research reports
University of Bradford
2001/2
Yorkshire Cancer Research Laboratory of Drug Design
The Tom Connors Cancer Research Centre
Director: Professor T.C. Jenkins
NQO2 enzyme-directed strategies for use in ENACT therapy
Prof. T.C. Jenkins, Ms S.L. Moores
ENACT (ENhanced Anti-Cancer Therapy) offers an opportunity for genuine clinical targeting of tumours by exploiting the overexpression of enzymes that can activate otherwise inert prodrugs by conversion to their active drug forms. A strong collaboration with Prof. R.J. Knox (Enact Pharma plc, Porton Down) has resulted in selection of an ENACT strategy using CB1954 (prodrug) and reduced nicotinamide riboside (NRH as triggering co-substrate) with the reducing enzyme NQO2 for a clinical trial to start in 2002 in Birmingham and Oxford.
In conjunction with Dr S.W. Doughty (University of Nottingham), Dr C.W. Wright (University of Bradford) and Dr J.L. Lisgarten (University of London) we have embarked upon a systematic evaluation of the mechanisms of activation by NQO2/NQO1 and the basis for strict accommodation and shuttling of substrate/co-substrate within the enzyme pocket. This study has identified an hitherto unknown molecular family of substrates for NQO2, an enzyme that is remarkably selective in terms of substrate. These agents are being synthesised and examined for differential activity in cells that have been engineered to overexpress the activating enzymes. Thermodynamic and kinetic profiles are being established for each component in the therapeutic cocktail in order to improve their clinical utility.
Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) as DNA-targeting agents
Prof. T.C. Jenkins, Dr L.J. Adams, Dr J.R.P. Arnold, Ms T. Brown, Mr G. Wilkinson
The PBD family of ligand molecules offers a unique profile of DNA recognition largely due to their covalent reaction only with guanines and mediated via the minor groove. As part of a collaboration with Prof. D.E. Thurston and his colleagues (Spirogen plc and School of Pharmacy, University of London), Dr P. Loadman (University of Bradford) and Dr S.P. Mackay (University of Strathclyde), we are probing the reactivity towards DNA duplexes and high-order DNA structures containing defined guanine-rich target sequences.
Molecular modelling and biophysical studies in our laboratories have now culminated in the selection of a tethered PBD dimer (SJG-136) for NCI-supported clinical trials in early 2003. In support, we are developing a sensitive HPLC-based assay to examine pharmacology and tumour biodistribution for this class of agent, where our studies have highlighted difficulties associated with cellular localisation of such reactive compounds. Clinical assays will be optimised to enable effective monitoring of drug levels in patients following administration.
DNA tetraplex-binding ligands as novel inhibitors of telomerase
Prof. T.C. Jenkins, Dr J.R.P. Arnold, Dr P.J. Perry, Dr T.A. Norris, Ms T. Brown
The four-stranded tetraplex structures formed by folding the single-stranded telomeric tail of chromosomal DNA are a viable target for chemotherapeutic intervention as this binding interferes with the function of telomerase, an enzyme that confers immortality in most tumours. In collaboration with Dr I. Haq (University of Sheffield), Dr D. Cairns and Dr R. Anderson (University of Sunderland) and Dr S.P. Mackay (University of Strathclyde) we are continuing our structural and thermodynamic studies of DNA tetraplexes and are developing new classes of recognition agent for their selective recognition within a tumour environment.
New classes of agent are being examined using isothermal titration and differential scanning calorimetry (ITC/DSC) methods, together with stopped-flow or kinetic CD, to probe DNA-ligand interactions at the DNA duplex/tetraplex interface. Drugs stemming from such work would have unique potential to recognise and interfere with cancer-related DNA assemblies, and offer prospects for a novel mode of biomolecular targeting.
Fused-ring tetracyclic compounds related to anthracene-9,10-diones with clinical application are being synthesised and evaluated using state-of-the-art biophysical techniques, including NMR, biocalorimetry and molecular modelling. We have identified a versatile tetraplex DNA-binding chromophore ligand that can be elaborated to deliver 'warhead' chemical functionality, so as to effect irreversible fixation of such high-order DNA structures. It is anticipated that this drug action will block processivity of telomerase and prevent unwanted enzyme function. Computer-assisted molecular modelling has highlighted directions for rational drug design, where this remains a major 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
p53 tumour suppressor activity is negatively regulated by the MDM2 oncoprotein. This interaction regulates the cellular response to genotoxic stress and, in part, can influence the success of anticancer therapies. Potent peptide inhibitors of the p53-MDM2 interaction have established 'proof of principle' and provided a level of target validation with the effect of such inhibitors mimicking that of genotoxic agents but without the induction of genetic damage. Following observations that certain chalcone derivatives also possess the ability to inhibit the p53-MDM2 interaction we are systematically studying the chalcone chromophore to establish the structural determinants required for activity. A large number of novel chalcone-based candidate inhibitors has been synthesised and these agents are undergoing evaluation in a modified ELISA assay (in collaboration with Dr S.M. Picksley, University of Bradford). In addition, molecular modelling and high-resolution NMR techniques are being used to further probe the molecular basis for interaction(s) with this class of inhibitor.
Mutagenic profiling of DNA-interactive anti-cancer drugs
Prof. T.C. Jenkins, Dr P.J. Perry
Absence of mutagenicity is an important factor in the pharmacological profile of any new drug destined for the clinic, and is critical for anti-cancer agents such as telomerase inhibitors that will require a lengthy or prolonged administration. Knowledge of the factors that dictate the mutagenicity of DNA-interactive agents is thus a vital requirement in the overall drug design process. Using a modification of established Ames and Comet assay procedures we are systematically probing the structural determinants that govern mutagenicity for a range of DNA-interactive agents now under evaluation (in collaboration with Prof. D. Anderson and Dr D. Harrington, University of Bradford). To date we have identified several agents that are essentially non-mutagenic towards various bacterial strains, with excellent correlation between findings from the Ames and Comet procedures. Further, a novel assay has been developed to examine the mutational spectrum of any drug or candidate compound. This assay is based upon analysis of restriction fragment length polymorphisms in the hisG46 gene of chemically-induced revertants of the Salmonella typhimurium strain TA100 subsequent to the digestion of PCR-amplified hisG46 revertant gene products with restriction endonucleases. Important structural factors are emerging that govern or influence the vital relationship between DNA-interactivity and mutagenicity. This information will benefit the design of non-mutagenic anti-cancer drugs and help to optimise their therapeutic development.
Microtubule inhibitors: chalcones as mimics of combretastatin A-4
Dr P.J. Perry, Dr T.A. Norris, Prof. T.C. Jenkins
Combretastatin A-4 is a natural product isolated from Combretum caffrum that is currently undergoing clinical trials in its water-soluble disodium phosphate prodrug form. This agent binds strongly to the colchicine-binding site of tubulin and shares many structural similarities to other tubulin-binding agents such as colchicine and podophylotoxin. Chalcone derivatives display a broad spectrum of biological activities including antimitotic potency. Preliminary molecular modelling studies have identified a family of chalcone analogues with a marked structural and conformational similarity to established inhibitors of microtubule assembly. Importantly, candidate agents have been shown to bind strongly to the colchicine-binding site and to effect displacement of bound colchicine. These minimal agents show remarkable cytotoxic potency towards a panel of human tumour cell lines, and represent powerful leads for potential application with our new tumour-specific targeting and delivery approaches.
CYP1B1-activated prodrugs in novel anti-cancer therapies
Dr P.J. Perry, Dr J.R.P. Arnold, Prof. T.C. Jenkins
Resveratrol is a naturally-occurring stilbene commonly found in red wine and known to have cancer preventative properties. We have recently demonstrated (in conjunction with Prof. G.A. Potter and colleagues at De Montfort University, Leicester) that this compound is selectively metabolised by the enzyme CYP1B1 to the known anti-cancer agent piceatannol, a potent broad-spectrum inhibitor of tyrosine kinases. The CYP1B1 enzyme is a member of the cytochrome P450 family and possesses selective aromatic hydroxylase activity. Importantly, CYP1B1 appears to be overexpressed in a broad variety of human tumours but not in normal somatic tissues. Expression of selective aromatic hydroxylase activity in tumour cells may thus provide a highly exploitable mechanism for the specific activation of prodrugs exclusively at the site of tumour. Chemical synthesis has provided a number of structurally-related prodrug/drug leads that show a demonstrably exquisite potency toward a wide range of human tumours upon enzymatic activation. To complement our drug synthesis, cellular pharmacology and in vivo models using primary human tumour xenografts are being established (in conjunction with Dr P.M Loadman, University of Bradford) to examine and validate the therapeutic viability of this strategy. Such approaches towards tumour-specific activation of otherwise non-toxic prodrugs are currently unexploited.
Novel mRNA-targeted therapeutic agents
Prof. T.C. Jenkins, Dr J.R.P. Arnold, Dr T.A. Norris, Dr P.J. Perry
Using a novel competitive equilibrium dialysis technique developed in our laboratory we have identified (in conjunction with Prof. J.B. Chaires, University of Mississippi) a number of small-molecule ligands capable of recognising and binding to single-stranded A-rich RNAs such as the 3'-poly(A) regions of processed mRNA. Such binding to mRNAs may have potential for interfering with the full processing of mRNAs by poly(A) polymerase (PAP). Neo-PAP, a structural homologue of PAP, is overexpressed in human tumours and may thus represent a highly tumour-specific target that can be exploited to develop a mew class of therapeutic agents. A concerted approach involving structural (high-field solution NMR) and thermodynamic (isothermal titration calorimetry/UV spectrophotometry) methodologies is being used to probe the molecular determinants and to characterise the nature of key interactions for mRNA-ligand recognition and binding.
Interactions of thymidylate synthase with RNA and clinical anti-tumour agents
Dr J.R.P. Arnold, Prof. T.C. Jenkins
Thymidylate synthase (TS) is an important chemotherapy target in many cancer treatments and is a target for clinical compounds such as fluorouridine and antifolates. A serious clinical complication is that treatment with such drugs often induces elevated levels of TS in cancer cells, which can lead to drug resistance, where the cause is not established. TS has recently been found to be an RNA-binding protein, with a high affinity for segments within its own mRNA, as well as the mRNAs and other important proteins in cancer biology such as p53 and c-myc. This suggests that TS may participate in many regulatory processes of significance in cancer cells although TS expression is tightly regulated. It is uncertain how RNA interactions with TS effect drug-protein binding and there is as yet no direct connection with drug-induced elevation of TS. Thus, TS-RNA-drug interactions need to be characterised in greater detail than has so far achieved. To this end, we are investigating the interactions between TS, RNA and TS-inhibitors as a basis for the design of new TS-binding drugs.
In collaboration with Dr J. Fisher (University of Leeds) and Dr J. Andrews (University of Manchester), 19F NMR spectroscopy has been used to characterise the interactions between human TS (hTS), 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) and other ligands. Under NMR conditions (i.e. high concentrations of hTS), FdUMP binding is weak (KD approx equal to 1 mM) but much tighter binding (KD approx equal to 1 mM) is evident at lower TS levels, such as those used in fluorescence measurements. In the binary TS-FdUMP complex, an internal equilibrium gives rise to a ca. 1:1 mixture of noncovalent versus covalent binding of FdUMP to hTS. The addition of antifolates such as methotrexate, folic or folinic acids disturbs this equilibrium so that only covalent binding of FdUMP is observed in the corresponding tertiary complexes. FdUMP binding remains weak, though enhanced in the presence of folate; the binding of the antifolates themselves is relatively strong. This behaviour is similar to that previously noted for TS from other mammalian sources but quite different to that for microbial TS.
Binding of hTS to a stem-loop region within its mRNA (which contains the start codon) has been confirmed. The binding of this RNA is non-competitive with respect to substrates and inhibitors, supporting the notion that the RNA binding site is distinct from, but communicates with, the active site. Studies using NMR-labelled RNA are currently in progress, as we seek to further characterise the hTS-RNA interaction. In addition, the cause of the relatively weak affinity of FdUMP under conditions of our NMR experiments is being pursued; this may be a manifestation of a mechanism for the regulation of hTS activity, as opposed to expression. Future studies will also include investigation of hTS interactions with other RNA motifs.
Antisense nucleic acid systems for therapeutic control
Dr J.R.P. Arnold
A specific mRNA can be inactivated by the introduction of a complementary, or antisense, oligonucleotide strand to which it will bind and then become unavailable for translation. This approach requires that the antisense oligonucleotide (or derivative) is stable in the cell, and binds both selectively and with a high affinity to the target RNA sequence, whilst also being as short as possible. With these considerations in mind, we are seeking to develop improved or next-generation antisense agents for chemotherapeutic use. In collaboration with Dr J. Fisher (University of Leeds) and Dr R. Cosstick (University of Liverpool) oligodeoxynucleotides have been investigated where modifications are introduced to improve affinity for the targeted RNA. The alterations need to be structurally conservative in order to preserve the ability of the antisense-RNA complex to be recognised by cellular RNase H, which then cleaves the target RNA. DNA containing a modification in which the oxygen atom attached to the 3'-carbon of the 2'-deoxyribose ring has been replaced by sulfur offer many potential advantages. We have incorporated this 3'-thiophosphothiolate link into a model DNA-RNA hybrid. In line with predictions, the hybrid is stabilised by this modification, whilst detailed 1H NMR analysis has shown that the structural effects of the modification are highly localised within the double-helical architecture, which itself is of an intermediate A/B helical nature. Evidently the A/B-type helix is forgiving in terms of accommodating chemical modifications, a characteristic also frequently observed in DNA B helices. Continued work will involve the concerted incorporation of several modifications to further optimise these antisense agents. We are also seeking a more complete structural characterisation of the DNA-RNA hybrid system.
Studies of nucleic acid structure and thermodynamics
Prof. T.C. Jenkins, Dr J.R.P. Arnold
In conjunction with Dr J. Fisher (University of Leeds) we are investigating the structure and thermodynamics of DNA duplexes that contain chemical modifications to mimic the damage caused, for example, by ionising radiation. Such lesions have a well-established linkage to oncogenesis and cancer progression, where damage can involve nucleotide disruption by loss of either a base or the base together with part of an attached sugar. Detailed 1H NMR analysis, and comparison with the corresponding undamaged DNA duplex, shows that the structural effects of such modifications are highly localised, becoming almost unnoticeable within 1-2 base pairs from the damaged site. In contrast, however, the effects upon thermodynamic behaviour are profound. Dissociation of the damaged duplex into separate single strands is far more readily achieved than for the undamaged or parent counterpart, as shown by UV thermal denaturation experiments. These observations will be reconciled from our structural and biophysical studies.
Development of ribozyme-based anti-cancer therapy
Dr J.R.P. Arnold
The specific inactivation or destruction of undesirable mRNA is emerging as a potentially important tool in anti-cancer therapy. Increased understanding of the cell biology of cancer is revealing further opportunities for chemotherapeutic intervention at the RNA level. In addition to the antisense approach (outlined above) we are also evaluating the application of ribozymes. The simplest ribozyme with significant activity is the so-termed 'hammerhead', where recent important advances have enabled an understanding of the underlying events and the catalytic mechanism(s). This raises an intriguing potential for the rational design of catalytic machinery for use in the cleavage of targeted RNA target sequences, i.e. an artificial ribonuclease. In this way a ribozyme analogue can be developed which itself is more robust in the cellular environment than would be a pure-RNA species, whilst retaining specificity and an adequate level of activity. Design and fundamental development work toward this goal is now in progress.
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