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J Med Chem
2014 Apr 24;578:3511-21. doi: 10.1021/jm500183r.
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Discovery of a potent and selective α3β4 nicotinic acetylcholine receptor antagonist from an α-conotoxin synthetic combinatorial library.
Chang YP
,
Banerjee J
,
Dowell C
,
Wu J
,
Gyanda R
,
Houghten RA
,
Toll L
,
McIntosh JM
,
Armishaw CJ
.
Abstract
α-Conotoxins are disulfide-rich peptide neurotoxins that selectively inhibit neuronal nicotinic acetylcholine receptors (nAChRs). The α3β4 nAChR subtype has been identified as a novel target for managing nicotine addiction. Using a mixture-based positional-scanning synthetic combinatorial library (PS-SCL) with the α4/4-conotoxin BuIA framework, we discovered a highly potent and selective α3β4 nAChR antagonist. The initial PS-SCL consisted of a total of 113 379 904 sequences that were screened for α3β4 nAChR inhibition, which facilitated the design and synthesis of a second generation library of 64 individual α-conotoxin derivatives. Eleven analogues were identified as α3β4 nAChR antagonists, with TP-2212-59 exhibiting the most potent antagonistic activity and selectivity over the α3β2 and α4β2 nAChR subtypes. Final electrophysiological characterization demonstrated that TP-2212-59 inhibited acetylcholine evoked currents in α3β4 nAChRs heterogeneously expressed in Xenopus laevis oocytes with a calculated IC50 of 2.3 nM and exhibited more than 1000-fold selectivity over the α3β2 and α7 nAChR subtypes. As such, TP-2212-59 is among the most potent α3β4 nAChRs antagonists identified to date and further demonstrates the utility of mixture-based combinatorial libraries in the discovery of novel α-conotoxin derivatives with refined pharmacological activity.
Figure 1. Design of the mixture-based PS-SCL based on the α4/4-conotoxin
framework. The conserved cysteine framework and native (globular)
disulfide bond connectivity are indicated. Conserved Gly, Ser, and
Pro residues are shown in gray. On is a single defined position, and X is
an equimolar mixture of 22 natural and non-natural l-amino
acids.
Figure 2. Initial screening
of the α4/4-conotoxin BuIA PS-SCL for α3β4
nAChR inhibition using the fluorescent membrane potential assay. The
library was screened in triplicate at 100 μM, and the percentages
of inhibition were calculated by comparing the potency of 10 μM
mecamylamine (MCA), which was defined as 100% inhibition. Residues
that were selected for the synthesis of a second generation library
of individual analogues are marked with an asterisk. Amino acids indicated
with a cross-hatch pattern correspond to native α-conotoxin
BuIA residues.
Figure 3. Representative
LC–MS analysis of TP-2212-59. (A) LC analysis
of TP-2212-59 samples. Samples were analyzed using a C18 column (50 mm × 4.6 mm i.d.) with a gradient of 0–60%
acetonitrile containing 0.1% formic acid over 12 min at a flow rate
of 0.5 mL/min and monitored at 214 nm. Crude samples (bottom) were
desalted in parallel using SPE cartridges prior to initial library
screening. Globular and ribbon isomers for further pharmacological
characterization (center and top, respectively) were synthesized using
a two-step regioselective folding approach and purified to >95%
homogeneity
as described in the Experimental Section.
(B) Representative electrospray ionization MS of TP-2212-59. The final
mass was calculated from the observed [M + 2H]2+ ion: calculated
mass, 1382.9; expected mass, 1382.5.
Figure 4. Fluorescent membrane
potential assay screening of the second generation
individual library at 10 μM. Compounds that exhibited >80%
inhibition
at 10 μM (indicated with a solid line and asterisk) were selected
for further chemical and pharmacological characterization.
Figure 5. Functional characterization
of selected individual α4/4-conotoxins
for α3β4 nAChR inhibition using the fluorescent membrane
potential assay.
Figure 6. Radioligand binding assays of BuIA and compounds 57–60 bound to the α3β4 nAChR
subtype.
[3H]Epibatidine was used as a hot ligand in the α3β4
nAChR ligand–receptor binding assay.
Figure 7. Functional
characterization of TP-2212-59 for α3β4
and α3β2 nAChR inhibition of acetylcholine evoked currents
in Xenopus oocytes. ACh (300 μM) was applied
as a 1 s pulse once per minute to Xenopus oocytes
expressing rat nAChRs. (A) TP-2212-59 (10 nM) was perfusion applied
to oocytes expressing α3β4 nAChRs until steady-state block
of the ACh current was achieved. TP-2212-59 was then washed out and
recovery of block measured. (B) Concentration response of TP-2212-59
on the α3β4 nAChR. The top and bottom of the curve were
constrained to 100 and 0, respectively. The IC50 value
is calculated as 2.3 nM. n = 3–5 oocytes for
each peptide concentration. (C) TP-2212-59 was applied as a 10 μM
static bath for 5 min to oocytes expressing rat α3β2 nAChRs
(1000 times higher peptide concentration than that used for α3β4
nAChRs). Representative traces are shown for each nAChR subtype.
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