Generation of candidate ligands for nicotinic acetylcholine receptors via in situ click chemistry with a soluble acetylcholine binding protein template

Article abstract of DOI:10.1021/ja3001858

Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide-alkyne cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole-AChBP complexes shows binding poses and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and antagonists for ligand-gated ion channels.

Full text of DOI:10.1021/ja3001858

Article
pubs.acs.org/JACS
Generation of Candidate Ligands for Nicotinic Acetylcholine
Receptors via in situ Click Chemistry with a Soluble Acetylcholine
Binding Protein Template
Neil P. Grimster,† Bernhard Stump,† Joseph R. Fotsing,† Timo Weide,† Todd T. Talley,‡
‡
‡,§
John G. Yamauchi, Akos Nemecz, Choel Kim,∥ Kwok-Yiu Ho,‡ K. Barry Sharpless,† Palmer Taylor,‡
́
and Valery V. Fokin*,†
†Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037,
United States
§
‡Department of Pharmacology, Skaggs School of Pharmacy & Pharmaceutical Sciences, and Department of Chemistry and
Biochemistry, University of California San Diego, La Jolla, California 92093, United States
∥Department of Pharmacology, The Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of
Medicine, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: Nicotinic acetylcholine receptors (nAChRs), which are responsible for mediating key physiological functions, are
ubiquitous in the central and peripheral nervous systems. As members of the Cys loop ligand-gated ion channel family, neuronal
nAChRs are pentameric, composed of various permutations of α (α2 to α10) and β (β2 to β4) subunits forming functional
heteromeric or homomeric receptors. Diversity in nAChR subunit composition complicates the development of selective ligands
for specific subtypes, since the five binding sites reside at the subunit interfaces. The acetylcholine binding protein (AChBP), a
soluble extracellular domain homologue secreted by mollusks, serves as a general structural surrogate for the nAChRs. In this
work, homomeric AChBPs from Lymnaea and Aplysia snails were used as in situ templates for the generation of novel and potent
ligands that selectively bind to these proteins. The cycloaddition reaction between building-block azides and alkynes to form
stable 1,2,3-triazoles was used to generate the leads. The extent of triazole formation on the AChBP template correlated with the
affinity of the triazole product for the nicotinic ligand binding site. Instead of the in situ protein-templated azide−alkyne
cycloaddition reaction occurring at a localized, sequestered enzyme active center as previously shown, we demonstrate that the in
situ reaction can take place at the subunit interfaces of an oligomeric protein and can thus be used as a tool for identifying novel
candidate nAChR ligands. The crystal structure of one of the in situ-formed triazole−AChBP complexes shows binding poses
and molecular determinants of interactions predicted from structures of known agonists and antagonists. Hence, the click
chemistry approach with an in situ template of a receptor provides a novel synthetic avenue for generating candidate agonists and
antagonists for ligand-gated ion channels.
for central nervous system disorders such as schizophrenia,
nicotine addiction, and Alzheimer’s disease among other
cognitive disorders.2,4,5 An extensive variety of synthetic and
naturally occurring ligands for nAChRs of variable selectivity
are known. Agonists and competitive antagonists bind in the
1. INTRODUCTION
Nicotinic acetylcholine receptors (nAChRs) belong to a
superfamily of neurotransmitter ligand-gated ion channels
characterized by a pentameric structure of Cys-loop-containing
subunits.1,2 This family of proteins is actuated upon binding of
a specific ligand and includes other key neurotransmitter
receptors, such as glycine, GABA-A, and 5-HT3 types.3 The
nAChRs are currently being considered as therapeutic targets
Received: January 7, 2012
Published: March 6, 2012
© 2012 American Chemical Society
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extracellular domain at the interface between subunits, and
agonists transmit ligand occupation through conformational
changes to open an internally located ion channel, effecting a
rapid depolarization.1,6
the dynamic molecular motion of the protein subunit interface,
in situ click chemistry facilitates the identification of potential
ligands for different conformational states of a protein without
the need to synthesize large screening libraries. We initially
prepared an artificial triazole-containing AChBP ligand via
Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) con-
ditions,20,21 and after confirmation of the triazole’s affinity, we
subsequently utilized the compound’s components to validate
the AChBPs as in situ click chemistry templates. The alkyne
component and subsequent in situ azide component that
formed the product with higher efficiency and affinity were then
used as leads for the refinement of building blocks to form
triazole structures. These derivatives should mimic the
properties of the natural nAChR neurotransmitter, acetylcho-
line (ACh), along with naturally occurring and synthetic
congeners, thereby providing a starting point for developing
selective ligands that address the binding site. Furthermore, we
demonstrate that multiple ligands may be formed when a
mixture of building blocks is screened simultaneously, with the
amount of the triazole product generated by the template being
proportional to its affinity for the binding site. Structurally we
demonstrate in situ product formation at a subunit interface
binding region of a multisubunit protein, the AChBP. This
receptor surrogate serves as a template for the generation of
nicotinic receptor ligands.
The acetylcholine binding proteins (AChBPs), which are
homologous to the extracellular domain of pentameric ligand-
gated ion channels, have been found to be expressed as soluble
pentamers in select gastropods and polychaetes.7−11 Homolo-
gous to the N-terminal ∼210 amino acids in the extracellular
receptor domain, AChBPs mimic the recognition properties of
nAChRs, providing a critical template describing the shape and
spatial disposition of residues contributing to the binding
site.7,12 The five acetylcholine binding sites of the AChBPs lie at
the subunit interfaces and are partially surrounded by a flexible
loop C found between sheets β9 and β10 that extends across
the subunit interface.
Structure-based drug design and screening of compound
libraries have been a laborious task in the case of nAChRs. The
membrane disposition of the nAChR, the diversity of receptor
subtypes, and the dynamic nature of the binding site complicate
classical drug discovery approaches. In contrast, target-guided
in situ synthesis is an attractive and efficient approach to drug
discovery because it directly employs the biological target for
the assembly of candidate leads from their own building blocks.
Independent of the knowledge of the geometry of the protein
target, it allows fast screening of potential ligands whose
building blocks are “templated” by the target protein. Although
this concept has been previously demonstrated using different
connecting reactions,13−16 the in situ click chemistry approach
is unique with its reliance upon the nearly bioorthogonal 1,3-
dipolar cycloaddition between azides and alkynes, a reaction
that is fully compatible with functional groups found in normal
physiological environments.
These building blocks readily react with each other when in
proximity but remain inert toward side chains and amide
backbones of proteins. This highly exergonic reaction produces
five-membered nitrogen heterocycles, anti- or syn-1,2,3-
triazoles, that are stable toward acidic and basic hydrolysis as
well as under severe redox conditions.17 A target protein,
employed as a template, can generate a high-affinity ligand from
monovalent building blocks using the in situ click chemistry
approach.18 The process begins with selective binding of anchor
molecules to specific areas of the protein binding site.
Subsequently, if two of the bound anchor molecules are in
proximity, they link together irreversibly within the confines of
their binding pockets and thereby allow the formation of an
energetically more favorable new complex with the protein.
The resultant compound can then be detected by liquid
chromatography/mass spectrometry (LC/MS).
Since this approach employs the biological target to assemble
its own ligands from a library of reagents that can be combined
in a multitude of different ways,18 rather than requiring the
synthesis, purification, and screening of each possible library
product, the use of in situ click chemistry promises greater
efficiency than traditional combinatorial chemistry or fragment-
based drug design. Moreover, the triazole product may induce
or select a conformation of the macromolecule so adapted in
conformation to the binding of the ligand.19 The template-
generated compounds are then further screened to assess their
binding affinity and specificity for the target.
2. EXPERIMENTAL METHODS
Synthetic Compound Preparations. All azides were synthesized
by the displacement of either a halide, mesylate, or tosylate with
sodium azide in N,N-dimethylformamide. Propargyl phenol ethers
were prepared using literature conditions.22 The 1,4-triazole products
2, 14, 15, 18, 19, 27, 28, and 29 were synthesized using CuAAC,
whereas 1,5-triazole 18a was synthesized using ruthenium-catalyzed
azide−alkyne cycloaddition (RuAAC;23,24 see the Supporting In-
formation for details).
Procedure for the in situ Formation of 2 from a Binary-
Component Mixture. Azide 4 [1 μL, 100 mM in 0.1 M aqueous
sodium phosphate buffer, pH 7.0 (PBS)] was added to 98 μL of a
solution of the protein of interest (∼1 mg/mL in PBS) in a microfuge
tube, followed immediately by alkyne 3 [1 μL, 50 mM in dimethyl
sulfoxide (DMSO)] to give final concentrations of 1 mM for azide 4
and 0.5 mM for alkyne 3. The reaction mixture was briefly vortexed
and then incubated at room temperature. In a separate microfuge tube,
azide 4 (1 μL, 100 mM in PBS) and alkyne 3 (1 μL, 50 mM in
DMSO) were diluted with water (87 μL) before aqueous copper
sulfate (1 μL, 0.05 M) and aqueous sodium ascorbate (10 μL, 0.1 M)
were added. The reaction mixture was briefly vortexed and then
incubated at room temperature. After 3 days, three samples (25 μL)
from each tube were directly injected into the LC/MS instrument to
perform LC/MS-SIM analysis under the following conditions: Zorbax
4.6 mm × 3 cm, SB-C18 (rapid resolution) reversed-phase column
preceded by a Phenomenex C18 guard column; flow rate, 0.5 mL/min;
gradient elution, (H2O + 0.05% TFA)/(MeCN + 0.05% TFA) from
100:0 to 0:100 over 15 min followed by 100% MeCN + 0.05% TFA
for 5 min with a post-run time of 5 min using the starting solvent ratio.
Detection was by electrospray ionization and mass spectrometric
detection with positive selected-ion monitoring tuned to the molecular
mass of 2 (M+). The cycloaddition product was identified by
comparison of its retention time with those determined from analysis
of the copper-catalyzed reaction and by its molecular weight. Control
experiments in the presence of bovine serum albumin (BSA, 1 mg/
mL) instead of the binding protein as well as in the presence of Ls and
the known receptor inhibitor methyllycaconitine (MLA, 1 mM final
concentration) were run as described above.
In this work, we used AChBPs from Lymnaea stagnalis (Ls),
Aplysia californica (Ac), and the Y55W Aplysia mutant
(AcY55W) as template surrogates for nAChRs. Because of
Procedure for the in situ Screening of Single-Component
Libraries. To generate library 1a, azides 4−8 were dissolved in PBS
(100 mM) and the solutions combined. The solution of azides (1 μL)
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was added to the protein of interest (∼1 mg/mL in PBS, 98 μL) in a
microfuge tube, followed immediately by alkyne 3 (1 μL, 50 mM in
DMSO). The reaction and controls were performed similarly to the
binary component mixture. After 3 days, the reactions were analyzed in
triplicate as described for the binary mixture, using mass spectroscopic
detection with positive selected-ion monitoring tuned to the five
expected molecular masses of the products (M+). The cycloaddition
products were identified by comparison of the retention times with
those determined from the analysis of the copper-catalyzed reaction
and by their molecular weights. Azide library 1b was screened using in
situ click chemistry as described above, substituting azides 4−8 with
azides 9−13. The alkyne library was again screened as described above,
using azide 9 (100 mM in PBS) and a combined DMSO solution of
alkynes 3 and 23−26 at a final concentration of 0.5 mM.
Procedure for the in situ Screening of Azide and Alkyne
Libraries. Azides 4−13 were dissolved separately in PBS (100 mM),
and 25 μL aliquots of these solutions were taken and combined.
Alkynes 3 and 23−26 were dissolved in DMSO (50 mM) and the
solutions combined. The combined solution of azides (10 μL) was
added to a solution of Ls (∼1 mg/mL in PBS, 980 μL) in a microfuge
tube, followed immediately by the combined solution of alkynes (10
μL). The reaction mixture was briefly vortexed and then incubated at
room temperature. After 10 days, triplicate analysis of the protein-
catalyzed reaction was performed using the chromatography
conditions described above. To improve the MS detection sensitivity,
10 injections (each 25 μL) were performed; each injection was tuned
to detect five of the expected 50 molecular weights. A control
experiment in the presence of BSA (1 mg/mL) instead of the binding
protein was performed. As some of the potential in situ click chemistry
products have the same molecular weights, the corresponding triazoles
were prepared by reacting each azide with the five alkynes under
CuAAC conditions. Therefore, azides 4−13 (1 μL, 100 mM in PBS)
were charged to 10 separate microfuge tubes, and water (87 μL),
combined alkyne solution (1 μL) as described above, aqueous copper
sulfate (1 μL, 0.05 M) and aqueous sodium ascorbate (10 μL, 0.1 M)
were added. The reaction mixtures were briefly vortexed and then
incubated at room temperature. After 10 days, analyses of the copper-
catalyzed reactions were performed as described above. The
cycloaddition products of the in situ screen were identified by their
molecular weights and by comparison of the retention times of the
formed products with the values determined by analysis of the copper-
catalyzed reactions.
saturating concentration (12.5 μM) of MLA (Tocris) to an identical
set of samples. Competition assays were conducted in a similar manner
except that the concentration of ( )-[3H]epibatidine was held
constant (5−20 nM final concentration) and varying concentrations
of competing ligand were added to the samples in a constant volume,
of either 50 μl or 100 μl. The resulting mixtures were allowed to
equilibrate at room temperature for a minimum of 1 h and measured
on a 1450 MicroBeta TriLux liquid scintillation counter (Wallac). The
data obtained were normalized, and the Kd was calculated from the
observed EC50 value28 using GraphPad Prism version 4.02 (GraphPad
Software, San Diego, CA, USA, www.graphpad.com). At least three
independent experiments performed in duplicate were used to
determine the reported Kd values.
Complex Formation and Crystallization. The 18−Ac AChBP
complex was formed by combining 2 μL of a solution of 18 (10 mmol
dissolved in DMSO) with 48 μL of purified concentrated protein at a
concentration of 5 mg/mL to achieve a stoichiometric excess of ligand
to binding sites. The 18−Ac AChBP complex cocrystals were prepared
by the vapor diffusion hanging drop method. Concentrated protein
complex was mixed in a 1 μL/1 μL ratio with a solution consisting of
0.1 M Tris-HCl (pH 8.0), 0.25 M MgCl2, and 20% (w/v) PEG 4000;
the mixture was incubated at 22 °C and suspended over 500 μL of the
solution. Crystals of final size 0.3 mm × 0.3 mm × 0.2 mm appeared
after a few weeks.
X-ray Diffraction Data Collection. 18−Ac AChBP complex
cocrystals were transferred to a cryoprotective harvest solution [0.1 M
Tris-HCl (pH 8.0), 0.25 M MgCl2, 12% (w/v) PEG 4000, and 10%
(v/v) glycerol] and flash-cooled directly in liquid nitrogen. A full set of
X-ray diffraction data was collected at 100 K on ALS beamline 8.2.2 at
Lawrence Berkeley National Laboratory. The data were processed
using the HKL2000 program.29 Data collection statistics are given in
Table S7 in the Supporting Information.
Structure Refinement. The 18−Ac AChBP complex structure
was solved by the molecular replacement method with the PHASER30
software using an ensemble of AChBP structures (PDB entries 2BYN,
2PGZ, 2BYP, 2BYR, 2BYS, and 3C79) as the search model. The
electron density maps were fitted with COOT,31 and structure
refinement used the program REFMAC5.32 Refinement statistics are
listed in Table S7. Atomic coordinates and structure factors of the
complex have been deposited in the Protein Data Bank (PDB entry
4DBM). The structural figures were generated using PyMOL33 and
Discovery Studio 3.0 (Accelrys).
Preparation of AChBPs. The AChBPs from Ls, Ac, and AcY55W
were expressed and purified as previously described.25,26 Briefly,
AChBPs were expressed with an amino-terminal FLAG epitope tag
and secreted from stably transfected HEK293S cells lacking the N-
acetylglucosaminyltransferase I (GnTI−) gene.27 The protein was
purified with FLAG-antibody resin and eluted with FLAG peptide
(Sigma). The affinity-purified protein was further characterized by
size-exclusion chromatography [Superose 6 10/300 GL column (GE
Healthcare) in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.02%
NaN3] to ascertain the pentameric association. Purified AChBP
pentamers were then concentrated in a YM50 Centricon ultrafiltration
unit (Millipore) to a final concentration of ∼5 mg/mL, removing
monomeric subunits and trace contaminants.
3. RESULTS
We hypothesized that substituting a 1,2,3-triazole unit for the
ester moiety of ACh would mimic its hydrogen-bond-acceptor
character (Figure 1), an interaction that has previously been
observed in triazole-containing peptidomimetics.34−36 Further-
more, the conservation of the trimethylammonium-containing
moiety of ACh would maintain the crucial cation−π interaction
with Trp147 (Ac AChBP numbering) and other proximal
aromatic amino acid side chains.25,37−40
Radioligand Binding Assays. A scintillation proximity assay
(SPA) was used to determine the apparent Kd value of the compounds
as reported previously.26 Briefly, AChBP (0.5−1.0 nM final
concentration in binding sites), polyvinyltoluene anti-mouse SPA
scintillation beads (0.17 mg/mL final concentration, GE Healthcare),
monoclonal anti-FLAG M2 antibody from mouse 1:8000 dilution
(Sigma), and ( )-[3H]epibatidine (5−20 nM final concentration, GE
Healthcare) were combined with PBS. A quick screen was performed
to determine the relative binding affinities of the compounds by
adding compound to the previously mentioned solution at a final
concentration of 10 μM. Apparent Kd values were then determined for
compounds that reduced the normalized counts per minute below
50%. Saturation binding of ( )-[3H]epibatidine was measured by
adding increasing concentrations of ( )-[3H]epibatidine in a constant
volume. Nonspecific binding was determined in parallel by adding a
Figure 1. Binding pose of acetylcholine (left) and proposed binding
pose of the triazole-based acetylcholine mimetic (right).
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In conformance with our hypothesis, azide 1 was reacted
with a range of alkynes under standard CuAAC reaction
conditions, and the resultant tertiary amines were quaternized
with methyl iodide (eq 1 in Scheme 1). Screening of the
control reaction showed that MLA, a nicotinic antagonist,
returned the product formation to background levels, thus
demonstrating that the flexible subunit interface ACh binding
site indeed served as the template for the cycloaddition
reaction. It is noted that the amounts of product formed in the
presence of the template were correlated with the affinities (1/
Kd, μM−1) determined for the tested AChBPs. This suggests
that product selectivity toward a specific target over closely
related receptor subtypes could be inferred from the
bioorthogonal triazole formation without the need to acquire
individual binding data. Unfortunately, the 1,4- (anti-) and 1,5-
(syn-) 1,2,3-triazole isomers proved to be inseparable by LC/
MS, despite considerable method development. Thus, sub-
sequent experiments considered both regioisomers together,
while comparisons were made to the 1,4-isomers because of
their ease of synthesis via CuAAC.20,21 The outcome of the in
situ triazole templation at a flexible binding site expands the
templation potential of in situ click chemistry to receptor-
relevant targets and intersubunit binding sites.
Triazole 2, the in situ-formed ligand with the dissociation
constant in the nanomolar range for Ls, was next used as a lead
for the discovery of analogues with improved affinity and
selectivity for the closely related AChBPs. Selective target-
catalyzed synthesis of a new ligand from a library of building
blocks simultaneously present in a single reaction mixture
would be a significant advantage in drug discovery, enabling
rapid screening of many building-block pools or combinations
and reducing the amount of protein required for each individual
analysis. To this end, azide building blocks containing a variety
of quaternary nitrogen centers were synthesized (Table 2). This
Scheme 1. Synthesis of a Triazole Library Using Standard
CuAAC Reaction Conditions
compounds (data not shown) against Ac, Ls, and AcY55W
AChBPs identified compound 2 as having a relatively strong
association with all three AChR surrogates (Table 1).
Table 1. Proof of Concept: Amounts of Triazole 2 Formed
in situ in the Presence of AChBP Templates Compared with
the Respective Affinities of the Triazole Product
Table 2. Azide Libraries 1a and 1b: in situ Click Chemistry
Screen against Alkyne 3
[1] BSA control protein was used in place of the AChBP template. [2]
MS counts were corrected for background via BSA controls and
normalized to Ls. [3] Structure of the cation detected by MS.
We first wanted to confirm that the flexible subunit interfaces
in the AChBPs were capable of catalyzing the formation of 2 in
situ, as most previous examples have relied upon well-defined
sites internal to the subunit.41 To validate our hypothesis, the
constituent alkyne 3 and azide 4 (eq 2 in Scheme 1) were
incubated in the presence of Ls, Ac, and AcY55W AChBPs in
PBS at room temperature for 3 days (see Experimental
Methods). In addition, two control reactions were performed:
incubation of the reactants in the presence of BSA, to
determine whether nonspecific protein catalysis of the
triazole-forming cycloaddition was occurring; and incubation
in the presence of Ls with a known competing ligand, MLA, to
confirm that the protein-templated reaction was occurring at
the ACh orthosteric binding site.
Triplicate samples of the reaction mixtures were analyzed by
LC/MS with selected-ion monitoring (detection window set to
the molecular weight corresponding to the most abundant
peak), and the retention times were compared with those of the
previously synthesized sample of 2. Analysis of these data
showed that Ls efficiently templated the formation of ligand 2
under the reaction conditions, while both Ac and AcY55W
AChBP also produced the product, but much less efficiently
(Table 1).
library was designed to probe the effect of the quaternary amine
systematically using ring systems of increasing complexity (4−
8, library 1a; and 9, library 1b; Table 2) while also extending
the linker between the azide and the amine for each iterative
compound (10−13, library 1b; Table 2).
The in situ reactions for libraries 1a and 1b were allowed to
proceed at room temperature for 3 days. LC/MS analysis of the
reaction mixtures (performed in triplicate) revealed several
interesting phenomena (Tables 3a and 3b). As demonstrated
above, Ls catalyzed the formation of the triazole products more
efficiently than the Ac and AcY55W AChBPs, while no products
were detected in the BSA control reaction. However, in
contrast to the binary mixture, there was no pronounced
increase in the formation of triazole 2 when library 1a was used.
This finding can be explained by the preferential binding of
azides 6 and 8 to the template, favoring the formation of
products 15 and 14, respectively.42 These compounds were
synthesized via CuAAC, and their Kd values were determined.
Comparison of the amounts of product formed in situ with the
Comparison of the in situ click chemistry reactions
performed in the presence of AChBPs with the BSA control
reaction confirmed that triazole formation was selectively
accelerated in the presence of the receptor surrogates. The
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After tropane derivative 18 had been identified as the
compound of highest affinity, a set of alkynes was designed and
reacted with azide 9. This library, comprising the previously
tested quinolinone derivative 3 and variously substituted aryl
propargyl ethers 23−26 (Table 4), was incubated with the Ls,
Table 3a. Templation and Binding Data for the Triazole
Derivatives of the in situ Click Chemistry Screen of Azide
Building Blocks (Library 1a)
Table 4. Alkyne Library 2 Utilized for an in situ Click
Chemistry Screen against Azide 9
Ac, and AcY55W AChBPs. LC/MS analysis of the reaction
mixtures (performed in triplicate) showed that all of the tested
alkynes underwent AChBP-templated cycloaddition reactions
with azide 9. As before, the reactions against the BSA control
produced no detectable products. Triazoles 27−29 were
synthesized using standard CuAAC conditions, and their Kd
values were determined (Table 5). Again it is worth noting that
[1] MS counts were corrected for background via BSA controls and
normalized. [2] n.d. represents no data, as the compound was not
synthesized to generate affinity values. [3] Structures of the cations
detected by MS.
Table 3b. Templation and Binding Data for the Triazole
Derivatives of the in situ Click Chemistry Screen of Azide
Building Blocks (Library 1b)
Table 5. Templation and Binding Data for the Triazole
Derivatives of the in situ Click Chemistry Screen of Alkyne
Building Blocks (Library 2)
[1] MS counts were corrected for background via BSA controls and
normalized. [2] n.d. represents no data, as the compound was not
synthesized to generate affinity values. [3] Structures of the cations
detected by MS.
[1] MS counts were corrected for background via BSA controls and
normalized. [2] n.d. represents no data, as the compound was not
synthesized to generate affinity values. [3] Structures of the cations
detected by MS.
affinities of these compounds (1/Kd, μM−1) revealed a clear
trend (Table 3a), which suggests that the amount of product
formed is related to its affinity for the specific AChBP relative
to those of the other members of the library. A similar
correlation was observed when azide library 1b (9−13) was
screened against alkyne 3 (Table 3b). Under those conditions,
triazole 18 was formed in the greatest amount by Ls and was
also shown to have the highest affinity (Kd = 0.96 0.22 nM),
consistent with the observations from library 1a. Pyrrolidinium
derivative 19, which was formed in lower amounts, had a
significantly lower affinity (Kd = 82 14 nM). In contrast, the
amount of triazoles 16, 17, and 20−22 were comparatively low,
and therefore, they were not synthesized. The capability to
identify only combinations that warrant larger-scale synthesis
and further investigation demonstrates another advantage of
the in situ click chemistry approach.
compounds 27−29 shared both comparable amounts of
product formation and similar magnitudes of the affinity.
Only triazole 30 was not synthesized in the presence of Ac or
AcY55W AChBPs, and only small amounts were detected in the
reaction catalyzed by Ls. The previously discovered triazole 18,
originating from alkyne 3 and azide 9, was formed in
significantly higher quantity and, as expected, exhibited a
substantially higher affinity for all of the binding proteins
(Table 5).
Having demonstrated that AChBPs selectively catalyze the
formation of high-affinity ligands from libraries of alkynes and
azides, we next examined whether the template could select the
most productive combinations (i.e., those resulting in the most
potent ligands) from a pool of various azides and alkynes
simultaneously present in the reaction mixture. To this end,
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azides 4−13 were reacted with alkynes 3 and 23−26 in a
combined mixture using Ls as the template (see Experimental
Methods for details). Ls was chosen because of the higher
yields obtained in the previous reactions shown in Tables 3a,
3b, and 5. To compensate for the reduced concentrations of the
individual building blocks, the incubation time was increased to
10 days. The results showed that several triazole products could
be detected in comparison with the BSA control reaction,
which exhibited no product formation (Figure 2).
we determined binding parameters of its 1,5-isomer, 18a, which
was prepared using RuAAC as shown in Scheme 2. Although
Scheme 2. RuAAC Synthesis of the 1,5-Triazole Isomer 18a
the Kd of 18a for Ls was only slightly higher than of 18, its
binding to Ac and AcY55W was significantly weaker, thus other
1,5-disubstituted triazoles were not examined further in the
present study.
To define the binding site determinants governing the
reaction, we were interested in deciphering the binding pose of
triazole 18, the most potent ligand found in this study. Since
our conditions of crystallization typically yielded higher-
resolution structures with Ac AChBP, we concentrated on
refining the structure of this complex, yielding a structure with a
resolution of 2.3 Å. The 3σ omit map of the best represented
ligand is shown in Figure 3A.
Figure 2. Templation data for the triazole derivatives of the in situ
screen of azide and alkyne building blocks on Ls AChBP (data have
been normalized to the largest peak).
We found the density for the quinolinone to be much weaker
than that of the triazole and tropane ring systems in most of the
binding sites. The faint density suggests a flexibility of the
quinolinone that is most likely due to the rotatable methylene
carbon bond that links the quinolinone to the triazole. Indeed,
the binding mode shows that the quaternary-bridged-nitrogen
forms cation−π interactions with the Trp147, Tyr188, and
Tyr93 side chains, a previously noted observation for
quaternary amines that bind nAChRs.38 Figure 3B and
Supplementary Figure 1C in the Supporting Information
show the distances between the quaternary tropane nitrogen
and the closest atoms in the aromatic side chains of Trp147,
Tyr93, and Tyr188 to be between 4.3 and 5.2 Å, as has been
noted to be optimal for cation−π interactions.43 The tight fit of
the ligand is achieved by the aromatic side chains, including
Y195 from the principal subunit and Y55 from the
complementary subunit, which surround the quaternary
nitrogen and provide an anchor for the azide building block
9 (Figure 3C). The propargyloxyquinolinone ring system stacks
between the vicinal disulfide bridge joining Cys190 and Cys191
of the β9−β10 linker (loop C) and Met116 (Figure 3B,D and
Supplementary Figure 1B). The triazole is positioned such that
N3 forms a hydrogen bond with a neighboring water molecule,
similar to that reported for the pyridine ring systems (Figure 3B
and Supplementary Figure 1A).37,39,44 This water molecule
forms a hydrogen-bond linkage with other water molecules
extending toward the complementary subunit and the vestibule
of the nAChR channel. The ether oxygen also forms a
hydrogen-bonding system with Arg79 and Tyr195. The
hydroxyl of Tyr195 approaches basally, while the protonated
nitrogen of Arg79 reaches down from the apical region. The
carbonyl of Val148, which binds to the complementary
hydrogen of Arg79’s protonated nitrogen, and a structural
water associated between Tyr195 and Glu193 completes the
hydrogen-bonding system (Supplementary Figure 1A).
The majority of the products formed in the presence of the
Ls template had been previously identified in simpler in situ
reaction pools. Triazole 18, the product of the cycloaddition
between alkyne 3 and azide 9, was again formed in the greatest
amounts, which is consistent with the affinity of this triazole for
Ls. Compound 14, with the second highest affinity discovered
in this study, was formed in the second largest amount. Similar
trends between the affinities of previously examined triazole
products and the amounts formed were also observed, despite
the increased complexity of the system. Interestingly, the
combination of quinuclidinium azide 8 and alkynes 25 and 26
and the cycloaddition products of piperidinium azide 6 with
alkynes 23, 24, and 25 were identified by LC/MS analysis.
These products were the result of combinations that had not
previously been screened. Although it is of note that these
compounds formed in situ, the relatively low amounts formed
suggests they have lower affinities and were therefore accorded
lower priority.
We have therefore demonstrated that it is possible to screen
libraries of azides and alkynes simultaneously and detect the
formation of triazole products while generating valuable
information regarding the affinity of the compounds for the
target protein. The increased number of building blocks
screened in a single reaction further increases the throughput
of the in situ click chemistry approach. Moreover, the observed
positive correlation between the amount of product formed and
its affinity for the target could be useful for subsequent
structure−activity studies, whether through traditional medic-
inal chemistry methods or further iterations of in situ target-
templated screening.
Having demonstrated that triazole 18 exhibited the highest
affinity of all the compounds detected throughout this study,
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Figure 3. Crystallographic analysis of triazole 18 in complex with Ac AChBP. The complementary subunit is represented with green labels, while the
primary subunit is denoted with black labels. (A) Surface representation of ligand interpolated charges and the Fo − Fc omit map at 3σ. (B) Triazole
18 in the principal subunit binding pocket highlights the interactions of the dimethyltropane aza nitrogen with the surrounding aromatic residues
Trp147, Tyr93, and Tyr188 in the principal subunit, viewed radially at the subunit interface. The interaction of the triazole nitrogen (N3) with a
structural water is also shown. Residues shown are within 4 Å. (C) Connolly surface representation of the quaternary tropane moiety as viewed from
the membrane side of the binding site. Stabilization is achieved by the aromatic nest of residues of the principal subunit noted in (B) and Tyr55 from
the complementary subunit. (D) Surface representation of the quinolinone, triazole, and tropane rings (dark gray sticks, white label) showing the
participation of Arg79, Met116, Ile118, and Tyr55 of the complementary subunit in addition to those noted in the principal subunit.
change in conformation that enhanced aromatic π−π
association at the peripheral site, whereas a change in
conformation did not occur with the lower affinity anti
isomer.19 Accordingly, one of the tenets of selectivity for the
in situ click chemistry reaction may arise from the precursors
driving a conformation preferred by the triazole reaction
product rather than accommodating a conformation of the
unbound protein. This bodes well for the ability of the
technique to select conformations of the ligand−protein
complex that differ from the unbound protein.
Further support for the concept that the triazole product
adopts a binding pose and selects a conformation most
compatible with the product association comes from an
examination of the crystal structure of triazole 18. Here we
observe a bound conformation and pose that has been
predicted from the conformation of other quaternary-amine-
containing ligands that bind to AChBP through cation−
quadrupole interactions involving the electron-rich aromatic
side chains (e.g., tryptophan). The positioning of the triazole
such that it may form a hydrogen bond within the pocket
supports the notion that precursors drive a conformation
change needed for the reaction between them. The in situ
reaction may be influenced by the binding strength of the
component containing the tertiary or quaternary nitrogen (in
our case, the quaternary tropane) and the ability of the
complementary component to form the triazole in the proper
orientation to allow for the formation of a highly selective
4. DISCUSSION
In situ click chemistry has previously been employed in the
development of high-affinity ligands for the active sites of
acetylcholinesterase, HIV protease, and carbonic anhy-
drase.18,41,45,46 These ligands bind within a gorge centrosym-
metric to a subunit in the oligomeric AChE, the shallow
binding site in the HIV protease, or the single subunit in
monomeric carbonic anhydrase, respectively. The AChBPs,
with their binding sites at the interface of two subunits, provide
a distinct template for in situ click chemistry, yet the
bioorthogonal reaction forming the heterocyclic triazole can
take place in the presumably more flexible and exposed subunit
interface.
In this study, we have also demonstrated that the efficiency of
the target-catalyzed synthesis of the ligands is partially
determined by the affinity of the reaction product, that is, the
amount of the formed triazole increases with the strength of its
binding to the target. This was also the case when we compared
the preference of anti and syn regioisomer formation on
acetylcholinesterase with the equilibrium affinities of the
respective triazoles.47 This correlation between preference of
formation and equilibrium affinity may suggest that the
transition state more closely resembles the reaction product
than the simple bimolecular associations of the building blocks
with the template.
With the acetylcholinesterase complexes, we demonstrated
that the higher-affinity syn regioisomer of the triazole induced a
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Notes
molecule. Comparison of precursor component conformations
with those of the final triazole compound will be of interest in
the future. However, accomplishing this task may be difficult, as
the precursor affinities are typically low, so their crystal
structures would not be well-resolved in the pocket of the
AChBP.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by USPHS Grants UO1 DA19320-05
and R01 GM18360-37. B.S. acknowledges a fellowship from the
́
Swiss National Science Foundation (SNF). J.G.Y. and A.N.
5. CONCLUSIONS
were supported by NIH Training Grant GM-07752. J.G.Y. was
also supported by NSF Grant GK-12 0742551. C.K. was
supported by NIH Grant R01 GM090161-01 and a Robert A.
Welch Foundation Chemistry and Biology Collaborative Grant.
V.V.F. acknowledges support by NIH (NIGMS) Grant R01
GM087620. The Berkeley Center for Structural Biology is
supported in part by the National Institutes of Health, National
Institute of General Medical Sciences, and the Howard Hughes
Medical Institute. The Advanced Light Source is supported by
the Director, Office of Science, Office of Basic Energy Sciences,
of the U.S. Department of Energy under Contract DE-AC02−
05CH11231.
We have successfully used a combination of CuAAC and in situ
click chemistry to achieve rapid conversion of the endogenous
ligand of nAChRs, acetylcholine, into compound 18, a highly
potent and selective ligand for Ls AChBP. This work represents
the first example of in situ click chemistry performed at a
flexible subunit interface of an oligomeric protein and thus
increases the number of biologically relevant targets that can be
investigated using this methodology. In addition, we have
demonstrated a positive correlation between the amount of
product generated in situ and its affinity for the target binding
protein. Therefore, this approach enables rapid refinement of
structures while providing valuable information about binding
and selectivity over closely related targets without the need to
synthesize and assay individual compounds. Furthermore, we
have shown that sets of building blocks can be screened in a
single reaction pool with 10 azides and 5 alkynes, allowing for
the possible generation and detection of the highest-affinity
compounds from several discrete sets of congeneric building
blocks. The variety of possible combinations enhances the
efficiency of the technique and reduces the amount of protein
required for the experiments. Finally, we have cocrystallized the
most active compound that emerged from these studies, 18,
with Ac-AChBP and shown that our initial hypothesis about
specific interactions was indeed valid. We are currently
continuing to expand the utility of the combination of these
methodologies for further refinement of our lead molecules for
both affinity and selectivity toward Ls AChBP and subsequently
investigating whether these properties can translate the ligand’s
affinity to human nAChRs. Historically, the AChBPs have
provided significant insights into the determinants of ligand
recognition by nAChRs. In particular, crystal structures of Ls−
and Ac−ligand complexes have elucidated and confirmed
residues involved in the ligand binding domain as well as
given rise to information regarding the global extracellular
domain. It has also been possible to modify the side chains of
AChBP so that its subunit interface more closely resembles that
of the homomeric α7 nAChR.48 This target-templated ligand
identification approach, coupled with a multiarray synthesis of
triazoles from azide and alkyne building blocks, offers an
efficient means of identifying and then refining candidate
ligands specifically engineered toward the nAChRs and other
flexible targets.
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