Investigation of banglenes as neurotrophic agents and development of a new fluorescent protein- based FRET pair
Creators: Gohil,Khyati
Publisher: name: University of Alberta Library
Year: 2022

<p>This thesis describes efforts towards understanding the neurotrophic activity of banglenes and the development of a new fluorescent protein-based FRET pair. <br>
Neurotrophic small molecule natural products are functional analogs of signaling proteins called neurotrophins, which cause a pro-growth, pro-survival, or pro-differentiation responses in neuronal cells. While these phenotypic responses are desirable to combat neurodegenerative disease progression, neurotrophin proteins possess pharmacokinetic properties that present challenges to their administration in whole organisms, whether in biomedical studies or as therapeutics. Therefore, neurotrophic small molecules such as the cis- and trans-banglenes offer attractive alternatives. <br>
The first part of this thesis describes the synthesis and testing of banglene derivatives to establish a structure-activity response for the banglene family. Further, it describes studies to provide insights into the mechanism of action of banglenes.<br>
Notably, trans-banglene demonstrates neuritogenic effect alone and substantially potentiates nerve growth factor (NGF) induced neuritogenesis. The neuritogenic studies demonstrate that (–) trans-banglene is primarily the active enantiomer, while its (+) enantiomer is minimally effective. Further, the structure-activity relationship studies show that select modifications on the cyclohexene ring of trans-banglene do not impair its neuritogenic activity.<br><br>
The combination of (–) trans-banglene and NGF induces NGF secretion in PC-12 cells which might be in part responsible for its neuritogenic effect. However, (–) trans-banglene’s potentiation of NGF-induced neuritogenesis was unaffected by the presence of kinase inhibitors designed to inhibit NGF-induced neurotrophic signaling. Collectively, these results suggest a dual-mode of action for (–) trans-banglene (neurotrophic alone and strong potentiating of NGF activity), and that its potentiating action is unaffected by the presence of inhibitors of specific kinases involved in canonical NGF signal transduction pathways.<br>
The second part of this thesis describes the development of new green-red fluorescent protein-based Förster resonance energy transfer (FRET) pair as an alternative to the classically employed cyan fluorescent protein-yellow fluorescent protein pair. This FRET pair constitutes of mScarlet-I and its blue shifted version gScarlet. Indicators to detect protease activity, Ca2+ and K+ were developed using the gScarlet–mScarlet-I pair.<br>
An attempt was made to increase the FRET efficiency of these indicators by increasing the intramolecular association between gScarlet and mScarlet-I. This strategy proved useful in improving the response of the protease indicator, but it did not improve the response of the Ca2+ and K+ indicators. <br>
Finally, the comparison of gScarlet–mScarlet-I indicators to the best existing FRET pairs- mClover3-mRuby3 and cpVenus-mScarlet-I demonstrates comparable responses, establishing gScarlet–mScarlet-I as a reliable FRET pair which can be utilised to create FRET-based indicators for various cellular applications.</p>