research report

University of Bradford

2005/6

Institute of Cancer Therapeutics
Director Professor L.H. Patterson

Matrix Metalloproteinases as targets for selective delivery of antitumour agents
Dr R.A. Falconer and Prof. L.H. Patterson

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are associated with the tumour vascular development and metastatic process. One role of these enzymes is to degrade components of the extracellular matrix in order to facilitate cell migration and invasion. Our objective is to design tumour selective agents that release a potent therapeutic when activated by over-expressed MMPs within the tumour microenvironment. A design and library synthesis process is on-going to select candidate compounds for proof-of principle studies. Our synthesis strategy is to derivatise the candidate antitumour agents by attachment to an amino acid, with subsequent immobilisation to the solid support through the amino acid side chain. Conventional solid phase peptide synthesis then follows, with resin cleavage producing the target agents in high yield. We are also currently incorporating design features into these agents to enhance their physicochemical (medicine-like) properties. In parallel, biology studies are on-going that are further validating the (MMPs) of interest in clinical samples, and identifying tumour models in vitro and in vivo to enable an efficient screening programme ultimately to select a lead agent.

In collaboration with Dr Jason Gill and Dr Paul Loadman at the Institute of Cancer Therapeutics we are investigating activation and stability of these novel MMP-activated agents. Results to date demonstrate that certain compounds are indeed selectively cleaved in the tumour to release the active drug. Crucially the results also suggest significant stability in plasma and liver, the main site of drug metabolism and inactivation. We have also successfully verified selective (sequence specific) activation of these agents by recombinant MMP-2 and MMP-9 enzymes.

Targeting the glycocalyx of tumour cells. 1. Polysialyltransferases
Dr R.A. Falconer and Prof. L.H. Patterson

The glycocalyx is the carbohydrate-decorated surface of cells and the cancer process often results in modifications that drive the tumour process. We are targeting several systems that contribute to carbohydrate synthesis in tumour cells in order to modulate their activity. Polysialic acid (PSA) is a linear a-2,8-linked polymer of up to 200 sialic acid residues which decorates the cell-surface of several tumours, notably small cell lung cancer. Specifically, it decorates the neural cell adhesion molecule (NCAM), and its synthesis is regulated by polysialyltransferases PST-1 (ST8SiaIV) and STX (ST8SiaII). Changes in the expression of PSA are associated with metastasis. We reason that inhibition of polysialyltransferases will interfere with the production of PSA found on the surface of these cancer cells. This would then alter the surface properties of the cell, preventing release and thus inhibiting the metastastic process. This is of considerable interest to the development of new therapies because it is metastatic disease to which patients circum.

We are designing and synthesising several libraries of unnatural sugar analogues and sugar mimetics as potential small-molecule inhibitors of PST-1 and STX. Our compound design has been informed by molecular modelling (in collaboration with Dr Mire Zloh, University of London) of the sialyltransferase structure. In collaboration with Prof Paul Smith (University of Cardiff) we are investigating the effects of our agents on cell-surface PSA assembly.

With Dr Paul Loadman at the Institute of Cancer Therapeutics and Prof Fukoda, Burnham Institute, California, we are developing methods to quantify polysialyltransferases-mediated assembly of PSA to enable a high through-put screening of the structural analogues in preparation. Promisingly, two of our hits have demonstrated the ability to a reduce PSA decoration in vitro.

Cytochrome P450 as a target for discovery of tumour selective molecular delivery devices
Dr K. Pors and Professor L.H Patterson

Cytochrome P450s (CYP's) are a superfamily of mixed function oxidases responsible for metabolising drugs and xenobiotics. Metabolism by the CYP 1-3 family is generally viewed as a route to drug detoxification and increased elimination but they also have the potential for tumour selective drug activation. There is considerable evidence demonstrating expression of a wide range of CYPs in all the major clinically derived solid tumours and even over-expression of selected isoforms including the CYP 1A1, CYP1B1 and CYP4W1. Spirocyclopropacyclohexadienone is the reactive unit of several families of natural products that promote stalling of replication forks, DNA double strand breaks and cell death, however, they are not tumour selective. We, in collaboration with Dr Mark Searcey, University of London, have synthesised two libraries of analogues lacking the hydroxyl group crucial to the initiation of DNA covalent binding. Several classes of agent Screens have been developed in collaboration with Dr Paul Loadman and Prof Mike C Bibby to identify regioselective hydroxylation by extrahepatic and tumour expressed CYPs and have identified several agents suitable for further development. A hit compound has CYP1A1 selectivity in in vitro and cell based screens and no activation in liver, the primary organ of drug metabolism. Molecular modelling studies with Dr Colin Fishwick, University of Leeds, to develop an in silico screen for more CYP-selective drug candidates. We are also developing xenograft tumour over-expressing CYP systems as models to screen in vivo the most promising hit compounds. Dr Friedberg at Dundee University is collaborating to identify a gene therapy approach using high Kcat mutant CYP variants to the library of agents currently under synthesis.

Tumour hypoxia-selective chemotherapeutics
Dr K Pors and Professor LH Patterson

Solid tumours make up more than 90% of all human cancers and can be considerably less oxygenated compared to normal tissues, a phenomenon that is associated with resistance to radiotherapy and chemotherapy in the clinic. The importance of cell killing in those regions of tumours with low oxygen tension (hypoxia) is crucial if cancer is to be effectively treated. As such we are developing agents that are activated by cytochromes P450 associated with the hypoxic tumour environment. Using this approach we have progressed AQ4N, a tumour hypoxia-selective topoisomerase II inhibitor, into on-going worldwide Phase 1/II clinical trials undertaken by Kudos Pharma and Novacea. We have now synthesised a library of N-oxides of alkylating anthraquinones that are rationalised to have high affinity for DNA and DNA processing enzymes but are completely inactive until reduced in hypoxic tumour cells. Screening data shows several hits that alkylate DNA only in hypoxic regions of tumours spheroids, a model system developed by Dr R.M. Phillips of the Institute of Cancer Therapeutics. Studies in vivo are underway in collaboration with Prof M.C. Bibby to identify a lead molecule for full preclinical evaluation.

Proteomic analysis of cytochromes P450 and matrix metalloproteinases
Dr. C. W. Sutton and Prof. L.H. Patterson

In September 2006 a proteomic facility was initiated at the Institute of Cancer Therapeutics under the direction of Dr Chris Sutton and Prof Patterson to complement the established molecular biology, chemistry and pharmacology groups. Even the most basic proteomics experiments require a large investment in state-of the-art equipment, bioinformatics and skilled scientific staff. During this funding year a process of evaluation of commercial products and in-depth discussion with vendors has enable the purchase of an array of instruments to form the cornerstone of the Proteomics Facility. A high performance MALDI TOF-TOF mass spectrometer, nano-HPLC and fraction collector, database search engine and LIMS system form the initial outlay for the Proteomics Facility. The following represent key functions that will be performed with the equipment (i) Protein identification by peptide mass fingerprinting (ii) Protein identification by LC MALDI MS/MS (iii) Peptide sequencing and protein characterisation (iv) Database management and sample tracking (v) Drug characterisation. Projects that are currently underway include development of methodology for proteomic analysis of drug target proteins, specifically Matrix metalloproteinases and cytochromes P450s to help fine-tune drug design.