Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2000-09-11
2002-11-19
Borin, Michael (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C702S019000, C435S006120
Reexamination Certificate
active
06484103
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the enzyme pantothenate synthetase, and in particular its crystal structure and the use of this structure in 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 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 bacteria and fungi but not to human or animal subjects infected by bacteria.
Of specific interest is pantothenate synthetase (D-pantoate: &bgr;-alanine ligase (AMP-forming); EC 6.3.2.1) This enzyme catalyses the condensation between &bgr;-alanine and pantoic acid, the final steps in pantothenic acid biosynthesis. Inhibitors (whether competitive, non-competitive, uncompetitive or irreversible) of pantothenate synthetase would be of significant technical and commercial interest.
Purification of pantothenate synthetase (PS) to homogeneity was achieved by Miyatake et. al, (
J. Biochem
., 79, (1976), 673-678). The enzyme was reported to require stoichiometric amounts of ATP as an energy source which is hydrolysed to AMP and inorganic pyrophosphate. The mechanism of the enzymic reaction involves pantoate adenylate as an intermediate.
However, until now no one has successfully determined the structure of PS. This has prevented PS inhibitors being developed via structure-based drug design methodologies. Knowledge of the structure of PS would significantly assist the rational design of novel therapeutics based on PS inhibitors.
SUMMARY OF THE INVENTION
The present invention is at least partly based on overcoming several technical hurdles: we have (i) produced PS crystals of suitable quality, including crystals of selenium atom PS derivatives, for performing X-ray diffraction analyses, (ii) collected X-ray diffraction data from the crystals, (iii) determined the three-dimensional structure of PS, and (iv) identified sites on the enzyme which are likely to be involved in the enzymic reaction.
In a first aspect, the present invention provides a crystal of PS having a monoclinic space group P2
1
, and unit cell dimensions of a=66.0±0.2 Å, b=78.1±0.2 Å, c=77.1±0.2 Å and &bgr;=103.7±0.2°. Preferably the PS is a dimer.
In a second aspect, the invention also provides a crystal of PS having the three dimensional atomic coordinates of Table 1.
In a third aspect, the invention provides a method for crystallizing a selenium atom PS derivative which comprises producing PS by recombinant production in a bacterial host (e.g.
E. coli
) in the presence of selenomethionine, recovering a selenium atom PS derivative from the host and growing crystals from the recovered selenium atom PS derivative.
Thus, the selenium atom PS derivative and PS produced by crystallising native PS (see the detailed description below) are provided as crystallised proteins suitable for X-ray diffraction analysis.
The crystals may be grown by any suitable method, e.g. the hanging drop method.
In a fourth aspect, the present invention provides a method for identifying a potential inhibitor of PS comprising the steps of:
a. employing a three-dimensional structure of PS, or at least one sub-domain thereof, to characterise at least one PS active site, the three-dimensional structure being defined by atomic coordinate data according to Table 1; and
b. identifying the potential inhibitor by designing or selecting a compound for interaction with the active site.
By “sub-domain” is meant at least one complete element of secondary structure, i.e. an alpha helix or a beta sheet, as described in the detailed description below.
If more than one PS active site is characterised and a plurality of respective compounds are designed or selected, the potential inhibitor may formed by linking the respective compounds into a larger compound which maintains the relative positions and orientations of the respective compounds at the active sites. The larger compound may be formed as a real molecule or by computer modelling.
In any event, the determination of the three-dimensional structure of PS provides a basis for the design of new and specific ligands for PS. For example, knowing the three-dimensional structure of PS, computer modelling programs may be used to design different molecules expected to interact with possible or confirmed active sites, such as binding sites or other structural or functional features of PS.
More specifically, a potential modulator of PS activity can be examined through the use of computer modelling using a docking program such as GRAM, DOCK, or AUTODOCK (see Walters et al.,
Drug Discovery Today
, Vol.3, No.4, (1998), 160-178, and Dunbrack et al.,
Folding and Design
, 2, (1997), 27-42) to identify potential inhibitors of PS. This procedure can include computer fitting of potential inhibitors to PS to ascertain how well the shape and the chemical structure of the potential inhibitor will bind to the enzyme.
Also computer-assisted, manual examination of the active site structure of PS may be performed. The use of programs such as GRID (Goodford,
J. Med. Chem
., 28, (1985), 849-857)n—a program that determines probable interaction sites between molecules with various functional groups and the enzyme surface—may also be used to analyse the active site to predict partial structures of inhibiting compounds.
Computer programs can be employed to estimate the attraction, repulsion, and steric hindrance of the two binding partners (e.g. the PS and a potential inhibitor). Generally the tighter the fit, the fewer the steric hindrances, and the greater the attractive forces, the more potent the potential modulator since these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a potential drug, the more likely it is that the drug will not interact with other proteins as well. This will tend to minimise potential side-effects due to unwanted interactions with other proteins.
Alternatively, step b. may involve selecting the compound by computationally screening a database of compounds for interaction with the active site. For example, a 3-D descriptor for the potential inhibitor may be derived, the descriptor including geometric and functional constraints derived from the architecture and chemical nature of the active site. The descriptor may then be used to interrogate the compound database, a potential inhibitor being a compound that has a good match to the features of the descriptor. In effect, the descriptor is a type of virtual pharmacophore.
Having designed or selected possible binding partners, these can then be screened for activity. Consequently, the method preferably further comprises the further steps of:
c. obtaining or synthesising the potential inhibitor; and
d. contacting the potential inhibitor with PS to determine the ability of the potential inhibitor to interact with PS.
More preferably, in step d. the potential inhibitor is contacted with PS in the presence of a substrate, and typically a buffer, to determine the ability of said potential inhibitor to inhibit PS. The substrate may be e.g. pantoic acid (or a salt thereof), &bgr;-alanine (or a salt thereof), or ATP. So, for example, an assay mixture for PS may be produced which comprises the potential inhibitor, substrate and buffer.
Instead of, or in addition to, performing e.g. a chemical assay, the method may comprise the further steps of:
c. obtaining or synthesising said potential inhibitor;
d. forming a complex of PS and said potential inhibitor; and
e. analysing said complex by X-ray crystallography to determine the ability of said
Abell Christopher
Blundell Tom L.
Von Delft Frank
Astex Technology Limited
Borin Michael
Galitsky Nikolai
Nixon & Vanderhye P.C.
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