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MedChemComm
CONCISE ARTICLE
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Bile acid amphip
aLaboratory of Nanotechnology and Ch
Biotechnology, 180 Udyog Vihar, Phase I, G
[email protected] and Infectious Disease Research C
Technology Institute, 496 Udyog Vihar Ph
[email protected] University, Manipal, India
† Electronic supplementary information (synthesis of amphiphiles. See DOI: 10.103
‡ These authors contributed equally.
Cite this:Med. Chem. Commun., 2014,5, 1761
Received 11th July 2014Accepted 31st August 2014
DOI: 10.1039/c4md00303a
www.rsc.org/medchemcomm
This journal is © The Royal Society of C
hiles with tunable head groups ashighly selective antitubercular agents†
Sandhya Bansal,‡a Manish Singh,‡a Saqib Kidwai,‡b Priyanshu Bhargava,a
Ashima Singh,a Vedagopuram Sreekanth,ac Ramandeep Singh*b and Avinash Bajaj*ac
Tuberculosis faces major challenges for its cure due to (a) long treatment period, (b) emergence of drug
resistance bacteria, and (c) poor patient compliance. Disrupting the membrane integrity of mycobacteria
as a therapeutic strategy has not been explored well as the rigid, waxy, and hydrophobic nature of
mycobacterial lipids does not allow binding and penetration of charged amtimicrobial amphiphiles and
peptides. Here, we present a new concept that fine-tuning of the charged head group modulates the
specificity of amphiphiles against bacterial membranes. We show that hard-charged amphiphiles interact
with mycobacterial trehalose dimycolates and penetrate through rigid mycobacterial membranes. In
contrast, soft-charged amphiphiles specifically inhibit the growth of both E. coli and S. aureus via
electrostatic interactions. These subtle variations between interactions of amphiphiles and bacterial
membranes could be explored further to design more specific and potent antimycobacterial agents.
Introduction
Tuberculosis (TB) remains a global health burden with �8.6million individuals infected with M. tuberculosis (Mtb) andaccounting for �2.0 million deaths in 2012.1 Major drawbacksof the current TB regimen targeting essential mycobacterialpathways is the 6 month long duration and poor complianceamong patients. This situation has further worsened due to theemergence of various MDR/XDR TB strains, HIV–TB co-infec-tion, and BCG vaccine failure to impart protection againstpulmonary TB.2 The advent of computational methods alongwith whole-cell and HTS-based assays have led to the identi-cation of various scaffolds that are currently in clinical trials.The newly identied scaffolds should have (i) improved activityagainst dormant bacteria, (ii) novel mechanism of action, (iii)specicity against mycobacteria, (iv) good pharmacokinetics/pharmacodynamics properties, and (v) compatibility withcurrent TB and retroviral therapies.3
Antimicrobial peptides (AMPs) induce non-receptor medi-ated disruptions in the target membranes of microorganisms byvirtue of their amphiphilic nature with a discrete cationic
emical Biology, Regional Centre for
urgaon-122016, Haryana, India. E-mail:
entre, Translational Health Science and
ase III, Gurgaon-122016, India. E-mail:
ESI) available: Fig. S1–S6 and Table S1,9/c4md00303a
hemistry 2014
charge.4 Amphiphiles mimicking these AMPs with variablecationic charge groups on hydrophobic moieties have beenexplored for their antimicrobial activities.5,6 These amphiphilesexert their antimicrobial activity through electrostatic interac-tions with the lipid components of bacterial membranes.7,8
These membrane-disruptive amphiphiles have the ability to (a)shorten the duration of treatment, and (b) eradicate drug-resistant bacteria.
Mycobacterial non-polar lipids present in the outermembrane of mycobacteria may not interact with polar chargedamphiphiles/peptides, and hence do not allow their insertionacross rigid hydrophobic mycolic lipids. Hence, disrupting theintegrity of mycobacterial membranes has not been extensivelyexplored to combat TB.9 Therefore, the present study was con-ducted to: (i) address the lack of specicity of AMPs andamphiphiles against microorganisms, (ii) explore the disrup-tion of membrane integrity as a mechanism to combat TB, and(iii) better understand interactions between amphiphiles andmycobacterial membranes.
Bile acids are inherently facial amphiphilic in nature due tothe stereochemical orientation of hydroxyl groups.10 Savage andco-workers have synthesized cholic acid derived cationic anti-microbials that possessed a higher affinity for lipid A of bacte-rial membranes as compared to polymyxin B.11 In thismanuscript, we propose that ne-tuning of charged headgroups on bile acid amphiphiles modulate their specicityagainst mycobacteria. We unraveled that hard-charged amphi-philes specically kills mycobacteria as the hydrophobic, rigid,waxy outer membranes of the mycobacteria can allow penetra-tion of these hydrophobic amphiphiles (Fig. 1). Contrastingly,so-charged amphiphiles specically kill Gram-positive/Gram-
Med. Chem. Commun., 2014, 5, 1761–1768 | 1761
Fig. 1 Schematic presentation of the study showing the selectivity of hard-charged amphiphiles for mycobacteria, and soft-charged amphi-philes for Gram-positive and Gram-negative bacteria.
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negative bacteria through electrostatic interactions with polarbacterial lipids.
Results and discussionDesign and synthesis of amphiphiles
We engineered 20 amphiphiles derived from bile acids withdifferent charged head groups using four bile acids, lithocholicacid (LCA), chenodeoxycholic acid (CDCA), deoxycholic acid(DCA), and cholic acid (CA) (Fig. 2). The polarity of that attachedhead groups varied from so-charged ammonium (AMM) tohard-charged trimethyl ammonium (TMA), N-methylpiperidine(PIP), pyridine (PYR), and dimethylaminopyridine (DMAP).Amphiphiles were synthesized by chloroacetylation of the cor-responding bile acid methyl ester10a followed by quaternizationwith the respective tertiary amines (Scheme 1, ESI†), and char-acterized by 1H-NMR, 13C-NMR and HRMS (ESI†).
Antibacterial activities against different microorganisms
We then evaluated activities of amphiphiles against mycobac-terial species, M. smegmatis (Msm), M. tuberculosis H37Rv (Mtb),and M. bovis BCG (Table 1). From structure–activity studies, weconcluded that (a) multiple-charged amphiphiles are morepotent as compared to single-charged amphiphiles; (b) amongmultiple-charged amphiphiles, hard-charged amphiphiles are
Fig. 2 Molecular structures of lithocholic acid (LCA)-, chenodeoxycholamphiphiles; different charged head groups were synthesized and studi
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more potent as compared to so-charged amphiphiles; (c) ingeneral, DMAP-derived amphiphiles (CDCA-DMAP2, DCA-DMAP2, and CA-DMAP3) were most potent against Mtb inhib-iting mycobacterial growth in the range of 0.78–6.25 mM; (d) CAderived amphiphiles followed the order of DMAP > PYR > PIP >TMA > AMM in terms of their anti-tuberculosis activity; (e)ammonium head group-derived so-charged amphiphilespossess no activity against mycobacteria.
To unravel the selectivity of amphiphiles, we determinedtheir activities against Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria (Table 2). MIC99 determination studiessuggested that: (a) single-charged LCA amphiphiles showed nogrowth inhibition up to 512 mM; (b) among multiple-chargedamphiphiles, so-charged amphiphiles (DCA-AMM2 and CA-AMM3) inhibited 99% growth at 16–64 mM; (c) hard-chargedmultiple-headed amphiphiles are less potent than so-chargedamphiphiles; (d) CA-derived hard-charged amphiphiles showedno growth inhibition even at 512 mM. Interestingly, the so-charged CA-AMM3 amphiphile was highly specic in its abilityto inhibit growth of E. coli/S. aureus, whereas the hard-chargedCA-DMAP3 amphiphile was highly specic for mycobacterialspecies. CA-DMAP3 possesses MIC99 of 1.56–6.25 mM formycobacteria, and a selectivity index of �40–320 over E. coli/S.aureus. The presence of 50% plasma also maintains its highpotency (MIC99 ¼ 6.25 mM) against mycobacteria.
ic acid (CDCA)-, deoxycholic acid (DCA)-, and cholic acid (CA)-baseded.
This journal is © The Royal Society of Chemistry 2014
Table 1 Antimycobacterial activities, hemolytic activities and therapeutic index of amphiphiles
Amphiphile
MIC99a (mM)
MHC50b (mM) TIc Amphiphile
MIC99a (mM)
MHC50b (mM) TIcBCG mc2155 H37Rv BCG mc2155 H37Rv
LCA-AMM1 >50 >50 >50 >1 <20 DCA-AMM2 25 >50 >50 >1 <20LCA-TMA1 50 6.25 50 >1 >20 DCA-TMA2 25 12.5 >50 >1 <20LCA-PIP1 50 6.25 50 >1 >20 DCA-PIP2 3.12 0.78 6.25 >1 >160LCA-PYR1 >50 25 >50 >1 <20 DCA-PYR2 6.25 1.56 12.5 >1 >80LCA-DMAP1 50 12.5 50 >1 >20 DCA-DMAP2 2.34 0.78 4.68 >1 >213CDCA-AMM2 37.5 >50 >50 >1 <20 CA-AMM3 18.75 >50 >50 >1 <20CDCA-TMA2 4.68 3.12 12.5 >1 >80 CA-TMA3 6.25 12.5 50 >1 >20CDCA-PIP2 6.25 1.56 12.5 >1 >80 CA-PIP3 6.25 6.25 25 >1 >40CDCA-PYR2 9.375 3.12 25 >1 >40 CA-PYR3 6.25 3.12 37.5 >1 >26CDCA-DMAP2 3.12 0.78 3.12 >1 >320 CA-DMAP3 3.12 1.56 6.25 >1 >160INH 0.78 >50 0.78 —d —d Levooxacin 0.39 0.58 0.78 —d —d
a MIC99 of amphiphiles against three mycobacterial strains. b MHC50 is minimum hemolytic conc. at which 50% hemolysis is observed.c Therapeutic index for M. tuberculosis H37Rv as ratio of MHC50/MIC99 (M. tuberculosis H37Rv).
d Not determined.
Table 2 Antibacterial activities of bile acid amphiphiles against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria; and toxicities ofbile acid amphiphiles against lung epithelial (A549) and macrophage (THP-1) cell lines
Amphiphile
MIC99a (mM) IC50
b (mM)
Amphiphile
MIC99a (mM) IC50
b (mM)
E. coli S. aureus A549 THP-1 E. coli S. aureus A549 THP-1
LCA-AMM1 >512 >512 24.6 � 8.8 14.0 � 0.2 DCA-AMM2 16 64 14.7 � 5.6 13.96 � 0.3LCA-TMA1 >512 >512 69 � 2.5 62 � 2.6 DCA-TMA2 256 256 157 � 2.26 105 � 7.3LCA-PIP1 >512 >512 42 � 6.5 77 � 6.0 DCA-PIP2 >512 128 50 � 8.1 77 � 8.3LCA-PYR1 >512 >512 68 � 1.3 89 � 5.0 DCA-PYR2 128 >512 78 � 5.0 124 � 8.7LCA-DMAP1 >512 >512 112 � 5.9 86 � 1.5 DCA-DMAP2 64 64 50 � 9.6 63 � 5.4CDCA-AMM2 64 128 14.8 � 9.5 13.7 � 0.8 CA-AMM3 32 64 75.9 � 1.3 38.2 � 5.1CDCA-TMA2 256 512 144 � 7.8 142 � 1.7 CA-TMA3 >512 >512 >200 80 � 6.02CDCA-PIP2 128 256 79 � 7.1 164 � 1.7 CA-PIP3 >512 512 >200 >200CDCA-PYR2 128 256 168 � 0.1 >200 CA-PYR3 >512 >512 >200 >200CDCA-DMAP2 128 128 154 � 4.8 137 � 2.1 CA-DMAP3 >512 256 >200 >200Polymyxin 0.5 1.0 —c —c — — — — —
a MIC99 of amphiphiles against Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. b IC50 is minimum conc. at which 50% celldeath is observed against lung epithelial cells (A549) and macrophages (THP-1). c Not determined.
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Cytotoxicity studies against mammalian cells
Cytotoxicity studies of amphiphiles against mammalian A549(human lung epithelial cell line) and THP-1 (human monocytecell line) cells showed that single-charged LCA amphiphiles arethemost cytotoxic, and so-charged amphiphiles are more toxicas compared to hard-charged amphiphiles (Table 2, Fig. S1 andS2, ESI†). A highly sensitive propidium-iodide-based cell-cycleanalysis also showed no change in the cell-cycle phase of A549cells on treatment with 100 mM of CA based-amphiphiles(Fig. S3, ESI†). Hemolytic studies against chicken and sheepblood RBCs showed that all the amphiphiles possessed MHC50
values greater than 1 mM (Table 1 and Fig. S4, ESI†). Thera-peutic index (MHC50/MIC99) calculations suggested that hard-charged CA amphiphiles have a high therapeutic index overso-charged amphiphiles, and DMAP-derived multiple-chargedamphiphiles are the most potent with the highest therapeuticindex.
This journal is © The Royal Society of Chemistry 2014
Mechanism of action
The above studies concluded that so-charged CA-AMM3
selectively inhibits the growth of E. coli/S. aureus, whereas hard-charged CA-DMAP3 is highly selective for mycobacteria. Weobserved that the killing of E. coli/S. aureus by CA-AMM3 wasdose-dependent and bactericidal in nature, whereas no growthinhibition of E. coli/S. aureus was observed on exposure to CA-DMAP3 (Fig. 3a and S5, ESI†). Incubation of Msm with CA-DMAP3 inhibited bacterial growth by 10 000–100 000 fold(Fig. 3b), whereas no such growth inhibition was observed onexposure to CA-AMM3. Similarly, we observed perturbations inE. coli/S. aureus membranes only on incubation with CA-AMM3
(Fig. 3c), whereas CA-DMAP3 specically inhibited the ability ofMsm to generate proton motive force (Fig. 3d).
Next, we performed AFM studies to determine bothmorphological and topological changes in E. coli and M. bovisBCG on treatment with CA-AMM3 and CA-DMAP3. Incubation of
Med. Chem. Commun., 2014, 5, 1761–1768 | 1763
Fig. 3 (a) Time-dependent killing of E. coli and S. aureus suggesting a bactericidal effect in the presence of CA-AMM3. (b) Time-dependent killingof Msm showing selective bactericidal activity by CA-DMAP3. (c) Membrane permeabilization of E. coli and S. aureus by propidium iodideshowing selective permeabilization of E. coli and S. aureus by CA-AMM3. (d) Effect of CA-AMM3 and CA-DMAP3 on the membrane potential ofMsm suggests selective activity of CA-DMAP3.
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E. coli with CA-AMM3 induced surface indentations, micelle-likestructures, and leakage of a large amount of cytoplasmiccontents (Fig. 4b and Table S1, ESI†). Incubation of M. bovisBCG with CA-DMAP3 led to pore and groove formation with arugged surface (Fig. 4f), whereas the morphology and topologyof CA-DMAP3-treated E. coli (Fig. 4c) and CA-AMM3 treated M.bovis BCG (Fig. 4e) were similar to their respective untreatedsamples (Fig. 4a and d).
To understand the mechanism of this differential specicity,we next determined the activity of these amphiphiles againstboth E. coli and S. aureus in the presence of EDTA, a knowndestabilizer of lipopolysaccharide (LPS) and proteoglycans.12
Fig. 4 (a–f) Atomic forcemicrographs of E. coli (a–c) and BCG (d–f) on insuggesting selective disruptions of E. coli membranes by CA-AMM3 (b) a
1764 | Med. Chem. Commun., 2014, 5, 1761–1768
Mechanistic studies showed that the presence of EDTAenhances the activity of CA-DMAP3 by 64-fold and by 16-foldagainst E. coli and S. aureus, respectively, whereas only �2-foldincrease in activity was observed for CA-AMM3. These resultsconclude that that presence of LPS and proteoglycans inhibitsthe interactions of CA-DMAP3 with E. coli/S. aureus bacterialmembranes, thereby accounting for lack of its activity againstthem.
Amphiphile–membrane interactions
We speculated that presence of hydrophobic mycolic lipids liketrehalose dimycolate (TDM)13 might be responsible for the
cubation with CA-AMM3 (b and e) and CA-DMAP3 (c and f) amphiphilesnd BCG membranes by CA-DMAP3 (f) respectively.
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Fig. 5 (a–c) Surface hydration of model membranes suggesting the dehydrated nature of PE : PG : TDM membranes; and maximum dehy-dration of PE : PG : LPS and PE : PG membranes on interactions with amphiphiles due to electrostatic interactions. (d–f) Membrane rigidity ofmodel bacterial membranes showing high rigidity of PE : PG : TDM membranes; and induction of membrane fluidity of PE : PG : TDMmembranes on interactions with CA-DMAP3.
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selective killing of mycobacteria by CA-DMAP3. We thereforeprobed the interactions of CA-AMM3 and CA-DMAP3 with modelbacterial membranes to quantify changes in the surfacehydration and rigidity of membranes. Using a Laurdan probe,14
we observed that TDM-dopedmycobacterial model membranes,PE : PG : TDM (1 : 1 : 2), are more dehydrated than E. colimodeled membranes, PE : PG : LPS (5 : 3 : 1) and PE : PG (1 : 1)(Fig. 5a–c).
Incubation of CA-AMM3 induced a higher amount of dehy-dration in PE : PG : LPS and PE : PG membranes (Fig. 5a–c) dueto strong electrostatic interactions, thereby accounting for theselective activity of CA-AMM3 against E. coli/S. aureus. Similarly,the smaller amount of dehydration of PE : PG : TDMmembranes by CA-AMM3 accounts for poor interactions and theinability of CA-AMM3 to kill mycobacteria. We did not observeany dehydration of PE : PG : TDMmembranes upon incubationwith CA-DMAP3 (Fig. 5c), thereby suggesting the presence ofhydrophobic interactions between CA-DMAP3 andPE : PG : TDM.
Membrane uidity studies using a DPH probe15 suggestedthat mycobacterial modeled membranes are more rigid incomparison to other membranes (Fig. 5d–f). Incubation ofPE : PG : LPS and PE : PG with CA-AMM3 does not induce anyuidity, suggesting poor interactions of CA-AMM3 with thehydrophobic regions of membranes. Therefore the observeddehydration without any alteration in the rigidity ofPE : PG : LPS and PE : PG conrms a carpet-like mechanism forthe activity of CA-AMM3 against E. coli/S. aureus.16 Contrast-ingly, the presence of hydrophobic, rigid mycolic lipids likeTDM prevents interactions of CA-AMM3 with mycobacterialmembranes. Interestingly, CA-DMAP3 increases the uidity ofmycobacterial (PE : PG : TDM) membranes as observed in caseof alamethicin17 by virtue of the insertion of CA-DMAP3 in
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hydrophobic membranes. We did not observe this enhancedmembrane uidity on incubation of PE : PG and PE : PG : LPSwith CA-DMAP3. These observations suggest that CA-DMAP3 hasthe ability to interact with rigid mycolic lipids and form pore-like structures in mycobacterial membranes.18
Conclusions
In summary, we demonstrated that ne-tuning charged headgroups on a bile acid scaffold modulates their specicity againstbacteria. So-charged primary amine amphiphiles interact withGram-positive and Gram-negative bacterial membranes,whereas hard-charged head groups provide effective and selec-tive interactions with hydrophobic mycobacterial membranes.In-depth mechanistic studies revealed that this specicity inmechanism of action for these amphiphiles was due to molec-ular differences in the cell wall architecture of mycobacterialand Gram-positive/Gram-negative bacteria. The present studywill help us in understanding the molecular basis of specicamphiphile–membrane interactions and in the design of morepotent 2nd-generation amphiphiles that are highly specic for aparticular bacterial species, which might be useful to combatthe problem of drug resistance.
Experimental sectionMaterials and methods
Bile acids were purchased from Sigma-Aldrich. CCCP, INH, andDiCO2 were purchased from Invitrogen corporation. All thesynthesized compounds were puried using Combi-ash chro-matography with a 230–400 mesh size silica gel. 1H NMR and13C NMR spectra were recorded using a Brucker 400 MHzspectrometer. Chemical shis (d) are reported in ppm with
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tetramethylsilane as the internal standard. High-resolutionmass spectra were measured on an AB-SCIEX-5600 mass spec-trometer. E. coli (MTCC 443) and S. aureus (MTCC 737) werepurchased from MTCC. The A549 cell line was purchased fromSigma Aldrich and THP-1 cells were a kind gi from NCCS,Pune. M. smegmatis and M. tuberculosis H37Rv were a kind gifrom Dr Clion E. Barry, NIH, and M. bovis BCG was kind gifrom Professor Anil K. Tyagi (Department of Biochemistry,UDSC, India).
Antibacterial activity
We determined the antibacterial activity of bile acid amphi-philes using a slight modication of a method mentioned byHancock et al.19 The bacterial strains were grown for 6 h anddiluted to 105 cfu mL�1. 150 mL of this bacterial suspension wasadded to a 96-well plate containing the required concentrationof amphiphiles. The plate was then incubated at 37 �C withcontinuous shaking for 12 h, and OD600 nm was measuredusing a molecular probe M5 microplate reader. MIC99 valueswere determined by taking the average of triplicate values foreach concentration and were performed in duplicate. Polymyxinwas used as the positive control in our assays. For experimentswith EDTA, both the bacterial cultures were grown in nutrientbroth containing 10 mM EDTA and MIC99 was determined asdescribed above in the presence of 10 mM EDTA. The anti-mycobacterial activity of these amphiphiles was determinedusing an inverted plate reader method as described previously.20
The plates were incubated at 37 �C and MIC99 values were readmicroscopically using an inverted plate reader aer 14 days forM. bovis BCG, Mtb, and aer 2 days for Msm. Each reading wasmade three independent times. Standard drugs such as isoni-azid and levooxacin were used as positive controls in ourassays. For killing curves early-log phase cultures were incu-bated with CA-AMM3 and CA-DMAP3 at designated time points.Bacterial enumeration was performed by plating 10-fold serialdilutions on MB7H10 plates, and the plates were incubated at37 �C.
Cytotoxicity assay21
The THP-1 cell line was maintained in RPMI media as perstandard protocol and differentiated in macrophages by theovernight addition of 50 ng mL�1 of PMA (phorbol 12-myristate13-acetate). For the cytotoxicity assay, 5 � 103 cells were seededper well in a 96-well plate. Aer 24 h, cells were overlayed withmedium containing various concentrations of amphiphiles.Aer overlaying macrophages for 48 h, 20 mL of 5 mgmL�1 MTT{3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide}was added to each well. Aer incubation for 4 h, cells were lysedby the addition of 200 mL of a 1 : 1 mixture of DMSO and MeOHto dissolve the formazen crystals and the absorption at 540 nmwas measured. Cell viability was calculated using the equation[{A540(treated cells) � background}/{A540(untreated cells) �background}] � 100.
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Cell cycle analysis
Cell cycle analyses against A549 cells for bile acid amphiphileswere performed according to published protocol.21
Hemolytic assay22
Hemolytic assays were performed using chicken and sheepblood. For RBC isolation, blood was centrifuged at 4000 rpm for5 min. For hemolytic assays, 195 mL of erythrocyte suspension(5% or 20% RBC) were added per well in a 96-well plate andincubated with facial amphiphiles at the desired concentra-tions. Aer incubation for 1 h, the plate was centrifuged at1200g for 15 min. The supernatant was diluted 1 : 100 in 1� PBSand absorption at 413 nm was measured. All our assay platesincluded positive (1% PBST), buffer (1� PBS) and solvent(MeOH) controls. The percentage of hemolysis was determinedfrom {(A � A0)/(Atotal � A0)} � 100, where A is the absorbance ofthe test well, A0 is the absorbance of the negative controls, andAtotal is the absorbance of 100% hemolysis wells at 413 nm. Allthe experiments were performed at least twice in duplicates.
Membrane permeabilization studies23
E. coli and S. aureus cells were grown till mid log phase. Cellswere harvested by centrifugation, washed with 1� PBS and re-suspended in 1� PBS. These washed E. coli and S. aureus bacillisuspensions were pre-incubated with amphiphiles at aconcentration of 80 mM or 160 mM, respectively, followed by theaddition of 15 mM propidium iodide (PI). The uptake of PI wasmeasured by the increase in uorescence of PI for 10 min as ameasure of membrane permeabilization using an excitationwavelength of 535 nm and an emission wavelength of 617 nm.
Membrane potential studies24
Msm was grown till an OD600 nm value of 1.0 and bacteria werepre-incubated with amphiphiles or INH or CCCP. Bacterial cellswere immediately exposed to 15 mM of DiCO2 (3,30-diethylox-acarbocyanine iodide) at room temperature. Aer 30 minutes oflabeling, cells were washed twice with 1� PBS, and green uo-rescence (Ex480 nm/Em530 nm) and red uorescence (Ex488 nm/Em610 nm) were measured in a 96-well plate reader (Bioteksynergy Hr). Cells treated with no drug were kept as the controlfor background uorescence. Membrane potential was calcu-lated as the ratio of red uorescence to green uorescence usingBiotek Synergy Hit and Gene5 soware.
Atomic force microscopy studies (sample preparation andimaging)25
Poly-L-lysine coated (1.0 mm uniform thickness) microscopicslides were obtained from Polysciences Inc. All the AFM-imaging experiments were done on a JPK NanoWizard® AFMhead using an AC air mode cantilever. The pyramidal tipcantilever used was purchased from ACTA made of silicon witha spring constant of 40 N m�1. All the data was processed usingJPKSPM data-processing soware. For AFM studies bacteriawere grown till an OD600 nm value of 1.0, harvested, washedtwice with 1� PBS and subsequently exposed to CA-AMM3 or
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CA-DMAP3 in a 96-well plate. Aer incubation for 24 h, sampleswere applied on poly-L-lysine coated slides and dried using aslow stream of nitrogen gas. Imaging was done using the AC air/tapping mode with 15 mm z-scale size. All images were obtainedwith a scan speed of 0.5 Hz and a resolution of 1024 � 1024pixels. The height, width, and 3D-toplogical information wasacquired and processed with JPKSPM data-processingsoware.
Amphiphile–membrane interactions
Membrane vesicles with the desired lipid ratios were preparedas described previously.26 Changes in the surface hydration ofvesicles were studied aer incubation of the vesicles with 10weight percent of facial amphiphiles at 25 �C for 4 h. Werecorded the generalized polarization of Laurdan in a 96-wellplate in a Molecular Devices M5 instrument. Fluorescence ofLaurdan was recorded using an excitation wavelength of 350nm and emission wavelengths of 440 nm and 490 nm. Thegeneralized polarization (GP) was calculated using the equa-tion GP ¼ (I440 � I490)/(I440 + I490). Similarly, we measuredchanges in steady-state anisotropy of DPH in a 96-well plateusing lex at 350 nm and lem of 452 nm aer incubation of thesevesicles with amphiphiles at 25 �C in a Molecular Devices M5instrument.
Acknowledgements
AB conceived the idea. AB and RS designed the experiments.MS, PB, AS synthesized the amphiphile molecules. SB per-formed all the experiments with E. coli and S. aureus. SB, SK, RSperformed experiments with mycobacteria. MS performed theLaurdan and DPH-based experiments with model membranes.VS performed AFM experiments. SB, MS, VS, RS, and ABanalyzed the data. AB and RS wrote the manuscript. AB and RSsupervised the overall research. The authors thank RCB,THSTI, DST and DBT for funding. SB thanks DBT for aResearch Fellowship. VS thanks RCB for a Research Fellowship.RS and AB thank DBT and DST for their Ramalingaswamy andRamanujan Fellowships, respectively. The authors are gratefulto Prof. Anil K. Tyagi, Delhi University, for access to BSL3facility.
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