Cytochromes P450 (CYPs) are a superfamily of mixed function oxidases of which CYP1-4 subfamily members are unique in their ability to oxidise drugs. CYP2W1 is expressed in a number of human tumour types including colorectal, breast and head & neck cancers.
Several clinically used cancer chemotherapeutics, notably the DNA alkylating oxazaphosphorines and nitrosoureas are known to be metabolised by CYPs to reactive intermediates. These agents were not designed as CYP-specific and are primarily activated in the liver. Their metabolism is associated with profound tissue toxicity and lack of tumour selectivity. A panel of duocarmycin prodrugs has been prepared that are converted to potent cytotoxins only at the site of CYP2W1 expression, thereby minimising systemic cytotoxicity. CYP2W1 is expressed in 30% of primary colorectal cancers (CRC) and 50% of patients with metastatic CRC. Furthermore, mutations in CYP2W1 gene may occur, which have a significant impact on the enzyme activity of the expressed protein. The development of methods for detecting specific CYP2W1 isoforms and determining the levels of active proteins are essential in order to identify patients for whom targeted therapy can be most effectively administered. In the absence of high quality antibodies that can detect the protein and enzyme assays that are sufficiently specific for CYP2W1, an activity-based proteomic probe approach will be used. Initially, methods for enrichment and mass spectrometric analysis will be based on affinity probes (comprising benzofuran non-competitive inhibitors linked to biotin), developed with collaborators from Durham. These probes are designed to react with, and enrich members of the CYP3A subfamily, using streptavidin chromatography, from established sources – recombinant in baculovirus, mouse liver and human hepatocyte sources.
Experience from CYP3A-targeted probe work will be used to inform research on biotinylated benzofuran-based duocarmycin analogues such as ICT2726, which will be synthesized for affinity capture of CYP2W1 from established cancer cell lines and xenografts. In addition to medicinal chemistry, the prospective student will gain skills in a range of protein handling, proteomic and mass spectrometric techniques. Hence, the main aim of this project is to develop an affinity enrichment approach for the mass spectrometric identification of an important therapeutic target, CYP2W1, which can be used to profile patient samples.