research report

University of Bradford

2007/08

Institute of Cancer Therapeutics
www.cancer.brad.ac.uk
Director Professor L.H. Patterson    


Selective delivery of anti-tumour agents using Matrix Metalloproteinases  
Dr R.A. Falconer and Prof L.H. Patterson    

The Matrix metalloproteinases (MMPs) are overexpressed in tumours and are crucial to many tumourigenic processes, particularly extracellular matrix breakdown, functionality of cell signalling receptors, tumour invasion and angiogenesis. We have focused our tumour-selective drug development strategy on the membrane-type MMP (MT-MMP) sub-family as a delivery device for vascular disrupting agents (VDA). Our hypothesis is that the increased expression and activity of degradative endoproteases within the tumour environment, relative to normal tissues, can be used to selectively release potent chemotherapeutics from non-toxic peptide conjugates. The result will be high levels of the active agent at the tumour and none or negligible drug levels in 'normal' tissues. Our current focus has been to design conjugates specifically targeted to the membrane type MMPs (MT-MMPs), specifically MMP-14. We have successfully synthesised conjugates with diversity at the peptide N-terminus to introduce enhanced physicochemical properties (aqueous solubility). Our synthetic 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.  In parallel, biological 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 to assist in the selection of 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 successfully verified selective (sequence specific) activation of these agents by the relevant recombinant enzymes. Our lead agent, ICT2588, leads to a significant tumour growth delay in vivo. This agent is currently the subject of advanced in vivo evaluation.  

Targeting the tumour cell glycocalyx

The surface of all cells is decorated by carbohydrate molecules that are crucial to cellular functions as well as the interaction between a cell and its environment. Altered patterns of this decoration are often associated with cancer and can therefore be used as markers to detect tumours and as the basis to develop therapeutic agents. Furthermore, many of these changes drive the growth and migration of cancer cells and prevention of their occurrence has a beneficial effect on cancer patients. We are devising chemical intervention strategies which are targeting several processes which contribute to the synthesis of these carbohydrate-decorated molecules in tumour cells.

1.      Polysialyltransferase as a target for the development of anti-metastatic agents 
Dr R.A. Falconer, Dr K. Afarinkia and Prof. L. H. Patterson  

Decoration of the neural cell adhesion molecule (NCAM) on the tumour cell surface by polysialic acid (PSA) is characteristic of a number of cancers of neural crest origin. Mechanistically, changes in PSA expression have been associated with the dissemination of cells from the primary tumour mass and consequently, increased PSA cell-surface decoration has been correlated with tumour progression and a poorer prognosis. The post-translational polysialylation of NCAM is controlled by two specifc α-2,8-polysialyltransferases, namely ST8SiaII (STX) and ST8SiaIV (PST). Our hypothesis is that modulation of PSA decoration on the tumour cell surface will suppress changes in cell-cell and cell-matrix adhesion, thereby diminishing the opportunity for phenotypic alterations associated with cell motility, migration, invasion and hence metastatic potential. Our initial efforts are focussed on small cell lung cancer (SCLC) and neuroblastoma which both show a strong correlation between PSA expression and disease progression. We are designing and synthesising small-molecule inhibitors of PST and STX. Our compound design is informed by molecular modelling of the sialyltransferase structure (in collaboration with Dr Colin Fishwick, University of Leeds). We have successfully completed the synthesis of two libraries of thio-linked sialoside compounds and have developed synthetic methodology for their preparation. These compounds will be the subject of in vitro evaluation in the coming period. In addition, we are developing non-sugar based compounds, which will have superior pharmacokinetic profiles. Development of an enzyme-based assay for PST inhibition is underway, with the successful expression and purification of STX. In collaboration with Prof Paul Smith (University of Cardiff) we continue to investigate the effects of our agents on cell-surface PSA assembly. With Dr Paul Loadman at the Institute of Cancer Therapeutics and Prof Fukuda, (Burnham Institute, USA) we continue to develop methodology to quantify polysialyltransferase-mediated assembly of PSA. In collaboration with Dr Catherine Cullinane (University of Leeds) we are examining PSA-NCAM, PST and STX expression in neuroblastoma clinical samples.  

2. Golgi mannosidase II inhibitors 
Dr. K. Afarinkia and Prof. L. H. Patterson  

Golgi a-mannosidase II (GMII), a class II retaining  glycosyl hydrolase of family 38, is a key enzyme in the biosynthesis of hybrid and complex type N-linked cell surface glycoproteins. GMII catalyzes the trimming of two mannose residues, one which is α-1,6 linked and one which is a-1,3-linked, from Man₅GlcNAc₂(Asn) to give Man₃GlcNAc₂(Asn) which is then further manipulated by various glycosyltransferases in the Golgi complex. GMII is an important target in cancer because its inhibition can lead to both a slowing down of the cell proliferation, and prevention of metastasis through removal of the signals leading to the breakdown of extracellular matrix. We have rationally designed and are developing to a series of aza-linked carbohydrate mimetics which specifically inhibit α-1,6-mannoside cleavage. We have also developed an in vitro assay for the assessment of our compound libraries and are currently in the process of determining their activity and selectivity.  

3. Heparanase inhibitors 
Dr. K. Afarinkia and Prof. L. H. Patterson  

Glycosaminoglycans, including heparan, chondroitin, keratan and hyluronan, regulate growth factor signaling, cellular behavior, inflammation, angiogenesis, and the ECM proteolytic activity. Dysregulated expression of glycosaminoglycans is strongly associated with cancer and is reported to correlate directly with clinical prognosis in several malignant neoplasms. Our expanding understanding of the biological roles these molecules play in cancer biology, tumor angiogenesis, and metastasis has promoted the development of drugs targeting them. Current chemical intervention strategies involve administration of low molecular weight heparins (LMWH) which have highly undesirable side effects. We have recently initiated a programme to prepare novel heparanase inhibitors. Informed by molecular modelling studies, we have already obtained molecules with moderate activity and are currently in the process of the design and synthesis of second generation inhibitors.  

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. Spirocyclopropacyclohexadienone is the reactive unit of several families of natural products that promote cell death via DNA damage, however, they are not tumour selective.  We, in collaboration with Dr Mark Searcey, University of East Anglia, have synthesised several libraries of analogues lacking the hydroxyl group crucial to the initiation of DNA covalent binding In vitro screens have been developed in collaboration with Dr Paul Loadman to identify regioselective hydroxylation by extrahepatic and tumour expressed CYPs and have identified several agents from the core libraries suitable for further development. A hit compound has been identified that possesses CYP1A1 selectivity in vitro and in vivo  but with no activation in the liver, the primary organ of drug metabolism. In collaboration with Dr Colin Fishwick, University of Leeds, in silico screen models are being developed to identify novel potential 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.   

Design and Synthesis of Chemokine Antagonists  
Dr. K. Afarinkia and Prof. L. H. Patterson  

Chemokine receptors are over-expressed and  recently demonstrated to be key players in tumour cell progression, angiogenesis and metastasis.  For example, both CXC chemokine receptor-4 (CXCR4) and CC chemokine receptor-7 (CCR7) are highly expressed in human primary and metastatic breast cancer, yet their expression is low or absent in normal breast epithelium. Furthermore, the expression of CXCL12, the natural ligand to CXCR4 is highest in lung, liver, bone marrow and brain, all major sites for breast cancer metastasis; while the expression of CCL19 and CCL21, the natural ligands to CCR7 is highest in lymph nodes, suggesting they control dissemination of breast cancer via the lymphatic system. Indeed there is evidence that the two axes may work in tandem.  We are currently engaged in a programme to design and synthesise novel small molecule inhibitors of CXCR4 and CCR7 to assess both their involvement in tumour metastasis and the therapeutic potential for inhibition of chemokine signalling. Supported by additional funding from Yorkshire Enterprise and White Rose Health Innovation Partnership, we expect to make significant advances in these projects in the near future.  

Tumour hypoxia-selective chemotherapeutics  
Dr K. Pors and Professor L. H. Patterson  

The importance of killing tumour regions 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 I/II clinical trials undertaken by Kudos Pharma and Novacea Pharma. 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 tumour spheroids, a model system developed by Dr R.M. Phillips of the Institute of Cancer Therapeutics.  Concurrently, we are investigating the use of our N-oxide concept as an entirely original smart platform technology applicable to a range of clinically available chemotherapeutics to enable (i) significant increase in the delivery of an effective concentration of specifically to the tumour cells and vascular (ii) produce minimal or even no debilitating side effects to normal tissue (iii) activate in the hypoxic compartments unique to tumours and kill hypoxic and through lateral controlled diffusion kill the surrounding oxygenated  malignant cells through a bystander effect.  

Proteomic analysis of drug targets  
Dr. C. W. Sutton and Prof. L. H. Patterson  

The proteomic facility at the Institute of Cancer Therapeutics was established in 2006 through generous support from YCR to complement the established chemistry and pharmacology groups and is under the direction of Dr Chris Sutton (Senior Lecturer in Proteomics and Mass Spectrometry) and Prof Laurence Patterson. The Proteomics Facility has state-of-the art MALDI TOF-TOF mass spectrometry, nano LC, conventional chromatography, 1D gel electrophoresis and bioinformatics capabilities. It is  expanded further to include 2D gel electrophoresis, gel image comparison software and proteomics interpretation software. The main aims of the Facility are: (1) the characterisation of target proteins from biological systems, which are used to test new drugs candidates, (2) the identification and characterisation of novel targets, and (3) changes in protein expression and activity (pharmacoproteomics) due to drug administration from preclinical and clinical trials.  

Over the last year, the main objective has been to recruit research staff and students to perform the expanding proteomics projects and collaborations. The number of collaborations have been extended over the last year: 1. with Dr. Richard Wheelhouse, Department of Pharmacy, UoB, - Ph. D. studentship - to identify a protein target for a novel compound, 2. with Dr. John Kelly, Bruker - Ph. D. studentship - cytochrome P450 profiling and quantitative proteomics applications, 3. with Dr. Kyriacos Kyriacou, Cyprus Institute of Neurology and Genetics (CING) - proteomics screening normal and tumour breast tissues (2 year project), 4. with Dr. Paul Loadman (ICT) and Dr. Malcolm Clench (Sheffield Hallam University) - MALDI imaging to look at drug localisation in tissues, 5. with Dr. Roger Phillips (ICT) - Ph. D. studentship - proteomics profiling of spheroids to identify new hypoxia markers, 6. with Dr. Andy Stapleton, Bio-Rad - PDRA - develop novel affinity capture materials that can be used to identify new disease markers (3 year project), 7. with Dr. Steve Shnyder (ICT) and Bio-Rad - determine changes in protein expression in cells grown in hollow fibre assays following drug administration, 8. with Dr. Jason Gill (ICT) and Professor Ron Grigg (University of Leeds) - characterise proteins that bind to novel inhibitors of histone deacetylases.


Institute of Cancer Therapeutics
University of Bradford  
Director: Prof Laurence H. Patterson        


Targeting of Cytochromes P450 overexpressed in tumours  
Dr K. Pors, Prof. L.H. Patterson and Dr S.D. Shnyder  

Several Cytochromes P450 (CYPs) are now described as over-expressed in tumour tissue and surrounding stroma compared to surrounding normal tissue, notably CYP1A1, 1B1, 2J2, 2S1, 2W1 and 4Z1. The functional significance of increased tumour CYP expression has not yet been fully elucidated, but several studies have demonstrated correlations between increased expression of specific CYPs and increased cellular proliferation. As a part of our drug discovery program, we are currently investigating CYP 1A1, 1B1, 2J2, 2S1 and 2W1 as targets to explore tumour-selective drug activation since these isoforms possess aryl oxidase activity towards xenobiotics. Tumours expressing high levels of active CYPs could induce their own demise via the conversion of otherwise inert agents into potent cytotoxins. We have identified the aryl-based chloromethylindolines as a novel class of agent and shown that they are inactive until metabolised to potent cytotoxins. This project concerns the synthesis of agents that are selectively activated by CYPs in tumours and explore their potential as cancer therapeutics using in vitro and in vivo models. Specifically, we have synthesised a novel series of chloromethylindolines, which have been modified at the 3-position of the alkylating subunit and at the 5, 6 or 7 position of the DNA minor groove binding unit. In vitro screening of these novel agents have shown that they are intrinsically inactive in a CYP1A1 null cell line but are >1000-fold more cytotoxic in a CYP1A1-overexpressing CHO cell line. The results indicate that functionalisation at the 3-position of the alkylating subunit lead to agents with enhanced potential for CYP1A1 metabolism and subsequent cytotoxic activity.


School of Pharmacy
Head of School: Professor J.R. Purvis  

Identifying and Drugging a Novel, Anti-tumour Molecular Target  
Dr R.T. Wheelhouse, Dr C.W. Sutton, Dr R.M. Phillips, Mr D. Rong and Miss H.R. Evans  

We have discovered a family of novel, drug-like small molecules with potent activity against colon carcinoma, leukaemia, NSC lung cancer and prostate cell lines.  The compounds interact poorly with DNA and this is unrelated to the observed chemosensitivity. COMPARE analysis of NCI data showed that the new agents act by a novel mechanism, related to the expression of genes of as-yet unknown function, independent of DNA binding and with a structure-activity relationship consistent with recognition of a specific molecular target.   

A proteomics-mass spectrometry programme is being undertaken to identify and characterise the molecular target(s) of the new agents by identifying proteins up-regulated in susceptible cell lines. Bespoke affinity matrices are being prepared that will be used to capture proteins that bind the novel compounds. Trapped proteins will be separated by two-dimensional gel electrophoresis and characterised by mass spectrometry.