Alison Ward

Crystal structure of human cytochrome P450 2C9 with bound warfarin

Cytochrome P450 proteins (CYP450s) are membrane-associated haem proteins that metabolize physiologically important compounds in many species of microorganisms, plants and animals. Mammalian CYP450s recognize and metabolize diverse xenobiotics such as drug molecules, environmental compounds and pollutants. Human CYP450 proteins CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are the major drug-metabolizing isoforms, and contribute to the oxidative metabolism of more than 90% of the drugs in current clinical use. Polymorphic variants have also been reported for some CYP450 isoforms, which has implications for the efficacy of drugs in individuals, and for the co-administration of drugs. The molecular basis of drug recognition by human CYP450s, however, has remained elusive. Here we describe the crystal structure of a human CYP450, CYP2C9, both unliganded and in complex with the anti-coagulant drug warfarin. The structure defines unanticipated interactions between CYP2C9 and warfarin, and reveals a new binding pocket. The binding mode of warfarin suggests that CYP2C9 may undergo an allosteric mechanism during its function. The newly discovered binding pocket also suggests that CYP2C9 may simultaneously accommodate multiple ligands during its biological function, and provides a possible molecular basis for understanding complex drug–drug interactions.

Expression, Purification and Characterisation of Full-length Histidine Protein Kinase RegB from Rhodobacter sphaeroides

The global redox switch between aerobic and anaerobic growth in Rhodobacter sphaeroides is controlled by the RegA/RegB two-component system, in which RegB is the integral membrane histidine protein kinase, and RegA is the cytosolic response regulator. Despite the global regulatory importance of this system and its many homologues, there have been no reported examples to date of heterologous expression of full-length RegB or any histidine protein kinases. Here, we report the amplified expression of full-length functional His-tagged RegB in Escherichia coli, its purification, and characterisation of its properties. Both the membrane-bound and purified solubilised RegB protein demonstrate autophosphorylation activity, and the purified protein autophosphorylates at the same rate under both aerobic and anaerobic conditions confirming that an additional regulator is required to control/inhibit autophosphorylation. The intact protein has similar activity to previously characterised soluble forms, but is dephosphorylated more rapidly than the soluble form (half-life ca 30 minutes) demonstrating that the transmembrane segment present in the full-length RegB may be an important regulator of RegB activity. Phosphotransfer from RegB to RegA (overexpressed and purified from E. coli) by RegB is very rapid, as has been reported for the soluble domain. Dephosphorylation of active RegA by full-length RegB has a rate similar to that observed previously for soluble RegB.

Collection and characterisation of bacterial membrane proteins.

A general strategy for the amplified expression in Escherichia coli of membrane transport and receptor proteins from other bacteria is described. As an illustration we report the cloning of the putative α‐ketoglutarate membrane transport gene from the genome of Helicobacter pylori, overexpression of the protein tagged with RGS(His)6 at the C‐terminus, and its purification in mg quantities. The retention of structural and functional integrity was verified by circular dichroism spectroscopy and reconstitution of transport activity. This strategy for overexpression and purification is extended to additional membrane proteins from H. pylori and from other bacteria.

Cloning, expression, purification and properties of a putative multidrug resistance efflux protein from Helicobacter pylori

Helicobacter pylori is a common pathogen of humans, which predisposes individuals to gastric inflammation and a variety of diseases including gastric cancer. Sequencing of the organisms genome reveals an extensive portfolio of predicted membrane transport proteins, although few of the proteins encoded by these genes have yet been isolated. We describe here the cloning and expression in Escherichia coli of the H. pylori gene hp1181 encoding a putative multidrug resistance membrane transport protein. Substantial overexpression was accomplished, and the protein was tagged with RGS(His)(6) at the C-terminus, which enabled its purification in mg quantities. Identification of the full-length protein was achieved by N-terminal amino acid sequencing and Western blotting using an antibody to the RGS(His)(6) epitope. The retention of structural integrity and occurrence of predicted alpha-helix in the protein were verified by circular dichroism spectroscopy.

Engineering a High-Affinity Anti-IL-15 Antibody: Crystal Structure Reveals an α-Helix in VH CDR3 as Key Component of Paratope

Interleukin (IL) 15 is an inflammatory cytokine that plays an essential role in the activation, proliferation, and maintenance of specific natural killer cell and T-cell populations, and has been implicated as a mediator of inflammatory diseases. An anti-IL-15 antibody that blocked IL-15-dependent cellular responses was isolated by phage display and optimised via mutagenesis of the third complementarity-determining regions (CDRs) of variable heavy (VH) and variable light chains. Entire repertoires of improved variants were recombined with each other to explore the maximum potential sequence space. DISC0280, the most potent antibody isolated using this comprehensive strategy, exhibits a 228-fold increase in affinity and a striking 40,000-fold increase in cellular potency compared to its parent. Such a wholesale recombination strategy therefore represents a useful method for exploiting synergistic potency gains as part of future antibody engineering efforts. The crystal structure of DISC0280 Fab (fragment antigen binding), in complex with human IL-15, was determined in order to map the structural epitope and paratope. The most remarkable feature revealed lies within the paratope and is a novel six-amino-acid α-helix that sits within the VH CDR3 loop at the center of the antigen binding site. This is the first report to describe an α-helix as a principal component of a naturally derived VH CDR3 following affinity maturation.

E. coli expression and purification of human and cynomolgus IL-15.

The physiological activities of Interleukin-15 (IL-15) suggest that it could be useful as an immunomodulator to activate the innate immune system, however, the expression and purification yields of recombinant mature IL-15 have typically been low. In this report, a method was optimised to generate milligram quantities of this cytokine. Human IL-15 with an N-terminal (His)(6)-tag was expressed in Escherichia coli as an insoluble protein. The IL-15 material was purified from other cellular proteins by dissolution in 6M guanidine HCl, followed by Ni-NTA chromatography in a buffer containing 8M urea. Use of a multi-component screen identified the optimal conditions for folding (His)(6)-tagged human IL-15 and the method was scaled up to produce milligram quantities of folded material in its native conformation, with two intra-molecular disulphides as determined by electrospray mass spectrometry. Mature IL-15 was generated by cleavage with recombinant enterokinase, which was subsequently removed by Ni-NTA chromatography. Identical methods were used to produce mature cynomolgus monkey (Macaca fascicularis) IL-15 in similar quantities. Human and cynomolgus IL-15 were both active in two IL-15 dependent assays; mouse CTLL2 cell proliferation and human and cynomolgus CD69 upregulation on CD3(-) CD8+ lymphocytes in whole blood. Despite being 96% identical at the amino acid level the human IL-15 was 10-fold more potent than the cynomolgus IL-15 in both assays. The methods described here are useful for producing both mature IL-15 proteins in sufficient quantity for in vivo and in vitro studies, including X-ray crystallography.

The Discovery of Mmp7 Inhibitors Exploiting a Novel Selectivity Trigger.

Matrilysin or matrix metalloproteinase 7 (MMP7) is a member of a class of zinc-dependent endopeptidases (MMPs) capable of degrading extracellular matrix proteins and thought to play an important role in tissue remodeling associated with various physiological and pathological processes. It has broad specificity and cleaves a number of matrix substances, including proteoglycans and collagen III/IV/V/IX/X/XI, which underlies a potential role for MMP7 inhibitors in the treatment of disease associated with tissue degradation/remodeling. In addition, MMP7 has been reported to have a potential role in tumor metastasis and inflammatory processes. It has also been reported to be expressed in osteoarthritic cartilage, where it is colocalized with the tetraspanin, CD151, which has been implicated in its pericellular activation. As a class, MMPs have been the subject of intense study within the pharmaceutical industry over the last decade, and inhibitors have been described for many of the individual MMPs. However, MMP selectivity remains a significant hurdle for most MMP inhibitors due to a reliance on zinc chelation to provide a significant component of the binding energy. Despite high therapeutic value, there has been a consistent lack of success with such inhibitors in the clinic, and this can largely be attributed to several factors: poor selectivity, poor target validation for the targeted therapy, and poorly defined predictive preclinical animal models for safety and efficacy. The selectivity problem is particularly true with compounds bearing groups that chelate strongly to zinc (e.g. , hydroxamate, reverse hydroxamate) because a large component of the binding energy is derived from a feature common to all MMPs. With this in mind, we initiated a program to identify selective MMP7 inhibitors employing a high-throughput screening approach, with MMP selectivity assessment very early in the screening process. To provide an initial indication of selectivity, actives were screened against MMP1 and MMP14. Compounds showing good selectivity against these two MMPs were then screened against MMP2, MMP12 and MMP13 to give an indication of wider MMP selectivity. As anticipated, potent hydroxamate and reverse hydroxamate inhibitors were identified but none of them showed selectivity over other MMPs. In fact, without exception, hydroxamate, reverse hydroxamate and also hydantoin inhibitors proved more active against many of the other MMPs than against MMP7. One active hit that emerged from these initial selectivity screens was the carboxylic acid compound I. The compound has low potency against MMP7, but there was no indication of activity against any of the other MMPs tested. The low potency limited a true assessment of selectivity but, nonetheless, this initial lead was pursued further, driven largely by two factors: ease of synthesis and crystallization. Firstly, we were able to successfully crystallize this compound within the MMP7 protein and determine the complex structure to enable an understanding of the binding mode. Secondly, the compound was relatively simple and amenable to parallel synthesis from commercial sulfonyl chlorides and amino acids, which would facilitate a rapid initial exploration of structure–activity relationships. Furthermore, the library design for this parallel chemistry could be directed by virtual docking experiments using the X-ray data generated from compound I. We also crystallized one of the nonselective reverse hydroxamate MMP7 inhibitors (compound II) to assess the binding mode of compounds possessing a large S1’ substituent. MMP7 is unusual in the MMP family in the sense that it possesses a very shallow S1’ selectivity pocket, and compound II was expected to provide a poor fit. What was seen from X-ray data is that this compound expands the S1’ pocket to accommodate the large side chain. Using this information, we were able to employ both the open and closed MMP7 binding sites for virtual docking experiments. The structural work carried out around compounds I and II (Figure 1) is described below. The results give some insight into the basis of the selectivity seen with compound I. Also described are the initial results from the library design based on the structural data to increase MMP7 potency whilst retaining selectivity over other MMPs. MMP7 is the smallest member of the MMP family, with the mature enzyme consisting of the catalytic domain alone. The MMP7 structure confirmed the open-faced a-b-sandwich fold (Figure 2), characteristic for most zinc-dependant endopeptidases. The active site is located in a groove adjacent to the central helix B and the antiparallel b-sheet, and it is delineated by the typical HEXGHXXGXXH sequence motif. Upon binding, the scissile peptide bond is positioned with the carbonyl oxygen towards the catalytic zinc ion, which is coordinated by the three histidine residues. The nearby glutamate acts as a general base, promoting nucleophilic attack by a catalytic water molecule and subsequently stabilizes the tetrahedral intermediate. In addition to the catalytic zinc, the structure contains two calcium ions and one additional zinc ion, important for maintaining the structural integrity of the b-sheet. [a] Dr. K. Edman, C. Johansson, Dr. J. Petersen, Dr. A. Tervo, L. Wissler Global Structural Chemistry, AstraZeneca Mçlndal Pepparedsleden 1, 43183 Mçlndal (Sweden) Fax: (+ 46) 31-776-3700 Karl.Edman@astrazeneca.com [b] Dr. M. Furber, P. Hemsley, Dr. G. Pairaudeau, Dr. M. Stocks, Dr. A. Ward, Dr. E. Wells Departments of Medicinal Chemistry and BioSciences AstraZeneca Charnwood Bakewell Road, Loughborough, Leicestershire LE11 5RH (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000550.

Identification of a potent anti-IL-15 antibody with opposing mechanisms of action in vitro and in vivo

BACKGROUND AND PURPOSE Interleukin‐15 (IL‐15) is important in the activation and proliferation of lymphocytic cell populations and is implicated in inflammatory disease. We report the characterization of a novel monoclonal antibody DISC0280 which is specific for human IL‐15.

Development of a Homogeneous High-Throughput Screening Assay for Biological Inhibitors of Human Rhinovirus Infection

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule–1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti–ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.