- Title
- Disruption of clathrin-mediated endocytosis through small molecule inhibition of dynamin and clathrin
- Creator
- MacGregor, Kylie Anne
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2012
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Since the first evidence of clathrin-mediated endocytosis was reported almost 50 years ago, extensive research has been devoted to understanding the mechanisms of this process. Whilst molecular tools have contributed significantly to elucidating the mechanism of CME and the protein interaction network that underlies it, these tools suffer from pitfalls that limit their utility. Chemical inhibitors of endocytosis have been proposed as an attractive alternative to the current methods for disrupting protein function, but despite the extensive structural and biochemical knowledge about CME that is available, the development of chemical tools to interfere with this process is still in its infancy. The development of novel inhibitors to use in conjunction with existing inhibitors will assist in the molecular and functional dissection of the endocytic pathway, resulting in an increased understanding of many physiological phenomena and disease processes that rely on this pathway. Such understanding may also contribute to the rational discovery and development of novel, targeted therapeutic agents. This thesis focused on the development of novel series of compounds that specifically targeted the endocytic proteins dynamin and clathrin. Parent compounds for further development were sourced from both virtual screening and high-throughput screening strategies. Using an approach combining focused library development and molecular modelling-guided drug design, preliminary structure-activity profiles were generated, and a number of noteworthy analogues were identified. The cellular effects of selected dynamin and clathrin inhibitors were also investigated, specifically their effects on clathrin-mediated endocytosis and cell proliferation. The 1,4-benzoquinone derivative 2,5-bis(2-carboxyanilino)-1,4-benzoquinone (31-1) was selected as a parent compound for the development of novel dynamin inhibitors based on the benzoquinone scaffold. Compound 31-1 was found to possess a good level of dynamin inhibitory activity (IC₅₀: 22 ± 5 μM), and was predicted to bind to the GTP-binding site of dynamin, and possess a GTP-competitive mechanism of action. Examination of the binding conformation of 31-1 revealed that the inhibitory activity of this compound is due to a favourable hydrogen bonding, electrostatic and hydrophobic interactions with the binding site. The synthesis of four discrete analogue libraries revealed a number of structure-activity relationships, including a preference for polar substituents that are capable of increased electrostatic and hydrogen bonding interactions with the binding site. The most potent and noteworthy analogue in this series was 31-7, which exhibited a dynamin I inhibitory activity 4-fold greater than that of the parent compound. Further biological evaluation of 31-7 revealed that this compound could inhibit RME in cells, presumably via inhibition of dynamin. Through the synthesis of discrete analogue libraries based on 4-amino-3-sulfo-N-(2-hydroxyethyl)-1,8-naphthalimide (D1), a family of naphthalimides with dynamin inhibitory activity were developed. Biological evaluation of these analogue libraries allowed for the development of a preliminary structure-activity relationship profile for dynamin inhibitors based on the naphthalimide scaffold. Of the 61 analogues synthesised and subjected to biological evaluation, 21 were found to exhibit dynamin inhibitory activities comparable to, or better than, the parent compound. The most potent analogue, L1, exhibited a dynamin inhibitory activity 15-fold greater than the parent compound. The effects of selected inhibitors on transferrin uptake were evaluated, revealing that dynamin inhibitors based on the naphthalimide scaffold can inhibit endocytosis, provided the inhibitor is sufficiently lipophilic to enter the cell. Following a HTS and scaffold simplification approach, C12 was identified as an inhibitor of the interaction between clathrin-TD and amphiphysin B/C (IC₅₀: 18 μM). Co-crystallisation of C12 in complex with the clathrin-TD revealed that the binding site of C12 largely overlaps that used by clathrin-box-motif-containing accessory proteins. Synthesis of two discrete analogue libraries based on C12 revealed that the presence of a 3-sulfonate moiety is pivotal to activity, whilst hydrophobic substituents on the benzyl imide moiety improved activity due to increased hydrophobic interactions with the binding site. Examination of the cellular effects of selected inhibitors revealed an inability to inhibit RME, and this was attributed to poor membrane permeability. However, intracellular application of C12 by microinjection resulted in an inhibition of SVE, indicating that inhibition of clathrin-TD can result in inhibition of clathrin-mediated endocytosis.
- Subject
- clathrin-mediated endocytosis; endocytosis; inhibitors; targeted therapeutic agents; dynamin; 1,4-benzoquinone
- Identifier
- http://hdl.handle.net/1959.13/935828
- Identifier
- uon:12145
- Rights
- Copyright 2012 Kylie Anne MacGregor
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT02 | Thesis | 9 MB | Adobe Acrobat PDF | View Details Download |