Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids and Synthetic Agonist Analogs

All the members of the Dougherty lab were essential to my success at Caltech. Nyssa Puskar is the sole representative of the fifth-year class in the Dougherty lab and is a longtime friend and confidant.

CHAPTER
2:
The
Nicotinic
Pharmacophore:

The
Pyridine
N
of

 Nicotine
and
Carbonyl
of
ACh
Hydrogen
Bond
Across
a
Subunit

CHAPTER
3:
Residues
that
Contribute
to
Binding
of

CHAPTER
4:
Stereochemical
Preferences
of
the
Nicotinic
Receptor

CHAPTER
5:
Evidence
for
an
Extended
Hydrogen
Bond
Network

 in
the
Binding
Site
of
the
Nicotinic
Receptor:
Concerning
the
Role

CHAPTER
6:
New
Approaches
to
Photochemical
Cleavage
of

CHAPTER
7.
Progress
Toward
Small­Molecule
Activators
of
Voltage

APPENDIX
1:
Characterizing
the
Pharmacophore
Binding

APPENDIX
2:

Full
Collection
of
Data
for
the
Backbone
Ester
Mutation

 at
L119
in
the
α4β2
and
Muscle­Type
Nicotinic
Acetylcholine

APPENDIX
3:
Synthetic
Routes
Considered
for
the
Preparation

LIST
OF
FIGURES

LIST
OF
TABLES

LIST
OF
SCHEMES

CHAPTER
1:
Introduction

• Chemical
Signaling
in
the
Brain
• Using
 Physical
 Organic
 Chemistry
 to
 Study
 Ion
 Channels:
 The
 Power
 of
 Unnatural
Amino
Acids
• Incorporation
of
Unnatural
Amino
Acids
Through
Nonsense
or
Frameshift
 Suppression
Methodology
• Electrophysiology
as
an
Assay
of
Receptor
Function
• Mutant
Cycle
Analysis
• The
Nicotinic
Pharmacophore
• Summary
of
Dissertation
Work
• REFERENCES

Further evidence of the usefulness of unnatural amino acids can be seen in the study of hydrogen bonding interactions with the amide bond in a protein's backbone. An overview of the nonsense and frameshift suppression methods used to incorporate unnatural amino acids (UAAs).

Figure 1.1. Topology of a Cys-loop receptor subunit.

CHAPTER
2:
The
Nicotinic
Pharmacophore:

The
Pyridine
 N
of
Nicotine
and
Carbonyl
of
ACh
Hydrogen
Bond
Across
a

• ABSTRACT
• INTRODUCTION
• RESULTS
• DISCUSSION
• EXPERIMENTAL
SECTION
• ACKNOWLEDGEMENTS
• REFERENCES

Regarding the second component of the pharmacophore, the hydrogen bond acceptor, the AChBP structure yielded interesting results. Note that these studies do not establish that the interaction between the hydrogen bond acceptor component of the agonists and the backbone NH of.

Figure 2.1. Key structures considered in the present work. (A) Structures of agonists used

CHAPTER
 3:
 Residues
 that
 Contribute
 to
 Binding
 of
 the
 Nicotinic
 Pharmacophore
 in
 the
 Muscle­Type
 Nicotinic

• ABSTRACT
• INTRODUCTION
• RESULTS
• DISCUSSION
• EXPERIMENTAL
SECTION
• ACKNOWLEDGEMENTS
• REFERENCES

The α subunits contribute the major component of the agonist binding site, which binds to the cationic end of agonists. The complementary component of the agonist binding site is formed by non-α subunits, and recent work has shown that it makes a hydrogen bond interaction with the hydrogen bond acceptor of agonists. The present work focuses on the complementary component of the agonist binding site of the muscle-type nAChR.

The cationic N binds to the main component of the agonist binding site in the α subunit, and the hydrogen bond acceptor binds to the complementary, non-α subunit. The backbone ester strategy used here allows us to probe both components of the hydrogen bonding system. Taken together, these data provide a clearer picture of the agonist binding mechanisms of the muscle-type nAChR.

Figure 3.1. Depiction of binding interactions of the nicotinic pharmacophore as predicted by AChBP structures

CHAPTER
 4:
 Stereochemical
 Preferences
 of
 the
 Nicotinic
 Receptor:
 
 Pharmacophore
 Binding
 Interactions
 of

• ABSTRACT
• INTRODUCTION
• RESULTS
• DISCUSSION
• EXPERIMENTAL
SECTION
• ACKNOWLEDGEMENTS
• REFERENCES

In this model, the cationic center of the pharmacophore engages in a cation-π interaction with an aromatic box residue, TrpB. Another component of the pharmacophore, the hydrogen bond acceptor, forms a hydrogen bond to the NH L119 backbone of the complementary β2 subunit. These studies use nonsense suppression methodology to examine the pharmacophore binding interactions for each enantiomer of epibatidine and N-methyl epibatidine in the α4β2 receptor expressed in Xenopus oocytes.

Similar to the trend seen in studies of the cation-π interaction, the N-methyl derivs. showed nearly identical shifts in EC50 in response to. Surprisingly, we found that both enantiomers of the N-methyl derivative are almost identically sensitive to disruption of the three interactions. We find that all agonists participate in the pharmacophore interactions—a cation-π interaction to Trp B, a hydrogen bond to the backbone CO of TrpB, and a hydrogen bond to the backbone NH of L119 in the complementary subunit.

Figure 4.1. The binding interactions of the nicotinic pharmacophore shown for (+)-epibatidine

CHAPTER
5:
Evidence
for
an
Extended
Hydrogen
Bond
 Network
in
the
Binding
Site
of
the
Nicotinic
Receptor

Concerning
the
Role
of
the
Vicinal
Disulfide
of
the
α1
 Subunit ∗

ABSTRACT

INTRODUCTION

α nAChR subunits typically contain the major component of the agonist binding site and are distinguished by a unique adjacent disulfide in the agonist binding site formed by residues canonically designated as C192 and C193. In the context of a ring formed by a neighboring disulfide, the gauche(+) and gauche(−) forms produce energetically distinct (diastereomeric) structures. Combined backbone and disulfide conformational combinations result in four different conformers for the adjacent disulfide ring.

While the sulfurs of the disulfide can come into contact with small molecules that bind to the acetylcholine-binding protein (AChBP),23, 24 a very useful model of the nAChR binding site, any noncovalent interactions of this kind are expected to be quite weak will be. Instead, we and others have speculated about a role in receptor gating involving cis-trans isomerization of the amide and/or gauche(+)/gauche(−) interconversion of the disulfide. Conventional mutagenesis studies are expected to produce severely attenuated receptors, and so it has been challenging to design unambiguous probes of disulfide function. Here we use subtle structural variations made possible by unnatural amino acid mutagenesis to evaluate the possible functional role of the vicinal disulfide of nAChR α.

Figure 5.1. Vicinal disulfide structure. For model calculations of CH 3 CO-[Cys-Cys]-NH 2 , R 1 = CH 3 and R 2 = H

RESULTS

To test this model, we looked for modifications of the vicinal disulfide that would alter the inherent cis-trans bias of the system. If the cis-trans isomerization of the vicinal disulfide amide is functionally important, the modifications in question—the incorporation of an ester into the framework and amide N-methylation—should have opposite effects on receptor function. While they differ in their expected effect on cis-trans isomerization, the two backbone mutations are similar in another respect: both abolish the hydrogen bond-donating ability associated with the NH of the parent amide.

An inspection of AChBP structures reveals a potential acceptor for such a hydrogen bond donor: the carbonyl of residue Y190, a conserved member of the agonist binding site. This finding supports the existence of a new 3-residue hydrogen bond network, in which the NH of C193 hydrogen binds to the carbonyl of the peptide bond between Y190 and S191. Note that the introduction of the α-hydroxy acid at S191 weakens the hydrogen bond acceptor for the hydrogen bond with C193 and removes the hydrogen bond donor for the hydrogen bond with γD174/δD180.

Table 5.1. EC 50 , Hill coefficient (± standard error of the mean) and ∆G° values for mutations made to α1 2 β1γδ

DISCUSSION

We propose that the role of the vicinal disulfide is to shape the structure of the β-turn between Y190 and C193. The resulting distorted β-turn may then help position the backbone NH of S191 to form the second hydrogen bond. In the closed state of the receptor, the β-turn hydrogen bond is formed, but the intersubunit hydrogen bond (S191 to γD174/δD180) is not (consistent with the AChBP crystal structures).

The β-turn hydrogen bond is essential for proper formation of the agonist binding site (recall that Y190 is residue C1 of the highly conserved cluster of aromatic amino acids that delineate the agonist binding site). Activation involves formation of the intersubunit hydrogen bond, an event facilitated by proper positioning of the backbone NH of S191 by the vicinal disulfide and the β-turn hydrogen bond. In this model, the vicinal disulfide of the α-subunit distorts the β-turn of the C-loop to better position the backbone NH of S191 for optimal formation of an intersubunit hydrogen bond.

EXPERIMENTAL
SECTION

The powder was suspended in 3 mL of water and then an additional 1 mL of dioxane. 59 mg, 0.56 mmol) was added and the mixture was allowed to stir for five minutes before the addition of 6-nitroveratrioxycarbonyl chloride (NVOC-chloride) (92 mg, 0.33 mmol) in 4 mL of dioxane. After stirring at room temperature for 48 h, the solution was diluted with 10 mL CH2Cl2 and 10 mL water.

The solution was diluted with 15 mL of water and the organic layer was extracted with CH2Cl2 (×3), dried over Na2SO4 and concentrated to give a yellow oil. The aqueous layer was washed with 30 mL of ether and the organic layer was discarded. The solution was diluted with 15 mL of water and the organic layer was extracted with CH2Cl2 (×3), dried over Na2SO4.

ACKNOWLEDGEMENTS

Structure construction and subsequent ab initio calculations on CH3CO-[Cys-Cys]-NH2 were performed using the Gaussian 0362 software package by Kristin Rule Gleitsman63 at the B3LYP/6-31++G(d,p) level of phased theory of gas. Cis and trans isomers of a model peptide of the form CH3CO-[Cys-Cys]-NH2 with disulfide S-S torsion angles of ±90 were constructed using the GausView molecule construction tools. The geometric parameters for these starting structures were derived from the lowest energy conformers from previous ab initio calculations on a similar model peptide.21 Energy minimizations were performed on the four starting structures of the model peptide.

The lowest energy structures from these model peptide calculations then served as scaffolds to construct the initial ester and N -methyl structures, which were subsequently subjected to energy minimization calculations. In total, the energies of twelve geometry-optimized structures were calculated with four conformers for each model system. Additional details are given in Kristin Rule Gleitsman's thesis.63 Calculations on simple cyclic amides and related structures were performed with SPARTAN.64.

Kurasaki, M.; Takahashi, H.; Morimoto, Y.; Hirose, T.; Inayama, S.; Takahashi, T.; Kuno, M.; Numa, S., Location of functional regions of the acetylcholine receptor alpha subunit by site-directed mutagenesis. Hudaky, I; Gaspari, Z.; Carugo, O.; Cemazar, M.; Pongor, S.; Perczel, A., Vicinal disulfide bridge conformers by experimental methods and by ab initio and DFT molecular calculations. Capasso, S.; Mattia, C.; Mazzarella, L.; Puliti, R., Structure of a cis-peptide unit - molecular conformation of cyclic disulfide L-cysteinyl-L-cysteine.

Miyazawa, A.; Fujiyoshi, Y.; Stowell, M.; Unwin, N., Nicotinic acetylcholine receptor at 4.6 Å resolution: transverse tunnels in the canal wall. A., [28] In vivo incorporation of unnatural amino acids into ion channels in the Xenopus oocyte expression system.

CHAPTER
 6:
 New
 Approaches
 to
 Photochemical
 Cleavage
 of
Peptide
and
Protein
Backbones ∗

• ABSTRACT
• INTRODUCTION
• RESULTS

Selenide is one of the most potent nucleophiles known, and at physiological pH a selenol (pKa ~5–6) should be predominantly in the selenide form. We describe the synthesis and characterization of aliphatic (1) and aromatic (2) variants of the design along with mechanistic characterization. These studies used in vitro nonsense suppression methodology to incorporate 1 and 2 into the α1 subunit of the muscle-type nicotinic acetylcholine receptor (nAChR) at residue Met243 (located in the M2 transmembrane helix).

In these experiments, Npg was expressed in the N-terminal domain of the Drosophila Shaker B K + channel at residues Leu47 and Pro64.3. Channel inactivation occurs when one of the four beads of the homotetrameric protein clogs the channel pore. Depiction of the topology of the Shaker B K+ channel and the location of sites used to incorporate Npg.

Figure 6.1. Npg and the second-generation SNIPP unnatural α-hydroxy acids, 1 and 2.

Compounds 1 and 2 were successfully incorporated into the ShB protein expressed in Xenopus ooctyes using frameshift nonsense suppression methodology as

• DISCUSSION
• EXPERIMENTAL
SECTION
• ACKNOWLEDGEMENTS
• REFERENCES
• ABSTRACT
• INTRODUCTION
• PROGRESS

The pH of the solution was then increased to ~6 by adding 10 wt. % NaOH. The organic layer (colorless) was discarded, and the pH of the aqueous layer was lowered to pH 2 by the addition of 6 N HCl. The pH of the aqueous layer was then lowered to pH 2 by the addition of 6 M HCl.

Aliquots of the in vitro translation reactions (5 ml) were used for photolysis and base hydrolysis experiments. Madsen, R.; Roberts, C.; Fraser-Reid, B., The pent-4-enoyl group: a new amine protecting group that is readily cleaved under mild conditions. The macula is located in the center of the retina, the light-sensitive tissue that lines the back of the eye.3 The retina consists of several layers of neurons connected by synapses.

Figure 6.3. Proposed photochemical cleavage strategy using caged aniline 17.