Key Research Interests and Expertise

Our research programs embrace a diverse range of disciplines such as Synthetic Chemistry, Biotransformations, Enzymology, Biochemistry and Molecular/Microbiology. This facilitates the design, synthesis and application of novel chemical tools to address complex biological/medicinal challenges. Traversing these disciplinary boundaries provides a stimulating interdisciplinary research culture, and excellent research training/experience, across the full spectrum of the chemical/life sciences interface from the extremes of Synthetic Organic Chemistry to Cell Biology and beyond.

Bacillithiol: A unique low molecular weight thiol cofactor amongst many low G+C Gram positive bacteria
Glutathione (GSH) is the major low molecular weight (LMW) thiol in eukaryotes and many gram negative bacteria. GSH plays a critical role in disulfide stress management and maintaining an intracellular reducing environment via the chemical and/or enzymatic reduction of toxic oxidants. Protein glutathionylation (reversible formation of GS-S-protein disulfides) is also an important post-translational modification for regulating protein function and protecting exposed cysteine (Cys) residues from irreversible damage during oxidative stress.[1] Glutathione-S-transferases mediate xenobiotic metabolism/detoxification via S-conjugation with GSH. Gram positive bacteria lack GSH, but instead produce other LMW thiols (eg. mycothiol in Actinomycetes), which have functions analogous to GSH.[2] Recently, as part of an international and interdisciplinary research collaboration we helped characterise Bacillithiol (BSH) as the major, cysteine-containing, low molecular weight thiol amongst many low G+C Gram positive bacteria, which do not produce MSH or GSH.[3] These include Bacillus anthracis (anthrax), B. cereus (food poisoning), Staphylococcus aureus (bacterial sepsis), B. subtilis (soil bacterium and genetic model for the Bacilli) and Deinococcus radiodurans (a polyextremophile and the world’s toughest bacterium). The major focus of our recent[4][5] and ongoing research efforts is to elucidate the fundamental aspects of BSH biosynthesis and its roles in redox regulation, xenobiotic detoxification and metal ion homeostasis. Many of these projects also involve extensive collaborations with research groups with complementary expertise in microbiology, structural biology and proteomics. We have recently completed total syntheses of BSH, its symmetrical disulfide (BSSB) and all of its biosynthetic precursors.[4] Synthetic BSH has already been used to establish that the fosfomycin resistance protein (FosB)[5] found in low G+C gram positive bacteria is a bacillithiol-S-transferase for which bacillithiol is a preferred thiol substrate compared to cysteine or GSH.[4]

References: [1] Free Rad. Biol. Med. 2007, 43, 883; [2] Nat. Prod. Rep. 2008, 25, 1091; [3] Nat. Chem. Biol. 2009, 625-627; [4] Biochemistry, 2010, 49, 8398-8414; [4] Angew. Chemie Int. Ed. 2011, 50, 7101-7104. [5] J. Bacteriol. 2001, 183, 2380-2383. BSH is now commercially available from JEMA Biosciences http://jemabiosciences.com.

Garlic-derived diallyl-polysulfides: Mechanistic studies and their application as antifungal, antimicrobial and nematocidal agents
There is considerable interest in the development of new pesticides based on organosulfur metabolites from garlic, since such food-based products are environmentally benign. Garlic (Allium sativum) contains a wide range of such organosulfur metabolites with distinct biological activities. When garlic is crushed, the enzyme alliinase converts alliin into allicin, a highly reactive thiosulfoxide. Several bioactive degradation products are subsequently formed such as diallyl polysulfides (DAPS). Little is known about the mode(s) of action of these compounds. We are investigating how their bioactivity can be triggered and/or neutralised by low molecular weight thiols within prokaryotic/eukaryotic cells, how this impacts on the intracellular redox status and how this relates to their observed nematicidal, antifungal and antibacterial activities. A more detailed knowledge of the intracellular redox reactions induced by different DAPS will help to distinguish between the mode of action of different DAPS and can result in an improved use of the compounds in agriculture and medicine.

This project is supported and being pursued in close collaboration with ECOspray Ltd. (http://www.ecospray.com) who have developed and registered several, environmentally benign, agricultural/horticultural/amenity pesticides formulated from garlic extracts.

References: [1] Org. Biomol. Chem. 2007, 1505. [2] E. Bloc. Garlic and other alliums, The lore and the science, Royal Society of Chemistry, Cambridge, ISBN: 978-85404-190-9.

Mycothiol Disulfide Reductase (Mtr)
Mycothiol (MSH) is the major small molecule thiol found in M. tuberculosis which plays a key role in oxidative stress management.[1] The NADPH-dependent oxidoreductase mycothiol disulfide reductase (mtr) is responsible for recycling the disulfide (MSSM) back to MSH and thereby maintaining an intracellular reducing environment.[2] Further studies into the biological significance of the mycothiol redox cycle are currently hampered by the limited availability of mycothiol. We have recently developed an efficient synthesis of simplified mycothiol disulfide analogues that can be used as alternative substrate substrates for mtr[3] that are suitable for substrate-economic mycothiol disulfide reductase assays.[4] These assays are now being used to investigate the biological mechanisms of anti-tubercular natural products isolated from native flora in South Africa and Tanzania. References: [1] Nat. Prod. Rep. 2008, 25, 1091-1117; [2] Biochemistry, 2001, 40, 5119-5126; [3] Org. Biomol. Chem. 2008, 6, 385-390; [4] Analytical Biochem. 2009, 388, 91-96
Kinetic/Inhibition studies of B. anthracis pantothenate kinase

CoenzymeA (CoASH) is an essential biological cofactor (eg. as the major acyl-group carrier) whose biosynthetic pathway is also highly conserved across all organisms. The first obligate step in CoASH biosynthesis is the phosphorylation of pantothenate (Pan) by pantothenate kinase (PanK). To date, three bacterial PanK isoforms (PanKs-I-III) have been classified based on primary sequence and substrate/inhibitor specificities. Sequence similarity amongst PanKs I–III is N-5-pantothenamide 1 as an alternative substrate, but only type-I PanKs are susceptible to feedback inhibition by CoASH.1 The type-III enzymes cannot phosphorylate 1, nor are they inhibited by CoASH.2 The pantothenate binding pocket of type I-II PanKs includes a hydrophobic cavity that enables them to accommodate the extended alkyl chain of 1 and to use it as an alternative substrate. Type-III PanKs lack this hydrophobic pocket hence they cannot bind and phosphorylate 1. Structures of type-II S. aureus kinase (saPanK)3 and type-III kinase utilised by Bacillus anthracis kinase (baPanK)4have been determined and pantothenate mimetics 1-5 have also been reported as inhibitors/substrates of saPanK.5    

We are specifically interested in the B. anthracis type-III panK enzyme and are currently studying the substrate/inhibitor properties of known pantothenate mimetics (1-5) and novel analogues (prepared in-house). In Collaboration with Prof. Al Claiborne (Wake Forest University, N. Carolina) and Asinex (Moscow)  

References [1] Strauss et al, J. Biol. Chem. 2002, 277, 48205; [2] Brand et al, J. Biol. Chem. 2005, 280, 20185; [3] Hong et al, Structure, 2006, 14, 1251; [4] Nicely et al Biochemistry, 2007, 46, 3234. [5] Virga et al, Bioorg. Med. Chem., 2006, 14, 1007

Research Funding

Research Enterprise & Engagement UEA Proof of concept fund
Scale-up preparation and distribution of Bacillithiol
C. Hamilton
May-11 to Jul-11
£9595
Marie Curie ITN & ECOspray Ltd

Antifungal, antimicrobial and nematocidal studies of garlic-derived diallyl-polysulfides
C. Hamilton
Oct-10 to Sep-13
£25,395

Research Enterprise & Engagement UEA Proof of concept fund
Synthesis of expensive fine chemicals with therapeutic potential.
C. Hamilton, C. Brearley
Aug-10 to Jul-11
£3960

Nuffield Foundation undergraduate research bursary
Synthesis and Evaluation of Competitive Inhibitors of Pantothenate Kinase.
T. Powell-Davis, C. Hamilton
Jul-10 to Aug-10
£1,440

Wellcome Trust vacation scholarship
Kinetic inhibition studies of BshA, the glycosyltransferase, which catalyses the first biosynthetic step of the unique thiol cofactor, bacillithiol, amongst pathogenic Bacilli
C. Mitrofan, C. Hamilton
Jun-10 to Jul-10
£1,440

BBSRC Responsive Mode
Bacillithiol and its unique drug resistance pathways in Bacilli
C. Hamilton
Apr-10 to Sep-12
£348.618

Nuffied Foundation
Synthesis & evaluation of a selective colorimetric disulfide titrant
C.J. Hamilton
Jul 2007 – Aug 2007
£ 1360

Wellcome Trust
Substrate/inhibition kinetics of pantothenate analogues against Bacillus Anthracis pantothenate kinase
C.J. Hamilton
Jul 2007 - Aug-2007
£1400

Royal Society
Unveiling the mycothiol proteome
M. Rawat, C.J. Hamilton
Jul 2007 - Aug 2007
£ 2496

EPSRC
A chemoselective ligation approach to glycosyltransferase bisubstrate mimetics
C.J. Hamilton
Jul 2007 - Jun 2009
£ 228,532

Marie Curie Host Fellowships
New methods to synthesise unnatural nucleosides, known and putative antiproliferative agents
M. Migaud, C.J. Hamilton, D. Boyd, J. Mann
2006-2010
€ 626,507

Wellcome Trust
CMP-Neu5Ac synthetase catalysed synthesis of sugar-NMPs
C.J. Hamilton
Jul 2005 – Aug 2005
£ 1320

Royal Society / NRF
Scientific validation of South African plants for anti-TB, anti-diabetes and anti-tyrosinase activity
N. Lall, J.J. Marion, P. Houghton, C.J. Hamilton
R 175000

Wellcome Trust
13C-labelled acrylonitriles for mechanistic studies of covalent inhibitors of disulfide reductases
C.J. Hamilton
Jul 2004 – Aug 2004
£ 1,280

Nuffield Foundation
Synthetic/mechanistic studies of 13C-labelled time-dependent inhibitors of trypanothione reductase
C.J. Hamilton
Jul 2003 –Aug 2003
£ 1,596

Wellcome Trust
Cell Permeable NTP Analogues as Tools for Chemotherapy, Cell Biology and Chemical Genetics
C.J. Hamilton
Apr 2003 – Sep 2005
£ 125,000

Wellcome Trust
A Substrate-Economic High-Throughput Inhibition Assay for Mycothione Reductase
C.J. Hamilton
Jul 2002 – Aug 2002
£ 1160

Guildford Bench Methodology Fund
A Substrate-Economic High-Throughput Inhibition Assay for Mycothione Reductase
C.J. Hamilton
Jul 2002 – Aug
2002
£ 920

Research Group Membership

Miriam Arbach (Plant Biologist/Biochemist)
Dr Alexandra Roberts (Microbiologist/Biochemist)
Dr Sunils Sharma (Synthetic Chemist)

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