Diphenylethylenediamine-Based Potent Anionophores

Subscriber access provided by Kaohsiung Medical University

Biological and Medical Applications of Materials and Interfaces

Diphenylethylenediamine-Based Potent Anionophores: Transmembrane Chloride Ion Transport and Apoptosis Inducing Activities Nasim Akhtar, Abhishek Saha, Vishnu Kumar, Nirmalya Pradhan, Subhankar Panda, Sudhir Morla, Sachin Kumar, and Debasis Manna ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06664 • Publication Date (Web): 17 Sep 2018 Downloaded from http://pubs.acs.org on September 18, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Diphenylethylenediamine-Based Potent Anionophores: Transmembrane Chloride Ion Transport and Apoptosis Inducing Activities Nasim Akhtar,§,† Abhishek Saha,§,† Vishnu Kumar,§,‡ Nirmalya Pradhan,† Subhankar Panda,† Sudhir Morla,‡ Sachin Kumar,‡ and Debasis Manna*,† †

‡

Department of Chemistry, Indian Institute of Technology Guwahati, Assam 781039, India

Department of Bioscience and Bioengineering, Indian Institute of Technology Guwahati, Assam 781039, India

KEYWORDS: Apoptosis • caspase-pathway • Cl─ ion transport • ion carrier • membranes

ABSTRACT

Synthetic anion transporters have been recognized as one of the potential therapeutic agents for the treatment of diseases including cystic fibrosis, myotonia and epilepsy that originate due to the malfunctioning of natural Cl─ ion transport systems. Recent studies showed that the synthetic Cl─ ion transporters can also disrupt cellular ion-homeostasis, and induce apoptosis in cancer cell lines, leading to a revived attention for synthetic Cl─ ion transporters. Herein, we report the development of conformationally controlled 1,2-diphenylethylenediamine based bis(thiourea) derivatives as a new class of selective Cl─ ion carrier. The strong Cl─ ion binding properties (Kd = 3.87-6.66 mM) of the ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 50

bis(thiourea) derivatives of diamine-based compounds correlates well with their transmembrane anion transport activities (EC50 = 2.09-4.15 nM). The transport of Cl─ ions via Cl─/NO3─ antiport mechanism was confirmed for the most active molecule. Perturbation of Cl─ ion homeostasis by this anion carrier induces cell death by promoting the caspase-mediated intrinsic pathway of apoptosis.

INTRODUCTION Protein-mediated ion transport systems assist ions in overcoming the hydrophobic lipid bilayer to readily pass through the cellular membranes.1 A network of noncovalent interactions originating from the backbone and side chains of these carrier or channel forming proteins allow them to recognize these selective ions. Explicitly, the transport of ions like Na+, K+, Ca2+, Cl─ and others across the membranes is essential in maintaining ion and pH-homeostasis, that regulates signaling pathways, proliferation, and other cellular processes.1-4 Chloride is one of the most abundant anions under the normal physiological conditions. Selective conductance of Cl─ ions (potential gradient of 30–60 mV for eukaryotic cell membranes) is associated with various biological processes including blood pressure regulation, chloride reabsorption, salt and fluid secretion, and electrical response generation.5-7 Malfunctioning of Cl─ ion transport proteins, primarily due to mutation(s), induce pathological conditions including myotonia, Barter’s syndrome, epilepsy, cystic fibrosis and others.8-12 It is anticipated that similar to synthetic cationophores, the anionophores might also exhibit several beneficial biological activities. Successful introduction of synthetic anionophores for fighting against cystic fibrosis has incited researchers in exploring their other probable biological applications. However, pertinent anion transporters have only recently been developed and their biological activities beyond the “channel replacement therapy” have not been well explored. Recent studies revealed that the transport of Cl─ ion is associated with the induction of apoptosis of the cancer cells by either disrupting the ionic homeostasis or altering the intracellular pH. It is presumed that the synthetic ion transporter can prevent the drug resistance capability of the cancer cells, which is because of the reversal of pH and Cl─ ion concentration in the cytosolic and extracellular region of the cancer cells in comparison with ACS Paragon Plus Environment

Page 3 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that of the normal cells. The anticancer activity of natural product prodigiosin and its analogues is directly related with their Cl─ transport efficiency.13-14

The squaramide based synthetic Cl─ ion

transporters also showed disruption in autophagy and elevation of apoptosis by perturbing Cl─ ion concentration in cancer cells.15 In this regard, the development of synthetic Cl─ ion transporters adept of mimicking the cellular functions of natural Cl─ ion transporters is anticipated to be one of the most promising approaches to fight against diseases associated with the malfunctioning of natural Cl─ ion channel systems and others. Most of the reported small molecule based artificial anion transporters contain simple surrogate, whose structure can be easily altered to achieve higher specificity/selectivity. These hydrophobic molecules generally contain urea, thiourea, squaramide, amide, hydroxyl, sulphonamide and other ionrecognition moieties.11-12,

16-25

Electrostatic force or non-covalent interactions including hydrogen

bonding, halogen bonding, anion-π/dipole are the driving force for their ion recognition and transport properties.16,

26-29

Thiourea is one of the common Cl─ ion recognition and transport moieties. The

lipophilicity of the thiourea moiety generates an incessant shield of lipophilic surface for the compounds within the membrane interior, assisting the ionophores with superior Cl─ ion transport activity. Thioureas are also good hydrogen bond donor for Cl─ ion and have lower pKa values of the NH protons.30 To have strong regulation capability over the localized concentration and suitable positioning of the lipophilic ion-recognition moieties, preorganized scaffolds such as cholapods, diureidodecalins, tris(aminomethyl)benzene and others have been employed as synthetic Cl─ ion transporters.22, 24, 31-33 Presence of these preorganized scaffolds enhances the anion transport efficiency of these thiourea derivatives by many-fold, because of its fixed binding site to form stable complex and lowering of ∆G value of the ion binding process. However, investigation involving the variation of preorganized scaffold and its consequences on anion transport efficacy is still in its infancy. A series of preorganized and flexible diamine-based compounds were already developed and their Cl─ ion transport

ACS Paragon Plus Environment


Page 4 of 50

properties were explored.22, 26, 34-36 Whereas, the roles of conformationally controlled diamine scaffold in Cl─ ion transport properties have not been explored. Herein, we report a new class of bis(thiourea) containing anion transporter with conformationally controlled

1,2-diphenylethylenediamine

(DPEN)

moiety.

Thiourea

derivatives

of

ortho-

phenylenediamine, and 1,2-diaminocyclohexane were also synthesized to understand the role of DPEN scaffold.34, 36 In addition, position and number of substituent on the aryl ring attached with the thiourea moiety were also altered to study the effect of substitution on the acidity of N-H proton as well as lipophilicity. The potent thiourea derivatives showed markedly higher selectivity for Cl─ ion in comparison with other tested biologically relevant anions. Mechanistic studies revealed that these compounds follow Cl─/NO3─ antiport pathway and carrier mechanism. These anionophores also induces measureable Cl─ transport efficiency in egg-yolk phosphatidylcholine (EYPC)/cholesterol (6:4) liposomes at level down to transporter: lipid ratio of 1: 750000. Transportation of Cl─ ion by these compounds into the intracellular region of the cancer cells induces apoptosis through caspase-dependent pathway.

RESULT and DISCUSSION Design and Synthesis of the bis(thiourea) Derivatives ─ The structures of the bis(thiourea) derivatives discussed herein are shown in Table 1. The use of DPEN scaffold provides a well-defined conformationally restrained environment. A small series of bis(thiourea)-based compounds (1a-f) with stereo-chemical variations of the DPEN scaffold were synthesized. To understand the importance of DPEN scaffold on anion recognition and transport activities bis(thiourea) derivatives of orthophenylenediamine (2), and 1,2-diaminocyclohexane (3) scaffold were also synthesized.34-36 Most of the reported artificial ion transporters are either structurally complex or require multi-steps for their synthesis.12, 16, 37 For prevalent use of these compounds, single-step synthesis is evidently desirable. The compounds 1a-f were synthesized through the condensation of DPEN with corresponding


Page 5 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


isothiocyanates.38-39 A similar reaction method was followed for the synthesis of compounds 2 and 3 from ortho-phenylenediamine and 1,2-diaminocyclohexane, respectively.35,

38-39

The characterizations

as well as the purity of the compounds were scrutinized by recording the 1H NMR,

13

C NMR and

HRMS spectroscopic data. The cLogP values of the compounds were calculated (cLogP = 7.11-8.80, 8.13 and 7.43 for compounds 1a-f, 2 and 3, respectively) using DataWarrior program (Table 1).40 Table 1. Structures and chemical properties of the compounds.

Compound

cLogPa

pKa1 (N-H)b 18.92

pKa2 (N-H)b 9.20

1b (R, R), R1 = CF3; 7.11 R2 = H

18.92

9.20

─

1c (S, S), R1 = CF3; 7.11 R2 = H

18.92

9.20

9.17

1d (Rac. Mix.), R1 = 8.80 H; R2 = CF3

18.94

8.97

6.62

1e (R, R), R1 = H; 8.80 R2 = CF3

18.94

8.97

─

1f (S, S), R1 = H; R2 8.80 = CF3

18.94

8.97

3.87

8.13

8.92

7.59

13.29

3 (Rac. Mix.), R1 = 7.43 H; R2 = CF3

19.18

8.96

22.37

1a (Rac. Mix.), R1 = 7.11 CF3; R2 = H

2, R1 = H; R2 = CF3

Kd (mM)c 13.32

a

cLogP values of the compounds were calculated using DataWarrior program. bpKa1 and pKa2 were calculated using the MarvinSketch 5.3.1 program. cKd values of the compounds were calculated using 1 HNMR titration.



Page 6 of 50

Anion Binding Studies of the Bis(thiourea) Derivatives ─ To investigate the strength and mode of interactions of the compound with the Cl─ ion the 1H NMR titration measurements were carried out in DMSO-d6 solvent. Tetrabutylammonium chloride (TBACl) was used as the source of Cl─ ion during these NMR titration measurements. Analysis of the 1H NMR titration spectra in the presence of increasing concentration of TBACl showed that, the signals of Hb and Hc protons of the compounds shifted towards downfield (Figure 1 and S1−6). These spectral changes indicate the presence of NHb…. Cl─ and N-Hc…. Cl─ interactions between the compound and Cl─ ion. The chemical shifts () for N-Hc proton were used to calculate the binding constant and stoichiometry of binding. The  for NHb proton were lower under the similar experimental conditions, which could be because of weaker interaction with the N-Hb protons. However, the binding affinity pattern calculated using the N-Hb proton is similar as the N-Hc proton (Table S1). In solution state the binding stoichiometry was determined by a continuous variation method of Job‟s plot, where the chemical shifts of N-Hc proton was used to calculate stoichiometry of binding (Table S2). This was done by varying the mole fractions (x) of the compounds.11 The chemical shift () of N-Hc proton was plotted against the mole fraction of compounds. The maximum interaction was observed at x = 0.5, which indicated a 1:1 binding (host/guest) stoichiometry of the selected compound (1d) with the Cl─ ion (Figure S7). Accordingly, the binding constants for the interaction of these compounds with Cl─ ion were calculated using WinEQNMR2 programme and the titration curves were fitted with inbuilt 1:1 binding model.41 The values obtained for the dissociation constants (Kd) of the compounds were within the range of 3.8722.37 mM. These dissociation constant values clearly suggest that the presence of trifluoromethyl group on the Ar-moieties was primarily responsible for the increasing acidity of the N-H proton as well as binding affinity for the Cl─ ion. It is well documented that the meso isomer of compound 3 showed poor Cl─ ion recognition capability over the R,R and S,S enantiomers.35 In this regard the Cl─ ion binding ability of the meso isomers of compounds were not investigated. We hypothesized that the compounds


Page 7 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


would remain as monomer in DMSO-d6 solvent. The Kd values from the 1H NMR titrations would not be significantly different among the enantiomers. Hence, the 1H NMR titration measurements of the R,R enantiomers were not performed. Overall, the ion recognition abilities of these compounds also indicate the importance of the presence of a well-defined conformationally restrained environment of the thiourea derivatives. Additional, Cl─ vs. NO3─ selectivity of the potent compound (1f) was also investigated using 1H NMR titration experiment (Figure S8).42 The proportional change in  values of N-Hb/c protons confirmed negligible/nonspecific binding of NO3─ ion (Kd could not be determined because of the unattainability of signal saturation even in the presence of >10 equiv. of TBANO3 salt) with the compounds in comparison with the Cl─ ion. The higher charge density of Cl─ ion in comparison with NO3─ ion could be the driving force for their stronger selectivity.42 Hence, these dissociation constant values of the compounds indicate that compound 1f have stronger binding affinity for Cl─ ion, due to presence of both DPEN and 1,3-bis(trifluoromethyl)phenyl moieties.

Figure 1. Representative 1H-NMR (600 mHz, room temperature) titration curve for compound 1f (5.3 mM) with TBACl in DMSO-d6 solvent. The amounts of added TBACl are shown on the spectra.



Page 8 of 50

X-ray Crystallographic Analysis ─ The Cl─ ion recognition properties and their mode of interactions with these compounds was further corroborated from the X-ray crystal structure analysis. The crystallization was performed with compound 1d (Rac. Mix) in the presence of excess TBACl salt as the source of Cl─ ion in DMSO solvent system at room temperature. However, the structural analysis showed that in a single asymmetric unit, two Cl─ ions interact with three units of the meso-isomer of the compound 1d and TBA+ (Table S3 and Figure S9). The observed coordination number of the Cl─ ion of this complex was found to be four. The space-filling model of the meso-compound 1d also demonstrated that two symmetric equivalent of the molecule were responsible for the recognition of the Cl─ ion (Figure 2). It is important to mention that the solid-state interaction pattern of the compound is quite different than that in the solution state (1:1 bind model). Further structural analysis revealed that the N-H groups of thiourea moiety were responsible for Cl─ ion recognition and all the measured bond distances were well within the distance of hydrogen bonding (all N-H-Cl bond distances were in the range of 2.370-2.390 A°). An interaction of Cl─ ion with the C-H bond of TBA+ was also observed within the hydrogen bond distance (C-H-Cl distance 2.909 A°). This X-ray crystallographic analysis provides the direct proof of Cl─ ion binding to the designed compounds.

Figure 2. Wireframe view of chloride and TBA encapsulated complex of compound 1d (A). Space-fill view of chloride ion (green ball) within the molecule-created cavity (B).


Page 9 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Chloride Ion Transport Activities of the bis(thiourea) Derivatives ─ The Cl─ ion binding properties and X-ray co-crystal structure of these compounds with TBACl prompted us to investigate their ion transport activity across the membrane bilayer of large unilamellar vesicles (LUVs). The transmembrane Cl─ ion transport efficacy of the compounds was evaluated using the well-established fluorescence-based assay.11-12,

43-45

The vesicles were prepared using egg-yolk phosphatidylcholine

(EYPC) and cholesterol (6:4 molar ratio) lipids with entrapped 8-hydroxypyrene-1,3,6-trisulfonate (HPTS, a pH sensitive dye, pKa = 7.2) dye in the presence of 20 mM HEPES buffer, pH 7.2, containing 100 mM NaCl to get EYPC/CHOL-LUVs⊃HPTS.11-12,

43

The compounds were added in the

extravesicular solution at compound/ lipid ratios of 1:25,000 and allowed to equilibrate with the vesicles. For HPTS assay the transport kinetics was initiated by generating a pH gradient (∆pH = ~0.5) using NaOH in the extravesicular solution and the increased fluorescence intensity of the deprotonated HPTS dye was monitored at λem = 510 nm (λex = 450 nm). To get the maximum HPTS fluorescence intensity, the vesicular arrangements of the lipids were completely disrupted by using Titron X-100. The Cl─ influx/efflux affected by H+ efflux or OH- influx contributes to the enhancement of pH at the intravascular environment of the vesicles, resulting in an increase of HPTS fluorescence intensity. So, the higher HPTS fluorescence intensity is the measure of higher Cl─ transport efficacy (Figure 3). Similarly, we performed lucigenin assay to measure the Cl─ ion transport efficacy of the compounds. The EYPC/CHOL-LUVs⊃lucigenin were prepared by encapsulat ing the lucigenin (a halide sensitive dye) within the LUVs composed of EYPC and cholesterol (6:4 molar ratio) in the presence of 20 mM HEPES buffer, pH 7.2, containing 100 mM NaNO3.44-45 For lucigenin assay the chloride transport kinetics was initiated by addition of NaCl in the extravesicular solution and the lucigenin florescence was monitored at λem = 506 nm (λex = 455 nm). The Cl─ influx resulted in replacement of NO3─ from the lucigenin and quenching of the fluorescence intensity of the dye. Maximum lucigenin fluorescence quenching was obtained by disrupting the vesicular arrangements of the lipids using Titron X-100.



Page 10 of 50

Figure 3. Schematic representation of fluorescence based ion transport activity assay using EYPC/CHOL-LUVs⊃HPTS (A) and EYPC/CHOL-LUVs⊃lucigenin (B). Comparisons of ion transport activity of 1a-1f (C) and 1f, 2 and 3 (E) across EYPC/CHOL-LUVs⊃HPTS. Comparisons of ion transport activity of 1a-1f (D) and 1f, 2 and 3 (F) across EYPC/CHOLLUVs⊃lucigenin. Compound/lipid ratio of 1:25000.


10

Page 11 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hence, the higher lucigenin fluorescence quenching is the measure of higher Cl─ ion transport efficacy (Figure 3). The HPTS assay for Cl─ ion transportation was performed at different ratios of compound/lipid, using EYPC/cholesterol (6:4) liposomes. Figure S14-15 showed that compounds 1e and 1f are capable of inducing measureable Cl─ transport efficiency even at the compound/lipid ratio of 1:7,50,000. However, compound/lipid ratio of 1:25,000 was selected for further studies because of their maximum transportation efficacy under the similar experimental conditions. All tested compounds showed significant fluorescence intensity change for both HPTS and lucigenin dyes, indicating their transport efficiency across the bilayer of the vesicles. The change in both HPTS and lucigenin fluorescence signals revealed that, bis(trifluoromethyl) substituted compounds have much higher Cl─ ion transport efficacies than the other tested compounds under the similar experimental conditions. The higher transport efficacy of 1d-f in comparison with the other tested compounds indicates the role of lipophilicity and acidity of N-H protons on Cl─ ion transport efficiency. The compounds 1d-f are stereoisomers and they showed very similar Cl─ ion binding and transport activities, hence only 1f was selected for further ion transport activity studies. It is important to mention that the potent compounds follow exponential decay kinetics for their transmembrane transportation of Cl─ ion (Figure 3C-F). However, a rapid initial increase/decrease in fluorescence intensity, followed by slower kinetics was repeatedly observed for both HPTS and lucigenin assay which could be due to the higher lipophilicity and stronger acidity of N-H protons (Table 1). It is well documented that, anionic lipids are highly abundant both in the membranes of eukaryotic and prokaryotic cells.46-48 In this regard, additional Cl─ transport

efficacy

of

the

compounds

was

investigated

using

EYPC/DPPS/CHOL-

LUV⊃lucigenin (EYPC/DPPS/cholesterol at a molar ratio of 4:2:4) to understand their ion


11


Page 12 of 50

transport efficacy under the physiological conditions (Figure 4). The kinetics parameters were calculated according to the reported method.11-12. The half-life and rate constants were calculated using first-order exponential decay kinetics.11-12 The initial rate constants were compared to investigate the efficiency of the Cl─ ion transport ability of the tested compounds. Analysis of the transport kinetics behavior in the presence of compound 1f showed around 1.7-fold decrease in the Cl─ ion transport rate when the anionic lipids were introduced in the liposomes (Table S4). These transport kinetic parameters of compound 1f in the absence and presence of anionic lipids suggests that these compounds would have moderate Cl─ ion transport activity under the physiological conditions. A similar Cl─ ion transport behavior was observed for compound 1e in the presence of anionic lipids (Figure S16 and Table S4).

Figure 4. Comparisons of ion transport activity of compound 1f (Compound/lipid ratio of 1:25000) across EYPC/CHOL-LUVs⊃lucigenin and EYPC/DPPS/CHOL-LUV⊃lucigenin. Dose-dependent HPTS fluorescence assays were used to calculate the EC50 value and Hill coefficient (n, number of compounds associated with a single ion) for the compounds (Figure


12

Page 13 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S19-26). The calculated EC50 values of the compounds were found to be very low and in the nanomolar range (Table 2). The Hill coefficients were found to be around 1, indicating the Cl─ ion recognition capabilities of the compounds (1:1 binding) in the solution phase (Table 2). The EC50 values of the compounds correlate well with the Kd and cLogP values of the compounds (Table 1 and 2). The bis(trifluoromethyl) substituted compounds with better Cl─ binding ability (Kd = 3.87-6.66 mM) and higher membrane permeability (cLogP = 8.80) showed also very strong Cl─ transport activity (EC50 = 2.09-4.15 nM). Whereas, ion transport activity of compound 3 (EC50 = 98 nM) was poor in comparison with 1d-f. Interestingly, the compounds 1d-f and 3 have identical Ar-moieties and comparable permeability (Table 1), but weaker Cl─ binding affinity (Kd = 22.37 mM). The acidity of N-H protons of the thiourea moieties is lower for compounds 1d-f in comparison with compounds 2 and 3. Table 2. Cl─ Ion transport activities of the compounds under liposomal conditions.

Entry

% Cl─ ion transport activity (lipid EC50 and compound ratio of 1 : 25,000)[a] (nM) HPTS assay Lucigenin assay

1a

20.46 ± 2.38

23.02 ± 4.56

48.43 ± 7.9

1.675

1b

25.74 ± 2.76

30.02 ± 3.41

52.39 ± 3.6

0.931

1c

27.78 ± 3.32

30.84 ± 2.51

45.89 ± 4.1

0.977

1d

74.98 ± 4.82

71.00 ± 2.70

2.09 ± 0.2

1.347

1e

80.83 ± 5.29

78.90 ± 3.00

4.15 ± 0.5

0.761

1f

82.63 ± 4.46

80.38 ± 4.60

2.51 ± 0.2

1.394

2

22.83 ± 2.40

33.43 ± 2.91

36.62 ± 4.5

0.950

3

9.17 ± 1.32

20.87 ± 2.03

97.52 ± 11.7 0.851

[a]

Hill Co-efficient (n value)

% Cl─ ion transport activities were compared at compound/lipid ratio of 1:25000.


13


Page 14 of 50

The stability of LUVs in the absence and presence of compounds was confirmed by transmission electron microscopy (TEM).46 Additional dynamic light scattering (DLS) measurements also support the stability of LUVs (Figure S27).46 Leaching test confirmed that the compounds exclusively reside in the lipophilic membrane-bilayer environment (Figure S28 and Table S5).49 Hence, the Cl─ ion transportation by these compounds indicates that the conformation control due to the presence of diphenyl rings in the diamine-scaffold is crucial for better Cl─ ion transport activity for compounds 1d-f. For the similar reason compounds 2 and 3 showed poor Cl─ ion transport activity than compounds 1d-f.

Relative Cation and Anion Selectivity Studies of the bis(thiourea) Derivatives ─ The higher Cl─ ion transport efficacy of the compounds inspired us to explore their Cl─ ion selectivity in comparison with various other biologically relevant cations and anions. Anion selectivity was examined by monitoring anion transport activity across EYPC/CHOL-LUVs⊃HPTS bilayer with NaCl at the intravesicular region and iso-osmotic buffer solution of sodium salt of various anions (Cl─, Br─, I─, NO3─, ClO4─, and SO42─) at the extravesicular region. Similarly, iso-osmotic solutions of the chloride salt of various cations (Li+, Na+, K+, Ca2+, and Mg2+) in the extravesicular region were used to investigate the selectivity of various cations. The relative change in the HPTS fluorescence signal in the presence of compounds 1e and 1f (at compound/ lipid ratios of 1:25,000) under the similar experimental conditions indicates their ion recognition and transmembrane transport activity. These tested compounds 1e and 1f follows an anion transport efficiency in the order of Cl─ > ClO4─ > I─ > Br─ > NO3─ > SO42─ (Figure 5 and S2930). This Cl─ ion transport selectivity of the compounds is in accordance with their respective ion binding affinity. We also observed that compound 1f showed higher transport selectivity for


14

Page 15 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cl─ ion over NO3─ ion. For Cl─ ion transportation the calculated ri = 0.0726 s-1 and t1/2 = 9.56 s, whereas for NO3─ ion transportation the ri = 0.0213 s-1 and t1/2 = 32.57 s under the similar experimental conditions. This Cl─ vs. NO3─ ion selectivity is in accordance with their binding affinities (measured from the 1H-NMR titration measurements). The results also showed that in the presence of compounds the monovalent (Li+, Na+, and K+) and divalent (Ca2+ and Mg2+) cations follow comparable transport rate regardless of the nature of the cation. This indicates no substantial role of these cations on Cl─ ion transportation. The moderate cations transport capabilities of these compounds suggest the possibility of nonspecific cations recognition through their alternative binding motif present in the molecular cavity.50 Anion „jump‟ assay also displayed the similar order of anion selectivity of the compounds (Figure S31).

Figure 5. Anion (A) and cation (B) transport selectivity of compound 1f across EYPC/CHOLLUVs⊃HPTS. Mechanism of Chloride Ion Transport activities of the bis(thiourea) Derivatives ─ In the lucigenin–based Cl─ ion transport assay a Cl─/NO3─ gradient was constructed and influx of Cl─ ion into the vesicles was measured by monitoring the decrease of lucigenin fluorescence. This transmembrane Cl─ ion transportation into the NO3─ containing vesicles incited us to investigate its transport mechanism. To retain the overall charge neutrality, the transportation of Cl─ ion may


15


Page 16 of 50

proceed through Cl─/Na+ symport or Cl─/NO3─ antiport pathway. In this regard, chloride salt of various cations (LiCl, NaCl, KCl, and RbCl) and sodium salt of various anions (NaBr, NaSCN, Na2SO4, NaClO4, and NaNO3,) were used in the extravesicular region to categorize the symport or antiport mechanistic pathway (Figure 6 and S32). Lucigenin assay of compound 1f in the

Figure 6. Influx of Cl─ ion across EYPC/CHOL-LUVlucigenin in the presence of compound 1f with intravesicular NaNO3 and extravesicular MCl (M = Li+, Na+, K+, and Rb+) salt (A). Efflux of Cl─ ion across EYPC/CHOL-LUVlucigenin in the presence of compound 1f with intravesicular NaCl and extravesicular NaxA (A- = Br─, SCN─, SO42─, ClO4─, NO3─; x = valency) salt (B). presence of these sodium salts of anions showed substantial difference of the Cl─ ion transport rates. However, similar experiments in the presence of these chloride salts of cations revealed trivial differences in the Cl─ ion transportation rates. These results substantiate the possibility of the Cl─/NO3─ antiport and rule out the prospect of Cl─/Na+ symport transport mechanistic pathway. For further validation of this Cl─/NO3─ antiport mechanistic pathway, we also performed lucigenin assay in the absence and presence of valinomycin (a K+ selective transporter).


16

Page 17 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EYPC/CHOL-LUV⊃lucigenin in HEPES buffer containing NaNO3 salt was used for this assay. The kinetic experiments were initiated with the addition of iso-osmolar KCl solution at the extravesicular region and the ion transport rate of the compound 1f was measured in the absence and presence of valinomycin. A significant increase in the anion transport rate in the presence of compound 1f could be due to the synergistic effect of the compound and valinomycin under the vesicular environment (Figure 7A). Numerical analysis showed that 10-fold increase in the Cl─ ion transport rate of compound 1f in the presence of valinomycin indicating their synergistic effect on Cl─ ion transportation across the bilayer (Figure 7B). This result clearly indicates the Cl─/NO3─ antiport mechanistic pathway. We hypothesize that the other potent compounds 1d and 1e would also follow similar transport mechanism.

Chloride Ion Transport Pathway of the bis(thiourea) Derivatives ─ Apart from the membrane lipids, most of the reported ion-binding small-molecules and supramolecules transport Cl─ ion across the biological membranes through mobile carrier or self-assembled channel pathway.1, 11-12, 14-16, 26, 44 To distinguish whether this bis(thiourea) derivative 1f transport anion through carrier or channel mechanism, lucigenin assay was performed using 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC) lipid at different temperatures. Transitions from the solid gel to liquid crystal phase are regarded to be a crucial biophysical property of lipids to investigate the thermal stability and small molecule/ion release profile of the vesicles. The randomness originated during phase transitions allow faster movement of the carriers, while the channels remain mostly unaffected.


17


Page 18 of 50

(B) Entry

Half-life (s)

Initial rate (s-1)

1f

168.17 ± 12.39

0.00412 ± 0.0012

16.80 ± 4.17

0.04126 ± 0.0003

1f + V[a] [a]

V = valinomycin

Figure 7. Comparision of Cl─ ion transport activity of compound 1f in the absence and presence of valinomycin (V) (A). Calculated half-life and initial rate of Cl─ transport activity of compound 1f (B). The phase transition temperature (Tm) of DPPC lipid is 41 °C, which indicates the increase in randomness of the lipid bilayer above this temperature.16, 46 The transport activity was measured in the absence and presence of compounds using DPPC-LUV⊃lucigenin in 20 mM HEPES buffer (at pH 7.2, containing NaNO3) at 25 °C and 45 °C (temperature below and above phase transition, respectively). A sharp increase in ion transport activity of compound 1f was observed when the temperature was raised from 25 to 45 °C (Figure 8 and S33). There is also a


18

Page 19 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Temperature dependent lucigenin assay to demonstrate the carrier-mechanistic pathway of Cl─ ion transport activity by the compound 1f across DPPC-LUV⊃lucigenin.

significant difference (around 4-fold) in the transport rates of the compound 1f at the two different temperatures (Table S6). A similar temperature dependent Cl─ ion transport activity was observed for compounds 1e (Figure S33). However, the Cl─ ion transport activity was poor in the absence of compounds. This strong correlation between Cl─ ion transport activity and membrane fluidity suggest its carrier-mechanistic pathway. We also performed additional cholesterol dependency assay to confirm Cl─ ion transport mechanism of the compounds (Figure S34).49 The results showed that an increase of cholesterol concentration in liposomes decreases the Cl─ ion transport activity, indicating the carrier movement of the compounds. Whereas in case of channel-based transport mechanism the Cl─ transport rate should remain unaffected.49

Ion Transport Activities under Cellular Environment ─ Disruptions in Cl─ ion transportation can alter cellular pH and lead to the onset of apoptosis and related diseases. Recent studies


19


Page 20 of 50

showed that synthetic Cl─ ion transporters induce apoptosis mediated activities in the cancer cells through interference of ionic homeostasis of the cell.25,

51

The strong Cl─ ion transportation

properties of these bis(thiourea) derivatives prompted us to explore their activity under the cellular environment. The viability of normal (baby hamster kidney BHK-21) and model cancer (human cervical cancer HeLa and human tongue carcinoma SAS) cell lines in the presence of these compounds correlates well with their Cl─ ion transport abilities. First, the viability of these cells was measured by MTT assay for compounds 1a-f, 2 and 3 at a fixed concentration (10 μM). For compounds 1e and 1f maximum cell death was observed. (Figure 9) The IC50 values of compounds 1e and 1f were in the range of 4.04-8.56 μM and 6.44-9.08 μM, respectively for all these tested cell lines (Figure S35 and Table S7). The differences in IC50 values of the compounds 1e and 1f among all the tested cell lines are very small. However, the anion transport efficacies of these compounds are very strong (EC50 values of the potent compounds < 10 nM). The faster growing cancer cells will presumably be more susceptible to these compounds (at lower concentration level) than will be the kidney and other normal cells, which grow more slowly. However, the IC50 values of compound 1e across the cell lines are slightly lower than compound 1f; hence it was selected for further cellular activity studies. To understand whether the cell viability of compound 1e is due to the transportation of Cl─ ion into the cells and change in intracellular Cl─ ion concentration, MTT assay was performed using HBSS (Hank‟s balanced salt solution) buffer in the absence and presence of Cl─ ion. MTT assay showed that the extent of viability in the presence of compound 1e is higher when the cells were suspended with Cl─ ion free HBSS buffer than the Cl─ ion containing HBSS buffer (Figure 10). This result suggests that intracellular transportation of Cl─ ion by compound 1e is directly related with the extent of cell death.


20

Page 21 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Cell viability of the compounds 1a-f, 2 and 3 was measured at a fixed concentration of 10 μM. Cell viability was measured in BHK-21, HeLa and SAS cells after 24 hours of compound treatment. All experiments were performed in triplicate. In general, Cl─ ion assisted cell death is recognized to initiate the cellular apoptosis pathway.1112, 23, 51-52

During apoptosis, a sequential change occurs in the cells and this apoptotic process can

be investigated by monitoring several cellular process e.g., (1) mitochondrial membrane integrity, (b) phosphatidylserine redistribution and plasma membrane integrity, (c) expression level of caspase family of proteins involved in caspase-dependent cascade pathways (d) nuclear fragmentation and DNA laddering of the cells.11-12, 23, 51-54 HeLa cells incubated with compound 1e show the morphological changes such as cell shrinkage and detachment from their neighboring cells (Figure S36). These observations indicate the cell death through apoptosis.55 Disruption of mitochondrial membrane potential (MMP) is considered as a pre-apoptotic indicator that can be monitored by using JC-1, a membrane potential sensitive dye.11-12, 25, 54 The decrease in red fluorescence signal of JC-1 dye due to the loss of its J-aggregates in the mitochondrial membrane indicates the disruption of MMP in the presence of compound 1e. The


21


Page 22 of 50

depolarization of the mitochondrial membrane results in diffusion of the JC-1 dye into the cytosol, resulting in green fluorescence.11-12, 25 HeLa cells were incubated with compound 1e (0-

Figure 10. Viability of HeLa cells in the absence and presence of Cl– ions in HBSS buffer upon dose dependent treatment of compound 1e (0-20 μM) after 24 h of incubation. Each bar represents the mean intensity of three independent experiments and the differences in mean intensities are statistically significant (P < 0.001 for according to two-way analysis of variance (ANOVA). 20 µM) for 24 h and the JC-1 dye was used to stain the cells (Figure 11 and S37). A stepwise reduction of the red fluorescence and enhancement of the green fluorescence signals of the JC-1 dye were observed in a concentration dependent manner indicating the disruption of MMP in the presence of compound 1e. Nuclear condensation, fragmentation, and formation of apoptotic bodies are also considered as hallmark of the late phase of apoptosis.56-57 The nuclear morphology of HeLa cells in the presence and absence of compound 1e (10 µM) shows significant changes that were investigated


22

Page 23 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


using Hoechst 33342 dye (Figure 12 and S38). The presence of nuclear condensation and fragmentation indicated the death of cells due to apoptosis.

Figure 11. Representatives images of HeLa cells in the presence of 0 (A), 5(B), 10 (C) and 20 μM (D) of compound 1e after 24 hours of incubation. Cells were stained with JC-1 dye. Both red and green channels were merged in each image. This decrease in MMP instigates the release of cytochrome c to the cytoplasm where it binds to apaf-1 protein to form apoptosome.11-12,

53-54

Caspases have proteolytic activity and once

activated they irreversibly induce the programmed cell death. These proenzymes are generally divided into two categories, the initiator and effector caspases. The initiator caspases leads to a cascade of downstream signal activation. Whereas, the activation of caspase 9 by cytochrome c forms apoptosome leading to intrinsic pathway of apoptosis.11-12, 53, 58-60 Apoptosis can also be initiated by a caspase-8 mediated extrinsic pathway.60-61 However, both caspase 8 and 9 activate caspase-3, a common effector and essential intermediate among all caspases. In this regard, the


23


Page 24 of 50

Figure 12. Compound 1e induced apoptosis leads to nuclear fragmentation and release of apoptotic body in HeLa cells. Nuclear fragmentation of HeLa cells in the presence of compound 1e stained by Hoechst 33342 dye after 24 h incubation (hpi). The nucleus is intact in mockinfected cells (A), while it showed fragmentation following infection with 10 μM of compound 1e (B and C). The DNA laddering pattern in HeLa cells infected with compound 1e (D). expressions of the caspase enzymes in the HeLa cells were examined by immunoblot analysis for further understanding of the mitochondria-dependent pathway of apoptosis. The expression level of initiator caspase-9, caspase-3 and caspase-8 were investigated in the presence of compound 1e in a dose-dependent manner (0-20 µM). Presence of cleaved caspase-9 and degradation of procaspase-3 indicates the caspase-dependent intrinsic pathway of apoptosis in the presence of compound 1e (Figure 13). However, there is no activation of caspase-8 and no degradation of procaspase-8 which overlooks the prospect of extrinsic pathway of apoptosis (Figure 13). In apoptotic cells, active caspase-3 mediated proteolysis cleaves protein substrates into fragments. Poly(ADP-ribose) polymerase (PARP) family of proteins is involved in number of


24

Page 25 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cellular functions such as DNA repair, expression of essential genes during inflammation and activation of chromatin remodeling enzymes and apoptosis.62 The presence of cleaved PARP is considered to be a hallmark of apoptosis in the presence of active caspase-3.63 Presence of inactive PARP (89 kDa) in HeLa cells (Figure 13) supports the caspase-mediated apoptosis in the presence of compound 1e (0-20 µM). Activation of caspase-3 also activates the cytosolic endonuclease CAD (or DFF40) by catalyzing the cleavage of its associated inhibitor ICAD (or DFF45). The active CAD enzyme enters into the nucleolus and degrades the DNA. Apoptotic DNA fragmentation is considered as one of the key features of the apoptosis and DNA laddering assay is being used for identification of apoptotic cells.64-66 Activation of the endogenous nucleases of the apoptotic cells cleaved the DNA into internucleosomal fragments in the multiples of about 180-bp oligomers, which appears as ladder on the agarose gel.64 Figure 12 demonstrates the classical DNA fragmentation in HeLa cells in the presence of compound 1e. Phosphatidylserine generally sequestered in the innerplasma membrane, but localize to the outer plasma membrane during apoptosis and activates non-inflammatory phagocytic recognition of the apoptotic cell.11,

53

Compound 1e induced

apoptotic property of HeLa cells was further reinforced by phosphatidylserine staining using fluorescein isothiocyanate-conjugated annexin-V, under the flow cytometer. Compound 1e (10 μM) treated HeLa cells showed that 4.06% and 11.42% cells were present in early and late apoptotic stages, respectively. Whereas, only 0.21% and 2.17% HeLa cells were present in an early and late apoptotic stages, respectively in the absence of the compound 1e (Figure S39). It delineates the apoptotic like mode of death in HeLa cells following the treatment of compound 1e (10 µM) for 12 h.


25


Page 26 of 50

Figure 13. Immunoblot analysis of HeLa cells in the presence of compound 1e after 24 h of incubation with different concentrations (0, 5, 10 and 20 μM). L represents mock-infected HeLa cells. Antibody against poly(ADP-ribose) polymerase (PARP), Procaspase-8, pro-caspase-3, cleaved caspase-9 and beta-actin were used to develop the blot. CONCLUSION Herein, we described that bis(thiourea) derivatives of conformationally controlled 1,2diphenylethylenediamin scaffold can function as anionophores with remarkable activities. The X-ray crystallographic study and 1H NMR titrations confirmed their strong anion recognition capability. However, in solution state the interaction pattern was found to follow 1:1 binding model. The Cl─ ion transport properties of the potent compounds are very significant with EC50 values in the range of 2.09-4.15 nM. The rapid and stronger Cl─ ion transport activity of these potent compounds could be due to their conformationally controlled structure and higher membrane permeability. The potent compounds are highly anion selective and transported Cl─ ion across the lipid bilayer via Cl─/NO3─ antiport mechanism. The dependence of high transport


26

Page 27 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activity on membrane rigidity indicates the carrier pathway of Cl─ ion transport under the liposomal environment. Viability of different cell lines indicates that Cl─ transport activity is inversely related with their IC50 values. Additional MTT assay comparing the cell viability in the absence and presence of Cl– ions in extracellular media confirmed the influx of Cl─ ion in the intracellular region of cells by the most active anionophore. The perturbation of Cl─ ion homeostasis of the cells altered the mitochondrial membrane potential indicating pre-apoptotic pathway of cell death. Immunoblot analysis of the expressed family of caspases and cleaved PARP validated the mitochondria-dependent intrinsic apoptotic pathway of cell death. The cellular expression level of caspase-9 and caspase-3 proteins indicate the intrinsic pathway of cell death in the presence of most active molecule. Caspase-dependent nuclear fragmentation by this anionophore was also observed as the post-apoptosis process. Hence, this bis(thiourea) derivatives of 1,2-diphenylethylenediamin scaffold are potent synthetic Cl─ ion transporters and act as apoptosis inducing agents, which could be an useful tool in understanding the complex relationship between Cl─ ion transport and various disease associated with chloride dysfunction.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental procedures, compound characterization data and biological activity data (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; Fax: +91 03 612582349; Tel: +91 03 612582325.


27


Page 28 of 50

ORCID Debasis Manna: 0000-0002-6920-9000 Author Contributions §

N.A., A.S. and V. K. contributed equally to this work.

Funding Sources The authors gratefully acknowledge Department of Biotechnology, Govt. of India (MED/2015/04) and Science and Engineering Research Board, Govt. of India (EMR/2016/005008) for financial support. Notes The authors declare no competing financial interest.. ACKNOWLEDGMENT The authors are thankful to Central Instrument Facility and Department of Chemistry for instrumental support. The authors also thank Mr. Kartick Chandra Majhi and Dr. Sreeparna Das of IIT Guwahati for their valuable discussions. ABBREVIATIONS DPEN, 1,2-diphenylethylenediamine; TBACl, Tetrabutylammonium chloride; HPTS, 8hydroxypyrene-1,3,6-trisulfonate; EYPC, egg-yolk phosphatidylcholine. REFERENCES (1) Gamper, N.; Shapiro, M. S. Regulation of Ion Transport Proteins by Membrane Phosphoinositides. Nat. Rev. Neurosci. 2007, 8, 921-934.


28

Page 29 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Diamond, J.; Wright, E. Citation-Classic - Biological-Membranes - the Physical Basis of Ion and Nonelectrolyte Selectivity. Cc/Life Sci. 1987, 36, 18-18. (3) Pinto, L. H.; Dieckmann, G. R.; Gandhi, C. S.; Papworth, C. G.; Braman, J.; Shaughnessy, M. A.; Lear, J. D.; Lamb, R. A.; DeGrado, W. F. A Functionally Defined Model for the M-2 Proton Channel of Influenza a Virus Suggests a Mechanism for Its Ion Selectivity. Proc. Natl. Acad. Sci. 1997, 94, 11301-11306. (4) Song, L. Z.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. Structure of Staphylococcal Alpha-Hemolysin, a Heptameric Transmembrane Pore. Science 1996, 274, 1859-1866. (5) Davis, A. P.; Sheppard, D. N.; Smith, B. D. Development of Synthetic Membrane Transporters for Anions. Chem. Soc. Rev. 2007, 36, 348-357. (6) Choi, J. Y.; Muallem, D.; Kiselyov, K.; Lee, M. G.; Thomas, P. J.; Muallem, S. Aberrant Cftr-Dependent Hco3- Transport in Mutations Associated with Cystic Fibrosis. Nature 2001, 410, 94-97. (7) Duran, C.; Thompson, C. H.; Xiao, Q. H.; Hartzell, H. C. Chloride Channels: Often Enigmatic, Rarely Predictable. Annu. Rev. Physiol. 2010, 72, 95-121. (8) Vaughan-Jones, R. D.; Spitzer, K. W.; Swietach, P. Intracellular PH Regulation in Heart. J. Mol. Cell. Cardiol. 2009, 46, 318-331. (9) Dworakowska, B.; Dolowy, K. Ion Channels-Related Diseases. Acta Biochim. Pol. 2000, 47, 685-703.


29


Page 30 of 50

(10) Jentsch, T. J.; Hubner, C. A.; Fuhrmann, J. C. Ion Channels: Function Unravelled by Dysfunction. Nat. Cell Biol. 2004, 6, 1039-1047. (11) Saha, T.; Hossain, M. S.; Saha, D.; Lahiri, M.; Talukdar, P. Chloride-Mediated ApoptosisInducing Activity of Bis(Sulfonamide) Anionophores. J. Am. Chem. Soc. 2016, 138, 7558-7567. (12) Saha, T.; Dasari, S.; Tewari, D.; Prathap, A.; Sureshan, K. M.; Bera, A. K.; Mukherjee, A.; Talukdar, P. Hopping-Mediated Anion Transport through a Mannitol-Based Rosette Ion Channel. J. Am. Chem. Soc. 2014, 136, 14128-14135. (13) Sessler, J. L.; Eller, L. R.; Cho, W. S.; Nicolaou, S.; Aguilar, A.; Lee, J. T.; Lynch, V. M.; Magda, D. J. Synthesis, Anion-Binding Properties, and in Vitro Anticancer Activity of Prodigiosin Analogues. Angew. Chem. Int. Ed. 2005, 44, 5989-5992. (14) Gale, P. A.; Perez-Tomas, R.; Quesada, R. Anion Transporters and Biological Systems. Acc. Chem. Res. 2013, 46, 2801-2813. (15) Busschaert, N.; Park, S. H.; Baek, K. H.; Choi, Y. P.; Park, J.; Howe, E. N. W.; Hiscock, J. R.; Karagiannidis, L. E.; Marques, I.; Felix, V.; Namkung, W.; Sessler, J. L.; Gale, P. A.; Shin, I. A Synthetic Ion Transporter That Disrupts Autophagy and Induces Apoptosis by Perturbing Cellular Chloride Concentrations. Nat. Chem. 2017, 9, 667-675. (16) Behera, H.; Madhavan, N. Anion-Selective Cholesterol Decorated Macrocyclic Transmembrane Ion Carriers. J. Am. Chem. Soc. 2017, 139, 12919-12922. (17) Valkenier, H.; Davis, A. P. Making a Match for Valinomycin: Steroidal Scaffolds in the Design of Electroneutral, Electrogenic Anion Carriers. Acc. Chem. Res. 2013, 46, 2898-2909.


30

Page 31 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Li, H. Y.; Valkenier, H.; Judd, L. W.; Brotherhood, P. R.; Hussain, S.; Cooper, J. A.; Jurcek, O.; Sparkes, H. A.; Sheppard, D. N.; Davis, A. P. Efficient, Non-Toxic Anion Transport by Synthetic Carriers in Cells and Epithelia. Nat. Chem. 2016, 8, 24-32. (19) Busschaert, N.; Kirby, I. L.; Young, S.; Coles, S. J.; Horton, P. N.; Light, M. E.; Gale, P. A. Squaramides as Potent Transmembrane Anion Transporters. Angew. Chem. Int. Ed. 2012, 51, 4426-4430. (20) Sidorov, V.; Kotch, F. W.; Kuebler, J. L.; Lam, Y. F.; Davis, J. T. Chloride Transport across Lipid Bilayers and Transmembrane Potential Induction by an Oligophenoxyacetamide. J. Am. Chem. Soc. 2003, 125, 2840-2841. (21) Berezin, S. K.; Davis, J. T. Catechols as Membrane Anion Transporters. J. Am. Chem. Soc. 2009, 131, 2458-2459. (22) Valkenier, H.; Judd, L. W.; Li, H.; Hussain, S.; Sheppard, D. N.; Davis, A. P. Preorganized Bis-Thioureas as Powerful Anion Carriers: Chloride Transport by Single Molecules in Large Unilamellar Vesicles. J. Am. Chem. Soc. 2014, 136, 12507-12512. (23) Ko, S. K.; Kim, S. K.; Share, A.; Lynch, V. M.; Park, J.; Namkung, W.; Van Rossom, W.; Busschaert, N.; Gale, P. A.; Sessler, J. L.; Shin, I. Synthetic Ion Transporters Can Induce Apoptosis by Facilitating Chloride Anion Transport into Cells. Nat. Chem. 2014, 6, 885-892. (24) Valkenier, H.; Dias, C. M.; Goff, K. L. P.; Jurcek, O.; Puttreddy, R.; Rissanen, K.; Davis, A. P. Sterically Geared Tris-Thioureas; Transmembrane Chloride Transporters with Unusual Activity and Accessibility. Chem. Commun. 2015, 51, 14235-14238.


31


Page 32 of 50

(25) Ko, S. K.; Kim, S. K.; Share, A.; Lynch, V. M.; Park, J.; Namkung, W.; Van Rosso, W.; Busschaert, N.; Gale, P. A.; Sessler, J. L.; Shin, I. Synthetic Ion Transporters Can Induce Apoptosis by Facilitating Chloride Anion Transport into Cells. Nat. Chem. 2014, 6, 885-892. (26) Busschaert, N.; Caltagirone, C.; Van Rossom, W.; Gale, P. A. Applications of Supramolecular Anion Recognition. Chem. Rev. 2015, 115, 8038-8155. (27) Deng, G.; Dewa, T.; Regen, S. L. A Synthetic Ionophore That Recognizes Negatively Charged Phospholipid Membranes. J. Am. Chem. Soc. 1996, 118, 8975-8976. (28) Izzo, I.; Licen, S.; Maulucci, N.; Autore, G.; Marzocco, S.; Tecilla, P.; De Riccardis, F. Cationic Calix[4] Arenes as Anion-Selective Ionophores. Chem. Commun. 2008, 26, 2986-2988. (29) Jentzsch, A. V.; Hennig, A.; Mareda, J.; Matile, S. Synthetic Ion Transporters That Work with Anion-Pi Interactions, Halogen Bonds, and Anion-Macrodipole Interactions. Acc. Chem. Res. 2013, 46, 2791-2800. (30) Valkenier, H.; Mora, N. L.; Kros, A.; Davis, A. P. Visualization and Quantification of Transmembrane Ion Transport into Giant Unilamellar Vesicles. Angew. Chem. Int. Ed. 2015, 54, 2137-2141. (31) McNally, B. A.; Koulov, A. V.; Lambert, T. N.; Smith, B. D.; Joos, J. B.; Sisson, A. L.; Clare, J. P.; Sgarlata, V.; Judd, L. W.; Magro, G.; Davis, A. P. Structure-Activity Relationships in Cholapod Anion Carriers: Enhanced Transmembrane Chloride Transport through Substituent Tuning. Chem. Eur. J. 2008, 14, 9599-9606. (32) Hussain, S.; Brotherhood, P. R.; Judd, L. W.; Davis, A. P. Diaxial Diureido Decalins as Compact, Efficient, and Tunable Anion Transporters. J. Am. Chem. Soc. 2011, 133, 1614-1617.


32

Page 33 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(33) Marti, I.; Rubio, J.; Bolte, M.; Burguete, M. I.; Vicent, C.; Quesada, R.; Alfonso, I.; Luis, S. V. Tuning Chloride Binding, Encapsulation, and Transport by Peripheral Substitution of Pseudopeptidic Tripodal Small Cages. Chem. Eur. J. 2012, 18, 16728-16741. (34) Karagiannidis, L. E.; Haynes, C. J. E.; Holder, K. J.; Kirby, I. L.; Moore, S. J.; Wells, N. J.; Gale, P. A. Highly Effective yet Simple Transmembrane Anion Transporters Based Upon Ortho-Phenylenediamine Bis-Ureas. Chem. Commun. 2014, 50, 12050-12053. (35) Karagiannidis, L. E.; Hiscock, J. R.; Gale, P. A. The Influence of Stereochemistry on Anion Binding and Transport. Supramol. Chem. 2013, 25, 626-630. (36) Moore, S. J.; Haynes, C. J. E.; Gonzalez, J.; Sutton, J. L.; Brooks, S. J.; Light, M. E.; Herniman, J.; Langley, G. J.; Soto-Cerrato, V.; Perez-Tomas, R.; Marques, I.; Costa, P. J.; Felix, V.; Gale, P. A. Chloride, Carboxylate and Carbonate Transport by Ortho-PhenylenediamineBased Bisureas. Chem. Sci. 2013, 4, 103-117. (37) Share, A. I.; Patel, K.; Nativi, C.; Cho, E. J.; Francesconi, O.; Busschaert, N.; Gale, P. A.; Roelens, S.; Sessler, J. L. Chloride Anion Transporters Inhibit Growth of Methicillin-Resistant Staphylococcus Aureus (Mrsa) in Vitro. Chem. Commun. 2016, 52, 7560-7563. (38) Wu, M. Y.; He, W. W.; Liu, X. Y.; Tan, B. Asymmetric Construction of Spirooxindoles by Organocatalytic Multicomponent Reactions Using Diazooxindoles. Angew. Chem. Int. Ed. 2015, 54, 9409-9413. (39) Bian, G. L.; Fan, H. J.; Yang, S. W.; Yue, H. F.; Huang, H. Y.; Zong, H.; Song, L. A Chiral Bisthiourea as a Chiral Solvating Agent for Carboxylic Acids in the Presence of Dmap. J. Org. Chem. 2013, 78, 9137-9142.


33


Page 34 of 50

(40) Sander, T.; Freyss, J.; von Korff, M.; Rufener, C. Data Warrior: An Open-Source Program for Chemistry Aware Data Visualization and Analysis. J. Chem. Inf. Model 2015, 55, 460-473. (41) Hynes, M. J. Eqnmr - a Computer-Program for the Calculation of Stability-Constants from Nuclear-Magnetic-Resonance Chemical-Shift Data. J. Chem. Soc. Dalton 1993, 2, 311-312. (42) Yang, Y. F.; Wu, X.; Busschaert, N.; Furuta, H.; Gale, P. A. Dissecting the ChlorideNitrate Anion Transport Assay. Chem. Commun. 2017, 53, 9230-9233. (43) Gorteau, V.; Bollot, G.; Mareda, J.; Perez-Velasco, A.; Matile, S. Rigid Oligonaphthalenediimide Rods as Transmembrane Anion-Pi Slides. J. Am. Chem. Soc. 2006, 128, 14788-14789. (44) Gale, P. A.; Davis, J. T.; Quesada, R. Anion Transport and Supramolecular Medicinal Chemistry. Chem. Soc. Rev. 2017, 46, 2497-2519. (45) Valkenier, H.; Haynes, C. J. E.; Herniman, J.; Gale, P. A.; Davis, A. P. Lipophilic Balance - a New Design Principle for Transmembrane Anion Carriers. Chem. Sci. 2014, 5, 1128-1134. (46) Saha, A.; Panda, S.; Paul, S.; Manna, D. Phosphate Bioisostere Containing Amphiphiles: A Novel Class of Squaramide-Based Lipids. Chem. Commun. 2016, 52, 9438-9441. (47) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane Lipids: Where They Are and How They Behave. Nat. Rev. Mol. Cell Bio. 2008, 9, 112-124. (48) Manna, D.; Albanese, A.; Park, W. S.; Cho, W. Mechanistic Basis of Differential Cellular Responses

of

Phosphatidylinositol

3,4-Bisphosphate-

and

Phosphatidylinositol

3,4,5-

Trisphosphate-Binding Pleckstrin Homology Domains. J. Biol. Chem. 2007, 282, 32093-32105.


34

Page 35 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(49) Lisbjerg, M.; Valkenier, H.; Jessen, B. M.; Al-Kerdi, H.; Davis, A. P.; Pittelkow, M. Biotin[6]Uril Esters: Chloride-Selective Transmembrane Anion Carriers Employing C-H Center Dot Center Dot Center Dot Anion Interactions. J. Am. Chem. Soc. 2015, 137, 4948-4951. (50) Bochenska, M.; Biernat, J. F.; Topolski, M.; Bradshaw, J. S.; Bruening, R. L.; Izatt, R. M.; Dalley, N. K. Solvent-Extraction Studies of New Bis-Sulfonamide Group-Containing Podands. J. Inclusion Phenom. 1989, 7, 599-611. (51) Tsukimoto, M.; Harada, H.; Ikari, A.; Takagi, K. Involvement of Chloride in Apoptotic Cell Death Induced by Activation of Atp-Sensitive P2x(7) Purinoceptor. J. Biol. Chem. 2005, 280, 2653-2658. (52) Soto-Cerrato, V.; Manuel-Manresa, P.; Hernando, E.; Calabuig-Farinas, S.; MartinezRomero, A.; Fernandez-Duenas, V.; Sahlholm, K.; Knopfel, T.; Garcia-Valverde, M.; Rodilla, A. M.; Jantus-Lewintre, E.; Farras, R.; Ciruela, F.; Perez-Tomas, R.; Quesada, R. Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells. J. Am. Chem. Soc. 2015, 137, 15892-15898. (53) Kumar, U.; Kumar, S. Molecular Characterization of an Apoptotic Strain of Newcastle Disease Virus Isolated from an Outbreak in India. Cancer Gene Ther. 2015, 22, 402-409. (54) Ly, J. D.; Grubb, D. R.; Lawen, A. The Mitochondrial Membrane Potential (∆Ψm) in Apoptosis; an Update. Apoptosis 2003, 8, 115-128. (55) Nunez, R.; Sancho-Martinez, S. M.; Novoa, J. M. L.; Lopez-Hernandez, F. J. Apoptotic Volume Decrease as a Geometric Determinant for Cell Dismantling into Apoptotic Bodies. Cell Death Differ. 2010, 17, 1665-1671.


35


Page 36 of 50

(56) Park, S. H.; Choi, Y. P.; Park, J.; Share, A.; Francesconi, O.; Nativi, C.; Namkung, W.; Sessler, J. L.; Roelens, S.; Shin, I. Synthetic Aminopyrrolic Receptors Have Apoptosis Inducing Activity. Chem. Sci. 2015, 6, 7284-7292. (57) Busschaert, N.; Wenzel, M.; Light, M. E.; Iglesias-Hernandez, P.; Perez-Tomas, R.; Gale, P. A. Structure-Activity Relationships in Tripodal Transmembrane Anion Transporters: The Effect of Fluorination. J. Am. Chem. Soc. 2011, 133, 14136-14148. (58) Chu, Z. L.; Pio, F.; Xie, Z. H.; Welsh, K.; Krajewska, M.; Krajewski, S.; Godzik, A.; Reed, J. C. A Novel Enhancer of the Apaf1 Apoptosome Involved in Cytochrome C-Dependent Caspase Activation and Apoptosis. J. Biol. Chem. 2001, 276, 9239-9245. (59) Henderson, C. J.; Aleo, E.; Fontanini, A.; Maestro, R.; Paroni, G.; Brancolini, C. Caspase Activation and Apoptosis in Response to Proteasome Inhibitors. Cell Death Differ. 2005, 12, 1240-1254. (60) Ashkenazi, A. Directing Cancer Cells to Self-Destruct with Pro-Apoptotic Receptor Agonists. Nat Rev Drug Discov. 2008, 7, 1001-1012. (61) de Vries, E. G. E.; Gietema, J. A.; de Jong, S. Tumor Necrosis Factor-Related ApoptosisInducing Ligand Pathway and Its Therapeutic Implications. Clin. Cancer Res. 2006, 12, 23902393. (62) Boulares, A. H.; Yakovlev, A. G.; Ivanova, V.; Stoica, B. A.; Wang, G. P.; Iyer, S.; Smulson, M. Role of Poly(Adp-Ribose) Polymerase (Parp) Cleavage in Apoptosis - Caspase 3Resistant Parp Mutant Increases Rates of Apoptosis in Transfected. Cells J. Biol. Chem. 1999, 274, 22932-22940.


36

Page 37 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(63) Tewari, M.; Quan, L. T.; Orourke, K.; Desnoyers, S.; Zeng, Z.; Beidler, D. R.; Poirier, G. G.; Salvesen, G. S.; Dixit, V. M. Yama/Cpp32-Beta, a Mammalian Homolog of Ced-3, Is a Crma-Inhibitable Protease That Cleaves the Death Substrate Poly(Adp-Ribose) Polymerase. Cell 1995, 81, 801-809. (64) Liu, X. S.; Zou, H.; Slaughter, C.; Wang, X. D. Dff, a Heterodimeric Protein That Functions Downstream of Caspase-3 to Trigger DNA Fragmentation During Apoptosis. Cell 1997, 89, 175-184. (65) Liu, X. S.; Li, P.; Widlak, P.; Zou, H.; Luo, X.; Garrard, W. T.; Wang, X. D. The 40-Kda Subunit of DNA Fragmentation Factor Induces DNA Fragmentation and Chromatin Condensation During Apoptosis. Proc. Natl. Acad. Sci. USA 1998, 95, 8461-8466. (66) Sakahira, H.; Enari, M.; Nagata, S. Cleavage of Cad Inhibitor in Cad Activation and DNA Degradation During Apoptosis. Nature 1998, 391, 96-99.

Table of Contents (TOC)


37


Representative 1H-NMR (600 mHz, room temperature) titration curve for compound 1f (5.3 mM) with TBACl in DMSO-d6 solvent. The amounts of added TBACl are shown on the spectra. 218x178mm (300 x 300 DPI)


Page 38 of 50

Page 39 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Wireframe view of chloride and TBA encapsulated complex of compound 1d (A). Space-fill view of chloride ion (green ball) within the molecule-created cavity (B). 290x137mm (300 x 300 DPI)



Schematic representation of fluorescence based ion transport activity assay using EYPC/CHOL-LUVs⊃HPTS (A) and EYPC/CHOL-LUVs⊃lucigenin (B). Comparisons of ion transport activity of 1a-1f (C) and 1f, 2 and 3 (E) across EYPC/CHOL-LUVs⊃HPTS. Comparisons of ion transport activity of 1a-1f (D) and 1f, 2 and 3 (F) across EYPC/CHOL-LUVs⊃lucigenin. Compound/lipid ratio of 1:25000. 314x365mm (300 x 300 DPI)


Page 40 of 50

Page 41 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Comparisons of ion transport activity of compound 1f (Compound/lipid ratio of 1:25000) across EYPC/CHOLLUVs⊃lucigenin and EYPC/DPPS/CHOL-LUV⊃lucigenin. 151x132mm (300 x 300 DPI)



Anion (A) and cation (B) transport selectivity of compound 1f across EYPC/CHOL-LUVs⊃HPTS. 288x85mm (300 x 300 DPI)


Page 42 of 50

Page 43 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Influx of Cl─ ion across EYPC/CHOL-LUV⊃lucigenin in the presence of compound 1f with intravesicular NaNO3 and extravesicular MCl (M = Li+, Na+, K+, and Rb+) salt (A). Efflux of Cl─ ion across EYPC/CHOLLUV⊃lucigenin in the presence of compound 1f with intravesicular NaCl and extravesicular NaxA (A- = Br─, SCN─, SO42─, ClO4─, NO3─; x = valency) salt (B). 95x34mm (300 x 300 DPI)



Comparision of Cl─ ion transport activity of compound 1f in the absence and presence of valinomycin (V) (A). 165x206mm (300 x 300 DPI)


Page 44 of 50

Page 45 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Temperature dependent lucigenin assay to demonstrate the carrier-mechanistic pathway of Cl─ ion transport activity by the compound 1f across DPPC-LUV⊃lucigenin. 170x128mm (300 x 300 DPI)



Cell viability of the compounds 1a-f, 2 and 3 was measured at a fixed concentration of 10 µM. Cell viability was measured in BHK-21, HeLa and SAS cells after 24 hours of compound treatment. All experiments were performed in triplicate. 148x122mm (300 x 300 DPI)


Page 46 of 50

Page 47 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Viability of HeLa cells in the absence and presence of Cl– ions in HBSS buffer upon dose dependent treatment of compound 1e (0-20 µM) after 24 h of incubation. Each bar represents the mean intensity of three independent experiments and the differences in mean intensities are statistically significant (P < 0.001 for according to two-way analysis of variance (ANOVA). 156x135mm (300 x 300 DPI)



Representatives images of HeLa cells in the presence of 0 (A), 5(B), 10 (C) and 20 µM (D) of compound 1e after 24 hours of incubation. Cells were stained with JC-1 dye. Both red and green channels were merged in each image. 277x207mm (72 x 72 DPI)


Page 48 of 50

Page 49 of 50 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 1e induced apoptosis leads to nuclear fragmentation and release of apoptotic body in HeLa cells. Nuclear fragmentation of HeLa cells in the presence of compound 1e stained by Hoechst 33342 dye after 24 h incubation (hpi). The nucleus is intact in mock-infected cells (A), while it showed fragmentation following infection with 10 µM of compound 1e (B and C). The DNA laddering pattern in HeLa cells infected with compound 1e (D). 308x239mm (72 x 72 DPI)



Immunoblot analysis of HeLa cells in the presence of compound 1e after 24 h of incubation with different concentrations (0, 5, 10 and 20 µM). L represents mock-infected HeLa cells. Antibody against poly(ADPribose) polymerase (PARP), Procaspase-8, pro-caspase-3, cleaved caspase-9 and beta-actin were used to develop the blot. 231x122mm (300 x 300 DPI)


Page 50 of 50

Diphenylethylenediamine-Based Potent Anionophores

Recommend Documents