Thesis
Visualization of the subcellular localization of inhaled PI3Kδ inhibitors using bioorthogonal approaches
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2020
- Thesis identifier
- T17552
- Person Identifier (Local)
- 201690765
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- In contrast to oral delivery, administering small molecules by the inhaled route is inherently complex and relatively limited information is available on the regional localisation of drug retention following inhalation. Therefore, new tools and methodologies that can help to correlate molecular properties of inhaled drug molecules to their cellular distribution are of considerable interest. The programme of research described in this thesis uses bioorthogonal chemistry approaches to provide molecular tools that can contribute to the understanding of the cellular localisation of inhaled drug molecules via cellular imaging. More specifically, a tagging methodology based on bioorthogonal chemistry and the inverse electron demand Diels-Alder (IEDDA) reaction between trans-cyclooctene-(TCO)-tagged chemical probes and fluorescent tetrazine reporters was utilised to enable the visualization of drug subcellular localisation at high resolution. This strategy has been applied for the cellular imaging of chemical probe molecules derived from nemiralisib and GSK2292767, two inhaled clinical candidates inhibiting the lipid kinase PI3Kδ, currently being investigated for the treatment of respiratory diseases such as asthma and COPD. In rat lung tissue studies by MALDI imaging mass spectrometry, these two molecules exhibited distinct deposition and distribution profiles, and were therefore an ideal case for such studies. The syntheses of these bioorthogonal chemical probes required the development of robust synthetic routes for which a novel common intermediate was designed, thereby enabling the access of probes derived from both nemiralisib and GSK2292767 in a more convergent approach. Novel monomers were also derivatised to allow the late-stage installation of the trans-cyclooctene (TCO) tag at tolerated sites. This strategy relied on design of optimal linkers to ensure that the TCO tag would not disrupt the binding of the chemical probe to the target, whilst ensuring that it would remain available for IEDDA reaction with the tetrazine partner. Based on their properties, several chemical probes were selected for subsequent profiling in the cellular imaging assay. These experiments highlighted different subcellular localisation for the chemical probes derived from the inhaled PI3Kδ inhibitors, along with varying degrees of non-specific accumulation in acidic and membrane-rich subcellular organelles such as the endoplasmic reticulum and the Golgi apparatus. Interestingly, this aggregation was much more prominent for probes derived from nemiralisib and could not be observed for the probe derived from an oral PI3Kδ inhibitor. In addition, this cellular behaviour was shown to be independent from PI3Kδ as a similar accumulation outcome was observed in cells which do not express the target. Therefore, these findings suggest that the observed accumulation is independent from the target binding and could be caused by compound properties. To confirm that the subcellular localisation of the TCO probes derived from inhaled PI3Kδ inhibitors is a true reflection of the cellular behaviour of the parent molecules due to their intrinsic properties, rather than driven by the TCO tag and its influence on the physico-chemical properties of the functionalised chemical probes, a complementary strategy based on a relatively new mass spectrometry imaging technique called NanoSIMS was conducted. This approach relied on the design and synthesis of analogues of the inhaled compounds labelled with stable isotopes (15N). These complementary investigations corroborated the observations of the TCO bioorthogonal chemical probes and helped to explore the potential and limitation of these two orthogonal cellular imaging methods. Overall, these observations were key to correlate the molecular properties of inhaled PI3Kδ inhibitors to their lung distribution and aid in the understanding of their lung retention mechanisms. Thus, the PI3Kδ chemical probes developed here have proven to be very useful tools supporting the design of efficacious inhaled therapies. This is particularly important since several PI3Kδ inhibitors for delivery by both the inhaled and oral route are currently undergoing clinical development for various therapeutic indications. In addition, selective TCO-probes provide new ways to further study the biology of PI3Kδ by enabling specific visualization of the target localization and thereby overcoming the low selectivity problem of available antibodies. Overall, the demonstrated high impact of this strategy makes it an important tool for the development of inhaled therapies that could be applied to other targets. Finally, even though the IEDDA reaction between trans-cyclooctenes and tetrazines has become one of the most widely employed bioorthogonal reactions for the cellular imaging of biomolecules, and has proven to be impactful in this study, there is still a need for optimized transformations that would overcome some of the limitations associated with the TCO tag. In this context, additional strained alkenes such as cyclopropenes are of significant interest and have recently been investigated as alternative bioorthogonal “mini-tags”. To enable direct comparison of both tags, the synthesis of a cyclopropene chemical probe was successfully carried out and the suitability of cyclopropenes to act as a bioorthogonal reagent in the IEDDA cycloaddition was investigated. The kinetics of the IEDDA reaction with cyclopropene was evaluated by comparison of the second-order rate constants for different types of tetrazines synthesized in the course of this work. However, this alternative “mini-tag” for IEDDA reaction has not proven to be successful, therefore confirming the significant advantage of the chosen TCO strategy, whilst emphasizing a need to further develop small and reactive bioorthogonal reagents.
- Advisor / supervisor
- Taylor, Jonathan, 1981-
- Jamieson, Craig, 1973-
- Henley, Zoë
- Resource Type
- Note
- The confidentiality statement on each page of this thesis DOES NOT apply.
- Previously held under moratorium in the Chemistry Department (GSK) from 18th May 2020 until 11th December 2025.
- DOI
- Funder
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