Hulme, Alison

Brunton, Valerie

Sepp, Kristel

Engineering and Physical Sciences Research Council (EPSRC)

2021-07-20T17:07:31Z

2021-07-20T17:07:31Z

2021-07-31

Despite advancements in the drug discovery process, drug attrition rate in the late stages of clinical trials still remains high. Better understanding of drug efficacy in the pre-clinical stage would potentially translate into increased clinical trial success rate and reduce the economic burden of failed trials. However, visualising intracellular drug uptake and distribution at high resolution to improve the pre-clinical drug discovery process is still a challenge for scientists. Stimulated Raman scattering (SRS) microscopy represents a powerful imaging tool for visualising drug molecules inside cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing accurate determination of intracellular drug concentrations. Ponatinib is a clinically approved tyrosine kinase inhibitor that targets BCR-ABL and is used to treat chronic myeloid leukaemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. In this thesis, SRS microscopy was used to image intracellular ponatinib label-free with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. SRS imaging of ponatinib was optimised in Chapter 3, enabling ponatinib imaging at nanomolar treatment concentrations as well as determination of absolute ponatinib concentrations in both ponatinib sensitive and resistant CML cells. In Chapter 4, it was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not resensitise cells to ponatinib, confirming a BCR-ABL independent resistance mechanism in this CML cell model. To demonstrate further utility of SRS microscopy, it was applied to spheroid imaging in Chapter 5. CML cell lines formed three-dimensional (3D) cell ‘aggregates’ rather than spheroids with tight cell-cell contacts, and could not be used for SRS imaging. However, spheroid growth was successfully optimised in a breast cancer cell line T47D; and live T47D spheroids were imaged using SRS microscopy.

https://hdl.handle.net/1842/37766

http://dx.doi.org/10.7488/era/1042

en

The University of Edinburgh

Sepp, K.; Lee, M.; Bluntzer, M. T. J.; Helgason, G. V.; Hulme, A. N.; Brunton, V. G. Utilising stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells. J. Med. Chem. 2020, 63, 2028-2034

Raman microscopy

drug imaging

ponatinib

chronic myeloid leukaemia

Ponatinib imaging

intracellular localisation

Utilising stimulated raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy