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Protein-Small Molecule Interactions in Neocarzinostatin, the Prototypical Enediyne Chromoprotein Antibiotic

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Protein-Small Molecule Interactions in Neocarzinostatin, the Prototypical Enediyne Chromoprotein Antibiotic 704 DOI: 10.1002/cbic.200600534 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2007, 8, 704 – 717 DOI: 10.1002/cbic.200600534 Protein–Small Molecule Interactions in Neocarzinostatin, the Prototypical Enediyne Chromoprotein Antibiotic James R. Baker,*[a] DerekN. Woolfson, [b] FrederickW. Muskett, [c] Rhys G. Stoneman,[a] Michael D. Urbaniak,[d] and Stephen Caddick*[a] The enediyne chromoproteins are a class of potent antitumour spread interest in this area. Notable developments include the antibiotics comprising a 1:1 complex of a protein and a nonco- discovery of non-natural ligands for the apoproteins and the ob- valently bound chromophore. The protein is required to protect servation that multiple binding modes are available for these li- and transport the highly labile chromophore, which acts as the gands in the binding site. Mutation studies on the apoproteins cytotoxic component by reacting with DNA leading to strand have revealed much about their stability and variability, and the cleavage. A derivative of the best-studied member of this class, application of an in vitro evolution method has conferred new neocarzinostatin (NCS), is currently in use as a chemotherapeutic binding specificity for unrelated ligands. These investigations in Japan. The application of the chromoproteins as therapeutics hold great promise for the application of the apoproteins for along with their unique mode of action has prompted wide- drug-delivery, transport and stabilisation systems. 1. Introduction 2. The Enediyne Chromoproteins Interactions between proteins and small molecules are central In 1965 a proteinaceous substance, neocarzinostatin (NCS), to many biological processes, and provide targets for the ma- was isolated from a culture of Streptomyces carzinostaticus,[1] jority of drug-discovery programmes. Usually, tight and specific and was found to possess broad-spectrum antibiotic activity. It binding depends on the formation of well-defined 3D protein was also discovered to exhibit antiproliferative activity against structures that incorporate highly selective binding pockets. a number of tumour cell lines, both in vitro and in vivo.[2] Early Our understanding of protein–small molecule interactions is in- biological studies were carried out before its true creasing rapidly, particularly through the combination of chem- composition was known. It was not until 1979 that NCS was ical synthesis, protein mutagenesis and structural techniques identified as a 1:1 noncovalent complex of a labile chromo- such as X-ray crystallography and NMR spectroscopy. In turn, phore and a protein.[3] The chromophore was found to be re- such studies are enabling the exploration of many systems in sponsible for the cytotoxic nature of this complex, although the search for new ligands for proteins and macromolecular because of its instability to nucleophiles, heat, UV light and ex- complexes of interest in fundamental research and as potential tremes of pH,[4] its structure remained elusive until 1985.[5] The leads for the pharmaceutical industry. key features of NCS chromophore are a core nine-membered The enediyne chromoproteins represent an unusual example of protein–ligand interactions in that the protein serves to pro- tect and transport a highly unstable small-molecule chromo- [a] Dr. J. R. Baker, R. G. Stoneman, Prof. S. Caddick University College London, Department of Chemistry phore. These chromophores contain reactive enediyne func- Christopher Ingold Laboratories tionalities and are potent cytotoxic compounds exerting their 20 Gordon Street, London, WC1H 0AJ (UK) effect by reacting with DNA to cause strand cleavage. As a Fax: (+ 44)207-679-7463 consequence, the chromoproteins are potent antibiotics, and E-mail: [email protected] highly effective antitumour agents. This review is not intended [b] Prof. D. N. Woolfson School of Chemistry, University of Bristol as an exhaustive overview of the large amount of research car- Cantock’s Close, Bristol, BS8 1TS (UK) ried out on the enediyne chromoproteins. Rather, it will give [c] Dr. F. W. Muskett an introduction to these intriguing complexes, and then dis- Biological NMR Centre, Department of Biochemistry cuss some of the reported studies on probing the apoproteins Henry Wellcome Building, University of Leicester with new ligands and mutagenesis. Neocarzinostatin provides University Road, Leicester, LE1 9HN (UK) the central focus for the discussion as it is by far the most [d] Dr. M. D. Urbaniak Division of Biological Chemistry and Molecular Microbiology studied of the chromoproteins and is representative of the School of Life Science, University of Dundee class. Dundee, DD1 5EH (UK) ChemBioChem 2007, 8, 704 – 717 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 705 J. R. Baker, S. Caddick et al. enediyne ring, fused to a cyclopentene with pendent naph- 2.1 The NCS chromophore and DNA damage thoate and sugar groups (Figure 1A). The 3D structure of the holocomplex was then reported by Rees and Myers in 1993 The biological activity of NCS derives from its ability to cleave (Figure 1B).[6] DNA.[7] Its mechanism of action involves the naphthoate group James Baker completed his MSci at the Rhys Stoneman graduated with a BSc in University of Nottingham in 2000. He Medicinal Chemistry and a DPhil in then went to work as a research chemist Chemistry from the University of Sussex at Novartis before commencing a PhD at under the supervision of Profs. Caddick the University of Bristol working with Prof. and Woolfson. Following a postdoc posi- Kevin Booker-Milburn. His thesis consid- tion with Prof. Caddick at UCL, he worked ered the application of a photocycloaddi- for Dainippon Sumitomo Pharma Europe, tion reaction to the total synthesis of ste- then at Roche Products in the operational moamide. He received his PhD in 2005 at side of clinical research. He currently which point he moved to UCL to carry works for ICON Clinical Research as a out postdoctoral work with Prof. Stephen Medical Writer. Caddick towards the total synthesis of the neocarzinostatin chromo- phore. He has recently been awarded an RCUK fellowship at UCL. His current interests include the development of novel synthetic method- ology and the discovery of new ligands for proteins of biological sig- nificance. Dek Woolfson gained a BA in Chemistry Mick Urbaniak received his D.Phil from at the University of Oxford and a PhD in the University of Sussex under the supervi- Chemistry at the University of Cambridge. sion of Profs. Caddick and Woolfson. For He carried out postdoctoral research at his postdoctoral work, he moved to the the Universities of Cambridge, London University of Dundee to study the parasite and Berkeley (USA). He was appointed to Trypanosoma brucei with Prof. Mike Fer- Lecturerships in Biochemistry at the Uni- guson, where he is currently a postdoctor- versity of Bristol, and then at the Universi- al associate. He has continued to use an ty of Sussex where he was promoted to interdisciplinary approach to study galac- Professor of Biochemistry. In August 2005 tose metabolism and GPI biosynthesis in he returned to the University of Bristol to T. brucei and has developed assays for take up a joint Chair in Chemistry and Biochemistry. His research in- HTS of key T. brucei enzymes as drug targets in the Dundee Drug terests have always been at the interface between chemistry and biol- DiscoveryACHTUNGRE for Tropical Disease initiative. His current interests are in ogy, with his main focus being on understanding sequence–structure the application of chemical biology techniques to the study of para- relationships in proteins and applying this knowledge to rational pro- site protein kinases. tein design. Fred Muskett received his PhD from the Stephen Caddick completed his BSc at University of Edinburgh and then went on Paisley College. After two years in the to complete a Masters degree in Biologi- agrochemical industry he carried out his cal NMR Spectroscopy at the University of PhD at the University of Southampton Dundee. After five years of postdoctoral and then postdoctoral work at Imperial work in structural biology, he joined the College. In 1991 he joined Birkbeck Col- MRC Biomedical NMR Centre at the lege London and then moved to the Uni- NationalACHTUNGRE Institute for Medical Research versity of Sussex in 1993, where he rose to (London, UK). In 2003, he joined the the rank of full professor. In 2003 he Henry Welcome Laboratories for Structur- joined University College London (UCL) as al Biology (University of Leicester) as the Vernon Professor of Organic Chemistry NMR Centre manager. His current research interests are use of NMR and Chemical Biology. His research interests include natural products methods to study protein–ligand interactions. synthesis and development of new methodologies for organic synthe- sis. Major areas of biological and biomedical research interests in- clude heart failure, functional role of methylarginine processing en- zymes, total and semi-synthesis of proteins, NMDA receptor–ligand interactions, drug design and delivery. 706 www.chembiochem.org 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2007, 8, 704 – 717 Protein–Small Molecule Interactions in Neocarzinostatin glutathione might function as this thiol in cells.[11] This attack leads to opening of the epoxide yielding an unstable cumu- lene species 1, which has been observed at low temperatures by 1H NMR spectroscopy.[12,13] Cycloaromatisation of this cumu- lene gives the diradical 2, which abstracts hydrogen atoms
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