hand, the lateral heterocyclic moiet-ies are able to direct the BQACs to other targets, such as DNA,as was found with the bisnaphthalimides.

Although nonspecific interactions with membranes was re-garded for a long time to be the main mechanism of QAC andBQAC action, closer investigations into the mode of action re-vealed similar and different targets for this class of chargedcompounds. Particularly in the case of antimalarial BQACs, itwas shown that members of this compound class are able toaddress very specific targets. None of the newly reported com-pounds are currently in clinical use; however, some of themare in, or are close to entering, clinical trials.

Acknowledgements

We thank Henri Vial (Universit� Montpellier II, France) for helpfuldiscussions. G.P. acknowledges funding by the Emmy NoetherProgram of the Deutsche Forschungsgemeinschaft ; U.H. , K.O. ,and M.T. acknowledge funding by the Collaborative ResearchGrant (SFB 630) of the Deutsche Forschungsgemeinschaft.

Keywords: ammonium compounds · cations · dequalinium ·drug discovery · malaria

[1] G. Domagk (Alba Pharmaceuticals Co.), US002108765, 1938.[2] G. McDonnell, A. D. Russell, Clin. Microbiol. Rev. 1999, 12, 147 – 179.[3] M. Babbs, H. O. J. Collier, W. C. Austin, M. D. Potter, E. P. Taylor, J. Pharm.

Pharmacol. 1956, 8, 110 – 119.[4] V. Della Casa, H. Noll, S. Gonser, P. Grob, F. Graf, G. Pohlig, Arzneim.-

Forsch. 2002, 52, 699 – 705.[5] M. T. Gutierrez-Lugo, H. Baker, J. Shiloach, H. Boshoff, C. A. Bewley, J.

Biomol. Screening 2009, 14, 643 – 652.[6] D. C. Jones, I. Hallyburton, L. Stojanovski, K. D. Read, J. A. Frearson, A. H.

Fairlamb, Biochem. Pharmacol. 2010, 80, 1478 – 1486.[7] C. K. Ng, V. Singhal, F. Widmer, L. C. Wright, T. C. Sorrell, K. A. Jolliffe,

Bioorg. Med. Chem. 2007, 15, 3422 – 3429.[8] G. Gamboa-Vujicic, D. A. Emma, S. Y. Liao, C. Fuchtner, A. Manetta, J.

Pharm. Sci. 1993, 82, 231 – 235.[9] M. J. Weiss, J. R. Wong, C. S. Ha, R. Bleday, R. R. Salem, G. D. Steele, Jr. ,

L. B. Chen, Proc. Natl. Acad. Sci. USA 1987, 84, 5444 – 5448.[10] A. I. Garc�a-P�rez, E. Galeano, E. Nieto, P. Sancho, Leuk. Res. 2011, 35,

1395 – 1401.[11] P. Sancho, E. Galeano, E. Nieto, M. D. Delgado, A. I. Garc�a-P�rez, Leuk.

Res. 2007, 31, 969 – 978.[12] E. Galeano, E. Nieto, A. I. Garc�a-P�rez, M. D. Delgado, M. Pinilla, P.

Sancho, Leuk. Res. 2005, 29, 1201 – 1211.[13] C. Abeywickrama, S. A. Rotenberg, A. D. Baker, Bioorg. Med. Chem. 2006,

14, 7796 – 7803.[14] D. Qin, R. Sullivan, W. F. Berkowitz, R. Bittman, S. A. Rotenberg, J. Med.

Chem. 2000, 43, 1413 – 1417.[15] J. R. Rodrigues, N. D. Gamboa, Parasitol. Res. 2009, 104, 1491 – 1496.[16] J. R. Rodrigues, N. Gamboa de Dominguez, Exp. Parasitol. 2007, 115, 19 –

24.[17] M. T. Makler, (Portland Veterans Administration Medical Center, Portland,

OR, USA), US004946849, 1990.[18] D. Galanakis, J. A. D. Calder, C. R. Ganellin, C. S. Owen, P. M. Dunn, J.

Med. Chem. 1995, 38, 3536 – 3546.[19] D. Galanakis, C. A. Davis, B. Del Rey Herrero, C. R. Ganellin, P. M. Dunn,

D. H. Jenkinson, Bioorg. Med. Chem. Lett. 1995, 5, 559 – 562.

[20] D. Galanakis, C. A. Davis, C. R. Ganellin, P. M. Dunn, J. Med. Chem. 1996,39, 359 – 370.

[21] D. Galanakis, C. A. Davis, B. D. R. Herrero, C. R. Ganellin, P. M. Dunn, D. H.Jenkinson, J. Med. Chem. 1995, 38, 595 – 606.

[22] D. Galanakis, C. R. Ganellin, S. Malik, P. M. Dunn, J. Med. Chem. 1996, 39,3592 – 3595.

[23] C. R. Ganellin, D. Galanakis, J. C. Rosa, S. Malik, P. M. Dunn, D. H. Jenkin-son, Book of Abstracts, 211th ACS National Meeting, New Orleans, LA(USA), March 24 – 28, 1996, MEDI-002.

[24] J. C. Rosa, D. Galanakis, C. R. Ganellin, P. M. Dunn, J. Med. Chem. 1996,39, 4247 – 4254.

[25] J. W. Park, I. H. Lee, J. S. Hahn, J. Kim, K. C. Chung, S. R. Paik, Biochim.Biophys. Acta Gen. Subj. 2008, 1780, 1156 – 1161.

[26] L. Spngberg, K. E. Safavi, A. Kaufman, E. A. Pascon, J. Endod. 1988, 14,175 – 178.

[27] D. S. Wilkinson, Hautarzt 1970, 21, 114 – 116.[28] D. Caldwell, W. A. Cox, P. F. D’Arcy, L. R. Rowe, J. Pharm. Pharmacol.

1961, 13, 554 – 564.[29] W. A. Cox, Appl. Microbiol. 1965, 13, 956 – 966.[30] W. B. Hugo, M. Frier, Appl. Microbiol. 1969, 17, 118 – 127.[31] W. L. Bodden, S. T. Palayoor, W. N. Hait, Biochem. Biophys. Res. Commun.

1986, 135, 574 – 582.[32] T. C. Rowe, V. Weissig, J. W. Lawrence, Adv. Drug Delivery Rev. 2001, 49,

175 – 187.[33] M. R. J. Salton, J. Gen. Microbiol. 1951, 5, 391 – 404.[34] M. R. J. Salton, R. W. Horne, V. E. Cosslett, J. Gen. Microbiol. 1951, 5,

405 – 407.[35] Z. Baker, R. W. Harrison, B. F. Miller, J. Exp. Med. 1941, 74, 611 – 620.[36] S. P. Denyer, Intl. Biodeterioration Biodegradation 1995, 36, 227 – 245.[37] M. R. Brown, E. Tomlinson, J. Pharm. Sci. 1979, 68, 146 – 149.[38] H. H. Locher, D. Ritz, P. Pfaff, M. Gaertner, A. Knezevic, D. Sabato, S.

Schroeder, D. Barbaras, K. Gademann, Chemotherapy 2010, 56, 318 –324.

[39] A. D. Russell, G. W. Gould, J. Appl. Bacteriol. 1988, 65, S167 – S195.[40] D. O. Kolawole, FEMS Microbiol. Lett. 1984, 25, 205 – 209.[41] S. J. Broadley, P. A. Jenkins, J. R. Furr, A. D. Russell, Lett. Appl. Microbiol.

1991, 13, 118 – 122.[42] A. Leelaporn, I. T. Paulsen, J. M. Tennent, T. G. Littlejohn, R. A. Skurray, J.

Med. Microbiol. 1994, 40, 214 – 220.[43] T. G. Littlejohn, D. Diberardino, L. J. Messerotti, S. J. Spiers, R. A. Skurray,

Gene 1991, 101, 59 – 66.[44] I. T. Paulsen, M. H. Brown, T. G. Littlejohn, B. A. Mitchell, R. A. Skurray,

Proc. Natl. Acad. Sci. USA 1996, 93, 3630 – 3635.[45] M. F. BraÇa, J. M. Castellano, M. Moran, M. J. Perez de Vega, C. R. Romer-

dahl, X. D. Qian, P. Bousquet, F. Emling, E. Schlick, G. Keilhauer, Anti-Cancer Drug Des. 1993, 8, 257 – 268.

[46] M. F. BraÇa, M. Cacho, M. A. Garci�, B. de Pascual-Teresa, A. Ramos, M. T.Dom�nguez, J. M. Pozuelo, C. Abradelo, M. F. Rey-Stolle, M. Yuste, M.B�Çez-Coronel, J. C. Lacal, J. Med. Chem. 2004, 47, 1391 – 1399.

[47] T. M. Menzel, M. Tischer, P. Francois, J. Nickel, J. Schrenzel, H. Bruhn, A.Albrecht, L. Lehmann, U. Holzgrabe, K. Ohlsen, Antimicrob. Agents Che-mother. 2011, 55, 311 – 320.

[48] J. Gallego, B. R. Reid, Biochemistry 1999, 38, 15104 – 15115.[49] L. Gonz�lez-Bulnes, J. Gallego, J. Am. Chem. Soc. 2009, 131, 7781 – 7791.[50] H. Jenssen, P. Hamill, R. E. Hancock, Clin. Microbiol. Rev. 2006, 19, 491 –

511.[51] T. Schneider, T. Kruse, R. Wimmer, I. Wiedemann, V. Sass, U. Pag, A.

Jansen, A. K. Nielsen, P. H. Mygind, D. S. Raventos, S. Neve, B. Ravn,A. M. Bonvin, L. De Maria, A. S. Andersen, L. K. Gammelgaard, H. G. Sahl,H. H. Kristensen, Science 2010, 328, 1168 – 1172.

[52] World Malaria Report 2010, World Health Organization http://www.who.int/malaria/world_malaria_report_2010/worldmalariare-port2010.pdf (accessed November 8, 2011).

[53] D. A. Bullough, E. A. Ceccarelli, J. G. Verburg, W. S. Allison, J. Biol. Chem.1989, 264, 9155 – 9163.

[54] C. Ben Mamoun, S. T. Prigge, H. Vial, Drug Dev. Res. 2010, 71, 44 – 55.[55] S. D�champs, S. Shastri, K. Wengelnik, H. J. Vial, Int. J. Parasitol. 2010,

40, 1347 – 1365.[56] N. Elabbadi, M. L. Ancelin, H. J. Vial, Biochem. J. 1997, 324, 435 – 445.[57] G. Pessi, G. Kociubinski, C. B. Mamoun, Proc. Natl. Acad. Sci. USA 2004,

101, 6206 – 6211.

30 www.chemmedchem.org � 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2012, 7, 22 – 31

MED U. Holzgrabe et al.

[58] M. L. Ancelin, M. Parant, M. J. Thuet, J. R. Philippot, H. J. Vial, Biochem. J.1991, 273, 701 – 709.

[59] G. A. Biagini, E. M. Pasini, R. Hughes, H. P. De Koning, H. J. Vial, P. M.O’Neill, S. A. Ward, P. G. Bray, Blood 2004, 104, 3372 – 3377.

[60] A. M. Lehane, K. J. Saliba, R. J. Allen, K. Kirk, Biochem. Biophys. Res.Commun. 2004, 320, 311 – 317.

[61] M. L. Ancelin, H. J. Vial, J. R. Philippot, Biochem. Pharmacol. 1985, 34,4068 – 4071.

[62] M. L. Ancelin, H. J. Vial, Antimicrob. Agents Chemother. 1986, 29, 814 –820.

[63] M. Calas, G. Cordina, J. Bompart, M. Ben Bari, T. Jei, M. L. Ancelin, H. Vial,J. Med. Chem. 1997, 40, 3557 – 3566.

[64] M. Calas, M. L. Ancelin, G. Cordina, P. Portefaix, G. Piquet, V. Vidal-Sail-han, H. Vial, J. Med. Chem. 2000, 43, 505 – 516.

[65] M. Calas, M. Ouattara, G. Piquet, Z. Ziora, Y. Bordat, M. L. Ancelin, R.Escale, H. Vial, J. Med. Chem. 2007, 50, 6307 – 6315.

[66] S. Ortial, S. Denoyelle, S. Wein, O. Berger, T. Durand, R. Escale, A. Pellet,H. Vial, Y. Vo-Hoang, ChemMedChem 2010, 5, 52 – 55.

[67] K. Wengelnik, V. Vidal, M. L. Ancelin, A. M. Cathiard, J. L. Morgat, C. H.Kocken, M. Calas, S. Herrera, A. W. Thomas, H. J. Vial, Science 2002, 295,1311 – 1314.

[68] M. L. Ancelin, M. Calas, A. Bonhoure, S. Herbute, H. J. Vial, Antimicrob.Agents Chemother. 2003, 47, 2598 – 2605.

[69] A. Hamz�, E. Rubi, P. Arnal, M. Boisbrun, C. Carcel, X. Salom-Roig, M.Maynadier, S. Wein, H. Vial, M. Calas, J. Med. Chem. 2005, 48, 3639 –3643.

[70] O. Nicolas, D. Margout, N. Taudon, S. Wein, M. Calas, H. J. Vial, F. M. Bres-solle, Antimicrob. Agents Chemother. 2005, 49, 3631 – 3639.

[71] S. A. Caldarelli, M. Boisbrun, K. Alarcon, A. Hamz�, M. Ouattara, X.Salom-Roig, M. Maynadier, S. Wein, S. Peyrottes, A. Pellet, M. Calas, H.Vial, Bioorg. Med. Chem. Lett. 2010, 20, 3953 – 3956.

[72] S. A. Caldarelli, J. F. Duckert, S. Wein, M. Calas, C. Perigaud, H. Vial, S.Peyrottes, ChemMedChem 2010, 5, 1102 – 1109.

[73] O. Berger, S. Wein, J. F. Duckert, M. Maynadier, S. El Fangour, R. Escale, T.Durand, H. Vial, Y. Vo-Hoang, Bioorg. Med. Chem. Lett. 2010, 20, 5815 –5817.

[74] K. Fujimoto, D. Morisaki, M. Yoshida, T. Namba, K. Hye-Sook, Y. Wataya,H. Kourai, H. Kakuta, K. Sasaki, Bioorg. Med. Chem. Lett. 2006, 16, 2758 –2760.

[75] K. Motoshima, Y. Hiwasa, M. Yoshikawa, K. Fujimoto, A. Tai, H. Kakuta, K.Sasaki, ChemMedChem 2007, 2, 1527 – 1532.

[76] D. Barbaras, M. Kaiser, R. Brun, K. Gademann, Bioorg. Med. Chem. Lett.2008, 18, 4413 – 4415.

[77] S. Bonazzi, D. Barbaras, L. Patiny, R. Scopelliti, P. Schneider, S. T. Cole, M.Kaiser, R. Brun, K. Gademann, Bioorg. Med. Chem. 2010, 18, 1464 – 1476.

[78] M. Tischer, L. Sologub, G. Pradel, U. Holzgrabe, Bioorg. Med. Chem.2010, 18, 2998 – 3003.

[79] G. A. Biagini, E. Richier, P. G. Bray, M. Calas, H. Vial, S. A. Ward, Antimi-crob. Agents Chemother. 2003, 47, 2584 – 2589.

[80] E. Richier, G. A. Biagini, S. Wein, F. Boudou, P. G. Bray, S. A. Ward, E. Pre-cigout, M. Calas, J. F. Dubremetz, H. J. Vial, Antimicrob. Agents Chemo-ther. 2006, 50, 3381 – 3388.

[81] K. G. Le Roch, J. R. Johnson, H. Ahiboh, D. W. Chung, J. Prudhomme, D.Plouffe, K. Henson, Y. Zhou, W. Witola, J. R. Yates, C. B. Mamoun, E. A.Winzeler, H. Vial, BMC Genomics 2008, 9, 513.

[82] B. Alberge, L. Gannoun-Zaki, C. Bascunana, C. Tran van Ba, H. Vial, R.Cerdan, Biochem. J. 2010, 425, 149 – 158.

Received: August 23, 2011Revised: October 28, 2011Published online on November 24, 2011

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