Quantification of intracellular drug concentrations using Surface Enhanced Raman Spectroscopy (SERS) – Thomas Rabl
Supervisors: Dr Kevin Read and Dr David McGloin
Penetration of potential novel drugs into the cell can sometimes be a significant challenge in early drug discovery. While many cells show problems herein, gram negative bacteria are of special interest due to their complex mechanisms to protect themselves from treatments. This behaviour, in cells as well as in bacteria, is often complicated by the uptake mechanisms, and protective efflux mechanisms present in the cell membrane/cell wall. While some molecules that seem too big to enter the cell are penetrating easily, others that seem small enough and have the correct physicochemical properties to enter the cell easily are being directly pumped out. For modern Drug Discovery Units (DDU’s) a robust approach to quantifying intracellular drug concentration would add significant value to the early drug discovery process, particularly when there is disconnect between activity in biochemical assays and the whole cell. Current approaches are based on Ultra performance liquid chromatography – tandem mass spectrometer (UPLCMSMS) and assessment of cell fraction unbound with a number of caveats to data produced.
At the moment one of the most promising alternative techniques for quantifying intracellular drug concentrations is Raman Spectroscopy (RS). Raman Spectroscopy has evolved into a whole field with vast impact on physics, chemistry, biology and life science. It offers quantitative imaging and monitoring of substances with Raman-active regimes as well as finger printing of many different chemicals. By using RS the possibility to monitor uptake as well as the drug transport within cells arise, and can offer novel insights into drug transport mechanisms of various cells.
This project will involve
- Calibration and building of the Raman spectroscopy system, including measuring of various substances for a compound library
- Investigating the applicability of Surface Enhanced Raman Scattering (SERS) techniques (particularly SERS enhanced with metal nano-particles) to the Drug Discovery Process
- Investigating the applicability of chemically enhancing drug targets with Raman-Active groups for enhancing the SNR
- Testing various cell types (HEK, THP1 and primary macrophages in the first instance)
- Enhancing protocol(s) for pre-treatment of cells, including the concentration, application time and special treatments such as dry freezing of cells
- Validation of enhanced Raman spectrum measured drug concentration using classical Mass Spectrometer (MS) methods and building a library
- Elaboration of diagnostic criteria and protocol(s) of intra cellular drug concentration including a double blinded test with a number of commercially available drug targets and in house targets
- Development of a reliable data acquisition program and protocol to be used in Drug Discovery Lead Optimisation programmes
[November 2017] D4.3 Validation of measurement with various cell-lines and building of a library.
[November 2017] D4.4: Development of a reliable data acquisition program and protocol to be used in an everyday Drug Discovery Setting.
Journal and Conference Papers and Posters
[May 2017] Intermediate phases during solid to liquid transitions in long-chain n-alkanes. Stella Corsetti, Thomas Rabl, David McGloin and Johannes Kiefer, Phys.Chem. Chem. Phys (2017) 19, 13941.
[August 2016] Using Raman spectroscopy for the detection of drug concentrations inside of cells. Oral presentation at the Biophotonic approaches: From molecules to living systems conference, Dundee, Scotland, 22nd August 2016.