Hulme, Alison

Brunton, Val

Lilienkampf, Annamaria

Steven, Craig Forrest

2024-08-23T16:09:28Z

2024-08-23T16:09:28Z

2024-08-23

Interest in stimulated Raman scattering (SRS) cellular imaging as a tool to aid preclinical drug testing has grown in recent years. Chapter 1 discusses the fundamental principles of SRS, examples of its utility in intracellular drug imaging and challenges within the field. One major challenge is that the complex nature of intracellular biological environments complicates drug tracking by SRS, frequently necessitating attachment of small chemical tags to aid visualisation. Unfortunately, poor signal intensity and solubility have hampered the widespread use of these tags. The poor solubility and high lipophilicity of Raman tags was addressed in Chapter 2. New molecules were synthesised using an optimised cross-coupling method. These tags were designed with greater consideration of medicinal chemistry parameters such as solvent partition coefficients (logP and logD) and fraction of sp3-hybridised carbons (Fsp3). The relative propensity of the tags to engage in intermolecular stacking, which is known to confer poorer solubility, was analysed by mass-spectrometry (MS) and X-ray crystallography. Whilst MS analysis found that tags with higher logP formed stronger associative interactions, determination of X-ray crystal structures revealed changes in tag crystal packing when Fsp3 was increased. These results suggest a combined analysis of logP and Fsp3 should guide future tag design. SRS imaging revealed that the tags were cell permeable, Raman activity was unaffected by structural modification and changes in physicochemical parameters did not significantly affect tag biodistribution. In Chapter 3, the new tags were attached to the PARP inhibitor olaparib to allow intracellular drug tracking. Rigorous in vitro testing of the new drug analogues was carried out to investigate their physicochemical properties, target protein inhibition and binding kinetics, and biological stability. It was determined that olaparib activity was retained after tagging, providing the necessary prerequisites for the transition to in cellulo testing. SRS imaging allowed determination of drug biodistribution and discrimination of the compounds based on their relative Raman activity and physicochemical properties. An unexpected cellular distribution was observed with no drug detected within the nucleus by SRS, inconsistent with the known nuclear localisation of PARP and previous fluorescence imaging of olaparib. This warranted further study to confirm the biological effects of Raman tagging. The effects of tagged drug analogues on cancer cells were studied in Chapter 4. The drug analogues were found to reduce cell viability in a range of cancer cell lines and a 3D cell model. Using a live-cell phenotypic readout, this loss of viability was shown to correlate with increased DNA damage when compared to olaparib, thus suggesting enhanced PARP inhibition. The origin of the increased potency was explored by identifying new biological pathways activated in response to exposure of the drug analogues. It was determined by protein expression analysis, gene profiling and proteomics that Raman tagging of olaparib induced degradation of PARP and activation of the unfolded protein response, which could be linked to cell death. These studies have allowed the intracellular tracking of a PARP inhibitor by Raman microscopy for the first time and provide foundations for the future development of 2-in-1 theranostic Raman probes.

https://hdl.handle.net/1842/42129

http://dx.doi.org/10.7488/era/4851

en

The University of Edinburgh

C. F. Steven, E. Chiarparin, A. N. Hulme, V. G. Brunton, in Stimulated Raman Scattering Microscopy: Techniques and Applications, ed. J. Cheng, W. Min, Y. Ozeki, D. Polli, Elsevier, 2022, pp. 403–419

C. F. Steven, M. Lee, Gary S. Nichol, Paul R. J. Davey, E. Chiarparin, A. N. Hulme, V. G. Brunton, Eur. J. Org. Chem., 2022, 30, e202200393

C. F. Steven, M. P. Ravindra, M. Lee, Paul R. J. Davey, E. Chiarparin, A. N. Hulme, V. G. Brunton, Proc. SPIE, 2023, 123570G

M. P. Ravindra, M. Lee, S. Dimova, C. F. Steven, M. T. J. Bluntzer, V. G. Brunton and A. N. Hulme, Chem. Eur. J., 2023, 29, e202300953.

Raman-Active Chemical Probes

Cancer Cell Imaging

Medicinal Chemistry

stimulated Raman scattering (SRS) cellular imaging

solvent partition coefficients (logP and logD)

fraction of sp3-hybridised carbons (Fsp3)

mass-spectrometry

X-ray crystallography

Raman-active chemical probes for cancer cell imaging and medicinal chemistry

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy