1 2013 RSC Bio-Organic Group Postgraduate Symposium Manchester Institute of Biotechnology, University of Manchester Thursday 11 th April 2013 10:15 Registration, coffee, poster set-up 10:55 Welcome 11:00 – 12:30 Sarah Lovelock (University of Manchester) Anabaena variabilis Phenylalanine Ammonia Lyase: An investigation into the catalytic mechanism and applications as a biocatalyst. Cristina Marculescu (University College London) The Development of a New Class of Maleimides as Reagents for Protein Modification Simge Davulcu (University of Bath) Catalytic conversion of unactivated nitriles into N-substituted amides Jon Ashley (National University of Singapore) Hybridised-SELEX: a capillary electrophoresis based method for maximising the number of aptamers screened 12:30 – 13:40 Lunch, poster session 13:40 – 15:10 Ryan Beattie (University of Bristol) A Versatile Synthetic Approach to Novel Deoxy Sugar Analogues Matthew Styles (University of Manchester) Tailoring Complex Natural Products by Altering the Biosynthetic Machinery that Builds Them Oscar Cascón (Cardiff University) Study of (+)-δ-cadinene synthase mechanism using farnesyl diphosphate analogues. John M. Wadsworth (University of Edinburgh) The natural product inhibitor myriocin displays a unique dual mode of action against serine palmitoyltransferase 15:10 – 15:45 Coffee, poster session 15:45 – 16:30 Plenary lecture: Prof. Nigel Scrutton, Manchester Institute of Biotechnology Addressing controversies in tunnelling and dynamics in enzyme catalysed H transfer 16:30 – 17:15 Prizes, refreshments 17:30 Close We are grateful to the following organisations for sponsoring this event:
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1
2013 RSC Bio-Organic Group Postgraduate Symposium
Manchester Institute of Biotechnology, University of Manchester
Thursday 11th April 2013
10:15 Registration, coffee, poster set-up
10:55 Welcome
11:00 – 12:30 Sarah Lovelock (University of Manchester)
Anabaena variabilis Phenylalanine Ammonia Lyase: An investigation into the catalytic
mechanism and applications as a biocatalyst.
Cristina Marculescu (University College London)
The Development of a New Class of Maleimides as Reagents for Protein Modification
Simge Davulcu (University of Bath)
Catalytic conversion of unactivated nitriles into N-substituted amides
Jon Ashley (National University of Singapore)
Hybridised-SELEX: a capillary electrophoresis based method for maximising the number
of aptamers screened
12:30 – 13:40 Lunch, poster session
13:40 – 15:10 Ryan Beattie (University of Bristol)
A Versatile Synthetic Approach to Novel Deoxy Sugar Analogues
Matthew Styles (University of Manchester)
Tailoring Complex Natural Products by Altering the Biosynthetic Machinery that Builds
Them
Oscar Cascón (Cardiff University)
Study of (+)-δ-cadinene synthase mechanism using farnesyl diphosphate analogues.
John M. Wadsworth (University of Edinburgh)
The natural product inhibitor myriocin displays a unique dual mode of action against
serine palmitoyltransferase
15:10 – 15:45 Coffee, poster session
15:45 – 16:30 Plenary lecture: Prof. Nigel Scrutton, Manchester Institute of Biotechnology
Addressing controversies in tunnelling and dynamics in enzyme catalysed H transfer
16:30 – 17:15 Prizes, refreshments
17:30 Close
We are grateful to the following organisations for sponsoring this event:
2
Talk 1
Anabaena variabilis Phenylalanine Ammonia Lyase: An investigation into the catalytic mechanism and
applications as a biocatalyst.
Authors: Sarah Lovelock, Rachel Heath, Richard Lloyd and Nicholas Turner.
Presenting author affiliation: University of Manchester
Other affiliations: CoEBio3
OH
O
R
PAL+NH3
-NH3
OH
O
RNH2
Figure 1: The amination of substituted cinnamic acids to yield L-amino acids is catalysed by phenylalanine ammonia lyase
In Nature, the enzyme phenylalanine ammonia lyase (PAL) catalyses the deamination of L-phenylalanine to
yield trans-cinnamic acid and ammonia.1 However, the reaction is reversible under conditions of high
ammonia concentration and synthetically useful conversions in the amination direction can be obtained.
PALs therefore represent potentially attractive biocatalysts for the synthesis of enantiomerically pure L-
amino acids from substituted cinnamic acids and ammonia (figure 1). The broad use of PALs as biocatalysts
for the synthesis of non-natural amino acids is currently limited by their relatively narrow substrate range.
The application of eukaryotic PALs as biocatalysts for the amination of cinnamic acid analogues has been
described previously.2 However, the activity of prokaryotic PALs towards non-natural substrates has not
been investigated. The bacterial PAL from Anabaena variabilis(AvPAL) has recently received attention as a
potential therapeutic enzyme for the metabolic disorder phenylketonuria.3 An available crystal structure of
this enzyme makes it a suitable target for structure guided directed evolution.4 We have examined the
activity and enantioselectivity of AvPAL towards a broad range of non-natural substrates and compared this
activity with the eukaryotic PALs from the yeast Rhodotorula glutinis (RgPAL) and parsley Petroselinum
crispum (PcPAL). AvPAL shows significantly higher activity towards a series of non-natural substrates than
previously described eukaryotic PALs. Interestingly, some non-natural substrates also led to significant
formation of D-amino acids and the catalytic mechanism has been investigated.
Key references:
(1) N. J. Turner, Curr. Opin.Chem. Bio., 2011, 15, 234; J. Ward and R. Wohlgemuth, Curr. Org. Chem, 2010,
14, 1914
(2) A. Gloge, J. Zón, Á. Kövári, L. Poppe, and J. Rétey, Chem. Eur. J., 2000, 6, 3386; C. Paizs, A. Katona and J.
Rétey, Eur. J. Org. Chem, 2006, 5, 1113; A. Gloge, B. Langer, L. Poppe and J. Rétey, Arch. Biochem. Biophys.,
1998, 359, 1; S. Bartsch and U. T. Bornscheuer P.E.D.S., 2010, 23, 929.
(3) M. C. Moffitt, G. V. Louie, M. E. Bowman, J. Pence, J. P. Noel and B. S. Moore, Biochemistry, 2007, 46,
1004
(4) L. Wang, A. Gamez, H. Archer, E. E. Abola, C. N. Sakissian, P. Fitzpatrick, D. Wendt, Y. Zhang, M. Velard, J.
Bliesath, S. M. Bell, J. F. Lemontt, C. R. Scriver and R. C. Stevens, J. Mol. Biol., 2008, 380, 623.
3
Talk 2
THE DEVELOPMENT OF A NEW CLASS OF MALEIMIDES AS REAGENTS FOR PROTEIN MODIFICATION
Authors: Cristina Marculescu, Dr. James Baker, Dr. Rachel Morgan, Dr. Lyn Jones.
Presenting author affiliation: University College London, 20 Gordon St., London, WC1H 0AJ.
Other affiliations: Pfizer
In 2009, the Baker group reported on the bromomaleimides as the first of a new class of reagents
that could be efficiently used for the highly selective and reversible modification of cysteine and for the
bridging of disulfide bonds in proteins.1,2,3 Aiming to prove that these transformations are not restricted to
bromomaleimides, the present work presents a library of novel analogues, bearing different leaving groups
on the double bond. By controlling the chemistry of this class of compounds we were able to tune
properties such as selectivity, reactivity, solubility, and cross reactivity with reducing agents.
Fig. 1 Mono- and disubstituted maleimide analogues.
The utility of the novel monosubstitued analogues as protein labelling reagents was shown using a
single cysteine mutant of protein Grb2 (L111C) as a model system. The disubstitued analogues were tested
as cystine bridging reagents therefore the model system used was somatostatin, a 14-aminoacid peptide
containing a disulfide bridge. A novel dual modification of peptides method will be presented.
Fig. 2 Reactivity profiles of mono and di-substituted maleimides with the model systems.
Key references:
(1) Tedaldi, L. M.; Smith, M. E. B.; Nathani, R. I.; Baker, J. R., Chem. Commun. 2009, 6583.
(2) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick,
S.; Baker, J. R. J. Am. Chem. Soc. 2010, 132, 1960.
(3) Schumacher, F. F.; Nobles, M.; Ryan, C. P.; Smith, M. E. B.; Tinker, A.; Caddick, S.; Baker, J. R. Bioconj.
Chem. 2011, 22, 132.
4
Talk 3
Catalytic conversion of unactivated nitriles into N-substituted amides
Authors: Simge Davulcu, Jonathan M J Williams
Presenting author affiliation: University of Bath
The amide bond is essential to sustain life, making up the peptide bonds in proteins such as enzymes. It is
found in numerous natural products and biologically active molecules (Scheme 1). Despite their
importance, no currently used industrial methods for constructing the amide bond are particularly
“practical” or atom–efficient.
Scheme 1. Amide bonds in natural products and biologically active molecules
We have developed a zinc triflate and hydroxylamine hydrochloride catalysed methodolgy for direct
conversion of unactivated nitriles into N-substituted amides (Scheme 2). The reaction proceeds in
environmentally friendly water and provides a straightforward, atom-efficient methodology to synthesise
secondary and tertiary amides from nitriles which is a rarely reported transformation in the literature.
Scheme 2. Amide synthesis from nitriles and amines in water
The zinc triflate in combination with hydroxylamine hydrochloride salt efficiently catalyses the direct
conversion of unactivated nitriles into N-substituted amides with both primary and secondary amines.
Possible mechanisms for this reaction are discussed and evidence for initial amidoxime and amidine
formation pathways are reported. Isolated yields vary from 25-96%.
Key references:
(1) S. Davulcu, C. L. Allen, K. Milne and J. M. J. Williams, ChemCatChem 2013, 5, 435-438.
(2) C. L. Allen and J. M. J. Williams, Chemical Society Reviews 2011, 40, 3405-3415.
(3) C. J. Cobley, M. van den Heuvel, A. Abbadi and J. G. de Vries , Tetrahedron Letters 2000, 41, 2467-2470.
5
Talk 4
Hybridised-SELEX: a capillary electrophoresis based method for maximising the number of aptamers
screened
Authors: Jon Ashley, Sam Fong Yau Li
Presenting author affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive 3
Singapore 117543
Aptamers are ssDNA or ssRNA which specifically bind to biomolecules such as proteins, small molecules and
even whole cells. DNA aptamers are usually selected from a library of random sequences using a method
called Systematic evolution of ligands by exponential enrichment (SELEX) (Ellington and Szostak 1990). A
number of post SELEX modifications have appeared in the literature to address the issues associated with
the use of SELEX such as reducing the number of rounds of selection needed. Capillary electrophoresis
based methods such as CE-SELEX and Non-SELEX are attractive alternatives due to the high separation
efficiency, which can allow a selection to be carried out in <5 rounds. Also CE-SELEX and Non-SELEX are
carried out in free solution removing the necessity to immobilize the target and allowing the aptamers to
bind with the whole target (Mendonsa and Bowser 2004; Berezovski, Musheev et al. 2006). Moreover Non-
SELEX removes the need for intermittent amplification reducing the likelihood of contamination. However
due to the small sample sizes associated with CE, the number of sequences screened is limited, reducing
the likely hood of finding aptamers with high binding affinity. In this research, we propose a method which
significantly increases the number of sequences screened during the aptamer selection using capillary
electrophoresis. One round of selection using nitrocellulose filter was combined with subsequent rounds of
selection using Non-SELEX (Fig. 1).
Fig. 1 A general scheme for the hybridized SELEX procedure
The NC filter removes all non-binding sequences in the first round leaving non-specific binding sequences
and the target bound sequences. Non-specific binding sequences are then removed in the capillary
partitioning steps. Aptamers towards Cholesterol esterase were selected in less than 3 rounds of selection.
An Aptamer with a binding affinity KD of 116.6 ± 4.7 nM and good specificity was successfully selected.
Key references:
Ellington, A. D. and J. W. Szostak (1990). "IN VITRO SELECTION OF RNA MOLECULES THAT BIND SPECIFIC LIGANDS."
Nature 346(6287): 818-822.
Mendonsa, S. D. and M. T. Bowser (2004). "In vitro evolution of functional DNA using capillary electrophoresis."
Journal of the American Chemical Society 126(1): 20-21.
Berezovski, M., M. Musheev, et al. (2006). "Non-SELEX selection of aptamers." Journal of the American Chemical
Society 128(5): 1410-1411.
6
Talk 5
A Versatile Synthetic Approach to Novel Deoxy Sugar Analogues
Authors: Ryan J. Beattie, Christine L. Willis, M. Carmen Galan
Presenting author affiliation: School Of Chemistry, University of Bristol
Carbohydrates are one of the most abundant and important class of biomolecules, implicated in roles as
energy sources, structural frameworks (e.g. cellulose) and glycobiology as motifs for intracellular
recognition, for example in immune response and metastasis. A subclass within this family are the deoxy
sugars, which are components of a number of important biologically active natural products.1 However
studies into their roles of mediating pharmacological activity are limited by a lack of rapid and versatile
access to defined carbohydrates within this important class of compounds.2
To this end, we have developed methodology based upon Prins cyclisations3 to access stereodefined
tetrahydropyran scaffolds towards deoxy sugars targets and their analogues. A novel cyclisation involving
homoallylic alcohols and silyl acetals has been established, which, with subsequent Tamao-Fleming
oxidation affords lactols that can be converted in the same pot to O-acetates. By varying the reaction
conditions for the cyclisation, different groups (X) may be introduced. The approach is ideally suited for the
divergent syntheses of libraries of analogues for biological evaluation and these studies will be described.
Key references:
(1) Weymouth-Wilson, A. C., Nat. Prod. Rep. 1997, 14, 99.
(2) Kirschning, A.; Jesberger, M.; Schoning, K. U., Synthesis-Stutt. 2001, 507.
(3) Olier, C.; Kaafarani, M.; Gastaldi, S.; Bertrand, M. P., Tetrahedron, 2010, 66, 413.
7
Talk 6
Tailoring Complex Natural Products by Altering the Biosynthetic Machinery that Builds Them
Authors: Matthew Styles
Presenting author affiliation: University of Manchester
Natural products are often referred to as ‘privileged’ structures in that they are predisposed for protein
binding or other biological activity. This high propensity for bioactivity makes them an important source of
drug scaffolds in modern drug design. Nonribosomal peptides represent an important class of secondary
metabolite natural products biosynthesised by huge assembly line enzymes known as nonribosomal
peptide synthetases. These synthetases are capable of producing extremely complex products, containing
not just a huge range of natural and non-proteinogenic amino acids, but a large number of other
modifications such as halogenations and glycosylations which would be practically unattainable from a
purely synthetic standpoint.
The genes responsible for these elaborate systems tend to be clustered together in the organisms that
produce them. This gene organisation, coupled with the modular nature of the nonribosomal peptide
synthetases, has led to the conclusion that it would be possible to gain access to this biochemical space
through manipulation of the genes themselves. It is conceivable to envisage the redesign of such systems in
order to carry out complex biotransformations and produce altered or ‘unnatural’ natural products which
may have optimised or novel activity.
In this work it is shown how we can manipulate two antibiotics, calcium dependent antibiotic (CDA) and
enduracidin. In CDA biosynthesis we modify the adenylation domain responsible for the incorporation of
the polar 3-methyl-glutamate, so that it instead accepts the non-polar 3-methyl-glutamine, an unnatural
non-proteinogenic amino acid not observed in nature.
The biosynthesis of enduracidin is altered by the integration of a putative mannosyl transferase gene,
ram29, taken from the producer of the antibiotic ramoplanin, onto the chromosome of Streptomyces
fungicidicus, the enduracidin producing organism. This results in new variants of enduracidin being
produced with an additional mannosyl group attached to a specific residue.
(1) J. Thirlway, R. Lewis, L. Nunns, M. Al Nakeeb, M. Styles, A.-W. Struck, C. P. Smith and J. Micklefield,
Angewandte Chemie International Edition 2012, 51, 7181-7184.
CDA
CDA3a: R1=H, R
2=OH
CDA4a: R1=CH3, R
2=OH
CDA3a-10Q: R1=H, R
2=NH2
CDA4a-10Q: R1=CH3, R
2=NH2
8
Talk 7
Study of (+)-δ-cadinene synthase mechanism using farnesyl diphosphate analogues.
Authors: Oscar Cascón, Juan A. Faraldos, Veronica Gonzalez, David J. Miller, Rudolf K. Allemann*
Presenting author affiliation: School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Park
(+)-δ-Cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent
cyclisation of (E,E)-farnesyl diphosphate (FDP) to the sesquiterpene δ-cadinene, the parent
hydrocarbon of prominent cotton phyotoalexins such as gossypol. In contrast to other
sesquiterpene cyclases, DCS catalyses the reaction with exquisite fidelity producing > 98% of the
final bicyclic hydrocarbon, but as a consequence, the enzyme leaves no mechanistic traces of its
mode of action.
Here we report a detailed investigation of the catalytic mechanism of DCS using a variety of FDP
analogues as mechanistic probes including isotopically labelled FDPs, fluorinated FDPs and C2,C3
double-bond isomers of FDP and 2F-FDP. Analysis of the reaction products arising from the
incubation of these mechanistic probes with DCS gave results that were not consistent with either
proposed catalytic mechanism; rather they suggest a mechanism for the DCS catalyzed
conversion of FDP to δ-cadinene that involves a 1,6-cyclization of FDP to α-bisabolyl cation.
9
Talk 8
The natural product inhibitor myriocin displays a unique dual mode of action against serine
palmitoyltransferase.
Authors: John M. Wadswortha, David J. Clarkea, Stephen A. McMahonb, Ashley E. Beattiea, Pat
Langridge-Smitha, Teresa Dunnc, James H. Naismithb and Dominic J. Campopianoa
Presenting author affiliation: aEaStChem, School of Chemistry, University of Edinburgh, Scotland, EH9 3JJ Other affiliations: aEaStChem, School of Chemistry, University of Edinburgh, Scotland, EH9 3JJ bEaStChem, School of Chemistry, University of St Andrews, Scotland, KY16 9ST cBiochemistry & Molecular Biology, Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland, 20814
Sphingolipids (SLs) and ceramides are essential components of cellular membranes and important signalling
molecules. Because of a growing appreciation of their diverse biological roles, understanding of the
biosynthesis and regulation of SLs has recently become a key goal in drug discovery. Serine
palmitoyltransferase (SPT) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyses the
condensation between L-serine and a long-chain (C16) acyl thioester. This first step in SL biosynthesis is
conserved in all organisms studied to date, from microbes to man. Myriocin, a natural product from the
fungus Mycelia sterilia, is a potent inhibitor of SPT, however, the molecular details of inhibition are not fully
understood. Myriocin contains a long alkyl chain and a polar head group so displays features of both SPT
substrates. Thus, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic
of a key transition state of the catalytic mechanism. We have used a combination of UV-vis spectroscopy,
mass spectrometry, x-ray crystallography and enzyme inhibition assays to study the interaction between S.
paucimobilis SPT and myriocin. We show that myriocin initially forms an inhibitory PLP:myriocin aldimine
complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is succeptable to
unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a
retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This
aldehyde is then capable of covalently modifying the active site lysine, forming a second (suicide) inhibitory
complex and rendering the enzyme catalytically inactive. This novel mechanism has implications on the SAR
and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and
further expands the range of reactions catalysed by this important enzyme.
10
Poster 1
The Development of Biophysical Assays for Protein-Protein Interactions: HIF-1α/p300 and eIF4e/eIF4g
Authors : George Burslem, Hannah Kyle, Thomas Edwards, Adam Nelson, Stuart Warriner, Andrew Wilson
Presenting author affiliation: University of Leeds
Historically medicinal chemistry and drug discovery have targeted enzyme/substrate interactions to meet a
clinical need however protein-protein interactions (PPIs) are implicated in virtually all biological pathways
and are emerging as new targets for medical intervention. Enzyme inhibition assays are often biochemical
in nature and are based on the measurement of either the substrate or product of the enzyme catalysed
process. Often for PPIs no such process exists so only biophysical assays which determine the energy of
interaction (isothermal titration calorimetry) or the proximity of two proteins (fluorescence anisotropy or
surface plasmon resonance) can be used.
Herein we report the development of novel biophysical assays for 2 PPIs of interest to the oncology field,
which could potentially be used to identify inhibitors. We have developed and validated fluorescence
anisotropy assays for the eukaryotic initiation factor (eIF) 4e/eIF4g interaction and the hypoxia inducible
factor (HIF) 1α/p300 interaction as well as developing orthogonal biophysical assays. These assays have also
allowed us to further elucidate the contributions of certain regions of the protein to the interaction affinity,
which could potentially inform the design of inhibitors.
Key references: (1) T.A. Edwards and A.J. Wilson, Amino Acids, 2011, 41, 743 (2) C.J. Brown, C. S. Verma,
M.D. Walkinshaw and D.P. Lane, Cell Cycle, 2009, 8,1905 (3) S.J. Freedman, Z-Y. J. Sun, F. Poy, A.L. Kung,
D.M. Livingston, G. Wagner and M.J. Eck, P. Natl. Sci. USA., 2002, 8, 5367
11
Poster 2
Re-engineering Riboswitches as Novel Gene Expression Tools
Authors: Neil Dixon, Christopher J. Robinson , Phillip Lowe, Ming-Cheng Wu, Helen Vincent, James Leigh
and Jason Micklefield
Presenting author affiliation: The University of Manchester
Other affiliations: BBSRC
The ability to independently control the expression of multiple genes in response to small molecule
inducers or repressors is highly desirable in the study of metabolic pathways; has numerous applications in
the fields of drug discovery and synthetic biology; and has commercial potential in the production of
therapeutic proteins. In light of this, we have developed an entirely novel method of controlling gene
expression by re-engineering naturally occurring RNA regulatory elements known as riboswitches, so that
they no longer respond to intracellular metabolites, but instead can be controlled by synthetic small
molecules which are not naturally present in the cell. Riboswitches are structured regulatory genetic
elements, which specifically bind small molecule metabolites to regulate gene expression through
conformational mechanisms, including transcriptional termination, translational initiation, pre-mRNA
splicing and mRNA self-cleavage.
We chose to study the adenine-responsive riboswitch from the add gene of Vibrio vulnificus, which induces
gene expression upon ligand binding by releasing the ribosome-binding site from a repressor stem. We
selectively mutated residues which contact the adenine ligand, these mutant riboswitches were then
screened for in vivo control of gene expression using a library of over 100 heterocyclic molecules. Mutant
riboswitch-ligand pairs were identified, which were no longer responsive to adenine, but which responded
to specific synthetic small molecules, allowing for highly responsive, dose-dependent and dynamic control
of gene expression in vivo. We have also shown that mutually orthogonal riboswitches can be deployed
within the same bacterial cell to independently control the co-expression of multiple genes in a dose-
dependent fashion, in response to distinct small molecule inducers. Building on these principles, we have
subsequently re-engineered the PreQ1-binding riboswitch from the preC gene of Bacillus subtilis, to
generate a riboswitch which prematurely terminates mRNA transcription upon addition of a novel synthetic
ligand, thereby demonstrating the general applicability of our approach for expanding the repertoire of
novel gene expression tools.
Key references:
1. Dixon N, Duncan J, Geerlings T, Dunstan M, Leys D, Micklefield J. (2010) Re-engineering
orthogonally selective riboswitches. Proceedings of the National Academy of Sciences of the United
States of America. 2010; 107(7): 2830-2835.
2. Dixon N, Robinson C, Geerlings T, Duncan J, Drummond S, Micklefield J. (2012) Orthogonal
riboswitches for tuneable co-expression in bacteria. Angewandte Chemie (International ed. in
English). 2012 April; 51(15): 3620-4.
12
Poster 3
Investigating the Programming of Iterative Polyketide Synthases
Authors: Douglas Roberts, David Ivison, Russell Cox
Presenting author affiliation: University of Bristol
Other affiliations: Leibnez Universität Hannover
Highly-reducing iterative type I polyketide synthases (iPKS) produce a diverse range of chemical structures.
iPKS are able to do this by condensing acetate units together and then further reductive domains are able
to instill additional functionality into the molecule. iPKS differ from other classes of polyketide synthase as
they only have a single copy of each reductive domain. In each round of chain extension these domains
have to be switched on and off, this commonly known as programming. Programming of iPKS is not fully
understood and it remains unclear the role that individual domains have.
Scheme 1: The mechanism of polyketide chain extension and the active domains required for the synthesis of 1
In this project the ER domain of squalestatin tetrakedie synthase (that produces 1) was isolated as a soluble
active protein and was assayed in vitro with synthesized mimics of polyketdide biosyntheis. This
methodology enables a thorough examination of which structural features triggers programming.
Figure 1: Results of in vitro assays of the isolated ER domain of SQTKS with substrate mimics as percentage turnover against time
(mins)
Figure 1 shows results of some of the assays, this shows that the isolated ER domain is unable to turnover
the tetraketide substrate mimic. Assays with further substrate mimics have shown that the programming is
likely to be casused by methylation in the backbone and not by chain-length. It has also been shown by
NMR experiments that the domain is unable to place the correct stereochemistry at the α-position. The
exact reason for this remains unclear but further assays have been designed to test this further.
Key references:
(1) Cox, R.J. Org Biomol Chem, 2007, 5, 2010
13
Poster 4
Approach Towards a Novel Mimetic Heparanase Inhibitor
Authors Garrett T. Pottera, Gavin J. Millera, Gordon C. Jaysonb, John M. Gardinera
Presenting author affiliation: aManchester Institute of Biotechnology - The University of Manchester
Other affiliations: bThe Patterson Institute for Cancer Research
Heparanase (Hpa1) is an enzyme present in nearly all forms of cancer, usually found on the growth front of
tumours.1 It plays a key role in cancerous formation, growth, angiogenesis, metastasis, and resulting
adhesion.2 Hpa1 has a natural affinity for Heparan Sulfate (HS) chains that are ubiquitous to the surface of
cells, where the enzyme docks to and cleaves the chains to release growth factors necessary for tumour
growth. The region of binding requires a specific sulfation and sugar pattern not yet utilised by drug
developers, though other sugar-based heparanase inhibitors are in advanced clinical trials (i.e. PI-88). We
suggest that, by following a more strict interpretation of sulfation and sugar sequencing, by imitating that
which is naturally occuring and favoured by Hpa1, we will increase binding affinity and form a potentially
more effective cancer treatment.
Through the culmination of research done by various groups for over three decades, the trisaccharide core
necessary for recognition and binding by Hpa1 has been elucidated, as has its sulfation pattern.3
Additionally, computer modelling has suggested this core is accurate for enzyme recognition and binding.4
We propose a synthesis of this core with a functional handle attached that could then potentially be
adapted for ADMET purposes and used in chemical biological investigations (Figure 1). This synthesis uses,
in part, selectively protected monomers developed from precursors made within our group.5
Key references:
(1) N. Ilan, M. Elkin, and I. Vlodavsky, Int. J. Biochem. Cell Biol., 2006, 38, 2018–2039.
(2) D. Liu, Z. Shriver, G. Venkataraman, Y. El Shabrawi, and R. Sasisekharan, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 568–573.
(3) D. Liu and R. Sasisekharan in Chemistry and Biology of Heparin and Heparan Sulfate, ed. H. G. Garg, R. J. Linhardt, and Charles A.
Hales, Elsevier, Amsterdam, 2005, pp. 699–725.
(4) N. Sapay, É. Cabannes, M. Petitou, and A. Imberty, Biopolymers, 2012, 97, 21–34.
(5) S. U. Hansen, G. M. Miller, M. Baráth, K. R. Broberg, E. Avizientye, M. Helliwell, J. Raftery, G. C. Jayson, and J. M. Gardiner, J. Org.
Chem., 2012, 77, 7823–7843.
14
Poster 5
The next generation of CtBP inhibitors and their anti-tumour effects
Authors: Charlotte Mardle, Jeremy Blaydes, Ali Tavassoli
Presenting author affiliation: University of Southampton, School of Chemistry, SO17 1BJ
Other affiliations: University of Southampton, Faculty of Medicine
C-terminal binding proteins (CtBP1 and CtBP2) are transcriptional co-repressors with roles in cell-cycle
regulation and development. They have been identified as a potential target for cancer therapeutics as
their repression targets include pro-apoptotic genes and essential adhesion molecules. The repression of
these genes can promote tumour-cell survival, epithelial-to-mesenchymal transition and the migration of
tumour cells1. The function of CtBPs is dependent on its oligomeric state, as it is CtBP homo-and hetero-
dimer complexes that recruit the transcription factors necessary to mediate transcription. CtBPs dimerise
upon binding to NADH. The increase in NADH in tumour cells, caused by hypoxia and/or increased aerobic
glycolysis, means that they are particularly dependent on CtBP dimerisation compared to normal cells.
Inhibiting CtBP dimerisation will therefore directly target the stress-response pathways in cancer cells.
A small-molecule inhibitor of CtBP dimerisation, which works at low concentrations, could be a useful tool
in the investigation of the roles of CtBPs in cancer and potentially used as a basis for the design of a
therapeutic agent targeting CtBPs.
A cyclic peptide inhibitor of CtBP1 homo-dimerisation has been identified via high-throughput-screening of
a 3.2 million compound library generated by split-inteins (SICLOPPS)2 and a bacterial reverse-two hybrid
system (RTHS). A FRET assay has been developed, based on previously reported work3, to test the ability of
the inhibitor to prevent the binding of CtBP1 to NADH. Currently alanine analogues of the peptide inhibitor
are being synthesised to identify the active motif using the FRET assay and further in vitro techniques.
Furthermore screening to identify specific inhibitors to CtBP2 homo-dimerisation is being undertaken using
(2) Tavassoli A, Benkovic SJ. Nat Protoc 2007, 2(5): 1126-1133.
(3) Fjeld CC, Birdsong WT, Goodman RH. Proceedings of the National Academy of Sciences 2003,
100(16): 9202-9207.
15
Poster 6
Regioselective N-7 methylation of purine derivatives
Authors: Honorine Lebraud, Benoit Carbain, Bernard T. Golding, Celine Cano, Ian R. Hardcastle, and Roger J. Griffin.
Presenting author affiliation: Northern Institute for Cancer Research at the Newcastle Cancer Centre, School of
Chemistry, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK
As part of a project directed towards the synthesis of purine-based irreversible inhibitors of Nek2,1 a cell
cycle-associated kinase implicated in cancer, control compounds were required to assess the selectivity of a
lead series of 2-aminoaryl-6-ethynylpurines in cellular assays. In particular, derivatives bearing a methyl
substituent at the purine N-7 position were required, as this modification abrogates Nek2-inhibitory activity
without imposing dramatic structural changes elsewhere. Although a number of literature procedures
describe selective N-7 methylation of purines,2 these were found to be of limited value for purines bearing
a diverse range of substituents at the 2- and 6-positions.
New methodology has been developed for the regioselective methylation of substituted purines, inspired
by the N-7 alkylation of guanine residues in DNA following exposure to alkylating agents.3, 4 In the absence
of repair of these lesions by glycosylases, depurination occurs to generate potentially mutagenic abasic
sites.5 We reasoned that analogous N-7 alkylation of purines bearing a suitable N-9 protecting group, would
facilitate cleavage of the N-9 substituent to give the requisite N-7 substituted purines. To this end, the
introduction of a para-methoxybenzyl (PMB) group at the purine N-9 position was readily achieved
employing PMB-chloride in N,N-dimethylformamide. Methylation at the purine N-7 position with
trimethyloxonium tetrafluoroborate, followed by heating in 2,2,2-trifluoroethanol (TFE) under microwave
conditions in a ‘one-pot’ reaction, furnished the target N-7 methylpurines in excellent yields. This new
method was found to have broad applicability, and was also amenable for the introduction of ethyl
substituents at the purine N-7 position utilising triethyloxonium tetrafluoroborate.
This approach enabled the preparation of a defined series of N7-methylpurine derivatives, varied with
respect to substituents at the 2- and 6-positions, for subsequent biological studies. The synthesis and
biological activity of 6-substituted 2-arylamino-N7-methylpurines will be discussed.
References 1. Fry, A. M., The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 2002, 21 (40), 6184-94. 2. (a) Dalby, C.; Bleasdale, C.; Clegg, W.; Elsegood, M. R. J.; Golding, B. T.; Griffin, R. J., Regiospecific alkylation of 6-chloropurine and 2,6-dichloropurine at N7 by transient protection of N3/N9 by methylcobaloxime. Angew. Chem. Int. Ed. Engl. 1993, 32 (12), 1696-1697; (b) Kotek, V.; Chudikova, N.; Tobrman, T.; Dvorak, D., Selective Synthesis of 7-Substituted Purines via 7,8-Dihydropurines. Org. Lett. 2010, 12 (24), 5724-5727; (c) Kohda, K.; Baba, K.; Kawazoe, Y., Chemical reactivity of alkylguanines. I. Methylation of O6-methylguanine derivatives. Tetrahedron Lett. 1987, 28 (50), 6285-6288. 3. Barrows, L. R.; Magee, P. N., Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro. Carcinogenesis 1982, 3 (3), 349-351. 4. Boysen, G.; Pachkowski, B. F.; Nakamura, J.; Swenberg, J. A., The formation and biological significance of N7-guanine adducts. Mutat. Res. 2009, 678 (2), 76-94. 5. Gates, K. S.; Nooner, T.; Dutta, S., Biologically Relevant Chemical Reactions of N7-Alkylguanine Residues in DNA. Chem. Res.
Toxicol. 2004, 17 (7), 839-856.
16
Poster 7
SYNTHESIS AND APPLICATION OF GLUCURONIDATED
3'-DEOXY-3'-[19F]FLUOROTHYMIDINE ([19F]FLT)
Suzannah J. Harnor, Tommy Rennison, Roger J. Griffin, David R. Newell,
Céline Cano and Bernard T. Golding
Newcastle Cancer Centre, Northern Institute for Cancer Research, School of Chemistry, Bedson Building,
Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
Design and Synthesis of Stapled α-Helix Mimetics: New Synthetic Tools to Investigate the Role of
HDACs in Cancer
Authors: Naomi S. Allaway*, Peter J. Watson, John W.R. Schwabe, Andrew G. Jamieson
Presenting author affiliation: University of Leicester
In recent years, histone deacetylase (HDAC) enzymes have emerged as important oncology drug targets because they have been linked mechanistically to the pathogenesis of cancer and can be regulated by small molecule inhibitors.1 Indeed, a number of HDAC active site inhibitors are currently in clinical trials and two drugs, vorinostat and romidepsin have been approved for the treatment of cutaneous T-cell lymphomas. However, these inhibitors are not isoform selective and so selective HDAC inhibitors are urgently required to investigate HDAC isoform phenotypes and provide targeted therapeutics.
Class 1 HDACs are generally recruited into large multi-subunit co-repressor complexes for maximal activity. However, little attention has been paid to targeting the disruption of these protein-protein interactions (PPIs) and so represents a novel approach to develop more specific therapeutics and chemical probes. Until Schwabe and co-workers solved the structure of HDAC3 bound to the deacetylase activation domain (DAD) from the human SMRT co-repressor (Figure 1),2 little was known about the structure of HDAC co-repressor complexes. Using this structural knowledge, we initiated a study to determine if a synthetic DAD mimetic could be designed to disrupt the HDAC3-DAD PPI and thus HDAC activity by using a stapled α-helix.
In this poster, we will present our most recent results, including the design, synthesis and biological
evaluation of a novel hydrocarbon stapled α-helix proteomimetic.
(1) B. Venugopal, T. R. J. Evans.; Developing histone deacetylase inhibitors as anti-cancer therapeutics.
Curr. Med. Chem. 2011, 18, 1658-1671.
(2) Watson, P. J.; Fairall, L.; Santos, G. M.; Schwabe J. W. R.; Structure of HDAC3 bound to co-repressor
and inositol tetraphosphate. Nature 2012, 481, 335-340.
(3) Blackwell, H.E. & Grubbs, R.H.; Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by
Figure 2. General synthetic scheme of stapled peptide mimetic.3,4
19
Poster 10
Analysis of Exopolysaccharides Produced by Bifidobacteria
Authors: Sohaib Sadiq, Patricia Ruas-Madiedo & Andrew P. Laws
Presenting author affiliation: University of Huddersfield, Queensgate, Huddersfield, West Yorkshire, HD1 3DH
Other affiliations: Spanish Dairy Industry, Instituto de Productos Lacteos de Asturias – Consejo Superior de Investigaciones Cientificas (IPLA – CSIC) Villaviciosa, Asturias, Spain
A number of bifidobacteria are believed to be probiotic organisms which, when consumed, provide a
potential health benefits. It is thought the exopolysaccharides generated by these organisms contribute to
their biological activity. Our work is focused on characterising the exopolysaccharides from Bifidobacteria.
hexose, 1,6-linked hexose, N-acetyl sugars and 1,3,4-linked hexoses.
Key references:
(1) Laurence, D.M., Bronwen G.S., Determination of Uronic Acid content of plant cell walls using colorimetric assay, Current Protocols in Food Analytical Chemistry (2001), E3.3.1-F3.3.4, John Wiley and Sons,Inc.
(2) Wilson, D.J., Gabriel, E, Gee, S., Tracing the source of Campy, PLoS Genetics (2008), vol.4, issue 9, e1000203
20
Poster 11
Biosynthesis of [FeFe]-hydrogenase Cyanide Ligands
Authors: Rebecca C. Driesener, Benjamin R. Duffus, Eric M. Shepard, Joan B. Broderick, John W. Peters,
Peter L. Roach
Presenting author affiliation: University of Southampton, Chemistry, Southampton, SO17 1BJ (UK)
Other affiliations: Montana State University, Bozeman, MT 59717 (US)
The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG catalyses L-tyrosine cleavage to yield p-cresol
and the the cyanide and carbon monoxide ligands for assembly of the [FeFe]-hydrogenase cofactor1, 2
(Figure). The HydG primary sequence contains two highly conserved cysteine motifs. These include the
canonical radical AdoMet CX3CX2C motif which coordinates a [4Fe4S] cluster for cosubstrate AdoMet
binding and the C-terminal CX2CX22C motif, thought to coordinate a second iron sulfur cluster. Mutations
in or absence of this C-terminal motif impair cyanide and carbon monoxide synthesis3.
The objective of this project is to investigate the role of the C-terminal cysteine motif in L-tyrosine cleavage
by spectroscopic and kinetic characterisation of wild-type and mutant HydG proteins. Using a Clostridium
acetobutylicum HydG C96/100/103A mutant unable to coordinate the N-terminal AdoMet cluster, we use
UV-Vis and EPR spectroscopy to unambiguously show that the C-terminal C386/389/412 cysteine motif
coordinates a second [4Fe4S] cluster. Spectroscopic comparison to a C-terminal deletion mutant (ΔCTD)
harbouring only the N-terminal cluster shows that both clusters have similar UV-Vis and EPR spectral
properties, but that AdoMet binding can only occur at the N-terminal cluster. A single C386S mutant can
partially coordinate both [4Fe4S] clusters. We further optimised pre-column derivatisation and HPLC
analyses to determine that the C386S and ΔCTD mutant HydG variants bind AdoMet with equal affinity
while the respective affinity for L-tyrosine is 7 and 45 fold reduced compared to wild-type HydG. Under
optimised in vitro conditions, the ΔCTD mutant was found to be completely impaired in cyanide formation,
while L-tyrosine cleavage was not affected. This strongly suggests that HydG harbours two distinct metallo-
active sites; the N-terminal cluster for tyrosine cleavage to p-cresol and the C-terminal cluster for synthesis
of cyanide and carbon monoxide.
(1) Driesener, R. C.; Challand, M. R.; McGlynn, S. E.; Shepard, E. M.; Boyd, E. S.; Broderick, J. B.; Peters, J. W.; Roach, P.
L. Angew. Chem. Int. Ed. 2010, 49, 1687 (2) Shepard, E. M.; Duffus, B. R.; George, S. J.; McGlynn, S. E.; Challand, M. R.;
Swanson, K. D.; Roach, P. L.; Cramer, S. P.; Peters, J. W.; Broderick, J. B. J. Am. Chem. Soc. 2010, 132, 9247 (3) Nicolet,
Y.; Martin, L.; Tron, C.; Fontecilla-Camps, J. C. FEBS Lett. 2010, 584, 4197
21
Poster 12
Aromatic Halogenases for Biocatalysis
Authors: Sarah Shepherd, Chinnan Velmurugan Karthikeyan, Mark Thompson, Matthew Styles, Anna-
Winona Struck and Jason Micklefield.
Presenting author affiliation: University of Manchester
Halogenated aromatics are key components of pharmaceuticals, polymers, pesticides and many other
products of major industrial importance. Current methods to produce haloaromatics rely on toxic
halogenating agents, deleterious solvents and catalysts. More efficient and sustainable methods of
producing haloaromatics are required that utilise benign inorganic halides, aqueous media and natural
catalysts. These new methods would overcome problems associated with regiocontrol and environmental
issues.
Biohalogenases include heme- and vanadium-dependent haloperoxidases, α-ketoglutarate dependent
halogenases and flavin dependent halogenases. [1] The enzymes that are currently being studied are flavin-
dependent halogenases involved in the formation of Pyrrolnitrin, PrnA [2] as well as PyrH involved in
Pyrroindomycin biosynthesis [3] and a halogenase enzyme found in the Streptomyces toxytricini strain,
STTH.[4]
These enzymes have been used to chlorinate and brominate novel non-indolic substrates and the
regioselectivity of the products has been determined. Mutations have been made to improve activity and
regioselectivity of a less active substrate and work is ongoing to expand the range of substrates accepted
via mutagenesis.
Key references:
1. van Pee, K.H., et al., Biological halogenation has moved far beyond haloperoxidases. Advances in Applied Microbiology, Vol 59, 2006. 59: p. 127-157.
2. Dong, C.J., et al., Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science, 2005. 309(5744): p. 2216-2219.
3. Zehner, S., et al., A regioselective tryptophan 5-halogenase is involved in pyrroindomycin biosynthesis in Streptomyces rugosporus LL-42D005. Chemistry & Biology, 2005. 12(4): p. 445-452.
4. Zeng, J. and J.X. Zhan, Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini. Biotechnology Letters, 2011. 33(8): p. 1607-1613.
22
Poster 13
Modulating protein-protein interactions – EB1 and SxIP proteins
Authors Teresa Almeida, Andrew Carnell, Igor Barsukov, Neil Berry
Presenting author affiliation: University of Liverpool
Protein-protein interaction inhibition is currently one of the major challenges in Drug Discovery.1 The
surface of interaction between two proteins is usually large, flat or moderately convex and cannot be
covered with a small molecule with drug like properties. Unlike other drug targets like enzymes, G-protein-
coupled receptors and ion channels there is no convenient natural substrate to act as a starting point for
small molecule design.2 As such discovery of molecules that modulate protein:protein interactions is a
contemporary challenge.
Microtubules are cellular polymers fundamental to many essential processes in eukaryotic cells including
maintenance of cell structure, accurate chromosome segregation, intracellular transport, and cell division.3
EB family members bind to microtubule growing ends and mediate the binding of other microtubule plus
end tracking proteins (+TIPs). Therefore, EB1 family members are considered as a key regulator of dynamic
+TIPs interaction networks at growing microtubule ends. A 4-residue sequence was shown to be
fundamental for this interaction – Ser, X, Ile, Pro – the SxIP motif.4
Working with the crystal structure of the EB1 – MACF complex, a struture-based molecular design approach
has been adopted to identify molecules that can modulate this interaction. Using a combination of
pharmacophore searching, molecular docking, scoring and ranking a virtual screening process has been
performed to identify candidate compounds.
Top scored/ranked compounds, with suitable drug-like properties, will be synthesised and tested. EB1 was
expressed and purified for binding assays using NMR and ITC techniques. The information gathered from
these assays will then be used to identify and further optimise molecular scaffolds for this target.
References
(1) Koes, D. R.; Camacho, C. J. Bioinformatics 2012, 28, 1951. (2) J. Wilson, A. Chemical Society Reviews 2009, 38, 3289. (3) Kumar, P.; Wittmann, T. Trends in Cell Biology 2012, 22, 418. (4) Kumar, P.; Chimenti, M. S.; Pemble, H.; Schönichen, A.; Thompson, O.; Jacobson, M. P.; Wittmann,
T. Journal of Biological Chemistry 2012, 287, 17050.
23
Poster 14
Characterization of Polysaccharides and Sophorolipids produced by the Yeast
Candida Bombicola
Ammar AL-Jasim
The University of Huddersfield, Department of Chemical and Biological Sciences
The yeast Candida bombicola (formerly Torulopsis bombicola) is a non-pathogenic yeast strain which is able
to produce extracellular glycolipidic biosurfactant known as sophorolipids (Fig.1). These types of glycolipids
are produced as a mixture of acidic and lactonic forms when the yeast grown on mixtures of carbohydrate
and fatty acids. It has been shown that SLs are efficient spermicides, anti-HIV drugs, and effective anti-
cancer agents. In addition to their use as deodorants, anti-dandruff agents and in pharmaceuticals field [1-
2]. Sophorolipids are low molecular weight biosurfactants consisted of a hydrophilic portion, the dimeric
sugar known as sophorose and a hydrophobic portion which is usually long chain fatty acid with a hydroxyl
group on either the penultimate or terminal carbon.
Fig.1: Chemical structures of lactonic and acidic sophorolipids.
The building blocks for sophorolipids synthesis are glucose (hydrophilic substrate) and fatty acid
(hydrophobic) and both can be provided in the production medium during biosynthesis. Those building
blocks can either be directly supplemented in the production medium or can be supplied as a triglyceride or
a fatty acid methyl or ethyl ester which will undergo extracellular hydrolysis by a lipase.
Sophorolipids biosynthesis starts with the hydroxylation of fatty acid after conversion of fatty acid to a
hydroxy-fatty acid, then two UDP- activated glucose molecules are added in a serial way and linked
glycosidically to the fatty acid hydroxyl group.
The aims of this study include the purification and identification of sophorolipids structural forms by TLC
and the use of analytical separation techniques (silica gel column chromatography) to separate the possible
sophorolipidic forms produced by fermentation.
Also to characterise and elucidate the chemical structures of possible sophorolipids using various analytical techniques such as, 1H and 13C NMR which will be utilized for the identification of the molecular structures of sophorolipids: as it’s a powerful method which is able to identify functional groups as well as the position of them within the carbohydrate and lipid molecules. Finally to analyse and identify sophorolipids hydroxyl fatty acid moieties by acid methanolysis reaction
using GC-MS as well as the molecular weight distribution of sophorolipids using HPLC method.
24
Poster 15
Continued development of a fluorescence based assay in the search for novel inhibitors of aromatase
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street, Paisley, Renfrewshire, PA1 2BE.
The cytochrome P-450 enzyme aromatase (AR) catalyses the conversion of androgens and androgen precursors (i.e. testosterone and androstenedione) to their respective estrogens (namely, estradiol and estrone) involving the oxidation of the C(19) methyl moiety of the substrates using three mols of NADPH and oxygen; resulting in the release of the estrogen and water. The traditional method of biochemical evaluation of compounds against AR has involved the use of radiolabelled substrate and which therefore leads to the release of tritiated water. A fluorescence-based assay has been reported previously as a high throughput alternative (using AR supersomes), however, the use this technique has (in our hands) proved to be difficult and troublesome, we therefore considered the modification of the assay so as to improve its efficiency so as to be able to compare against radiolabelled assay using cells as the source of AR as opposed to supersomes. Here, we therefore report the results of our initial study to re-develop the fluorescence assay and its use in the biochemical evaluation of both standard inhibitors (namely anastrozole, letrozole and aminoglutethimide) and novel inhibitors of AR. The non-radiolabelled assay involved the use of JEG-3 cells, 7-methoxy-4-trifluoromethyl coumarin as the substrate (resulting in 7-hyroxy-4-trifluoromethyl coumarin as the fluorescent product), media and methanol as the termination solution. The substrate was therefore incubated with the JEG-3 cells followed by the addition of methanol to terminate the reaction. The solution was then read for fluorescence and the inhibitory activity determined from the intensity of the emission. As previously stated, the assay in our hands proved to be difficult and inefficient, we therefore initially considered the basis of the assay and discovered that the reported wavelength stated proved to be the initial problem. That is, emission at 530nm was reported for the detection of the product, however, when we considered the emission spectra of both the substrate and product, we discovered that both possessed similar emission spectra. We therefore considered the excitation wavelength and the emission wavelength in a number of solvents and discovered that the best results (with the substrate and product differing significantly in their emission spectra) were when the excitation was initiated using 340nm. The development of a cell-based assay therefore involved the incubation of the substrate with JEG-3 cells which showed initial product formation but required extensive optimisation so as to produce an operational assay system. Using the assay, standard and novel inhibitors were evaluated and were found to give similar initial screening trends as the radiolabelled assay system, namely that aminoglutethimide is a weak inhibitor of AR in comparison to the triazole-based compounds currently in the clinic. In conclusion, the cell-based fluorescence assay for AR has significant limitations but has proved to be useful in the determination of inhibitory activity of novel inhibitors of AR.
25
Poster 16
Synthesis and biochemical evaluation of sulfamoylated derivatives of thiosemicarbazone-based
compounds as novel inhibitors of estrone sulfatase (ES)
Authors: Vasireddy, V.R., Rudra, B.C., Owen, C.P., Ahmed, S.
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.
The enzyme estrone sulfatase (ES) catalyses the conversion of estrone sulfate to the bio-active estrogen estrone (E1) in adipose and, in particular, breast cancer tissue; E1 is subsequently converted to estradiol (E2) involving the enzyme 17β-hydroxysteroid dehydrogenase type 1. Furthermore, ES has been shown to become the major source of estrogens in postmenopausal women as a result of the decrease in activity of ovarian aromatase (AR) and the expression of ES within estrogen-dependent breast cancer cells. As such, the inhibition of ES in postmenopausal women would be expected to lead to a decrease in E1 biosynthesis and therefore to a decrease in the stimulation of hormone-dependent breast cancer tissue. This would therefore be expected to lead to a decrease in tumour mass. ES has therefore become a major target in the treatment of estrogen-dependent breast cancer and has resulted in the synthesis of a wide range of steroidal and non-steroidal compounds; the majority of these have contained the sulfamate moiety which has been shown to lead to the irreversible inhibition of ES through the formation of an imine moiety. We have, however, recently reported the modelling of novel thiosemicarbazone-based compounds as potential inhibitors of ES and which have been shown to possess alosteric inhibitory activity. Here, we report the synthesis and biochemical evaluation of a series of novel sulfamoylated derivatives of thiosemicarbazone-based compounds as potential inhibitors of ES which would be expected to possess both irreversible inhibition (via the sulfamate moiety) and uncompetitive inhibition (as a result of the thiosemicarbazone moiety). The synthesis of the target compounds involved the initial synthesis of 4-hydroxyphenone-based compounds using Friedal-Crafts acylation. Following the isolation and purification, the 4-hydroxyphenone-based compounds were then heated to reflux in a solution of thiosemicarbazide in absolute ethanol to give the target compounds. The resulting intermediates were then derivatised to the sulfamate containing compound involving a reaction between aminosulfonyl chloride and the thiosemicarbazone-based intermediate. The biochemical evaluation involved the use of radiolabelled estrone sulfate which was incubated with JEG-3 cells as the source of ES at 37oC to give the radiolabelled estrone which was placed in scintillant and counted for 5min. In general, the synthesis of the target compounds were achieved in moderate to excellent yield and without any major problems, however, as previously reported by us, the sulfamoylated derivatives proved to be chemically unstable when dissolved in solvents, including media, as such, the biochemical assays were undertaken with freshly prepared solutions of the target compounds. The evaluation of the compounds showed them to be weak inhibitors of ES in comparison to the standard compounds, estrone-3O-sulfamate (EMATE) which was found to possess 99% inhibiton at 2.5µM. The most potent compound within the current range was found to possess approximately 85% inhibitory activity (IC50=198.01nM). In conclusion, the compounds have proved to be weak inhibtors of ES but have provided us with a new
library of compounds for further study.
26
Poster 17
Synthesis and biochemical evaluation of potential novel inhibitors of carbonic anhydrase type II (CAII)
Authors: Nazeer, A., Bhamare, B., Rudra, B.C., Singh, S., Owen, C.P., Ahmed, S.
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.
Carbonic anhydrase (CA) family of enzymes have been shown to play a major role in malignant neoplasm development, in particular, they have been shown to be expressed in greater amounts within tumour cells. As a result, it has been proposed that the hypoxic microenvironment which has been shown to develop, as well as the associated increase in acidity of the microenvironment of the tumour. As a result, a number of compounds have been evaluated against the CA family of enzymes. In particular, acetazolamide (1) and sulfamate-based compounds, including estrone-3-O-sulfamate (EMATE), 667-COUMATE and COUMATE have all been evaluated and shown to be potent inhibitors of CAII. Here, we report the results of our initial study into the synthesis and evaluation of a series of a novel range of non-sulfamoylated compounds which we propose undergo chelation to the Zn2+ ion found within the active site of CAII, we therefore report the synthesis and biochemical evaluation of a range of thiosemicarbazone-based inhibitors of CA II. In the synthesis of the potential inhibitors of CAII, carbonyl containing starting material was heated to reflux with derivatives of thiosemicarbazide to give the target compounds; the products were found to crystallise out of the reaction solvent and were subsequently recrystallised from aqueous ethanol to give the target compounds. Biochemical evaluation involved a colourimetric assay where 4-nitrophenyl acetate (as the un-natural substrate) was incubated together with the inhibitor and enzyme (human CA II) in Tris-HCl buffer. From the biochemcial evaluation of the synthesised compounds, we observe that the potential inhibitors are weak inhibitors of CA II in comparison to the standard compounds; EMATE and 667-COUMATE were found to possess IC50 values of 9.36nM and 14.86nM respectively. Within the range of compounds synthesised the most potent inhibitors were found to possess IC50 values of 4.40µM and 5.33µM. The inhibitory activity observed within these compounds appear to support our initial hypothesis that the thiosemicarbazone-based compounds are indeed inhibitors of CAII. In conclusion, we have provided a novel series of inhibitors of CA II and therefore offer a new library of compounds in the treatment of a number of diseases including cancer.
27
Poster 18
Sulfamate derivatives of 4-hydroxycoumaric acid-based compounds as inhibitors of carbonic anhydrase II
and estrone sulfatase (ES)
Authors: Bhamare, B., Rudra, B.C., Owen, C.P., Ahmed, S.
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.
Breast cancer cells have been shown to produce estrogens via the action of estrone sulfatase (ES) which
catalyses the conversion of estrone sulfate to estrone, as such, the ES pathway is a source of estrone within
postmenopausal women. Hormone-dependent breast cancer cells continue to be stimulated by the
continued production of estrone and in particular, estradiol (the most potent estrogen). Studies on tumour
cells have also shown an increased expression of carbonic anhydrase (CA) family of enzymes and which has
been proposed to play an important role in tumour cell survival. Both ES and CA family of enzymes would
appear to therefore possess an important role in tumour cell progression. A number of compounds,
including acetazolamide and estrone-3-O-sulfamate (EMATE) have been used to target both the CA and ES
family of enzymes. In our search for potent dual-inhibitors of both CA and ES, we report the use of the
EMATE backbone in the design, synthesis and biochemical evaluation of a series of sulfamoylated
derivatives of a range of 4-hydroxycoumaric acid-based compounds.
Synthesis of the target compound involved the initial synthesis of the 4-hydroxycoumaric acid amide
followed by the conversion of the 4-hydroxy moiety to the sulfamate derivative. Biochemical evaluation of
the target compounds against CA II involved inhibitors dissolved in dimethylsulfoxide (DMSO) (final
[I]=100µM). The assay was carried out (in triplicate) using prepared substrate (4-nitrophenyl acetate),
inhibitor, Tris-HCl; the mixture was incubated at 25°C. The intensity of the colour was then read using
microplate reader at 405nm. The ES assay involved the use of radiolabelled estrone sulfate which was
incubated with JEG-3 cells as the source of ES at 37oC to give the radiolabelled estrone which was placed in
scintillant and counted for 5min.
The results of the compounds considered within the current study show that these compounds are weak
inhibitors of both ES and are good inhibitors of CA II; e.g. the most potent target compound was found to
possess an IC50 value of 6.86nM against CA II but only possessed ~33% inhibitory activity against ES
([I]=2.5µM). Under similar assay conditions, EMATE was found to possess 99% inhibitory activity against ES
and possessed 14.5nM against CAII. As such, the investigation has resulted in the discovery of excellent
lead compounds in the search for more potent and specific inhibitors of CA II.
28
Poster 19
Synthesis and biochemical evaluation of novel potent inhibitors of estrogen synthesis
Authors: Shah, P.S., Nazeer, A., Owen, C.P., Ahmed, S.
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.
The cytochrome P-450 enzyme aromatase (AR) catalyses the conversion of androstenedione to estrone. A number of triazole-based inhibitors have been introduced for use in the clinic, i.e. anastrozole and letrozole, however, there appears to be a lack of structure-activity relationship having been reported for these compounds, as such, the current study considers a series of compounds which use the phenylmethyl imidazole backbone with the phenyl moiety having been substituted with various functional groups so as to provide polar-polar interaction with the active site of AR (in addition to the Fe-azole dative covalent bond). We therefore report the initial results of our efforts to synthesise and evaluate a number of derivatives of phenylmethyl imidazole (1) against AR and their specificity having been determined through against the enzyme 17α-hydroxylase/17,20-lyase (P-45017α). The synthesis of the target compounds involved a reaction between the appropriate phenylmethyl bromide and imidazole using anhydrous potassium carbonate as base in anhydrous tetrahydrofuran (THF). The biochemical evaluation involved the modification of a literature-based AR assay (using 3H-androstenedione) [1], however, JEG3 cells were used as the source of AR; an organic phase (chloroform) was also used to partition out all organic components from the aqueous phase (which therefore contained the 3H2O produced) and counted for 3H. The compounds were also evaluated against P-45017α using 3H-progesterone using rat testicular microsome as previously reported by Owen et al [2]. The reactions proceeded in good yield and without any major problem. The biochemical evaluation of the synthesised compounds shows that a number of the derivatives of 1 proved to be highly potent inhibitors of AR. Anastrozole was found to possess 92% inhibition against AR resulting in an IC50 value of 21.54nM. The most potent compound within the current study was found to be 4-nitrobenzylimidazole which was found to possess 92% inhibitory activity and therefore was found to be equipotent to anastrozole. With regard to the inhibitory activity observed against P-45017α, the compounds were also found to be highly potent inhibitors of the lyase (in comparison to the hydroxylase) component. In conclusion, these compounds have proved to be potent inhibitors of AR, however, they are not suitable for further development as they lack specificity as demonstrated by the inhibitory activity against P-45017α. Key references:
(1) E. A. Thompson and P. K., Siterii, “Utilization of oxygen and reduced nicotinamide adenine dinucleotide
phosphate by human placental microsomes during aromatization of androstenedione” J. Biol. Chem., 1974,
249, 5373-5378.
(2) C.P. Owen, S. Dhanani, C.H. Patel, I. Shahid, S. Ahmed, “Synthesis and biochemical evaluation of a range
of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17α-
Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of
Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.
Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD3) enzyme has been shown to be responsible for the conversion of the C(17)=O moiety within androgens (e.g. androstenedione) to the C(17)-βOH (resulting in the biosynthesis of testosterone, the more potent androgen). In the design of novel inhibitors of 17β-HSD3, we argued that mimicking of the steroid structure may allow potential compounds to possess inhibitory activity. We therefore undertook the design, synthesis, biochemical evaluation and rationalisation of the observed inhibitory activity of a series of compounds based upon the backbone of progesterone, in particular, of progesterone. The target compounds and potential inhibitors were synthesised involving the initial oxidative cleavage of the A-ring of progesterone [it should be noted that the C(20) carbonyl moiety was required to be protected through the formation of the ketal]. The resulting progesterone ‘keto-acid’ was subsequently derivatised to the ester functionality involving the reaction (in the absence of an acid catalyst) with a range of alcohols so as to give the appropriate ester. The biochemical evaluation of the synthesised compounds was undertaken using literature procedure using microsomes from rat testes. The assay was quenched using diethyl ether and the substrate and products were separated using thin layer chromatography, each spot cut out and counted for tritium for 4min. The reactions proceeded in good yield and without any major problems. Consideration of the initial screening data shows that the compounds are, in general, weak inhibitors of 17β-HSD3. The most potent was the butyl derivative which was found to possess ~57% inhibitory activity at [I]=100µM, whilst the weakest inhibitory activity was observed with the octyl derivative which was found to possess ~16% inhibitory activity under similar conditions. In comparison, the two standard inhibitors used (namely 4-hydroxynonanophenone and 4-hydroxydecanophenone which have been previously reported by us) were found to possess ~71% and ~62% inhibitory activity under similar conditions. Using the previously derived transition-state for 17β-HSD3, we propose that the weak inhibitory activity observed within the compounds (containing large alkyl chains) is due to steric hindrance between the alkyl chain within the inhibitor and the active site; resulting in a decrease in inhibitory activity. The results of our study show that in the inhibition of 17β-HSD3 and the mimicking of the steroid backbone, in particular, the C(17)=O group within the inhibitors is a factor in the inhibition process. We have therefore provided a novel range of compounds which may be suitable for further development.
32
Poster 23
Anthracene tagged biomolecules for sensing
Authors: G A Bullen, A F A Peacock and J H R Tucker
Presenting author affiliation: University of Birmingham
Anthracene is a well studied aromatic compound with
interesting fluorescence and photodimerisation
properties. Within this study both of these aspects are
explored with respect to potential bio-sensing
applications. Firstly, anthracene has been introduced
into an oligonucleotide sequence to allow for the
identification of the nucleobase located opposite on the
complimentary strand of DNA, by monitoring the
change in fluorescence intensity upon duplex formation. This has been used to identify the nucleobase
present at a single nucleotide polymorphism (SNP) site thought to be associated with Alzheimer’s disease.
Various anthracene probes have been evaluated and initial trials have identified a probe that can allow for
the quantitative determination of the amount of SNP nucleobase and wildtype nucleobase present.
Recently we have begun using this probe to analyse real biological samples from Alzheimer patients. 1-2
A second project involves coupling anthracene to a small biological building block
and exploiting the photodimerisation properties of the anthracene. An anthracene
unit has been attached to the basic region of GCN4, the DNA binding domain of a
well-studied bacterial transcription factor. When the anthracene tagged peptide is
exposed to UV light it is found that no photodimerisation occurs. However, upon
templating to target DNA photodimerisation ought to occur resulting in a loss of
the fluorescence of the anthracene and a decrease in the absorbance of light.
When DNA is present which does not contain the target sequence, dimerisation is
no longer expected to occur allowing for the sensing of target DNA within a
sample. Furthermore the DNA binding domain of GCN4 has a weak affinity for
target DNA as a monomer, however, dimerisation results in significantly enhanced
DNA binding. Therefore the same anthracene-GCN4 adduct can be used as a light triggered DNA binding
agent.
Key references:
(1) J.-L. H. A. Duprey, Z.-y. Zhao, D. M. Bassani, J. Manchester, J. S. Vyle and J. H. R. Tucker, Chem. Commun.,
2011, 47, 6629-6631.
(2) Z.-y. Zhao, M. San, J.-L. H. A. Duprey, J. R. Arrand, J. S. Vyle and J. H. R. Tucker, Bioorganic Medicinal
Chemistry Letters, 2012, 22, 129-132.
A
C
33
Poster 24
Understanding Oligonucleotide Synthesis Authors: James L. Scotson, Andrew P. Laws, Michael I. Page, John H. Atherton and Ben Atherton
Presenting author affiliation: The University of Huddersfield
Oligonucleotides have many uses including DNA sequencing, artificial gene synthesis and as trheraputic
agents. A recent surge in the number of oligonucleotide therapies in stage 2 and 3 clinical trials has lead to
a hugely increased amount of research in the area. Currently, the most common method of oligonucleotide
synthesis is via the ‘phosphoramidite’ method which uses a solid support accross which reagents are
passed sequentially and upon which the oligonucleotide chain grows as detailed in the scheme below.
Synthesis of a phosphorthioate modified DNA oligonucleotide.
Though this method is widely and successfully used to create tailor made oligocnucleotides little is known
about the mechanisms and kinetics of the individual steps in the reactions and what mechanistic studies
there are are in very early stages. Many of the conditions used to generate oligonucleotides have only ever
been implemented on a gram scale in which large excesses of reagents are used to force equilibria and
increase reaction yeilds. This method is not viable if these therapies are to be used on a large scale both in
terms of cost of production and in terms of the economy of the reactions.
It is the focus of this work to take a physical-organic approach to improving oligonucleotide synthesis. The
project will study each reaction individually to elucidate exact mechanisms of reactions to reduce waste
and cost. This work will also aim to offer alternative routes to oligonucleotide synthesis, i.e. novel and more
efficient activators and sulfurising agents.
Key references: 1. M. A. Russell, A. P. Laws, J. H. Atherton and M. I. Page, Org. Biomol. Chem., 2008, 6,
3270-3275
2. J. Hanusek, M. A. Russell, A. P. Laws, P. Jansa, J. H. Atherton, K. Fettes and M. I. Page, Org. Biomol. Chem.,
2007, 5, 478-484
3. M. A. Russell, A. P. Laws, J. H. Atherton and M. I. Page, Org. Biomol. Chem., 2009, 7, 52-57
34
Poster 25
Biotechnological applications for phenylpropanoids derived from biorefining
Authors: Prof Robert Edwards, Keir Bailey
Presenting author affiliation: The University of York
Other affiliations: BBSRC IBTI Club
Biorefining involves "refining" multiple useful
products from biomass. The biorefining of plant
fibres produces by-products, such as
phenylpropanoids. These phenylpropanoid by-products
can be used to produce more useful, high-value
compounds such as curcuminoids.
Curcuminoids are diarylhepatanoids that give turmeric,
the popular curry spice, its distinctive yellow colour. Research has shown these
molecules to have anti-tumour, anti-cancer, anti-oxidant and anti-inflammatory effects as
well as providing neuroprotection.1 Therefore, the ability to sustainably produce these
compounds in high yields is very lucrative.
This project aims to produce a variety of curcuminoids and curcuminoid-like analogues using plant
polyketide synthase (PKS) enzymes and Saccharomyces cerevisiae, with the hope that curcuminoids can
eventually be produced on a large scale by microbial fermentation.2 This involves cloning the two PKS
enzymes, diketide CoA synthase (DCS) and curcumin synthase 1 (CURS1) which produce curcuminoids in
the turmeric plant (Curcuma longa) and expressing them in yeast.3 The engineered yeast will be fed using
various phenylpropanoids and the yield of different curcuminoids will be monitored. The focus will then be
to optimise this metabolic pathway and investigate ways of elaborating the chemical structure of
curcuminoids by adding new enzymes to the system such as glucosyltransferases.
Key references:
(1) B. Aggarwal, C. Sundaram, N. Malani and H. Ichikawa, Adv. Exp. Med. Biol., 2007, 595, 818.
(2) K. Hong and J. Nielsen, Cell Mol. Life Sci., 2012, 69, 2671.
(3) Y. Katsuyama, T. Kita, N. Funa, and S. Horinouchi, J. Biol. Chem, 2009, 284, 11160.
(3) Adams, S. E.; Parr, C.; Miller, D. J.; Allemann, R. K.; Hallett, M. B. MedChemComm, 2012, 3, 566-570.
36
Poster 27
Regiospecific isotopic labelling via de novo NADPH biosynthesis
Authors: William M. Dawson, Louis Y.P. Luk, E. Joel Loveridge, Rudolf K. Allemann
Presenting author affiliation: School of Chemistry, Cardiff University
Dihydrofolate reductase (DHFR) is a popular therapeutic target against cancer, malaria and bacterial
infections, as it catalyses the NADPH-dependent reduction of dihydrofolate in nucleic acid and amino acid
biosynthesis. Despite being a well-studied enzyme, knowledge of the transition state is still lacking, and this
has become particulary relevant due to the recent debate surrounding the role of enzyme dynamics on the
reaction coordinate. Measurement of kinetic isotope effects (KIEs) using isotopically labelled substrates
presents an incredibly effective method to characterise the transition state at an atomic-scale resolution.
However, incorporation of isotopic labels into NADPH still remains a challenging task that requires a long
synthetic route with low yields.1 To incorporate isotopic labels into regiospecific positions of the
nicotinamide ring, we have reconstructed the de novo biosynthetic pathway of NADPH in our laboratory.
This work has resulted in the biosynthesis and isolation of key intermediates in the pathway, starting from
simple starting materials.
Key references:
(1) Oberfrank et al.,1984, Eur J Biochem, 140, 157
37
Poster 28
Chemoenzymatic approaches to bio-active monoterpenoids
Authors: Zulfa Yoosuf-Aly, David J. Miller, Juan A. Faraldos and Rudolf K. Allemann
Presenting author affiliation: Cardiff University
Terpenoids are the largest and structurally most diverse family of natural products,1 many of which are a source of commercially valuable fragrances, flavours, drugs, agrochemicals and synthetic building blocks.2
-Pinene (2), a monoterpene found in turpentine, is a precursor to several high-value mono-oxygenated terpenoids such as verbenone (3).3 It is produced naturally through the Mg2+ -dependent transformation of geranyl diphosphate (GDP, 1 -pinene synthase (APS).4 Bark beetle infestations pose a major threat to conifer populations worldwide with millions of acres of pine trees killed by beetles such as Dendroctonus ponderosae.5 The male beetles use (+)--pinene produced by the pines for host selection and then convert it to verbenol, which acts as an attractant for female beetles. (+)-Verbenone (3) is then produced as a dispersal pheromone by the beetles in order to modulate the attack density. In a scheme to limit tree damage from beetle infestation, foresters hang small bags of synthetic verbenone on trees.5
Here a chemoenzymatic synthesis is described that generates (+)-verbenone from GDP in good yield and without the need to isolate the intermediate (+)--pinene.6
OP2O63-
Mg2+
GDP (+)-a-pinene
O
Cr(CO)6
ButOOH
C5H12/
CH3CN2 3(+)-verbenone
1
APS
This approach has been applied to the synthesis of verbenone analogues from synthetic GDP derivatives. These ‘unnatural’ verbenones may display enhanced biological activities.
Key references:
1. J. Degenhardt, T. G. Kollner and J. Gershenzon, Phytochem., 2009, 70, 1621-1637. 2. E. M. Davis, in Comprehensive Natural Products II, Elsevier, 2010, pp. 585-608. 3. S. G. Bell, R. J. Sowden and L. L. Wong, J. Chem. Soc. Chem. Commun., 2001, 635-636. 4. M. A. Phillips, M. R. Wildung, D. C. Williams, D. C. Hyatt and R. Croteau, Arch. Biochem. Biophys., 2003, 411, 267-
276. 5. R. Petkewich, Chem. Eng. News, 2008, 86, 36-37. 6. Z. Yoosuf Aly, J. A. Faraldos, D. J. Miller and R.K. Allemann, Chem. Commun., 2012, 48, 7040-7042.
38
Poster 29
Why isn’t P(III) pyrophosphate an inhibitor or a good substrate for P(V) pyrophosphatase?
Authors: Dharmit Mistry, Professor M.I. Page and Professor D.R. Brown
Presenting author affiliation: Innovative Physical Organic Solutions (IPOS), University of Huddersfield.
The reactions and stabilities of phosphate mono- and di-esters underpin the entire processes of life:- the storage and
manifestation of genetic information, energy transduction, signalling, regulation, differentiation,
compartmentalisation (ionic phosphate esters unable to cross membranes), substrate modification to facilitate
chemical reactions and structural components.
The amazing paradox of the previous paragraph is the remarkable variation from extreme stability – half-lives of
millions of years for some spontaneous hydrolyses, to millisecond turnovers for some enzyme-catalysed reactions.
Phosphate mono- and di-esters show remarkable resistance to spontaneous hydrolysis under normal physiological
conditions, hence their contribution to the stability of genes1. By contrast enzyme-catalysed phosphoryl groups
transfer reactions are highly efficient and show some of the largest enzymatic rate enhancement – up to 1020
. The key
to understanding enzyme catalysis is the charge and geometric complementarity between substrate and enzyme
expressed in the transition state. The processes of phosphorylation and dephosphorylation that occur by associative
type mechanisms usually occur via a transition state with a trigonal bipyramidal geometry, and in the case of
phosphoryl transfer effectively involve a trigonal planar metaphosphate anionic species, PO3-. The important feature
of mono- and di- phosphate esters is their invariable negative charge, so a major feature of enzymes catalysing their
reactions is the neutralisation of this charge in the transition state2.
We have been interested in H-phosphonates as phosphorylating agents and have been studying P(III)
pyrophosphonates. Compounds containing ionisable groups will associate/dissociate dependent on the pH of their
surroundings, inevitably causing the compound to change its characteristics. This will in turn affect the rates of any
reaction in which the substrate is involved. P(V) pyrophosphates contain four ionisable protons whereas the P(III)
pyro-di-H-phosphonate only contains two. We have studied the hydrolysis of these two compounds with the use of 31
P
NMR, a pH rate profile has been constructed for the P(III) compound enabling comparison with the P(V)
pyrophosphate. There is a large difference between the two compounds, the hydrolysis rate of the P(V)
pyrophosphate decreases with increasing pH- with increasing ionisation decreasing the rate. However, the P(III) pyro-
di-H-phosphonate shows a characteristic U-shaped pH rate profile with a reaction between the di-anion and
hydroxide.
Most chemical reactions or physical changes are accompanied by a change in heat or enthalpy. Isothermal Titration
Calorimetry (ITC) has been used as a novel method to measure the rates of pyrophosphate hydrolysis with the enzyme
pyrophosphatase (Ppase), which cannot be measured by 31
P NMR. Molar enthalpies of hydrolysis for P(V)
pyrophosphate and P(III) pyro-di-H-phosphonate have been obtained.
The enzyme Ppase uses Mg2+
as a cofactor for hydrolysing pyrophosphate. ITC and NMR have shown conclusively that
pyrophosphate binds in a 1:1 ratio with Mg2+
whereas pyro-di-H-phosphonate exhibits no detectable binding.
Key references:
(1) N. Powles, J. Atherton and M. I. Page, Organic & Biomolecular Chemistry, 2012, 10, 5940-5947.
(2) L. Yang, R.-Z. Liao, J.-G. Yu and R.-Z. Liu, The Journal of Physical Chemistry B, 2009, 113, 6505-6510.
39
Poster 30
Synthesis and Biochemical Evaluation of Molecular Probes for Label-Free Detection of Thrombin
Activity.
Authors: James Murray, Dominika Nowak, Steven Johnson and Robin S Bon.
Presenting author affiliation: School of Chemistry, University of Leeds, Woodhouse Lane, LS2 9JT
Other affiliations: Astbury Centre for Structural and Molecular Biology, University of Leeds, LS2 9JT.
Activity-based protein profiling is a powerful technique for studying enzymatic activities and
annotating their roles in physiological and pathological processes1.. What has yet to be realised, is the
use of activity-based probes to generate protein activity fingerprints of enzymes involved in disease
and incorporation of these probes into point-of-care, biomedical diagnostic devices. We envision that
by assembling activity-based probes, on a gold electrode, label-free detection of enzyme activity and a
route to such diagnostic devices may be achieved.
We shall report the design and synthesis of functionalised self-assembled monolayer (SAM) - forming
alkanethiol-PEG molecules. By changing the terminal group of these molecules, a wide range of
ligation strategies are made available: molecules terminated with amines, azides and alkynes have
been demonstrated for the immobilisation of small molecules and peptides- including, peptidic
thrombin substrates. The kinetic parameters of these peptides have been determined in solution by
the incorporation of a fluorogenic moiety. Surface-phase characterisation of functionalised SAMs has
been realised using surface-plasmon resonance (SPR), cyclic voltammetry (CV) and electrochemical
impedance spectroscopy (EIS). EIS and CV studies showed that our molecules form well-ordered and
stable SAMs. Finally, we shall discuss preliminary results of the use of functionalised SAMs for the
detection of thrombin activity.
(1) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W., Annu. Rev. Biochem., 2008, 77, 383–414.
40
Poster 31
An integrated computational and experimental approach to identify novel ligands that target BACE-1
Authors: Giorgia Magnatti, Colin Fishwick, Nigel Hooper, Adam Nelson
Presenting author affiliation: University of Leeds, School of Chemistry
Other affiliations: Astbury Centre for Structural Molecular Biology, University of Leeds
Introduction: There are numerous complementary approaches to facilitate the identification of novel
ligands for biological targets, including high throughput screening and fragment-based drug discovery.
Computational tools are also employed to predict binding pose and affinity of the new ligands (1). In this
poster an integrated computational approach to identify novel ligands is presented. The approach involves
the design of a virtual library of synthetically accessible lead-like molecules, followed by virtual high
throughput screening (vHTS) against a specific biological target.
Discussion: In our group, diversity-oriented synthesis (DOS) has been employed to explore chemical space
(2) and more recently was focused to explore the chemical space of lead-like molecules. BACE-1 protein is a
well known biological target of pharmaceutical interest involved in Alzheimer’s disease, whose structure is
accessible from the protein data bank. Our in silico approach to identifying novel BACE-1 ligands is
schematically represented as follows.
N
H2N
SO
O
virtual library of synthetically accessible lead-like molecules BACE-1
predictedLE > 0.28
vHTS
prioritised ligandsfor synthesis
N
H2N
ON
N
N
NHN
O
NH2
N
O
N
NH
O
N
F
FF
O
NOH
A virtual library of lead-like molecules was generated based on DOS methods established in our laboratory.
The library was virtually screened against BACE-1 by using eHiTS (3) and two families of potential ligands
were identified with high predicted ligand efficiency (LE). The synthesis of focused libraries based on these
scaffolds is underway.
Future work: An established assay will be employed to evaluate the biological activity of the ligands. The
assay will allow to verify the validity of the integrated approach in identifying new ligands for biological
targets.
Key references: (1) N. Y. Mok, N. M. Hooper, A. P. Johnson, C. W. G. Fishwick et al., J. Med. Chem. 2013, 56, 1843-1852. (2) C. Cordier, A. Nelson et al., Angew. Chemie. Int. Ed. 2009, 48, 104-109, P. MacLellan and A. Nelson, Chem. Commun. 2013, in press. (3) Z. Zsoldos, A. P. Johnson et al., Curr. Protein and Pept. Sc. 2006, 7, 421-435.