Symmetric dimeric adamantanes for exploring the structure of two viroporins: Influenza virus M2 and hepatitis C virus p7

Article abstract of DOI:10.2147/DDDT.S157104

Background: Adamantane-based compounds have been identified to interfere with the ion-channel activity of viroporins and thereby inhibit viral infection. To better understand the difference in the inhibition mechanism of viroporins, we synthesized symmetric dimeric adamantane analogs of various alkyl-spacer lengths. Methods: Symmetric dimeric adamantane derivatives were synthesized where two amantadine or rimantadine molecules were linked by various alkyl-spacers. The inhibitory activity of the compounds was studied on two viroporins: the influenza virus M2 protein, expressed in Xenopus oocytes, using the two-electrode voltage-clamp technique, and the hepatitis C virus (HCV) p7 channels for five different genotypes (1a, 1b, 2a, 3a, and 4a) expressed in HEK293 cells using whole-cell patch-clamp recording techniques. Results: Upon testing on M2 protein, dimeric compounds showed significantly lower inhibitory activity relative to the monomeric amantadine. The lack of channel blockage of the dimeric amantadine and rimantadine analogs against M2 wild type and M2-S31N mutant was consistent with previously proposed drug-binding mechanisms and further confirmed that the pore-binding model is the pharmacologically relevant drug-binding model. On the other hand, these dimers showed similar potency to their respective monomeric analogs when tested on p7 protein in HCV genotypes 1a, 1b, and 4a while being 700-fold and 150-fold more potent than amantadine in genotypes 2a and 3a, respectively. An amino group appears to be important for inhibiting the ion-channel activity of p7 protein in genotype 2a, while its importance was minimal in all other genotypes. Conclusion: Symmetric dimeric adamantanes can be considered a prospective class of p7 inhibitors that are able to address the differences in adamantane sensitivity among the various genotypes of HCV.

Full text of DOI:10.2147/DDDT.S157104

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O r i g i n a l r e s e a r c h
symmetric dimeric adamantanes for exploring the
structure of two viroporins: influenza virus M2
and hepatitis c virus p7
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
Yasmine M Mandour1
Ulrike Breitinger2
Chunlong Ma3
Background: Adamantane-based compounds have been identified to interfere with the ion-
channel activity of viroporins and thereby inhibit viral infection. To better understand the differ-
ence in the inhibition mechanism of viroporins, we synthesized symmetric dimeric adamantane
analogs of various alkyl-spacer lengths.
Jun Wang3
Methods: Symmetric dimeric adamantane derivatives were synthesized where two amantadine
or rimantadine molecules were linked by various alkyl-spacers. The inhibitory activity of the
compounds was studied on two viroporins: the influenza virus M2 protein, expressed in Xenopus
oocytes, using the two-electrode voltage-clamp technique, and the hepatitis C virus (HCV) p7
channels for five different genotypes (1a, 1b, 2a, 3a, and 4a) expressed in HEK293 cells using
whole-cell patch-clamp recording techniques.
Frank M Boeckler4
hans-georg Breitinger2
Darius P Zlotos1
1Department of Pharmaceutical
chemistry, german University in
cairo, 2Department of Biochemistry,
The german University in cairo,
cairo, egypt; 3Department of
Pharmacology and Toxicology,
University of Arizona,Tucson, AZ,
Usa; 4Department of Pharmaceutical
and Medicinal Chemistry, Eberhard
Karls University of Tübingen,
Tübingen, Germany
Results: Upon testing on M2 protein, dimeric compounds showed significantly lower inhibitory
activity relative to the monomeric amantadine. The lack of channel blockage of the dimeric aman-
tadine and rimantadine analogs against M2 wild type and M2-S31N mutant was consistent with
previously proposed drug-binding mechanisms and further confirmed that the pore-binding model
is the pharmacologically relevant drug-binding model. On the other hand, these dimers showed
similar potency to their respective monomeric analogs when tested on p7 protein in HCV geno-
types 1a, 1b, and 4a while being 700-fold and 150-fold more potent than amantadine in genotypes
2a and 3a, respectively. An amino group appears to be important for inhibiting the ion-channel
activity of p7 protein in genotype 2a, while its importance was minimal in all other genotypes.
Conclusion: Symmetric dimeric adamantanes can be considered a prospective class of p7
inhibitors that are able to address the differences in adamantane sensitivity among the various
genotypes of HCV.
Keywords: adamantane, dimers, viroporins, p7, M2, HCV
Introduction
Viroporins are small virus-encoded proteins that are able to permeabilize membranes
for ions and small molecules.1,2 This family of proteins includes M2,3 Vpu,4 the
E-protein channels of coronaviruses,5 and the p7 ion channel of hepatitis C virus
(HCV).6 Viroporins commonly are small transmembrane (TM) proteins that contain
several identical self-oligomerizing subunits forming functional ion channels. However,
sequence similarity among the viroporin family is low, and the functional channels
differ in subunit size, stoichiometry, and topology.7 Viroporins are crucial for viral
pathogenicity, owing to their involvement in several steps of the viral life cycle, but
their main activities are in cell entry, virion assembly, and release from infected cells.8
M2 protein of influenza virus equilibrates the pH across the viral membrane during cell
Correspondence: Yasmine M Mandour
Department of Pharmaceutical
chemistry, german University in cairo,
new cairo, cairo 11835, egypt
Tel +20 2 2759 0715
Fax +20 2 2758 1041
email yasmine.magdy@guc.edu.eg
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Mandour et al
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entry, facilitating RNA release,9,10 and across the trans-Golgi Structural studies have revealed two proposed structures
membrane of infected cells during viral maturation.11 The for the oligomeric form of p7. The first structure proposes a
p7 protein of HCV is believed to act as an intracellular pH funnel-like architecture, where the individual p7 monomer,
shunt, contributing to the deacidification of endosomes and i, interacts with i + 2 and i + 3 monomers, forming a hexameric
virion-loaded particles and assisting in assembly and release assembly.26 The second structure suggests a flower-shaped pro-
of virions,12 as well as promoting inflammatory signaling.13 tein architecture, where each unit interacts only with neighbor-
Owing to their role in the viral life cycle and their structural ing monomers.27 However, in both structures six adamantane
differences from human channels, these viral proteins are con- molecules are proposed to bind to six equivalent hydrophobic
sidered ideal antiviral drug targets.8 Several compounds that pockets formed by residues from two adjacent monomers.
block viroporin ion-channel activity have been identified. Two
Previous studies have shown dimeric camphor deriva-
adamantane based compounds, amantadine and rimantadine, tives, which have a cage-like structure similar to adamantane,
were the first US Food and Drug Administration-approved to possess high antiviral activity against influenza. It was pos-
drugs for treatment of influenza infections.14 These compounds tulated that two camphor moieties bind to two adjacent sites
inhibit the M2 protein by blocking its proton conductance.15,16 of neighboring chains of M2.28 However, these compounds
It has also been found that these adamantane-based com- were not tested on M2 protein, so the exact mechanism of
pounds can block HCV p7 ion-channel activity17–19 and abolish inhibition of these compounds is unknown. It is noted that
p7-mediated inflammatory signaling in liver macrophages.13 cage-shape molecules might inhibit influenza viruses through
Structure determination of viroporins under physiologi- M2-independent mechanisms.22 To further elucidate the M2
cal conditions has been difficult, due to the hydrophobic and drug-binding mechanism and rule out the possibility of the
dynamic nature of these proteins. The few available viroporin exterior interface-binding model, we hereby report the design
structures revealed unexpected architecture that helped in and characterization of dimeric amantadines and riman-
understanding the operation of these channels.8 In addition, the tadines. If the exterior interface-binding model is correct, the
binding mode of several ion-channel blockers was revealed, dimeric amantadines and rimantadines should have compa-
which provided guidance for designing other inhibitors.8 rable or better channel-blockage activity than the monomeric
Several structures of M2 protein in complex with adamantanes amantadine and rimantadine. If the pore-binding model is
have been solved. These structures showed M2 as a tetramer correct, the dimeric amantadines and rimantadines should not
and suggested two possible binding models for adamantanes: be able to fit in the channel and have no channel inhibition.
the pore-binding model and the exterior interface-binding In contrast, the structure of p7 protein suggests the presence
model.20,21 In the pore-binding model, one adamantane mol- of several identical adamantane-binding pockets where six
ecule physically blocks the ion channel, breaking the con- adamantane molecules are required to inhibit one p7 protein
tinuous water wires in the channel that are critical for proton molecule. Accordingly, combination of two adamantane
conductance.21 For the exterior interface-binding model, four moieties in one molecule might lead to supra-additive increase
adamantane molecules bind allosterically at an external, lipid- in potency compared to the corresponding monovalent
facing pocket between adjacent monomers, thus inducing con- ligands. To test this hypothesis, we synthesized symmetric
formational changes in the TM helices that favor the closed state dimeric adamantane analogs of various alkyl-spacer lengths
of the channel.20 It was later confirmed that the pore-binding to find the optimum distance that allows simultaneous binding
model is the pharmacologically relevant model that accounts of the two adamantane rings. Here, we describe the design
for the antiviral activity of amantadine and rimantadine.22,23 and synthesis of symmetric dimeric adamantane derivatives
Unfortunately, naturally occurring drug-resistant mutations of various alkyl-spacer lengths and the determination of
havegraduallydecreasedtheseadamantanes’inhibitionability. their inhibitory activity on two viroporins: M2 channels of
S31N is the predominant amantadine-resistant M2 form that influenza virus and the p7 protein of HCV.
represents a huge challenge to drug discovery.
Methods
The structure of p7 protein has been studied using
nuclear magnetic resonance (NMR) methods24 and molecular general chemistry
modeling,25 showing different potential topologies and con- All applied starting materials were commercially available
formations. P7 is a 63-residue protein with two TM domains from Alfa Aesar and Sigma-Aldrich and used as received.
(TMDs) that are separated by a short hydrophilic cytosolic Column chromatography: silica gel 60 (0.063–0.200 mm;
loop.24,25 The p7 protomer is able to oligomerize to hexameric21,24 Merck). Reaction and column-chromatography purifica-
and heptameric complexes,17 forming an ion channel. tion progress was monitored by TLC silica gel (60F154)
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Exploring influenza M2 and HCV p7 with adamantanes
aluminum sheets. Compound spots were visualized in an 1.53–1.58 (m, 2H, H-c), 1.60 (m, 6H, 4,6,10-adamantane
iodine chamber. Melting point (MP): capillary MP apparatus H), 1.93 (d, J=2.5 Hz, 6H, 2,9,8-adamantane H), 2.00 (br
1
(Sanyo Gallenkamp, UK), uncorrected. H and 13C NMR s, 3H, 3,5,7-adamantane H), 2.03 (t, J=6.4 Hz, 2H, H-b),
spectra were recorded on a Bruker AV-400 spectrometer (1H, 5.27 (s, 1H, NH). 13C NMR (CDCl3, 100 MHz) δ (ppm)
400.13 MHz; 13C, 100.61 MHz). Proton chemical shifts are 25.09 (C-c) 29.45 (3,5,7-adamantane C), 37.27 (C-b), 36.38
referenced to CHCl3 (7.26 ppm) or methanol-d4 (3.31 ppm). (4,6,10-adamantane C), 41.66 (2,8,9-adamantane C), 51.87
Coupling constants (J) are given in hertz. Carbon chemical (1-adamantane C), 172.11 (C-a). EI-MS: m/z 412.25 [M]+.
shifts are referenced to CDCl3 (77 ppm) or methanol-d4
N,N′-bis-(adamantan-1-yl) suberic acid diamide (1c)
Compound 1c (330 mg, 54%) was obtained as a white solid;
MP: 228°C. 1H NMR (CD3OD, 400 MHz) δ (ppm) 1.33–1.40
(m, 2H, d H), 1.55–1.63 (m, 2H, H-c), 1.73 (m, 6H, 4,6,10-
adamantane H), 2.04 (br s, 6H, 2,9,8-adamantane H), 2.06
(br s, 3H, 3,5,7-adamantane H), 2.11 (t, J=7.5 Hz, 2H, H-b).
13C NMR (CD3OD, 100 MHz) δ (ppm) 25.65 (C-d), 28.49
(C-c), 29.52 (3,5,7-adamantane C), 36.12 (4,6,10-adamantane
C), 36.48 (C-b), 40.97 (2,8,9-adamantane C), 51.87
(1-adamantane C), 172.3 (C-a). ESI-MS: m/z 441.30 [M+H]+.
(49 ppm) (Figures S1–S12). Electrospray ionization (ESI)
mass spectrometry (MS) was determined on an Agilent
1100 liquid-chromatography MS system in a positive mode.
Elemental analyses were performed by the microanalytical
section of the Institute of Inorganic Chemistry, Würzburg
University, and the results were within ±0.4% of the theo-
retical values.
Procedure a for synthesis of compounds
1a–e
To a stirred solution of the respective dicarboxylic acid
(1.4 mmol) in dry chloroform (20 mL), oxalyl chloride
(0.48 mL, 5.6 mmol) was added dropwise, followed by
addition of two drops of dry dimethylformamide. The reac-
tion mixture was left to stir at room temperature (RT) for
4 hours. Toluene (20 mL) was added to the reaction mixture
and evaporated under reduced pressure. The resultant acid
chloride was dissolved in dry CHCl3 (3 mL) and the solution
added dropwise to a stirred solution of amantadine (0.6 g,
4.0 mmol) and dry triethylamine (0.5 mL, 3.305 mmol) in
CHCl3 (30 mL). The reaction mixture was left to stir for
16 hours at RT under inert conditions and washed with diluted
HCl (2×30 mL). The combined organic layers were washed
with water, dried over sodium sulfate, and concentrated under
reduced pressure. The resulting crude product was pure
enough to be used directly for the next step without further
purification, as indicated by 1H NMR spectra.
N,N′-bis-(adamantan-1-yl) sebacic acid diamide (1d)
Compound 1d (430 mg, 66%) was obtained as a white solid;
1
MP: 228°C. H NMR (CDCl3, 400 MHz) δ (ppm) 1.22
(br s, 4H, H-d,e), 1.47–1.55 (m, 2H, H-c), 1.60 (m, 6H, 4,6,10-
adamantane H), 1.92 (d, J=2.7 Hz, 6H, 2,9,8-adamantane
H), 2.03 (t, J=7.4 Hz, 5H, 3,5,7-adamantane H, H-b), 5.04
(s, 1H, NH). 13C NMR (CDCl3, 100 MHz) δ (ppm) 25.73
(C-e), 29.09 (C-d), 29.14 (C-c), 29.46 (3,5,7-adamantane C),
36.39 (4,6,10-adamantane C), 37.77 (C-b), 41.73 (2,8,9-
adamantane C), 51.78 (1-adamantane C), 172.33 (C-a).
EI-MS: m/z 468.30 [M]+.
N,N′-bis-(adamantan-1-yl) dodecanedioic acid
diamide (1e)
Compound 1e (560 mg, 82%) was obtained as a white solid;
MP: 179°C. 1H NMR (CD3OD, 400 MHz) δ (ppm) 1.34 (br s,
6H, d-f H), 1.56–1.64 (m, 2H, H-c), 1.74 (m, 6H, 4,6,10-
adamantane H), 2.05 (d, J=2.5 Hz, 6H, 2,9,8-adamantane H),
2.08 (s, 3H, 3,5,7-adamantane H), 2.17 (t, J=6.4 Hz, 2H, H-b).
13C NMR (CD3OD, 100 MHz) δ (ppm) 27.28 (C-f ), 30.13
(C-e), 30.36 (d C), 30.48 (C-c), 30.91 (3,5,7-adamantane C),
37.46 (4,6,10-adamantane C), 37.62 (C-b), 42.25 (2,8,9-
adamantane C), 51.78 (1-adamantane C), 172.33 (C-a).
ESI-MS: m/z 497.4 [M+H]+.
N,N′-bis-(adamantan-1-yl) succinic acid diamide (1a)
Compound 1a (400 mg, 74%) was obtained as a white solid;
MP: 210°C. 1H NMR (CD3OD, 400 MHz) δ (ppm) 1.61–1.67
(m, 6H, 4,6,10-adamantane H), 1.96 (d, J=2.4 Hz, 6H,
2,9,8-adamantane H), 1.99 (br s, 3H, 3,5,7-adamantane H),
2.52 (br s, 2H, H-b). 13C NMR (CD3OD, 100 MHz)
δ (ppm) 30.84 (3,5,7-adamantane C), 32.05 (C-b), 37.29
(4,6,10-adamantane C), 41.90 (2,8,9-adamantane C), 51.78
(1-adamantane C), 175.85 (C-a). EI-MS: m/z 384.25 [M]+.
Procedure B for synthesis of compounds
3a–e
N,N′-bis-(adamantan-1-yl) adipic acid diamide (1b)
Dicarboxylicacid(0.7mmol),1-ethyl-3-(3-dimethylaminopro-
Compound 1b (420 mg, 74%) was obtained as white pyl)carbodiimide (EDCI) HCl (0.423 g, 2.1 mmol), hydroxy-
1
solid; MP: 268°C. H NMR (CDCl3, 400 MHz) δ (ppm) benzotriazole (0.283 g, 2.1 mmol), and diisopropylethylamine
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(0.97 mL, 5.6 mmol) were dissolved in sufficient amount (m, 8H, 2,8.9-adamantane H, H-c), 1.92 (br s, 3H, 3,5,7-
of dry tetrahydrofuran (THF; 20 mL). After stirring for adamantane H), 2.10 (t, J=7.5, 2H, H-b), 3.65 (m, 1H,
30 minutes, a solution of rimantadine hydrochloride (0.604 g, CH-11), 5.25 (br s, 1H, NH). 13C NMR (CDCl3, 100 MHz)
2.8 mmol) in dry THF (10 mL) was added dropwise and the δ (ppm) 14.65 (12-C), 25.65 (d C), 28.32 (3,5,7-adamantane
reaction mixture stirred overnight at RT under inert condi- C), 28.78 (C-c), 35.71 (1-adamantane C), 36.99 (b-C), 37.07
tions. A saturated solution of NaHCO3 (100 mL) was added (4,6,10-adamantane C), 38.43 (2,8,9-adamantane C), 52.74
and the product extracted with ethyl acetate (2×40 mL) (C-11), 172.31 (C-a). EI-MS: m/z 497.35 [M]+.
The combined organic layers were washed with water and
concentrated under reduced pressure to remove the solvent. N,N′-bis-(1-adamantan-1-yl-ethyl) sebacic acid
The residue was dissolved in CHCl3 (30 mL) and the solu- diamide (3d)
tion extracted with concentrated HCl solution (2×50 mL). Compound 3d (200 mg, 82%) was obtained as a yellow oil.
The chloroform phase was washed with water and dried over 1H NMR (CDCl3, 400 MHz) δ (ppm) 0.94 (d, J=6.9 Hz, 3H,
sodium sulfate. The solvent was evaporated under reduced H-12),1.20–1.30(m,4H,H-d,e),1.44(dd, J=27.6,12.7Hz,6H,
pressure and the residue purified by column chromatography 4,6,10-adamantane H), 1.51–1.68 (m, 8H, 2,8,9-adamantane
(dichloromethane:MeOH, 10:0.5).
H, H-c), 1.92 (br s, 3H, 3,5,7-adamantane H), 2.10 (t, J=7.5,
2H, H-b), 3.65 (m, 1H, H-11), 5.20 (br s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) δ (ppm) 14.65 (C-12), 25.88 (C-e), 28.32
(3,5,7-adamantane C), 29.10 (C-d), 29.20 (C-c), 35.71
N,N′-bis-(adamantan-1-yl-ethyl)-succinic acid
diamide (3a)
Compound 3a (200 mg, 97%) was obtained as a white solid; (1-adamantane C), 37.07 (4,6,10-adamantane C), 37.19
1
MP: 191°C. H NMR (CDCl3, 400 MHz) δ (ppm) 0.94 (C-b), 38.43 (2,8,9-adamantane C), 52.72 (C-11), 172.45
(d, J=6.9 Hz, 3H, H-12), 1.43 (dd, J=26.3, 12.6 Hz, 6H, (C-a). EI-MS: m/z 525.30 [M+H]+.
4,6,10-adamantane H), 1.6 (m, 6H, 2,8,9-adamantane H), 1.91
(br s, 3H, 3,5,7-adamantane H), 2.42–2.52 (br s, 2H, H-b), N,N′-bis-(1-adamantan-1-yl-ethyl) dodecanedioic acid
3.61 (m, 1H, H-11), 5.86 (s, 1H, NH). 13C NMR (CDCl3, diamide (3e)
100 MHz) δ (ppm) 14.52 (C-12), 28.32 (3,5,7-adamantane Compound 3e (200 mg, 78%) was obtained as a yellow oil. 1H
C), 32.38 (C-b), 35.71 (1-adamantane C), 37.04 (4,6,10- NMR(CDCl3, 400MHz)δ(ppm)0.94(d, J=6.9Hz, 3H, H-12),
adamantane C), 38.34 (2,8,9-adamantane C), 53.15 (C-11), 1.19–1.28 (m, 6 H, H-d–f ), 1.44 (dd, J=27.4, 12.6 Hz, 6H,
171.69 (C-a). EI-MS: m/z 441.20 [M+H]+.
4,6,10-adamantane H), 1.67–1.51 (m, 8H, 2,8,9-adamantane
H, H-c), 1.92 (br s, 3H, 3,5,7-adamantane H), 2.10 (t, J=7.5,
2H, H-b), 3.65 (m, 1H, H-11), 5.22 (br s, 1H, NH). 13C NMR
(CDCl3, 100 MHz) δ (ppm) 14.65 (C-12), 25.95 (C-f), 28.43
N,N′-bis-(1-adamantan-1-yl-ethyl) adipic acid
diamide (3b)
Compound 3b (510 mg, 69%) was obtained as a white solid; (3,5,7-adamantane C), 29.27 (C-e), 29.38 (C-d), 29.69 (C-c),
1
MP: 205°C. H NMR (CDCl3, 400 MHz) δ (ppm) 0.94 35.71 (1-adamantane C), 37.07 (4,6,10-adamantane C), 37.24
(d, J=6.9 Hz, 3H, H-12), 1.44 (dd, J=26.3, 12.6 Hz, 6H, (C-b), 38.43 (2,8,9-adamantane C), 52.72 (C-11), 172.41
4,6,10-adamantane H), 1.67–1.51 (m, 8H, 2,8,9-adamantane (C-a). EI-MS: m/z 553.45 [M+H]+.
H, H-c), 1.91 (br s, 3H, 3,5,7-adamantane H), 2.15 (t, J=4.6,
2H, H-b), 3.65 (m, 1H, H-11), 5.39 (s, 1H, NH). 13C NMR
Procedure c for synthesis of compounds
(CDCl3, 100 MHz) δ (ppm) 14.60 (C-12), 25.23 (C-c), 28.32 2a–e, 4a–e, 5a
(3,5,7-adamantane C), 35.71 (1-adamantane C), 36.54 (C-b), Borane THF solution (1 M, 5 mL) was added dropwise to a
37.06 (4,6,10-adamantane C), 38.43 (2,8,9-adamantane C), stirred mixture of the indicated amount of the corresponding
52.87 (C-11), 172.07 (C-a). EI-MS: m/z 468.30 [M]+.
amide in dry THF (20 mL) and heated under reflux for 9 hours
under inert conditions. 2 M HCl (2 mL) was carefully added
under ice cooling and the reaction mixture heated under
reflux for 30 minutes. The mixture was left to cool at RT and
N,N′-bis-(1-adamantan-1-yl-ethyl) suberic acid
diamide (3c)
Compound 3c (200 mg, 87%) was obtained as a white basified with 25% aqueous ammonia under ice cooling. The
1
solid; MP: 191°C. H NMR (CDCl3, 400 MHz) δ (ppm) product was extracted with dichloromethane (2×20 mL), and
0.94 (d, J=6.9 Hz, 3H, H-12), 1.27–1.33 (m, 2H, H-d), 1.44 combined organic layers washed with water and dried over
(dd, J=26.3, 12.6 Hz, 6H, 4,6,10-adamantane H), 1.52–1.67 sodium sulfate. The solvent was evaporated under reduced
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Exploring influenza M2 and HCV p7 with adamantanes
pressure and the residue purified by column chromatography N,N′-bis-adamantan-1-yl-dodecane-1,4-diamine (2e)
(chloroform:MeOH:ammonia, 100:5:1).
Compound 2e (92 mg, 54%) was obtained as a white solid
1
from 178.83 mg of 1e; MP: 114°C. H NMR (CD3OD,
N,N′-bis-(adamantan-1-yl)-butane-1,4-diamine (2a)
400 MHz) δ (ppm) 1.22 (broad s, 8H, H-c–f), 1.33–1.41
Compound 2a (100 mg, 77%) was obtained as a white (m, 2H, H-b), 1.52–1.67 (complex m, 12H, 2,4,6,8,9,10-
solid from 128.34 mg of 1a; MP: 52°C. 1H NMR (CD3OD, adamantane H), 1.97 (br s, 3H, 3,5,7-adamantane H), 2.47
400 MHz) δ (ppm) 1.43–1.38 (m, 2H, H-b), 1.67–1.53 (t, J=7.6 Hz, 2H, H-a). 13C NMR (CD3OD, 100 MHz) δ (ppm)
(complex m, 12H, 2,4,6,8,9,10-adamantane H), 1.97 28.6 (C-f) 30.7 (C-c–e), 31.0 (3,5,7-adamantane C), 31.21
(br s, 3H, 3,5,7-adamantane H), 2.49 (t, J=7.02 Hz, 2H, H-a). (C-b), 37.73 (4,6,10-adamantane C), 41.182 (C-a), 42.75
13C NMR (CD3OD, 100 MHz) δ (ppm) 29.06 (C-b), 30.98 (2,8,9-adamantane C), 52.21 (1-adamantane C). ESI-MS:
(3,5,7-adamantane C), 37.69 (4,6,10-adamantane C), 41.0 m/z 469.40 [M+H]+.
(C-a), 42.72 (2,8,9-adamantane C), 52.21 (1-adamantane C).
EI-MS: m/z 357.4 [M+H]+.
N,N′-bis-(1-adamantan-1-yl-ethyl)-butane-1,4-
diamine (4a)
N,N′-bis-adamantan-1-yl-hexane-1,4-diamine (2b)
Compound 4a (100 mg, 53%) was obtained as a colorless
1
Compound 2b (75 mg, 54%) was obtained as a white oil from 158.53 mg of 3a. H NMR (CDCl3, 400 MHz)
1
solid from 138.43 mg of 1b; MP: 53°C. H NMR (CDCl3, δ (ppm) 0.87 (d, J=6.5 Hz, 3H, H-12), 1.38–1.42 (m, 2H,
400 MHz) δ (ppm) 1.3–1.22 (m, 2H, H-c), 1.43–1.35 (m, 2H, H-b), 1.42–1.55 (complex m, 6H, 4,6,10-adamantane H),
H-b), 1.63–1.50 (complex m, 12H, 2,4,6,8,9,10-adamantane 1.6 (dd, J=27.3, 11.8 Hz, 6H, 2,8,9-adamantane H), 1.90
H), 1.99 (br s, 3H, 3,5,7-adamantane H), 2.49 (t, J=7.31 Hz, (br s, 3H, 3,5,7-adamantane H), 1.98 (q, J=6.4, 1H, H-11),
2H, H-a). 13C NMR (CDCl3, 100 MHz) δ (ppm) 27.48 (C-c), 2.36 (dt, J=11.1, 6.3 Hz, 1H, H-a), 2.67 (dt, J=11.1, 6.3 Hz,
31.05 (C-b), 29.6 (3,5,7-adamantane C), 36.77 (4,6,10- 1H, H-a). 13C NMR (CDCl3, 100 MHz) δ (ppm) 13.59
adamantane C), 40.34 (C-a), 42.72 (2,8,9-adamantane C), (C-12), 28.2 (C-b), 28.61 (3,5,7-adamantane C), 36.14
52.21 (1-adamantane C). EI-MS: m/z 385.5 [M+H]+.
(1-adamantane C), 37.39 (4,6,10-adamantane C), 38.74
(2,8,9-adamantane C), 48.97 (C-a), 62.63 (C-11). ESI-MS:
m/z 413.3 [M+H]+.
N,N′-bis-adamantan-1-yl-octane-1,4-diamine (2c)
Compound 2c (113 mg, 75%) was obtained as a white
solid from 140.56 mg of 1c; MP: 55°C. 1H NMR (CD3OD, N,N′-bis-(1-adamantan-1-yl-ethyl)-hexane-1,6-
400 MHz) δ (ppm) 1.39 (br s, 4H, H-c,d), 1.48–1.59 (m, 2H, diamine (4b)
H-b), 1.68–1.77 (complex m, 12H, 2,4,6,8,9,10-adamantane Compound 4b (70 mg, 48%) was obtained as a colorless
1
H), 2.12 (br s, 3H, 3,5,7-adamantane H), 2.67 (t, J=7.6 Hz, oil from 169.83 mg of 3b. H NMR (CDCl3, 400 MHz)
2H, H-a). 13C NMR (CD3OD, 100 MHz) δ (ppm) 28.32 δ (ppm) 0.87 (d, J=6.5 Hz, 3H, H-12) 1.23–1.28 (m, 2H,
(C-d), 30.45 (C-c), 30.47 (C-b), 30.89 (3,5,7-adamantane C), H-c), 1.35–1.40 (m, 2H, H-b), 1.40–1.54 (complex m, 6H,
36.77 (4,6,10-adamantane C), 40.34 (C-a), 42.72 (2,8,9- 4,6,10-adamantane H), 1.60 (dd, J=28.1, 13.0 Hz, 6H, 2,8,9-
adamantane C), 52.21 (1-adamantane C). EI-MS: m/z 413.7 adamantane H), 1.98 (br s, 3H, 3,5,7-adamantane H), 1.97
[M+H]+.
(q, J=6.5 Hz, 1H, H-11), 2.34 (dt, J=11.4, 7.2 Hz, 1H,
H-a), 2.64 (dt, J=11.4, 7.1 Hz, 1H, H-a). 13C NMR (CDCl3,
100 MHz) δ (ppm) 13.59 (C-12), 27.41 (C-c), 28.62 (3,5,7-
N,N′-bis-adamantan-1-yl-decane-1,4-diamine (2d)
Compound 2d (47 mg, 50%) was obtained as a white solid adamantane C), 20.32 (C-b), 37.40 (4,6,10-adamantane C),
1
from 159.63 mg of 1d; MP: 59°C. H NMR (CD3OD, 36.14 (1-adamantane C), 38.75 (2,8,9-adamantane C), 49.05
400 MHz) δ (ppm) 1.39 (broad s, 6H, H-c-e), 1.49–1.57 (C-a), 62.56 (C-11). ESI-MS: m/z 441.4 [M+H]+.
(m, 2H, H-b), 1.67–1.80 (complex m, 12H, 2,4,6,8,9,10-
adamantane H), 2.12 (br s, 3H, 3,5,7-adamantane H), 2.68 N,N′-bis-(1-adamantan-1-yl-ethyl)-octane-1,8-
(t, J=7.7 Hz, 2H, H-a). 13C NMR (CD3OD, 100 MHz) δ (ppm) diamine (4c)
27.43 (C-e) 29.36 (C-d), 30.76 (C-c), 29.36 (C-b), 29.57 Compound 4c (90 mg, 47%) was obtained as a yellow
1
(3,5,7-adamantane C), 36.71 (4,6,10-adamantane C), 40.40 oil from 178.93 mg of 3c. H NMR (CDCl3, 400 MHz)
(C-a), 42.42 (2,8,9-adamantane C), 52.21 (1-adamantane C). δ (ppm) 0.96 (d, J=6.5 Hz, 3H, H-12) 1.31 (m, 4H, H-c,d),
EI-MS: m/z 441.88 [M+H]+.
1.42–1.48 (m, 2H, H-b), 1.49–1.63 (complex m, 6H,
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4,6,10-adamantane H), 1.68 (dd, J=27.1, 12.6 Hz, 6H, 2,8,9- 1H, H-a). 13C NMR (CDCl3, 100 MHz) δ (ppm) 13.41 (C-d)
adamantane H), 1.98 (br s, 3H, 3,5,7-adamantane H), 2.06 (q, 14.02 (C-12), 20.56 (C-c), 28.59 (3,5,7-adamantane C), 32.18
J=6.5 Hz, 1H, H-11), 2.42 (dt, J=11.3, 7.2 Hz, 1H, H-a), 2.72 (C-b), 35.98 (1-adamantane C), 37.34 (4,6,10-adamantane
(dt, J=11.3, 7.2 Hz, 1H, H-a). 13C NMR (CDCl3, 100 MHz) δ C), 38.68 (2,8,9-adamantane C), 48.68 (C-a), 62.69 (C-11).
(ppm) 13.56 (C-12), 27.41 (C-d), 29.56 (C-c), 28.61 (3,5,7- ESI-MS: m/z 236.16 [M+H]+.
adamantane C), 30.27 (C-b), 36.14 (1-adamantane C), 37.38
Biochemical analysis
(4,6,10-adamantane C), 38.73 (2,8,9-adamantane C), 49.10
(C-a), 62.61 (C-11). ESI-MS: m/z 469.38 [M+H]+.
generation of p7-expression constructs
The generation of genotype (GT)-specific cDNA constructs
for the p7 protein of GTs 1a–4a of HCV has been described
before.19
N,N′-bis-(1-adamantan-1-yl-ethyl)-decane-1,10-
diamine (4d)
Compound 4d (90 mg, 47%) was obtained as yellow oil from
188.93 mg of 3d. 1H NMR (CDCl3, 400 MHz) δ (ppm) 0.89
(d, J=6.5 Hz, 3H, H-12) 1.25–1.18 (m, 6H, H-c–e), 1.36–1.41
(m, 2H, H-b), 1.42–1.59 (complex m, 6H, 4,6,10-adamantane
H), 1.6 (dd, J=24.3, 10.8 Hz, 6H, 2,8,9-adamantane H),
1.90 (br s, 3H, 3,5,7-adamantane H), 2.01 (q, J=6.5 Hz, 1H,
H-11), 2.36 (dt, J=11.4, 7.3 Hz, 1H, H-a), 2.66 (dt, J=11.4,
7.2 Hz, 1H, H-a). 13C NMR (CDCl3, 100 MHz) δ (ppm) 13.43
(C-12), 27.41 (C-e) 29.51 (C-d), 29.53 (C-c), 28.59 (3,5,7-
adamantane C), 30.01 (C-b), 36.14 (1-adamantane C), 37.74
(4,6,10-adamantane C), 38.76 (2,8,9-adamantane C), 49.02
(C-a), 62.68 (C-11). ESI-MS: m/z 497.46 [M+H]+.
cell culture and transfection
HEK293 cells were cultured in 10 cm tissue-culture petri
dishes in MEM (Sigma-Aldrich, St Louis, MO, USA) supple-
mented with 10% FBS (Thermo Fisher Scientific, Waltham,
MA, USA) and penicillin–streptomycin (Sigma-Aldrich)
at 5% CO2 and 37°C in a water-saturated atmosphere. For
experiments, cells were plated on poly-l-lysine-treated
glass coverslips in 6 cm dishes and transfected 1 day after
passage using 1.5 µg p7 cDNA, 1.5 µg GFP cDNA and 3 µL
GenCarrier (Epoch Life Science, Sugar Land, TX, USA).
Measurements were performed 2–5 days after transfection.
electrophysiological recordings and data analysis
M2: Inhibitors were tested in a two-electrode voltage-clamp
assay using Xenopus laevis frog oocytes microinjected with
RNA expressing either the wild type (WT) or the S31N
mutant of the A/M2 protein, as previously reported.41 The
potency of the inhibitors was expressed as percentage inhibi-
tion of A/M2 current observed after 2 minutes of incubation
with 100 µM compounds, and we measured inhibition as the
average ± SD from three replicates.
P7: HEK293 cells, obtained from the Health Protection
Agency European Cell Culture Collection (Salisbury, UK),
on poly-l-lysine coated coverslips were transfected with
p7 cDNA constructs 2–4 days prior to electrophysiological
recordings. Current responses were measured at room tem-
perature (21°C–23°C) at a holding potential of -60 mV using
an EPC10 amplifier and Pulse software (Heka Electronics,
N,N′-bis-(1-adamantan-1-yl-ethyl)-dodecane-1,12-
diamine (4e)
Compound 4e (80 mg, 42%) was obtained as colorless oil
from 195.03 mg of 3e. 1H NMR (CDCl3, 400 MHz) δ (ppm)
0.87 (d, J=6.5 Hz, 3H, H-12) 1.22 (m, 8H, H-c–f ), 1.34–1.40
(m, 2H, H-b), 1.40–1.54 (complex m, 6H, 4,6,10-adamantane
H), 1.6 (dd, J=27.8, 12.8 Hz, 6H, 2,8,9-adamantane H),
1.90 (br s, 3H, 3,5,7-adamantane H), 1.97 (q, J=6.5 Hz, 1H,
H-11), 2.34 (dt, J=11.3, 7.3 Hz, 1H, H-a), 2.63 (dt, J=11.3,
7.2 Hz, 1H, H-a). 13C NMR (CDCl3, 100 MHz) δ (ppm) 13.59
(C-12), 27.46 (C-f) 29.60 (C-d,e), 29.62 (C-c), 28.62 (3,5,7-
adamantane C), 30.33 (C-b), 36.14 (1-adamantane C), 37.40
(4,6,10-adamantane C), 38.74 (2,8,9-adamantane C), 49.15
(C-a), 62.60 (C-11). ESI-MS: m/z 525.48 [M+H]+.
(1-Adamantan-1-yl-ethyl)-butyl-amine (5a)
Compound 5a (70 mg, 74%) was obtained as colorless oil Lambrecht, Germany). Recording pipettes were made from
from 110 mg of its precursor amide. 1H NMR (CDCl3, 400 borosilicate glass (World Precision Instruments, Berlin,
MHz) δ (ppm) 0.85 (t, J=7.3 Hz, 3H, H-d) 0.89 (d, J=6.5 Germany) using a P-97 horizontal puller (Sutter, Novato,
Hz, 3H, H-12) 1.22–1.32 (m, 2H, H-c), 1.36–1.41 (m, 2H, CA, USA). An OctaFlow system (NPI Electronics, Tamm,
H-b), 1.41–1.56 (complex m, 6H, 4,6,10-adamantane H), Germany) was used for fast perfusion of suspended single
1.60 (dd, J=25.2, 11.1 Hz, 6H, 2,8,9-adamantane H), 1.90 (br cells. The external buffer consisted of 90 mM N-methyl-d-
s, 3H, 3,5,7-adamantane H), 2.01 (q, J=6.5 Hz, 1H, H-11), glucamine, 3 mM CaCl2, 90 mM HEPES, and 90 mM 2-(N-
2.38 (dt, J=11.4, 7.3 Hz, 1H, H-a), 2.68 (dt, J=11.4, 7.2 Hz, morpholino) ethanesulfonic acid. The pH of the external
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Exploring influenza M2 and HCV p7 with adamantanes
buffer was adjusted to 8.5, 7.5, 6.5, 6.0, 5.5, 5.0 and 4.5 using the MMFF94x force field in MOE using a gradient of
using NaOH or HCl. Internal buffer was 90 mM N-methyl-d- 0.0001 kcal/(mol Å). Docking was performed using GOLD
glucamine, 10 mM ethylene glycol tetraacetic acid, 180 mM (version 5.3).43,44 Binding-site residues were defined by
HEPES, pH 7.5 (NaOH). For measurement of whole-cell p7 specifying the alpha carbon of Leu20 and using the default
currents, cells were perfused continuously with recording cutoff radius of 10 Å, with the “detect cavity” option enabled.
buffer during 1-minute recording intervals. Inhibitors were GOLD docking experiments were performed using the
diluted from stock solutions (10 mM) into pH 5.5 buffer. ChemPLP scoring function. The search efficiency of the
Inhibition measurements were started with baseline record- genetic algorithm was at 200% setting with the receptor kept
ings of extracellular buffer pH 7.5 (4–5 seconds), followed rigid. For each compound, 50 complexes were generated
by pH 5.5 (to induce channel opening). Then, inhibitors and clustered based on their RMSD with the threshold set at
(at pH 5.5) were applied, followed by final control applica- 0.75 Å using the complete linkage method. The best-ranked
tions. Sometimes, control solution (pH 5.5 without inhibitor) pose from the most populated cluster was selected as the
was perfused between inhibitor applications. Typical current final pose. Figures were prepared using PyMol (version 1.8;
amplitudes were 100–400 pA. Dose–response curves were Schrödinger, New York, NY, USA).
constructed from four to five cells per GT and inhibitor and
Results
IC50 values determined using a nonlinear fit to the equation
Iobs = Imax/[1 + ([I]/IC50)], where Iobs is the observed current chemistry
at any given concentration of inhibitor, Imax the maximum The target dimeric amantadines 2a–e and rimantadines 4a–e
current amplitude observed in the absence of inhibitor, and were prepared in a two-step approach involving diamide
[I] the concentration of inhibitor. IC50 for the inhibition of p7 formation with dicarboxylic acids HO2C-(CH2)n-CO2H (n=2,
channels was determined from the concentration-dependent 4, 6, 8, 10) and subsequent reduction using borane. In the
inhibition of p7-mediated TM currents. Values for inhibition amantadine series, the dicarboxylic acids were converted
of GTs 1a, 2a, 3a, and 4a by amantadine and rimantadine to the corresponding acid chlorides using oxalyl chloride.
have been previously reported.19 For this study, only a few The crude acid chlorides (1.4 mmol) were reacted with an
additional measurements were performed for confirmation. excess of amantadine (4 mmol) to give diamides 1a–e. The
target compounds 2a–e were obtained in 50%–75% yield by
Molecular modeling
The protein was prepared using the structure preparation
reduction with borane–THF complex (Figure 1).
Since rimantadine is commercially available only as a
wizard in MOE42 (version 2015.10) and saved as a mol2 file. hydrochloride, an alternative and more convenient one-pot
3-D structures of the compounds were built and minimized procedure for the amide formation was employed using
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Notes: Reaction conditions: (A) oxalyl chloride, catalytic dimethylformamide, CHCl3, 4 hours; (B) amantadine, triethylamine, CHCl3, 16 hours; (C) 1 M borane–tetrahydrofuran
complex, reflux 9 hours.
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Notes: Reaction conditions: (A) HO2C-(CH2)n-cO2H, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl, hydroxybenzotriazole, diisopropylethylamine in THF, 16 hours;
(B) butyric acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl, hydroxybenzotriazole, diisopropylethylamine in THF, 16 hours; (C, D) borane–THF complex in dry
THF, reflux 9 hours.
Abbreviation: ThF, tetrahydrofuran.
diisopropylethylamine to release the rimantadine base from at 100 µM concentration.29 Amantadine showed 91%±3%
its salt and using EDCI HCl–hydroxybenzotriazole to activate inhibition against WT M2 protein and was used as benchmark
the dicarboxylic acids. The rimantadine amides 3a–e were for M2 inhibitory activity. The newly synthesized dimeric
obtained in 69%–97% yield and reduced using borane–THF compounds showed significantly lower inhibitory activity
complex to give the target amines 4a–e in 42%–74% yield relative to the monomeric amantadine (Figure 3A). The
(Figure 2). For the monomeric reference compound 5a, a compounds’ M2-blocking activity showed no dependence on
similar procedure was employed, where butyric acid was alkyl-spacer length, with all dimeric compounds exhibiting
coupled to rimantadine followed by amide reduction using relatively similar inhibition. The inhibitory activity of the
borane–THF complex.
reference monomeric ligand 5a (40%±5%), whose structure
represents half the molecule of the rimantadine dimer 4c, was
approximately fourfold that of 4c, indicating that introduc-
tion of the extra bulky adamantane group was detrimental
Biological assays
M2-inhibitory activity
The inhibitory activity of all dimeric compounds was tested to blocking of M2 ion-channel activity.
on the influenza virus M2 ion channels expressed in Xenopus
Examination of the experimentally determined structures
oocytes using the two-electrode voltage-clamp technique of M2 protein shows an ion channel of limited pore size with
A
B
120
100
80
60
40
20
0
120
100
80
60
40
20
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Figure 3 M2-inhibitory activities of dimeric adamantanes.
Notes: Evaluation of inhibitory activity of dimeric adamantane compounds on wild-type (A) and S31N mutant (B) M2 proteins. M2 protein was expressed in Xenopus oocytes
and the compounds’ inhibitory activity measured using the two-electrode voltage-clamp technique at 100 µM concentration.
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Exploring influenza M2 and HCV p7 with adamantanes
its N-terminal end constricted by a hydrophobic Val27 valve. dimeric compounds had improved channel blockage against
The high-resolution X-ray crystal structure of M2 protein M2-WT when compared with amantadine. Our results are
(Protein Data Bank [PDB] 3LBW)30 shows three intercalated also in agreement with previous structure–activity relation-
water clusters, which are important not only for the stabil- ship studies where introduction of bulky substituents on the
ity and ion-channel activity of M2 protein but also for drug adamantane cage drastically decreased the compounds’ M2
binding. The upper layer of water molecules is stabilized inhibitory activity.32 The small diameter of M2 protein pores
by hydrogen bonds with the carbonyl groups of Gly34. The limits the size of ligands that can fit into the pores. As a result,
lower layer of water molecules lies deeper in the pore lumen, the second hydrophobic adamantane moiety introduced in our
forming hydrogen bonds with His37and Trp41.
dimeric compounds cannot be accommodated inside the M2
Overlaying the drug-free solid-state NMR structure of pore lumen, which explains the low inhibitory activity of our
M2 protein (PDB 2KQT)31 with the amantadine-bound X-ray dimeric compounds relative to the monomeric amantadine
structure (PDB 3LBW) showed amantadine present in the and ligand 5a.
pore lumen, with its adamantane cage placed in a hydro-
The inhibitory activity of these dimeric compounds was
phobic groove formed mainly by Ala30and Ser31 residues further evaluated against S31N M2 mutant form (Figure 3B).
(Figure 4A). When amantadine binds to the channel, it breaks The S31N represents the predominant amantadine-resistant
the continuous water wires in the channel, which are critical M2 form.33 All the dimeric compounds showed lower
for proton conductance. The positively charged ammonium inhibitory activity compared to amantadine. This could be
group appears to mimic the conducting hydronium ion, explained by the dimeric compounds lacking the adamantane-
forming hydrogen bonds with the backbone carbonyls of NH2+-CH2-aryl pharmacophore of M2-S31N inhibitors.34,35
Gly34 that are mediated by the upper layer of water mol- Nevertheless, the M2-inhibitory activity appears to be depen-
ecules. Importantly, amantadine binds to the M2-WT channel dent on the alkyl-spacer length within the dimeric amantadine
with its positively charged ammonium facing the C-termini series: an increase in inhibitory activity was observed with
of the channel, suggesting bulky substitutions on the amine increased spacer length, with the dimer linked by a ten-carbon
group will not be tolerated. Our dimeric compounds (2a–e, spacer (2d) exhibiting the highest inhibitory activity.
4a–e), with a secondary hydrophobic adamantane cage
A previous solution NMR structure of the inhibitor-bound
introduced in their structure, will not be expected to fit in the M2-S31N structure showed that the inhibitor binds to the
M2-WT channel. Indeed, as shown in Figure 3, none of the S31N mutant in a flipped orientation relative to that in the
Figure 4 Inhibition of the influenza M2 proton channel.
Notes: (A) Structure of amantadine (gray) in complex with M2 wild-type protein, as determined by solid-state nuclear magnetic resonance (Protein Data Bank [PDB]
2KQT),31 with the water molecules seen in the high-resolution crystallographic structure (PDB 3LBW)30 superimposed (red spheres). (B) Inhibition of amantadine-resistant
M2-S31N protein by an isoxazole drug (PDB 2LY0).36 The adamantane cage is shifted downward in the structure of the S31N-mutant form, due to the constricted pore size.
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M2-WT channel. Specifically, the amino group from the more potent than amantadine and equipotent to rimantadine.
S31N mutant-bound inhibitor is oriented upward toward the A positively charged amino group appears important for
N-terminal end.36 Up till now, compounds reported to possess binding to GT2a, as compound 2c with a secondary amine
inhibitory activity for M2-S31N mutant form share the gen- had an IC50 of 0.8 nM, whereas its corresponding amide ana-
eral adamantane-NH2+-CH2-aryl pharmacophore (Figure 4B). log 1c did not show any inhibitory activity (IC50.100 nM).
The positively charged ammonium group and the heteroaryl On the other hand, this positively charged amine group
moiety from the inhibitor form water-mediated interactions appears less important for inhibition of other GTs, as both
with the amides from the Asn31 residues. In our dimeric compounds 2c and 1c showed comparable potency in GTs
compounds, this polar aryl group is missing, which explains 1a, 1b, 3a, and 4a.
their lack of efficacy in inhibiting the M2-S31N channel.
Ourresultsshownodependencebetweenthep7-inhibitory
In summary, the lack of channel blockage of the dimeric activity of the dimeric compounds and alkyl-spacer length.
amantadine and rimantadine analogs against M2-WT and All dimeric compounds showed relatively similar potency
M2-S31N mutant is consistent with previously proposed in the tested GTs, suggesting that all these compounds have
drug-binding mechanisms and further confirms that the a similar binding mode. Despite the increasing amount
pore-binding model is the pharmacologically relevant drug- of structural data on p7, there is no consensus on which
binding model.
conformation exists during a natural infection. Because of
the structural impact, the proposed binding mode of our
compounds may differ depending on the 3-D model. Accord-
p7-inhibitory activity
The inhibitory activity of all compounds was tested on p7 ingly, we considered the two available NMR structures for
channels for GTs 1a, 1b, 2a, 3a, and 4a expressed in HEK293 p7 protein to develop hypotheses upon the binding mode of
cells (Table 1). Amantadine and rimantadine are known our second adamantane moiety. Two homology models for
inhibitors of p7 activity, blocking lysosomal deacidification one of the GTs under study (GT2a, strain J6) were previously
and lowering p7 currents in patch-clamp measurements,19 and constructed37 using the NMR structure of HCV p7 GT5a
thus were used as benchmarks for comparison. The dimeric (PDB 2M6X)26 as a template for model 1, and the NMR/MD
adamantanes showed similar potency to their respective structure of the monomeric form of GT1b as a template for
monomeric analogs in GTs 1a, 1b, and 4a. Testing of the model 2.38 Although the exact binding mode of amantadine–
inhibitory activity of our dimeric amantadine series on GT2a rimantadine is not clearly understood yet, it is generally
showed these compounds to be at least 700-fold more potent agreed that they bind in a membrane-facing pocket centered
than amantadine and eightfold more potent than rimantadine. on residue number 20. Structural examination of both models
For GT3a, the dimeric compounds were at least 150-fold showed the distance between the Cα atoms of Leu20 in two
Table 1 Inhibitory effect of the dimeric adamantane compounds on p7 protein in GTs 1a, 1b, 2a, 3a, and 4ac
Compound
IC50 (nM) ± SEM*
GT1a
GT1b
GT2a
GT3a
GT4a
amantadine
0.7±0.4 (5)
0.7±0.1 (5)
0.4±0.1 (5)
0.4±0.1 (4)
1.0±0.2 (7)
0.9±0.1 (3)
0.6±0.1 (4)
na
0.6±0.1 (6)
1.7±1.2 (8)
1.2±0.2 (3)
0.5±0.4 (3)
0.7±0.2 (4)
1.0±0.3 (4)
1.1±0.2 (4)
1.1±0.3 (3)
0.6±0.2 (4)
1.1±0.1 (3)
2.3±0.2 (4)
1.0±0.3 (7)
0.5±0.1 (4)
1.0±0.2 (6)
.1,000 (5)
24±4 (5)
2.8±0.3 (4)
0.9±0.1 (3)
0.8±0.1 (6)
0.6±0.1 (4)
0.4±0.1 (3)
na
.100 (4)
1.6±0.6 (5)
2.0±0.4 (4)
0.9±0.3 (4)
0.6±0.1 (9)
0.6±0.1 (4)
0.6±0.1 (5)
na
3.2±1.2 (5)
3.0±0.8 (4)
1.2±0.2 (4)
0.4±0.1 (5)
0.8±0.1 (7)
0.9±0.2 (4)
0.4±0.1 (6)
0.7±0.2 (4)
2.0±0.1 (3)
0.5±0.1 (4)
1.1±0.1 (4)
0.8±0.3 (6)
0.8±0.1 (4)
0.9±0.1 (4)
rimantadine
2a
2b
2c
2d
2e
4a
4b
4c
4d
4e
1c
5a
na
na
na
na
na
na
0.8±0.2 (3)
na
na
na
na
na
1.2±0.3 (4)
1.3±0.2 (4)
.100 (4)
na
1.0±0.1 (4)
na
Note: *Numbers in parentheses indicate the number of cells used for measurements.
Abbreviations: GT, genotype; NA, not applicable.
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Exploring influenza M2 and HCV p7 with adamantanes
adjacent adamantane-binding sites to be ~11 Å and 17 Å, so amantadine could not induce the conformational changes
respectively. The second adamantane moiety in compound 2a necessary for favoring the closed state of p7 protein. Previous
having the shortest spacer, cannot span this distance and NMR and MD simulation experiments of p7 protein reported
thus cannot bind in the neighboring adamantane-binding site the presence of a proline residue at position 49 that forms a
located in the adjacent monomer. This was also supported kink in the carboxy-terminal end of TMD2, tilting it toward
by preliminary docking studies, where the first adamantane TMD1 of the adjacent monomer and forming the proposed
ring in the dimeric compound 2a showed a similar binding adamantane primary binding pocket.39,40 Examination of the
mode as amantadine, while the second adamantane appeared p7 protein sequence showed both GTs 2a and 3a lacked this
protruding to another cavity (Figure 5A and B) which may proline (Figure 5C). A proline residue in a TM helix typically
suggest the presence of a secondary adamantane-binding changes the orientation of the helix, causing a kink or even
site. Future site-directed mutagenesis would be needed to breaking the helix. This common behavior results from its
confirm unambiguously the binding mode suggested by our inability to act as a hydrogen-bond donor (tertiary amide)
docking studies.
and steric interferences of its cyclic side chain with residues
The inhibitory activity of adamantanes varied between in the preceding helical turn. The lack of proline in GTs 2a
the different GTs under study. GT-dependent sensitivity and 3a could affect the kink angle of TMD2, causing possible
of p7 to multiple inhibitors was previously reported where changes in the size of the adamantane-binding pocket, which
amantadine could not inhibit p7 ion-channel activity in could explain the low inhibitory activity of amantadine. Our
GTs 2a and 3a,17,19 similarly to what we also observed here. dimeric compounds; being more bulky; could overcome
On the other hand, an extra ethyl group inserted between the amantadine’s resistance in GTs 2a and 3a, showing an even
adamantane ring and the amino group present in rimantadine higher potency than that of rimantadine.
led to at least 100-fold increase in potency compared to
The higher p7-inhibitory activity observed for our dimeric
amantadine, indicating a better fit in the adamantane pocket. amantadines relative to monomeric amantadine could be
This could suggest that the size of the adamantane pocket in attributed either to better fitting in this large adamantane
GTs 2a and 3a might be bigger than that of other GTs, and pocket or simultaneous binding of the two adamantane rings
Figure 5 J6 p7 hexameric structure model 1 (A) and model 2 (B).
Notes: Overlay of the docked poses of amantadine (gray) and compound 2a (magenta). Side-chain atoms of Leu20 are shown as sticks. (C) Sequence alignment of p7 protein
in GT5a and the five GTs under study – 1a, 1b, 2a, 3a, and 4a – showing the position of Pro49 indicated by the red arrow.
Abbreviation: gT, genotype.
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Mandour et al
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in the primary and secondary pocket inducing the conforma-
tional changes necessary for favoring the closed state of p7
protein. Future point-mutation studies introducing a proline
at position 49 in p7 protein in these resistant GTs should help
in better understanding the binding mode of these dimeric
compounds.
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