research report

University of Bradford

2002/3

Yorkshire Cancer Research Laboratory of Drug Design
The Tom Connors Cancer Research Centre
Director: Professor T.C. Jenkins

ENACT developments in NQO1/NQO2 enzyme-directed therapies
Prof. T.C. Jenkins, Ms S.L. Moores

New ENACT (ENhanced Anti-Cancer Therapy) strategies, where the YCR Laboratory of Drug Design has played a pivotal role in unravelling the key control events (with Prof. R.J. Knox, Enact Pharma plc, Porton Down), offer fresh and exciting opportunities for tumour targeting. This strategy exploits the tumour-selective over-expression of enzymes that can be used to activate inert prodrugs to their potently active drug forms (prodrug/drug switching). An ENACT-designed treatment, based upon the reducing enzyme NQO2, using CB1954 (prodrug) and reduced nicotinamide riboside (NRH) as the triggering co-substrate, will shortly be entering clinical trial.

The mechanisms of prodrug activation by NQO1 (DT diaphorase) and NQO2 enzymes have been evaluated (in conjunction with Dr S.W. Doughty, University of Nottingham), and a dual energy and kinetic model has been refined that explains the molecular differences in prodrug and co-substrate fitting for each protein. An in-house computer-assisted search algorithm has been used to score and identify enzyme-drug interactions for a series of related and unrelated candidate substrates, resulting in prodrugs that surpass the activity of our early CB1954 lead compound. Activation energy and kinetic pathway profiling has also defined the strict rules for prodrug/co-substrate accommodation within the enzyme reaction pockets. These results correlate with the enzyme reactivity toward each substrate, and this model has provided a superior basis for drug candidate selection and development.

Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) as DNA-targeted drugs
Prof. T.C. Jenkins, Ms T. Brown, Mr G.P. Wilkinson

PBDs act as remarkably potent alkylating agents towards double-stranded DNA, but reaction is confined to guanine residues accessed via the minor groove conduit. As part of a productive collaboration with Profs. D.E. Thurston and J.A. Hartley (Spirogen plc and School of Pharmacy/UCL Medical School, University of London) and Dr P.M. Loadman (Cancer Research Unit, University of Bradford), we have examined the DNA sequence targeting, biodistribution and pharmacokinetics of a reactive PBD dimer (SJG-136) and an unrelated monomer (DRH-417). The SJG-136 compound has recently been selected for NCI-supported clinical trials in the UK.

HPLC and fluorescence microscopy methods have been used to monitor the cellular uptake and nuclear penetration of these PBDs, and a reliable assay procedure has been developed to detect and quantitate their presence in blood or plasma at sub-nanomolar levels. Competing reactivity towards glutathione or non-protein thiols has been evaluated and used to develop a kinetic model for distribution and efficacy following intravenous drug administration. This assay is being validated for use in monitoring drug levels in patients. Molecular modelling has continued to provide a basis to understand the DNA recognition events and reactivity for these agents. A firm structure-activity relationship to understand their site and sequence preferences for alkylation and/or interstrand cross-linking has been refined, and shown to correlate with the biophysical behaviour and the time-scale for lesion formation in both cells and naked DNA.

Drug targeting of high-order DNA systems
Prof. T.C. Jenkins, Dr J.R.P. Arnold, Dr P.J. Perry, Dr T.A. Norris, Ms T. Brown

High-order DNA structures containing three or four strands continue to provide viable targets for chemotherapeutic intervention in tumour control, including telomerase function. Our structural and thermodynamic studies of DNA triplexes and tetraplexes have continued [in collaboration with Dr I. Haq (University of Sheffield), Dr D. Cairns and Dr R. Anderson (University of Sunderland), and Drs S.A. Jennings and R.T. Wheelhouse (University of Bradford)], and this wealth of information has been used to develop novel agents for structure-selective recognition in a tumour. Biocalorimetry and spectrophotometry have proved valuable and highlight the important role of structural interconversion between competing solution structures in biological media.

Next-generation heterocyclic agents related to clinically relevant anthracene-9,10-diones have been synthesised using this information and are being evaluated using biophysical techniques, including NMR, biocalorimetry and molecular modelling. A versatile DNA-binding chromophore ligand has been identified that can easily be manipulated to switch binding preference from duplex to triplex to tetraplex DNA (i.e. 2-, 3- or 4-strand systems), and hence be used to deliver actively-targeted chemical 'warheads'. Such 'smart' drugs could not have been anticipated using traditional medicinal chemistry methods. Computer-assisted molecular modelling and in-house biophysical methods have driven and informed this new phase of rational drug design, which remains a principal focus for our research activity.

Activation of p53 response through inhibition of the p53-MDM2 complex
Dr P.J. Perry, Dr J.R.P. Arnold, Prof. T.C. Jenkins

It is established that p53 tumour suppressor activity is negatively regulated by MDM2 and that such interaction can regulate the cellular response to genotoxic stress. As a consequence, p53-MDM2 interaction may often limit the clinical success of anti-cancer therapies. Inhibitors for this interaction can mimic the administration of genotoxic agents but do not trigger the onset of genetic damage. Certain chalcone compounds show ability to inhibit the p53-MDM2 interaction, and we are continuing a systematic evaluation of the structural features that confer this activity. As part of a wide drug synthesis programme, new classes of chalcone-based inhibitor have been prepared and these candidate anti-cancer agents are being evaluated (in collaboration with Dr S.M. Picksley, University of Bradford). Molecular modelling and high-resolution NMR techniques, augmented with parallel X-ray crystallography studies, are being used to probe fundamental aspects of the underlying biomolecular interaction events.

Mutagenic profiling and optimisation of DNA-targeted drugs
Prof. T.C. Jenkins, Dr P.J. Perry, Dr T.A. Norris

New agents developed for potential clinical application as anti-cancer drugs must not be inherently mutagenic, and we are continuing to apply this rule in all of our drug design strategies. In particular, where a lengthy drug exposure is likely to be necessary to elicit an optimal chemotherapeutic response, it is critical that this factor is effectively 'designed out' at an early stage. Using Ames-type and modern Comet assay procedures we are probing the key determinants of mutagenicity for a series of DNA-binding drugs under consideration for clinical use (in collaboration with Prof. D. Anderson and Dr D. Harrington, University of Bradford). Involvement in redox-cycling events, particularly for quinone-based or likely metal-chelating drugs, has emerged as an unwanted effect that can lead to unwanted mutagenicity. Key factors are emerging that modulate DNA-interactivity and can ultimately influence the mutagenic profile of a drug. Such molecular details will be valuable in on-going therapeutic developments.

Novel functionalised chalcones as microtubule inhibitors
Dr P.J. Perry, Dr J.R.P. Arnold, Prof. T.C. Jenkins

Chalcone derivatives offer a wide range of biological activity, including antimitotic potency, and are amenable to chemical modification and/or functionalisation in order to improve their biological efficacy. Further, certain chalcones share a structural and conformational similarity to established inhibitors of microtubule assembly, such as combretastatin A-4, an agent now undergoing clinical trials in a prodrug form. Our molecular modelling and biophysical studies show that particular chalcones bind strongly to the colchicine-binding site and can thus act as competitive ligands for this important location. Second-generation agents have now been developed that continue to show remarkable cytotoxicity towards human tumours, and these compounds are being further refined to establish structure-activity relationships in the modulation of both systemic bioavailability and drug stability.

CYP1B1-activated prodrugs for use in anti-cancer chemotherapy
Dr P.J. Perry, Prof. T.C. Jenkins

The cytochrome P450 enzyme displays selective aromatic hydroxylase activity that can be exploited for therapeutic gain in the design of a new class of anti-cancer agents. As CYP1B1 is overexpressed in a broad variety of human tumours, but not in normal tissues, this finding should be manifest in a differential response for potential tumour control. For example, selective aromatic hydroxylase activity offers a means to achieve tailored activation of inert or non-toxic prodrugs to potent drugs exclusively within the tumour. In this context, we have recently shown that resveratrol, a natural component of red wine with cancer preventative properties, is selectively metabolised by CYP1B1 to piceatannol, a known anti-cancer agent that acts as an inhibitor for important tyrosine kinases (in conjunction with Prof. G.A. Potter and colleagues at De Montfort University, Leicester). We have now used design strategies and molecular similarity methods to develop closely isostructural prodrug/drug leads based upon the resveratrol/piceatannol pair that offer exquisite potency toward a wide range of human tumours following activation by CYP1B1. To complement our drug synthesis programme, cellular pharmacology and in vivo models with human primary tumour xenografts have been established (in conjunction with Dr P.M. Loadman, University of Bradford) to validate the strategy. To our knowledge, such approaches remain unexplored for tumour-localised activation of prodrugs.

Structural analysis of chalcones by X-ray crystallographic, molecular modelling and solution NMR techniques
Prof. T.C. Jenkins, Dr P.J. Perry, Dr J.R.P. Arnold

Agents derived from the chalcone chromophore show a remarkably broad variety of chemotherapeutic effects and feature in our drug design programmes for many cancer-related protein biotargets. However, chalcones can often adopt a number of different structural conformations and/or isomeric configurations that may dramatically affect their interaction with their desired biotarget, and hence limit their biological efficacy. On this basis, we have carried out detailed structural analyses of this family of compounds using X-ray crystallographic, molecular modelling, and solution NMR techniques in order to characterise and maximise their targeted biointeractions. High-resolution structures have been obtained for five selected chalcone derivatives that highlight the effects of chemical substituents upon relative conformational flexibility and configuration (in conjunction with Dr I.J. Scowen, University of Bradford). Further, we have developed solution NMR methods that enable structural analysis and characterisation without the need for arduous X-ray crystal analysis. Such detailed information is proving invaluable in our on-going work to rationally design more potent and selective agents based on this important class of anti-cancer compound.

School of Pharmacy
Head of Department: Professor B. Costall

Structure-activity studies on novel DNA affinic compounds incorporating oestrogen-receptor ligand, polyamine and antineoplastic moieties
Dr. S. Carrington, Prof. J.E.Brown, Ms. H. Mackay

Healthy breast tissue requires a constant supply of oestrogens for growth and maintenance. Consequently it has been shown that up to 65% of breast cancers in pre-menopausal women are also dependent on oestrogens to sustain growth. We aim to target oestrogen receptors in such tumour cells with drug conjugates to produce selective therapies.

We have begun investigating structure-activity relationships between compounds consisting of an oestrogenic ligand, a polyamine and a cytotoxic DNA-binding moiety. Such conjugates are designed to target, and exhibit selective cytotoxicity towards breast cancers. We have already shown that linking oestrone to doxorubicin can confer selective cytotoxicity towards cells expressing oestrogen receptors (ER). By including a polyamine we aim to increase cellular uptake via the polyamine transport mechanism and to demonstrate increased cytotoxicity by enhanced polyamine associated DNA-binding. Compounds are being synthesised by established methodologies and then assayed in three areas: (i) Preclinical evaluation by in vitro MTT assay utilising ER-positive and ER-negative cell lines. (ii) Mechanistic studies including thermodenaturation, spectrophotometric titrations, and topoisomerase assays to characterise DNA interactions. (iii) Analytical HPLC and microscopy studies to determine compound stability and fate in the cell. Together the data from these assays will inform the design of further compounds.