Crystallization and structure determination of staphylococcus aureus NAD synthetase
AssigneePharmacia & Upjohn Company
InventorsTimothy E. Benson, Donald Bryan Prince
Life of this patent
Estimating the term…
Abstract
An unliganded form of Staphylococcus aureus NAD synthetase (S. aureus NadE) has been crystallized, and the three-dimensional x-ray crystal structure has been solved to 2.3 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of S. aureus NadE activity.
Claims43
A molecule or molecular complex comprising at least a portion of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket, wherein the substrate binding pocket comprises the amino acids listed in Table 3, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 1.1 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in Table 1.
A molecule or molecular complex that is structurally homologous to an S. aureus NAD synthetase molecule or molecular complex, wherein the S. aureus NAD synthetase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in Table 1.
A scalable three-dimensional configuration of points, at least a portion of said points derived from structure coordinates of at least a portion of an S. aureus NAD synthetase molecule or molecular complex listed in Table 1 comprising at least one of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket.
A scalable three-dimensional configuration of points, at least a portion of the points derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an S. aureus NAD synthetase molecule or molecular complex and comprises at least one of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket.
A machine-readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of at least one molecule or molecular complex selected from the group consisting of:
- (i) a molecule or molecular complex comprising at least a portion of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket comprising the amino acids listed in Table 3, the substrate binding pocket defined by a set of points having a root mean square deviation of less than about 1.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in Table 1;
- and
- (ii) a molecule or molecular complex that is structurally homologous to an S. aureus NAD synthetase molecule or molecular complex, wherein the S. aureus NAD synthetase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in Table 1.
A machine-readable data storage medium comprising a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein said first set of data comprises a Fourier transform of at least a portion of the structure coordinates for S. aureus NAD synthetase listed in Table 1;
- and said second set of data comprises an x-ray diffraction pattern of a molecule or molecular complex of unknown structure.
A method for obtaining structural information about a molecule or a molecular complex of unknown structure comprising:
- crystallizing the molecule or molecular complex;
- generating an x-ray diffraction pattern from the crystallized molecule or molecular complex;
- applying at least a portion of the structure coordinates set forth in Table 1 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex whose structure is unknown.
A method for homology modeling an S. aureus NAD synthetase homolog comprising:
- aligning the amino acid sequence of an S. aureus NAD synthetase homolog with an amino acid sequence of S. aureus NAD synthetase (SEQ ID NO:
- 1) and incorporating the sequence of the S. aureus NAD synthetase homolog into a model of S. aureus NAD synthetase derived from structure coordinates set forth in Table 1 to yield a preliminary model of the S. aureus NAD synthetase homolog;
- subjecting the preliminary model to energy minimization to yield an energy minimized model;
- remodeling regions of the energy minimized model where stereochemistry restraints are violated to yield a final model of the S. aureus NAD synthetase homolog.
A computer-assisted method for identifying an inhibitor of S. aureus NAD synthetase activity comprising:
- supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket, the substrate binding pocket comprising the amino acids listed in Table 3;
- supplying the computer modeling application with a set of structure coordinates of a chemical entity;
- and
- determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NAD synthetase activity.
A computer-assisted method for designing an inhibitor of S. aureus NAD synthetase activity comprising:
- supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket, the substrate binding pocket comprising the amino acids listed in Table 3;
- supplying the computer modeling application with a set of structure coordinates for a chemical entity;
- evaluating the potential binding interactions between the chemical entity and substrate binding pocket of the molecule or molecular complex;
- structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity;
- and
- determining whether the modified chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NAD synthetase activity.
A computer-assisted method for designing an inhibitor of S. aureus NAD synthetase activity de novo comprising:
- supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of an S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket, wherein the substrate substrate binding pocket comprises the amino acids listed in Table 3;
- computationally building a chemical entity represented by set of structure coordinates;
- and
- determining whether the chemical entity is an inhibitor expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NAD synthetase activity.
A method for making an inhibitor of S. aureus NadE activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus NadE activity, the chemical entity having been identified during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket;
- supplying the computer modeling application with a set of structure coordinates of a chemical entity;
- and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at a binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NadE activity.
A method for making an inhibitor of S. aureus NadE activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus NadE activity, the chemical entity having been designed during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket;
- supplying the computer modeling application with a set of structure coordinates for a chemical entity;
- evaluating the potential binding interactions between the chemical entity and a binding pocket of the molecule or molecular complex;
- structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity;
- and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at the binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NadE activity.
A method for making an inhibitor of S. aureus NadE activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus NadE activity, the chemical entity having been designed during a computer-assisted process comprising supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex, the molecule or molecular complex comprising at least a portion of at least one of a S. aureus NAD synthetase or NAD synthetase-like substrate binding pocket;
- computationally building a chemical entity represented by set of structure coordinates;
- and determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex at a binding pocket, wherein binding to or interfering with the molecule or molecular complex is indicative of potential inhibition of S. aureus NadE activity.
A method for crystallizing an S. aureus NAD synthetase molecule or molecular complex comprising:
- providing purified S. aureus NAD synthetase at a concentration of about 1 mg/ml to about 50 mg/ml;
- and
- crystallizing S. aureus NAD synthetase from a solution comprising about 5% by weight to about 50% by weight PEG and about 0% by weight to about 20% by weight DMSO.
A method for crystallizing an S. aureus NAD synthetase molecule or molecular complex comprising:
- providing purified S. aureus NAD synthetase at a concentration of about 1 mg/ml to about 50 mg/ml;
- and
- crystallizing S. aureus NAD synthetase from a solution comprising about 1% by weight to about 10% by weight PEG, about 0.1 M to about 5 M ammonium sulfate, and about 0% by weight to about 20% by weight DMSO, wherein the solution is buffered to a pH of about 6 to about 9.
A method for crystallizing an S. aureus NAD synthetase molecule or molecular complex comprising:
- providing purified S. aureus NAD synthetase at a concentration of about 1 mg/ml to about 50 mg/ml;
- and
- crystallizing S. aureus NAD synthetase from a solution comprising about 5% by weight to about 50% by weight PEG and about 0% by weight to about 20% by weight DMSO, wherein the solution is buffered to a pH of about 8 to about 11.
A crystal of S. aureus NAD synthetase.
Dependencies are inferred from the claim text.