ESR 3: William Palmer-Brown: October - November 2016

Title: Screening and assessment of microbial P450s in industry

Host:Almac, Craigavon, Northern Ireland, United Kingdom

Accommodation:Craigavon, Northern Ireland, United Kingdom

Science:Two fluorinated drugs, flurbiprofen and flutamide, were screened using cytochrome P450 (CYP) enzyme panels in an attempt to produce the major mammalian metabolites: 4’-OH-Flurbiprofen, 3’,4’-OH-Flurbiprofen for 1 and 2-Hydroxyflutamide for 2.

Figure 1. Drugs tested for P450 hydroxylation - Flurbiprofen (1) and Flutamide (2)

CYPs were screened as lyophilised powders in a 96 well format and the reactions were done in combination with a cofactor recycle system.

The screening results were analysed by UPLC for potential product formation. If a peak was observed, the samples were then analysed by LC-MS or GC-MS (still ongoing). The activity of the enzymes were assayed using the measurement of NAD(P)H consumption.

Recombinant E.coli strains with CYP clones of interest were expressed and assayed directly after cell lysis.

New Skills acquired

Cognitive Skills

  • Learnt to build and screen substrates using enzyme panels
  • Learnt about the high-throughput methodologies of industry
  • Learnt new methods for the scaled-up expression of CYP enzyme systems in E.coli

Technical Skills

  • Using high-throughput methods, such as 96-well plate screens and subsequent analysis using UPLC
  • Learnt a new method for testing the substrate-specific activity of CYPs

Interpersonal Skills

  • Working in a professional environment for a short period of time
  • Working within a specific time frame
  • Communicating effectively with people working in industry