Publication Date

8-2016

Date of Final Oral Examination (Defense)

6-20-2016

Type of Culminating Activity

Thesis - Boise State University Access Only

Degree Title

Master of Science in Chemistry

Department

Chemistry

Major Advisor

Owen McDougal, Ph.D.

Advisor

Tim Andersen, Ph.D.

Advisor

Don Warner, Ph.D.

Abstract

Ligand gated and voltage gated ion pore receptors are found throughout the mammalian body as either nicotinic acetylcholine receptors (nAChR) or voltage gated channels, and because malfunctioning of these receptors leads to a variety of diseases, these channels are heavily researched. Conotoxins have been studied for over 40 years, and have been discovered to selectively bind closely related isoforms of these ion pore receptors. Conotoxins, found in the venom of marine snails, are small cysteine rich peptides whose sequences and structural features have been found to dictate selectivity between closely related receptors (α-CTxMII[E11A] for the α6α4β2β3 α-CTxLvIA for α3β2-nAChR, α-CTxBuIA for α3β4-nAChR, and the µ-CTxGIIIA for Nav1.4). Chapter one discusses how wild-type and mutant conotoxins have been discovered and used to elucidate selectivity, and therefore tertiary binding paradigms for closely related ion pore receptor isoforms. Chapter One will provide an overview for the system explored computationally in subsequent chapters.

Due to low cost, speed, and unmatched ability to explore large numbers of compounds, high throughput virtual screening and molecular docking engines have become widely utilized by computational scientists. It is generally accepted that docking engines, such as AutoDock, produce reliable qualitative results for ligand-macromolecular receptor binding, and molecular docking results are commonly reported in literature in the absence of complementary wet lab experimental data. In this investigation, three variants of the sixteen amino acid peptide, α-conotoxin MII, were docked to a homology model of the α3β2-nAChR. The α-conotoxin MII and the α3β2-nAChR were chosen for study due to the literature precedent of this system, and due to the desire to compare computational results to future bioactivity studies, of which only the α3β2-nAChR is available for expression in Xenopus oocytes. DockoMatic version 2.0 was used to perform a virtual screen of each peptide ligand to the receptor for ten docking trials consisting of 100 AutoDock cycles per trial. The results were analyzed for both variation in the binding energy calculation obtained from AutoDock, and the peptide binding pose within the receptor. The results show that, while no clear correlation exists between consistent ligand binding pose and the calculated binding energy, AutoDock is able to determine a consistent peptide binding pose in the majority of trials when at least ten trials were evaluated. Chapter Two will discuss experimental parameters of HTVS, while providing insight into how to attain the most valuable results for this type of experimentation.

Nicotinic acetylcholine receptors (nAChRs) are found throughout the mammalian body in neuronal and muscle tissue; the distribution dependence is attributed to nAChR isoforms. The degradation of neuronal nAChR subtypes are prominent in various neurologic disorders, including Parkinson’s disease, Alzheimer’s disease, and Schizophrenia, making these receptors targets in the development of therapeutics. α-Conotoxin MII (α-CTxMII) is a 16 amino acid, disulfide rich peptide that selectively binds the α3β2-nAChR isoform. The strategy to discover a novel α-CTxMII analog with improved binding affinity for the α3β2-nAChR isoform has been performed using computational chemistry. A representative number of ligands from a database consisting of ~40 billion α-CTxMII mutant analogs was screened, and the best predicted mutant was identified. This peptide analog was compared to native α-CTxMII and α-CTxLvIA, which have experimentally reported IC50 values of 0.5-2.18 nM and 8.67 nM, respectively, for the α3β2-nAChR isoform. An iterative molecular dynamics process was used to refine the ligand to receptor interaction to identify the best predicted peptide sequence for binding the α3β2-nAChR; this peptide was identified to have the primary sequence WCCSHPGCYWSSHNWC with disulfide bonds bridging Cys2-Cys8 and Cys3-Cys16. This peptide was calculated to have a more favorable binding energy than either of the two native conotoxins tested, LvIA -84.30 kcal/mol, MII -41.25 kcal/mol, and rank_5 -89.76 kcal/mol.

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