X-ray or gamma ray systems or devices – Specific application – Diffraction – reflection – or scattering analysis
Reexamination Certificate
2001-03-27
2003-04-08
Bruce, David V. (Department: 2882)
X-ray or gamma ray systems or devices
Specific application
Diffraction, reflection, or scattering analysis
C435S106000
Reexamination Certificate
active
06546074
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the enzyme aspartate decarboxylase, and in particular the use of its crystal structure for drug discovery.
BACKGROUND OF THE INVENTION
Pantothenic acid (vitamin B
5
) is found in coenzyme A (CoA) and the acyl carrier protein (ACP), both of which are involved in fatty acid metabolism.
Pantothenic acid can be synthesised by plants and microorganisms but animals are apparently unable to make the vitamin, and require it in their diet. However, all organisms are able to convert pantothenic acid to its metabolically active form, coenzyme A.
The pathway for the synthesis of pantothenic acid in bacteria is shown in FIG.
1
. It provides a potential target for the treatment of infectious disease, since inhibitors of the pathway should be damaging to microorganisms but not to human or animal subjects infected by microorganisms.
Of specific interest is aspartate decarboxylase (L-aspartate-&agr;-decarboxylase (EC 4.1.1.1)). This enzyme catalyses the decarboxylation of L-aspartate to &bgr;-alanine, which then goes on to form pantothenate in a condensation reaction with D-pantoate. Inhibitors (whether competitive, non-competitive, uncompetitive or irreversible) of aspartate decarboxylase (ADC) would be of significant technical and commercial interest.
ADC was first isolated from
Escherichia coli
by Williamson et al. (
J. Biol. Chem
., 254, (1979), 8074-8082), who found indications that the protein was present in different processed states. The unprocessed enzyme is referred to as the n-chain and has 126 residues. Processing (see
FIG. 2
) splits the n-chain at the Gly24-Ser25 peptide bond into a larger C-terminal chain and a smaller N-terminal chain. A pyruvol group (for convenience termed Pv125) is generated from the serine residue (Ser25) at the end of the C-chain, and a carboxylate group is formed at the end of the glycine residue (Gly24) of the smaller N-terminal chain. Williamson et al. found that only a proportion of the enzyme chains were processed in this way.
Purification to homogeneity of overexpressed, recombinant ADC was achieved by Ramjee et al. (
J. Biochem
., 323, (1997), 661-669). The purified enzyme was found to be a tetramer which, after processing, contained three processed chains and one chain which was not fully processed.
Albert et al. (
Nature Structural Biology
, 5, (1998), 289-293) used X-ray crystallography to determine the structure of ADC to 2.2 Å resolution. They showed that the enzyme studied by Ramjee et al. has pseudo-fourfold rotational symmetry, each of the four tetramer subunits (each subunit or corresponding to a n-chain labelled A, B, C or D) having a six-stranded &bgr;-barrel capped by small &agr;-helices at each end. The binding cavities for aspartate decarboxylation are located between adjacent subunits. Three of the binding cavities have catalytic pyruvol groups resulting from respective processed n-chains. The other binding cavity has an ester which appears to be an intermediate in the processing reaction. The evidence points to an autocatalytic self-processing mechanism which did not lead to full processing of all the n-chains. The coordinates of the crystal structure determined by Albert et al. are available from the Protein Data Bank (Berman et al.,
Nucleic Acids Research
, 28, (2000), 235-242) under access code lAW8.
Albert et al. proposed a model of L-asparate binding, but did not suggest a mechanism by which ADC accomplishes aspartate decarboxylation. Until now very little was known about the enzyme's role in catalysis. This has impeded the development of ADC inhibitors via structure-based drug design methodologies. Knowledge of the mechanism would significantly assist the rational design of novel therapeutics based on ADC inhibitors.
DEFINITIONS
Specific residues are denoted herein by their conventional acronyms (e.g. Gly for glycine), and numbers corresponding to their position in the unprocessed n-chain counting from the N-terminal of the n-chain (e.g. Gly24). Moreover, because each binding cavity is formed from the residues of two n-chains, each residue is further denoted by a letter corresponding to the respective one of the n-chains (e.g. Gly24A or Lys9D). Below, we have used D and A to denote the two n-chains of a binding cavity, but in a tetramer with four equivalent binding cavities and subunits labelled A, B, C and D one could equally use A and B, B and C, or C and D instead.
In the following by “binding site” we mean a site, such as an atom or functional group of an amino acid residue, in the ADC binding cavity which may bind to an agent compound such as a candidate inhibitor. Depending on the particular molecule in the cavity, sites may exhibit attractive or repulsive binding interactions, brought about by charge, steric considerations and the like.
By “fitting”, is meant determining by automatic, or semi-automatic means, interactions between one or more atoms of an agent molecule and one or more atoms or binding sites of the ADC, and determining the extent to which such interactions are stable. Various computer-based methods for fitting are described further herein.
By “fully processed” ADC we mean a composition comprising an amount of ADC in which pyruvoyl groups are generated from at least 90%, preferably at least 95%, and more preferably at least 99% of the ADC Ser25 residues.
By “root mean square deviation” we mean the square root of the arithmetic mean of the squares of the deviations from the mean.
By a “computer system” we mean the hardware means, software means and data storage means used to analyse atomic coordinate data. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualise structure data. The data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems.
By “computer readable media” we mean any media which can be read and accessed directly by a computer e.g. so that the media is suitable for use in the above-mentioned computer system. The media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
SUMMARY OF THE INVENTION
The present invention is at least partly based on overcoming several technical hurdles: we have (i) produced fully processed crystals of ADC of suitable quality for performing X-ray diffraction analyses, (ii) formed ADC-ligand complexes by soaking the crystals in appropriate soaking solutions, (iii) collected X-ray diffraction data from the ADC-ligand complexes, (iv) determined the three-dimensional structures of the complexes, (v) identified regions of ADC which undergo conformational changes upon ligand binding and decarboxylation, and (vi) determined the likely mechanism by which ADC accomplishes aspartate decarboxylation.
In general aspects, the present invention is concerned with identifying or obtaining agent compounds (especially inhibitors of ADC) for modulating ADC activity, and in preferred embodiments identifying or obtaining actual agent compounds/inhibitors. Crystal structure information presented herein as useful in designing potential inhibitors and modelling them or their potential interaction with the ADC binding cavity. Potential inhibitors may be brought into contact with ADC to test for ability to interact with the ADC binding cavity. Actual inhibitors may be identified from among potential inhibitors synthesized following design and model work performed in silico. An inhibitor identified using the present invention may be formulated into a composition, for instance a composition comprising a pharmaceu
Abell Chris
Blundell Tom L.
Von Delft Frank
Astex Technology Limited
Bruce David V.
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