Crystallization and structure determination of staphylococcus aureus thymidylate kinase
AssigneePharmacia & Upjohn Company
InventorTimothy E. Benson
Life of this patent
Estimating the term…
Abstract
An unliganded form of Staphylococcus aureus thymidylate kinase (S. aureus TMK) 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 TMK activity.
Claims54
A molecule or molecular complex comprising at least a portion of an S. aureus thymidylate kinase or thymidylate kinase-like TMP binding pocket, wherein the TMP binding pocket comprises the amino acids listed in Table 1, the TMP binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in FIG. 2.
A molecule or molecular complex comprising at least a portion of an S. aureus thymidylate kinase TMP/ATP substrate binding pocket, wherein the TMP substrate binding pocket comprises the amino acids listed in Table 4, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in FIG. 2.
A molecule or molecular complex that is structurally homologous to an S. aureus thymidylate kinase molecule or molecular complex, wherein the S. aureus thymidylate kinase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in FIG. 2.
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 thymidylate kinase molecule or molecular complex listed in FIG. 2 comprising at least one of an S. aureus thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP 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 thymidylate kinase molecule or molecular complex and comprises at least one of an S. aureus thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP 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 thymidylate kinase or thymidylate kinase-like TMP binding pocket comprising the amino acids listed in Table 1, the TMP binding pocket defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2;
- (ii) a molecule or molecular complex comprising at least a portion of an S. aureus thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket comprising the amino acids listed in Table 4, the TMP/ATP binding pocket defined by a set of points having a root mean square deviation of less than about 2.1 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in FIG. 2;
- and
- (iii) a molecule or molecular complex that is structurally homologous to an S. aureus thymidylate kinase molecule or molecular complex, wherein the S. aureus thymidylate kinase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in FIG. 2.
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 structural coordinates for S. aureus thymidylate kinase listed in FIG. 2;
- 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 FIG. 2 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 thymidylate kinase homolog comprising:
- aligning the amino acid sequence of an S. aureus thymidylate kinase homolog with an amino acid sequence of S. aureus thymidylate kinase and incorporating the sequence of the S. aureus thymidylate kinase homolog into a model of S. aureus thymidylate kinase derived from structure coordinates set forth in FIG. 2 to yield a preliminary model of the S. aureus thymidylate kinase 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 thymidylate kinase homolog.
A computer-assisted method for identifying an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP binding pocket, the TMP binding pocket comprising the amino acids listed in Table 1;
- 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 thymidylate kinase activity.
A computer-assisted method for identifying an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding pocket comprising the amino acids listed in Table 4;
- 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 thymidylate kinase activity.
A computer-assisted method for designing an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP binding pocket, the TMP binding pocket comprising the amino acids listed in Table 1;
- 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 thymidylate kinase activity.
A computer-assisted method for designing an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding pocket comprising the amino acids listed in Table 4;
- 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 thymidylate kinase activity.
A computer-assisted method for designing an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP binding pocket, wherein the TMP substrate binding pocket comprises the amino acids listed in Table 1;
- 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 thymidylate kinase activity.
A computer-assisted method for designing an inhibitor of S. aureus thymidylate kinase 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 thymidylate kinase or thymidylate kinase-like TMP/ATP binding pocket, wherein the TMP/ATP binding pocket comprises the amino acids listed in Table 4;
- 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 thymidylate kinase activity.
A method for making an inhibitor of S. aureus TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus TMK 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 thymidylate kinase or thymidylate kinase-like TMP or TMP/AT 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 TMK activity.
A method for making an inhibitor of S. aureus TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus TMK 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 thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP 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 TMK activity.
A method for making an inhibitor of S. aureus TMK activity, the method comprising chemically or enzymatically synthesizing a chemical entity to yield an inhibitor of S. aureus TMK 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 thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP 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 TMK activity.
A method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex comprising:
- preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml;
- and
- crystallizing S. aureus thymidylate kinase from a solution comprising about 5 wt. % to about 50 wt. % PEG, about 0.05 M to about 0.5 M MgCl2, and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.
A method for crystallizing an S. aureus thymidylate kinase molecule or molecular complex comprising:
- preparing purified S. aureus thymidylate kinase at a concentration of about 1 mg/ml to about 50 mg/ml;
- and
- crystallizing S. aureus thymidylate kinase from a solution comprising about 2 mM to about 20 mM β,γ-difluoromethylene-bisphosphonate adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the solution is buffered to a pH of about 6 to about 7.
A crystal of S. aureus thymidylate kinase.
Dependencies are inferred from the claim text.