Exploring organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton channel

Article abstract of DOI:10.1021/ja2050666

We describe the use of organosilanes as inhibitors and structural probes of a membrane protein, the M2 proton channel from influenza A virus. Organosilane amine inhibitors were found to be generally as potent as their carbon analogues in targeting WT A/M2

Full text of DOI:10.1021/ja2050666

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pubs.acs.org/JACS
Exploring Organosilane Amines as Potent Inhibitors and Structural
Probes of Influenza A Virus M2 Proton Channel
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Jun Wang,†, Chunlong Ma,‡ Yibing Wu,|| Robert A. Lamb,^ Lawrence H. Pinto,‡ and William F. DeGrado*,†,
†Department of Chemistry and Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6059, United States
^Howard Hughes Medical Institute and Department of Biochemistry, Molecular Biology and Cell Biology and ‡Department of
Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208-3500, United States
S Supporting Information
b
Scheme 1. Common Strategies of Carbon-to-Silicon Switch
in Drug Designa
ABSTRACT: We describe the use of organosilanes as inhibi-
tors and structural probes of a membrane protein, the M2
proton channel from influenza A virus. Organosilane amine
inhibitors were found to be generally as potent as their carbon
analogues in targeting WT A/M2 and more potent against the
drug-resistant A/M2-V27A mutant. In addition, intermolecular
NOESY spectra with dimethyl-substituted organosilane amine
inhibitors clearly located the drug binding site at the N-terminal
lumen of the A/M2 channel close to V27.
nfluenza A virus is a serious human health threat that urgently
I
requires development of small-molecule antiviral agents.1
Drug resistance is the predominant issue in influenza pharma-
ceutical research, due to the rapid mutational rate and high
tendencies toward reassortment.2 Currently, there are four small-
molecule drugs used for the prevention and treatment of influenza A
virus infections in the United States. Oseltamivir and zanamivir are
neuraminidase inhibitors that block the release of progeny viruses
from the host cells; amantadine and rimantadine are M2 channel
blockers that inhibit the viruses’ uncoating process by preventing the
acidification of endosomally entrapped viruses. However, most
currently circulating viruses are resistant to amantadine and riman-
tadine, and the number of viruses resistant to oseltamivir and
zanamivir is on the rise.3 Thus, there is clearly a need to develop
novel antivirals that are able to combat drug-resistant viruses.
A/M2 forms a homotetrameric proton-selective channel in
viral membranes and plays an essential role in mediating viral
uncoating4 and budding.5 Additionally, it equilibrates the pH
across the Golgi apparatus to prevent the premature conforma-
tional change of hemagglutinin.6À8 A/M2 is more conserved
than other drug targets of influenza A virus, with only three
predominant drug-resistant mutations, S31N, V27A, and L26F,
observed in widely circulating viruses,9,10 all of which are located
in the transmembrane domain drug binding site.
a In path a, tert-butyl is substituted by trimethylsilane in p38 MAP kinase
inhibitor; in path b, silanedioles were designed to mimic the tetrahedral
hydrolysis intermediate in ACE inhibitor.
organosilanes can also be designed to mimic high-energy tetra-
hedral intermediates or novel scaffolds that are not accessible to
carbon analogues.17,18 The most common carbon-to-silicon switch
strategies fall into one of the two classes (Scheme 1). In the first
class, a quaternary carbon is replaced with a silicon to increase
hydrophobicity19 (Scheme 1a). In the second class, a carbonyl is
replaced with a sterically hindered silanediol to mimic the high-
energy intermediate of an amide-bound hydrolysis, providing
opportunities to inhibit proteins such as proteases20 (Scheme 1b).
Hydrophobicity plays a critical role in improving antiviral
potency in designing A/M2 inhibitors as anti-influenza drugs.21À23
The pore-facing residues of the A/M2 channel (V27, A30, S31,
and G34)24,25 form a hydrophobic binding pocket that favors
binding of hydrophobic molecules such as adamantane or spirane
amines. Our previous structureÀactivity relationship (SAR)
studies of spirane amine compounds showed a positive correlation
between hydrophobicity and antiviral potency.22,23 Considering
the increased size and hydrophobicity of silicon compared with
its carbon analogue, we therefore rationalized that replacement
of the quaternary carbon in spirane amine inhibitors with silicon
would increase their potency.
A carbon-to-silicon switch is a widely explored strategy in
developing and marketing organosilane pesticides.11,12 There is
also a continuing interest in the pharmaceutical industry to fine-
tune the pharmacological or pharmacokinetic properties of
marketed drugs using the same strategy.13À16 Silicon-containing
compounds generally have no heavy metal associated toxicities
and have metabolic profiles similar to those of their carbon
analogues.13,14 Apart from the increased size and hydrophobicity
of silicon compared to the corresponding carbon counterpart,
Received: June 1, 2011
Published: August 05, 2011
r
2011 American Chemical Society
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Table 1. Antiviral Activities of Silaspirane Amines against
WT A/M2 and A/M2-V27A Mutants
Figure 1. The smallest known organic M2 inhibitor.
Scheme 2. Synthesis of Silaspirane Amines
amantadine
7
6
8
5
WT A/M2a 90.8 ( 2.5 89.0 ( 1.5 94.5 ( 0.6 95.9 ( 0.9
93.9 ( 1.8
7.8 ( 0.6
47.8 ( 0.5
IC50 (μM)
A/M2-V27Aa
IC50 (μM)
16 ( 1.2 12.6 ( 1.1 13.7 ( 1.7 3.3 ( 0.2
0
NAb
53.2 ( 2.3 67.4 ( 1.1 25.2 ( 0.9
84.9 ( 13.6 31.3 ( 2.3 318.6 ( 57.3 96.3 ( 13.4
a Inhibition (%) at 100 μM inhibitor concentration. b NA = not available.
All compounds were also tested against S31N mutant and found to have
<20% inhibition at 100 μM.
inhibition of A/M2 current observed after 2 min of incubation with
100 μM compounds, and IC50 values were determined for selected
potent compounds. The results are summarized in Table 1. As
discussed previously, the potency in this assay reflects primarily the
kinetics of binding rather than true equilibrium due to the difficulty
of maintaining the oocytes for extended periods at low pH.21 Thus,
the IC50 values reflect upper limits of the true dissociation constant.
As expected, both silaspirane amines, 6 and 5, showed
potencies similar to those of their carbon analogues, 7 and 8, in
inhibiting WT A/M2 channel activity. All were more active than
amantadine. Noteworthy was an increase in antiviral potency of
silaspirane amine inhibitors against A/M2-V27A compared to
their carbon analogues. The IC50 of 6 against A/M2-V27A was
31.1 μM, which is more than 2.7-fold more potent than the
previously identified weak A/M2-V27A inhibitor, 7. Similarly, a
3.3-fold potency increase against the V27A mutant was seen when
the quaternary carbon in 8 was switched to silicon to give 5. The
dramatic antiviral potency increase against V27A by switching to
silicon might be due to the larger size and higher lipophilicity of
silicon compared with carbon, thus providing better hydrophobic
contact between the drug and the channel.31,32
Membrane proteins are characterized by high content of
aliphatic residues (Ala, Val, Leu, Ile),33 and this results in
crowded signal overlap at 0.5À1 ppm in the proton dimension of
their NMR spectra; also their large size and rapid relaxation render
traditional half-filtered experiments difficult. To map the drug
binding sites in membrane proteins, it is desired to have a small-
molecule inhibitor which shows characteristic signals beyond the
normal range of protein signals. To achieve this goal, two 4,4-
disubstituted silacyclohexane amine derivatives (10 and 14) andone
4,4-dimethyl-1,4-azasilepane (13) were designed and synthesized
(Table 2 and Supporting Information (SI) Scheme S1).
Another interesting aspect of organosilane compounds is that
CÀSi bond is polarized toward carbon due to the higher
electronegativity of carbon (2.50 for carbon and 1.74 for silicon).
This results in an upfield chemical shift of silicon R-protons to
∼0 ppm, which is generally well separated from the protein
background signals. In light of this unique property, we envisioned
that organosilane compounds can be applied to map the A/M2
channel drug binding site using simple nuclear Overhauser enhance-
ment spectroscopy (NOESY), thereby obviating the need for more
technically demanding and less sensitive half-filtered experiments.26
The minimal requirements for potent inhibition of A/M2 are a
basic group attached to an alkyl moiety of at least eight carbon
atoms with good steric fit to the A/M2 binding cavity.21 To test
the effectiveness of a carbon-to-silicon switch in A/M2 inhibitor
design, we first examined a commercially available organosilane
amine, (3-Aminopropyl)trimethylsilane hydrochloride (2,
Figure 1), which contains only six carbons and one silicon.
Encouragingly, it showed a 75.5 ( 0.5% inhibition against the
wild-type (WT) A/M2 at 100 μM concentration in a two-electrode
voltage clamp (TEVC) assay, which is comparable with the eight-
carbon analogue 1 (86.6 ( 1.1%).21 This is by far the smallest
molecule (less than eight carbons) that is active against A/M2.
In this study, two silaspirane amines, 5 and 6, were designed
and synthesized on the basis of the previously identified spirane
amine scaffold (Scheme 2). The synthesis started from dichlor-
osilane, and double substitution of the chlorides with vinylmag-
nesium bromide yielded the divinyl cyclosilane.27,28 Without
purification, it underwent hydroboration with 9-borabicyclo-
[3.3.1]nonane (9-BBN), followed by an exchange reaction with
boraneÀmethyl sulfide complex (BMS), and subsequent metha-
nolysis gave the silaborinane intermediate.29 Next, Brown’s
dichloromethyl methyl ether (DCME) process and oxidation
with 30% H2O2 furnished the spirosilicon ketone intermediates,
3 and 4, in a one-pot process with ∼40À50% overall yield.27,28
Finally, the ketone was converted to amines 5 and 6 using the two-
step hydroxylamine condensation/reduction with ∼75% yield.23
The synthesized inhibitors were tested in a TEVC assay using
Xenopus laevis frog oocytes micro-injected with RNA expressing
either the WT A/M2 or A/M2-V27A mutant protein.30 The
potency of the inhibitors was expressed as the percentage
4,4-Dimethyl silacyclohexane amines 10 and 14 showed a
high potency against the WT A/M2 and only minimal inhibition
against A/M2-V27A mutant (Table 2), which was expected, as
A/M2-V27A prefers binding molecules with extended conforma-
tions.23,32 Compound 13 was found to be less active than 10 and 14,
but slightly more active than its carbon analogue 16. The chemical
shifts of the methyl protons in all three organosilane amine
compounds were close to 0 ppm, which is distinguished from the
protein signals and is ideal for them to serve as structural probes for
intermolecular NOESY experiments.
With the structural probes (10, 14, and 13) in hand, we next
pursued 13C-edited NOESY spectra with WT M2TM (22À46)
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peptide reconstituted in DPC micelles. To facilitate assign-
ment, residues at positions V27, A30, and G34 were selectively,
uniformly 15N and 13C labeled. When 2 equiv of compound 10
(1 mM) was added to M2TM tetramer (0.5 mM), two strong
NOE cross-peaks were observed between one of the two
methyl groups from 10 and both of the γCH3 groups of V27
(Figure 2b); weaker NOEs were observed for the other methyl
group of 10 and both of the γCH3 groups of V27. This methyl
group also shows an NOE to the βCH3 of Ala30 (Figure 2b).
The nonequivalent cross-peaks between the two methyl
groups of 10 reflect the nonsymmetrical binding geometry of
the drug inside the channel. The substituent with stronger
NOE signals to V27 is assigned to the axial methyl from 10,
which in models is closer to V27 than A30;34,35 the methyl
group that showed cross-peaks to Ala30 was assigned to the
equatorial methyl from 10. No NOEs were found between the
methyl groups of 10 and Gly34, which lies lower in the structure of the
channel as viewed in Figure 2d. This binding model is consistent with
our earlier studies showing that the drug binds inside the channel with
amine pointing down toward H37 (Figure 2d).36 The same NOE
cross-peak patterns were observed when the experiment was repeated
with only 1 equiv of 10 (0.5 mM) (SI Figure S1). In comparison with
10, the two methyl groups in 13 are equivalent, showing a single peak
at 0.07 ppm (indicative of a lower barrier for interconversion of the
seven-membered ring). As a weaker inhibitor against WT A/M2
compared with 10, when 2 equiv (1 mM) of 13 was added to M2TM
tetramer (0.5 mM), two strong NOE cross-peaks were observed and
assigned to the γCH3 of V27 and the methyl protons of 13. In
addition, an unambiguous NOE was assigned to the βCH3 of A30
and the methyl protons of 13 (Figure 2f).
Both 16 and its silicon analogue 13 were crystallized by solvent
evaporation in CH2Cl2/CH3OH (3:1 v/v) and their structures
determined by X-ray crystallography. The SiÀC bond length
(1.87 Å) was found to be on average 22% longer than the
corresponding CÀC bond length (1.54 Å), which results in
nearly 50% volumeincrease atthe quaternary center (SITable S1).
The size expansion effect of the carbon-to-silicon switch might
contribute to the antiviral potency increase of organosilane against
A/M2-V27A, as the expanded organosilane amine is able to fill in
the extra space created by the bulky valine 27 to smaller alanine
mutation (Table 1). Moreover, silanes are more lipophilic than the
corresponding alkanes; the ClogPs for 16 and 13 are 2.99 and 4.15,
respectively. Thus, the improved antiviral potency of organosilanes
compared with their carbon analogues might be due to the
synergetic effects of size expansion and increased lipophilicity.
Biologically active organosilanes, discovered through either
rational design or high-throughput screening, are attractive
analogues of their carbon counterparts due to their unique
properties. Carbon-to-silicon switch retains the overall 3D con-
formation of the inhibitor, thus providing a minimal perturbation
Table 2. Antiviral Activities of Organosilane Structural
Probes against WT A/M2 and A/M2-V27A Mutants
15
10
14
16
13
WT A/M2a
IC50 (μM)
A/M2-V27Aa
IC50 (μM)
95.3 ( 1.1 95.4 ( 1.4 95.4 ( 0.4 75.4 ( 1.8 78.5 ( 1.2
2.4 ( 0.2 2.6 ( 0.2 5.7 ( 0.3 26.1 ( 2.2 19.4 ( 1.6
0
7.9 ( 1.1 19.1 ( 1.0 2.5 ( 0.4
NA NA NA
0
NAb
NA
a Inhibition (%) at 100 μM inhibitor concentration. b NA = not available.
All compounds were also tested against S31N mutant and found to have
<20% inhibition at 100 μM.
Figure 2. 13C-edited 1H NOESY spectra of M2TM(22-46)-V27A30G34 in DPC micelles in the presence of either 10 (spectra aÀc) or 13 (spectra eÀg).
10 and 13 are bound in the pore, as assessed from intermolecular NOEs circled in red. (a,e) Upfield region of 1D 1H NMR. (b,f) Upfield region of 2D
13C-edited 1H NOESY spectra with 100 ms mixing time. (c,g) Upfield region of 2D 13C-HSQC; complete assignment was reported earlier.36 (d,h)
Docking models of compounds 10 and 13 in the A/M2 channel. The poses of the drugs reflect the relative NOE signal intensities. Sample conditions:
2 mM A/M2(22-46) with residues V27A30G34 double-15N,13C-labeled, 100 mM DPC, 1 mM 10 or 13, and 50 mM pH 7.5 phosphate buffer in 10% D2O,
90% H2O. Spectra were recorded at 313 K on a Varian Inova 600 MHz (for 13) or a Bruker Avance 500 MHz spectrometer (for 10).
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of the binding between the ligand and the receptor. Additionally,
the larger covalent radius of silicon (1.17 Å) compared to that of
carbon (0.77 Å) (SI Table S1), and the higher hydrophobicity
renders the organosilane-based inhibitors with improved proper-
ties. Organosilanes are also often easier to synthesize than their
carbon analogues and, in certain cases, allow access to novel
scaffolds not accessible by standard carbon chemistry.17 In this
report, we explored a carbon-to-silicon switch in A/M2 inhibitors
design. The silaspirane amines were as potent as their carbon
counterparts against WT and were more potent in targeting drug-
resistant V27A, which highlights their promise for further optimiza-
tion. Moreover, this replacement shows promise for NMR spec-
troscopy; three organosilane structural probes were designed to
map the A/M2 drug binding site. Previously, a debate concerning
the location of the pharmacologically relevant A/M2 drug binding
site(s) was settled in favor of the pore-binding model using the
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’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
spectroscopic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
wdegrado@mail.med.upenn.edu
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’ ACKNOWLEDGMENT
This work was supported by the NIH (GM56423 and
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