- Title
- Development of Clathrin inhibitors from lead compound Pitstop® 2
- Creator
- Prichard, Kate L.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Clathrin is a key protein in clathrin-mediated endocytosis (CME), where it is the major component of vesicle coats that encapsulate and transport hormones, growth factors, receptors, nutrients, and other cellular cargo from the cell membrane into the cell. Additionally, it has a moonlighting role in mitosis, as a component of intermicrotubule bridges believed to stabilise microtubules. Clathrin is implicated in various diseases affecting humans: including cancers, viral entry, and a range of neurological disorders. Pitstop® 1 and Pitstop® 2 are two of the few currently known small molecule clathrin inhibitors and have been shown to bind into the clathrin terminal domain (CTD) at Site 1. The resultant clathrin inhibition, determined by an enzyme-linked immunosorbent assay (ELISA), gave IC50 values of 18 μM (in Pitstop® 1) and 1.9 μM (in Pitstop® 2). Pitstop® 2 represents a promising lead compound, and development of the Structure Activity Relationship (SAR) of Pitstop® 2, to increase potency and selectivity, may allow further exploration of the biological roles of this protein and its protein partners and will allow the potential of clathrin inhibitors as novel therapeutics to be explored. This thesis reports on the development of an extensive SAR analysis of Pitstop® 2, through the modification of three distinct regions of the compound; the aromatic head, the sulfonamide tail, and the core/ linker region. Further, a selection of chimeric analogues of Pitstop® 1 and Pitstop® 2 were explored, combining structural elements of both compounds in an effort to increase potential binding interactions in the CTD. SAR development of the aromatic head region of Pitstop® 2 (Chapters 3 and 4) found a preference (by ELISA) for 4-position substitutions, electron donating groups, and extending the length of the head region: both with phenylallylidene and biphenyl analogues. The most potent of these analogues include 2,3,4-trihydroxyphenyl 97 (ELISA IC50 0.94 μM), 4 nitrophenyl allylidene 133 (ELISA IC50 1.0 μM) and benzofuran 114 which showed improved ELISA (IC50 1.5 μM) and CME (IC50 6.8 ± 2.7 μM) inhibition compared to Pitstop® 2. Investigations into the sulfonamide tail of Pitstop® 2 (Chapters 5 and 6) elucidated a preference towards bulkier tails, halogenated phenylsulfonamides (specifically in the 4- and 2-positions) with the most potent analogues including a 2,6-dichlorophenylsulfonamide tail 352 (ELISA IC50 0.97 μM) and a 4-chlorophenylsulfonamide tail 339 (ELISA IC50 1.3 μM). Exploration into the core and linker regions of Pitstop® 2 (Chapter 7) found replacements including a N-glucosamine core and extended alkyl linker were poorly tolerated. An amide linker 378 (ELISA IC50 35.9 μM) was the most tolerated replacement, however still resulted in a significant loss of activity. Further to the exploration of Pitstop® 2, SAR developments of chimeric analogues of Pitstop® 1 and Pitstop® 2 (Chapter 7) found bulkier tail regions and phenyl linkers were favoured, as well as sulfonate and propyloxy substituents on the naphthalimide head region. The most potent chimeric analogues show improved activity on Pitstop® 1, although not improved on Pitstop® 2, and include 2-position phenyl linked analogues with a 1-naphthalenesulfonamide tail and varied substituents on the naphthalimide head (sulfonate 407, ELISA IC50 7.6 μM and propyloxy group 432 ELISA IC50 7.3 μM). This work provides invaluable insight into the SAR of Pitstop® 2, and demonstrates this lead to be a promising candidate for the development of clathrin inhibitors. The SAR developed within highlights a wide scope for variation while retaining activity, and demonstrates that extending the length of the compound both in the head or tail regions results in increased activity. A total of 235 analogues were synthesised to explore the SAR of Pitstop® 2 and the currently biological evaluations found 30 analogues to result in comparable or increased activity over the previous lead.
- Subject
- medicinal chemistry; Pitstop; Clathrin; Structure Activity Relationship (SAR)
- Identifier
- http://hdl.handle.net/1959.13/1492563
- Identifier
- uon:53362
- Rights
- Copyright 2023 Kate L. Prichard
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 37 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 1 MB | Adobe Acrobat PDF | View Details Download |