Chemical Biology

1. The function of the molecular chaperone Hsp90

Heat shock protein 90 (Hsp90) is an ATP dependant molecular chaperone essential for both creating and maintaining the active conformation of key regulatory proteins, such as certain hormone receptors, mitogen activated protein kinases and tumour suppressors such as p53 and Rb. In order to achieve its function, Hsp90 acts as the centre of a multi-protein chaperosome complex that includes an array of co-chaperones. The chaperosome complex has been highly conserved throughout the eukaryotic lineage, and we exploit yeast as a genetically tractable model system in which to investigate the contribution made by each co-chaperone to the overall function of the chaperosome. For example, we can assess activity of Hsp90 substrates - such as the glucocorticoid receptor and the pp60v-src oncogernic kinase - in genetic backgrounds deleted for the genes encoding the co-chaperones.

2. The role played by the Smc5/6 complex in maintenance of genome integrity

There are three Structural Maintenance of Chromosomes complexes. The core of all three is composed of a heterodimer. One complex, cohesin (Smc1/3), keeps sister chromatids together prior to mitosis. Condensin (Smc2/4), is responsible for the chromosome condensation that occurs during mitosis. The third, Smc5/6, has an essential yet poorly characterised role. We are trying to understand the role played by this complex in maintaining genome integrity. Smc5/6 is found in a complex containing at least five other proteins, Nse1-5 (Non Smc Elements). We are generating mutant alleles of the corresponding genes, in order to observe how they disturb genome structure, thereby generating insight into the function of the Smc5/6 complex itself.

3. Conditional mutants in QRI2/NSE4 arrest at the G2/M transition

Cells deleted for NSE4 bearing a vector-borne wild type allele (W+), or the nse4-4ts allele, were grown at 250C and shifted to 370C for 4 hours. Cells were fixed, followed by staining DNA and spindles with DAPI (B), and anti-tubulin (C), respectively. Panels A (phase), B & C are cells from the same field of view.

BV vectors as potential gene delivery vehicles

BV is an insect virus that has been exploited over many years to direct expression of foreign proteins in insect cell cultures. Because the range of post-translational modifications, e.g. glycosylation, mediated by insects are, in comparison to bacteria or yeast, more similar to those of mammalian cells the baculovirus expression vector system (BEVS) has gained considerable popularity. BV can also transduce and deliver a functional gene to the nuclei of a range of mammalian cells often with efficiencies >90%. The list of non-host cells transduced successfully has grown in recent years and includes several examples of human primary cells, e.g., hepatocytes, neural cells and pancreatic islet cells. BV exhibits several features that make it attractive as a vehicle for in vitro gene delivery to mammalian cells and as a potential alternative to more traditional viral vectors for in vivo gene therapy. The Chemical Biology group are modifying the genome of BV with the aim of developing it as a therapeutically viable gene therapy vector. In particular the interest is in targeting baculovirus to specific cell populations in the liver that regulate the extra-cellular matrix remodeling that is critical in the pathogenesis of cirrhosis. The lack of any effective small molecule anti-fibrotic drug class represents a critical and pressing unmet clinical need for this disease and we hope to develop alternative therapeutic strategies based on BV gene therapy.

Recombineering reporter fusions

In collaboration with the Hope lab (http://bgypc059.leeds.ac.uk/~web/), we have developed a strategy, based on recombineering with a counter-selection cassette, to directly, and seamlessly, modify genomic fosmid clones to generate fusion reporters for analyzing gene expression in C. elegans. The method was described in Dolphin CT, Hope IA. (2006). Further information, including a FAQs page, on the group recombineering protocol can be found here.

Gene targeting in C. elegans

Since its adoption as a genetically tractable model animal the nematode C. elegans has provided invaluable, detailed insight into numerous fundamental biochemical processes. Many of these discoveries were and are still made using classical forward "phenotype-to-gene" genetic screens. However, in contrast to several other models, such as mouse and yeast, it has not yet proved possible in C. elegans to generate precise sequence changes at genomic loci via homologous recombination (HR). Thus, a robust and facile gene-targeting (GT) method remains a highly desirable and sought-after functional genomics tool for worm researchers. The group is hoping to develop a new approach for GT in C. elegans based upon HR mediated by bacteriophage recombinases.

• Metabonomics “The quantitative measurement of the multivariate metabolic responses of multicellular systems to pathophysiological stimuli or genetic modification”. (GLOBAL SYSTEM ANALYSIS) Nicholson et al, Xenobiotica 1999. In particular for application in toxicity, disease and nutrition.  
• Fundamentals of liquid chromatography and applied liquid chromatography coupled to mass spectrometry (in particular micro and nano LC for high sensitive analysis of small molecules and peptides)  
• Capillary Electrochromatography/Electrophoresis and applications.  
• Ultra Performance liquid chromatography and applications.  
• NMR analysis (1D, 2D and MAS) of biofluids and tissues.  
• Advanced statistical analysis (chemometrics)  
• Chromatographic methods to calculate phase-analyte interactions (frontal analysis approach)  
• Molecularly imprinted polymers. MIPs  
• Validation of analytical methods

Dr Mountford has divided his time between two distinct areas of research.
Firstly, the development of new synthetic methods for the preparation of classes of molecule that are either poorly described in the literature or are currently not described. The rationale behind targeting these classes of compounds lies in both an ability to construct compounds to probe regions of a drug target’s active site that are currently inaccessible using the constructs currently available, and the improved IP position that would be obtained if these molecules were incorporated into a drug candidate.

The second area of research lies in the application of Drug Discovery methods to biological targets. Current targets lie in the fields of allergy, pain and inflammation, areas which Dr Mountford knows well from his time at CBT.

Current research is focused on a number of biological targets, both those that are well described in the literature and also potentially new drug targets arising from original research being carried out at King’s College London.

Currently specific areas of interest are in:

Novel piperidine architectures
Synthesis of antimalarial Natural Products
Novel multi-component reactions
Evaluation of possible new approaches to organocatalysis
Fragment based Drug Discovery
Drug targets for Asthma, inflammation and pain.

Prof David Thurston's research interests include the discovery of sequence-selective DNA-interactive agents as anticancer drugs, antibacterial agent, and as transcription factor inhibitors.

He is also interested in the discovery of novel protein-protein interaction inhibitors as anticancer agents, the development of novel bio-analytical methodologies, and the application of pharmacogenomics and personalised medicine approaches to anticancer drug discovery and the practise of pharmacy.

His research has been funded from a variety of sources including the Research Councils, Cancer Research UK, the British Council, the Commonwealth Fund and various pharmaceutical companies. In 1996 his funding from Cancer Research UK was awarded Programme Grant status.

Dr Wagner's main research interests are in medicinal chemistry and chemical biology – developing chemical tools to address biological and biomedical problems. Research in his laboratory sits at the interface of chemistry and biology and research projects in his group - such as the development of inhibitors and assays for therapeutically relevant enzymes - are generally very interdisciplinary. Dr Wagner and his group use a range of different methods, from organic synthesis, through protein biochemistry to various analytical techniques, particularly fluorimetry. The Wagner group collaborates extensively with external partners in the UK, Denmark and Germany.

Current projects:

Glycosyltrasferases as drug targets
Glycosyltransferases (GTs) are enzymes that catalyse the transfer of a monosaccharide from a glycosyl donor to a suitable acceptor, e.g. a glycan, peptide or lipid (see e.g. Annu. Rev. Biochem. 2008, 77, 521-555). GTs play a key role in many biological processes underpinning human health and disease, including glycoprotein and cell wall biosynthesis in human pathogens, carcinogenesis, and cellular adhesion. Individual GTs represent promising therapeutic targets, and the Wagner group is developing small molecular GT inhibitors as lead compounds for drug discovery, and as chemical tools for the investigation of glycosylation networks in living systems (Nat. Chem. Biol. 2010, 6, 321-323.)

Synthetic modifications of biomolecules in aqueous solution
Dr Wagner and his group have developed synthetic methodology for the direct structural modification of sensitive biomolecules in aqueous media, obviating the need for protecting groups and for lengthy synthetic sequences. The group has particular expertise in the Pd-catalysed cross-coupling of nucleosides, nucleotides, sugar-nucleotides and amino acids. This synthetic approach has proved very useful for the generation of novel fluorescent bioprobes.

Chemical tools for NAD-dependent enzymes
The dinucleotide NAD (nicotinamide adenine dinucleotide) is required as a cofactor not only by redox enzymes, but also by other enzyme classes which use NAD for covalent modifications. Many of these enzymes, such as the PARP enzymes, the sirtuins, and NAD-dependent ligases, play essential roles in cell signalling and transcription, and are exciting molecular targets for chemical biology and drug discovery. Dr Wagner and his group are currently developing structural analogues of NAD for the selective inhibition of individual enzymes and for the real-time imaging of their activity, e.g. in living cells.

Dr Thanou and her group prepare fluorescent nanoparticles and study their diffusion in biological samples using Multiple particle Tracking and fluorescent microscopy

Engineering targeted nanoparticles for cancer siRNA delivery

Dr Thanou and her group have developed a novel ligand for breast and prostate cancer that can functionally deliver siRNA to tumours. They studied and optimised the organisation of this ligand on the surface of Nanoparticles for optimum receptor recognition and endocytosis.

Developing Carbon Nanopipes as drug delivery systems

Dr Thanou and her group are developing novel carbon architectures that can be used safely for biomedical applications. These architectures show chemical versatility and the potential to be used in Drug Delivery.

Dr Rahman's research activities focus on the application of synthetic medicinal chemistry and chemical biology techniques to the design, synthesis and evaluation of novel anticancer and antibacterial agents, along with studies to understand their molecular and cellular mechanisms of action. The key research areas are -

Development of Novel Telomerase Inhibitors:

Inhibition of telomerase can force tumour cells to enter replicative senescence leading to programmed cell death or apoptosis. Research in this area will target telomerase using a small-molecule strategy by interfering with i) Telomeric G-quadruplex structure, and ii) DNA/RNA hybrid structures that are formed during the catalytic cycle of telomerase. Formation of this unique heteroduplex is a key step in the catalytic cycle of telomerase in its processing to extend the telomere. Small molecules that bind to this duplex may inhibit telomerase by either stabilizing the structure and preventing strand dissociation, or by sufficiently distorting the substrate duplex to cause misalignment of key catalytic groups

Discovery of Novel G-Quadruplex Targeting Agents to Modulate Expression of c-Myc, c-Kit and K-ras Oncogenes:

Oncogenic transcription factors are an increasingly important target for anticancer therapies, as inhibition could allow the “reprogramming” of tumour cells, leading to apoptosis or differentiation from the malignant phenotype. For example, the c-Myc transcription factor plays a key role in the progression of most human tumours, and is over-expressed in a wide variety of human cancers. The proto-oncogene c-Kit codes for a 145-160 kDa tyrosine kinase receptor which regulates several important signal transduction cascades that control cell growth and the proliferation of cancer cells. Similarly, mutation and over-expression of the K-ras oncogene is strongly associated with pancreatic cancer. Promoters of all three oncogenes (c-Myc, c-Kit and K-ras) contain sequences that have been shown to form G-quadruplex structures that control their transcriptional activity. Research in this area is focused on targeting these G-quadruplexes with small molecules in an attempt to modulate transcription.

Development of Novel Bio-Analytical Methodologies:

Research in this area has led to development of a number of novel analytical methodologies to evaluate the DNA interactions of sequence-selective molecules. One particular area of success has been the development of a HPLC-MS assay that has been used to measure the kinetics of reaction of novel agents with DNA, and to evaluate their sequence selectivity. The group is working to develop the scope of these assays to allow the monitoring of drug-genomic DNA interactions in cells, and to study the interactions of DNA-binding drugs with mitochondrial DNA.

Discovery of Novel DNA-Interactive Transcription Factor Inhibitors:

A combination of traditional synthetic chemistry and automated techniques is used to discover novel gene targeting agents capable of recognising specific gene sequences and inhibiting transcription factor binding in a selective manner. Work on C8-linked PBD conjugates have shown selective NFkB transcription factor inhibiting properties, and significant activity against breast and pancreatic cancer and leukaemia both in vitro and in vivo.

Discovery of Novel DNA-Interactive Antibiotics:

This research area involves the design, synthesis and evaluation of novel sequence-selective DNA-interactive agents as antibacterials, targeted toward specific sequences of DNA in the bacterial genome with a focus on MRSA, VRE and mycobacterium.

• Structure-function studies of the p53 family of transcription factors and their role in cancer and other human diseases

• Protein stability, protein (mis)folding, aggregation and disease

• Protein-protein, protein-DNA interactions.

Professor Peter Hylands has many years experience in natural product research and development, with an emphasis on the isolation and structural determination of novel bioactive compounds, both in academia and industry.

Professor Hylands has led multidisciplinary research programmes in Europe, the Americas and various Asian countries and has been an invited speaker all over the world.  Important aspects of his present research are innovative chemometric metabonomic approaches to natural product research, notably the standardisation of plant extracts.

• Combinatorial chemistry
• Anti-sense therapeutics
• Biomimetic catalysis