JP2003510250A - Crystallization and structure determination of Staphylococcus aureus elongation factor P - Google Patents

Abstract

(57) [Summary] Staphylococcus aureus elongation factor P S. aureus EF-P was crystallized, and its three-dimensional X-ray crystal structure was elucidated at atomic resolution (1.9 °). This X-ray crystal structure is useful for elucidating the structure of other molecules or molecular complexes and designing inhibitors of S. aureus EF-P.

Description

Detailed Description of the Invention

This application is a United States provisional application serial number 60 / filed August 6, 1999.
Claim the benefit of No. 147,851, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to the crystal and structure determination of Staphylococcus aureus elongation factor P (S. aureus EF-P).

[0003] Translation of the invention are fundamentally process biochemical and cellular total cells; thus, many of the antimicrobial agents not surprising that this process targets. Preparation for translation
It begins with the binding of mRNA to the ribosome, which places the AUG of the first codon in a position for interaction with fMet-tRNA. Translation is initiated by the binding of fMet-tRNA to the 30S subunit of the P site. The second tRNA is then carried to the ribosome by the GTP-dependent elongation factor-TU, which places the tRNA in the A site and the first peptide bond can be synthesized. After synthesis, the new free tRNA is E
The tRNA containing the growing amino acid chain moves to the P site, leaving the A site for the next aminoacyl-tRNA. This translocation step is catalyzed by the GTP-dependent elongation factor G.

Decades ago, another purified factor, EF-P, was introduced by Ganoza into fMet.
-Formylmethionylpuromycin from amino acid-tRNA, and aminoacyl tR
It was observed that its stimulation of the synthesis of puromycin, which functions as a mimic of NA, increases the rate of formation of the first peptide bond shown in the model system (MC Ganoza et al., Eur. J. Biochem; 146: 287-94 (1985); BR Gli
ck & MC Ganoza, Proc. Natl. Acad. Sci. USA; 72: 4257-60 (1975)
). Although the exact mechanism for stimulation by elongation factor P has not yet been determined, experiments have shown that G rather than large amino acids such as Phe, Met and Lys.
We have shown the selectivity of EF-P for stimulating the synthesis of peptide bonds at small to medium amino acids such as ly and Leu (BR Glick et al., Eur. J. Biochem; 97:
23-28 (1979)). The gene for EF-P from Escherichia coli has been cloned (H. Aoki et al., Nucl. Acid Res; 19: 6215-20 (1991)) and shown to be essential (H. Aoki et al., J. Biol. Chem. 272: 32254-59 (1997)).
The amount of EF-P in E. coli is about 1 EF-P molecule per 10 ribosomes, suggesting that it plays a catalytic role in translation (G. An, Can. J. Bioc.
hem; 58: 1312-14 (1980)). Although the sequence identity with higher organisms is quite low, this 1
The 93 amino acid protein has homologs throughout bacteria and eukaryotes.

[0005] In embodiments one Summary of the Invention, the present invention is from about 1mg / ml to about 50 mg / ml of S. aureus EF-P was purified by concentration prepared; then about 0% to about 50 wt%
Polyethylene glycol and 0 to about 20% by weight DMSO, about 3.5
To a method of crystallizing a S. aureus EF-P molecule or molecular complex comprising crystallizing S. aureus EF-P from a solution buffered to a pH of from about 5.5 to about 5.5.

In another aspect, the invention provides a crystalline form of S. aureus EF-P.
In one embodiment, it is provided that the crystals of S. aureus EF-P have the orthorhombic space group symmetry P2 ₁ 2 ₁ 2 ₁ .

In another aspect, the invention provides points in a scabable three-dimensional arrangement, wherein at least some of the points are preferably amino acids Val2.
9, Lys30, Pro31, Gly32, Lys33, Gly34, Ser3
5 and Ala36, derived from the structural coordinates of at least a portion of the S. aureus EF-P molecule or molecular complex listed in FIG. In one embodiment, at least a portion of the points represent S. aureus EF-P structural coordinates that represent at least the backbone atom positions of the amino acids that define the S. aureus EF-P or S. aureus EF-P-like binding surface. And the binding surface comprises amino acids selected from the surface residues listed in Table 1. In another embodiment, at least a portion of the points are L shown in Table 2.
Derived from the S. aureus EF-P structural coordinates representing the backbone atom of the amino acid within 4 Å of ys, preferably within 7 Å, more preferably within 10 Å and most preferably within 15 Å. In another embodiment, the invention provides a point having at least a portion of the points derived from the structural coordinates of at least a portion of the molecule or molecular complex that is structurally homologous to the S. aureus EF-P molecule or molecular complex. Provides a measurable three-dimensional arrangement of. On the molecular scale, in terms of origin from the molecule or molecular complex, preferably about 1 from the structural coordinates.
It has a root mean square deviation of less than .9Å.

In another aspect, the invention provides a molecule or molecular complex comprising at least a portion of a S. aureus EF-P binding surface. In one embodiment, the binding surface comprises amino acids selected from the surface residues listed in Table 1. In one embodiment, the binding surface is the amino acids Val29, Lys30, Pro31, Gly32, Lys33, Gly3 represented by the structural coordinates listed in FIG.
4, further defined by the set of points having a root mean square deviation of less than about 1.9Å from the points representing the skeletal atoms of Ser35 and Ala36. In another embodiment, the binding surface is within 4Å of Lys33 represented by the structural coordinates shown in Table 2 and listed in FIG. 4, preferably within 7Å of Lys33, more preferably within 10Å of Lys33 and Most preferably, it is further defined by a set of points having a root mean square deviation of less than about 1.9Å from the point representing the backbone atom of the amino acid within 15Å of Lys33.

[0009]

[Table 1]

[0010]

[Table 2]

In another aspect, the invention provides a molecule or molecular complex that is structurally homologous to a S. aureus EF-P molecule or molecular complex.

[0012] In another embodiment, the present invention provides a S. aureus EF including structurally equivalent structures as defined herein, particularly its binding surface or a similarly shaped homologous binding surface.
-Provides an instrument-readable storage medium containing structural coordinates of all or part of P molecules, molecular complexes, structurally homologous molecules or complexes. These data-encoded storage media can display, on a computer screen or similar display device, a three-dimensional graphic depiction of a molecule or molecular complex comprising a binding surface or a similarly shaped binding surface of homology.

In another embodiment, the present invention relates to a computerized modeling system.
Provide the coordinates of the molecule of S. aureus EF-P and identify the chemical moieties that are expected to bind or interfere with the molecule (eg, screen a small molecule library); Methods are provided for identifying inhibitors, ligands, etc. for S. aureus EF-P molecules by obtaining or synthesizing compounds or analogs derived therefrom for activity and then assaying. In another embodiment, the present invention provides a computerized modeling system for S. aureus EF-P.
An inhibitor by designing a chemical group that appears to bind or interfere with the molecule; and then optionally synthesizing the chemical group and assaying the chemical group for biological activity, A method for designing a ligand and the like is provided. In another aspect, the invention provides inhibitors and ligands designed or identified by the above methods. In one embodiment, a composition comprising an inhibitor or ligand designed or identified by the above method is provided. In another embodiment, the composition is a pharmaceutical composition.

In another aspect, the invention provides a method that includes molecular substitution to obtain structural information about a molecule or molecular complex of unknown structure. The method comprises crystallizing a molecule or molecular complex and producing an X-ray diffraction pattern from the crystallized molecule or molecular complex, wherein at least a portion of the EF-P structural coordinates shown in FIG. Applied to generate a three-dimensional electron density map of at least a portion of a molecule or molecular complex.

In another aspect, the present invention provides a method for homology modeling of S. aureus EF-P homologues.

[0016]   Definition   Two crystallographic data sets (with structure factor F) were

[0017]

[Equation 1]

If is less than about 35% for reflections between 8Å and 4Å, then it is considered isomorphic.

Abbreviations The following abbreviations are used throughout this disclosure: Staphylococcus aureus elongation factor P (S. aureus EF-P) isopropylthio-β-D-galactoside (IPTG) dithiothreitol (DTT). Dimethyl sulfoxide (DMSO). Multiple anomalous dispersion (MAD). Polyethylene glycol (PEG)

[0020]   The following amino acid abbreviations are used throughout this disclosure: A = Ala = alanine V = Val = Valine L = Leu = leucine I = Ile = Isoleucine P = Pro = proline F = Phe = phenylalanine W = Trp = tryptophan M = Met = methionine G = Gly = glycine S = Ser = Serine T = Thr = Threonine C = Cys = Cysteine Y = Tyr = tyrosine N = Asn = Asparagine Q = Gln = Glutamine D = Asp = aspartic acid E = Glu = glutamic acid K = Lys = lysine R = Arg = Arginine H = His = histidine

DETAILED DESCRIPTION OF THE INVENTION Crystal Form (s) and Process Applicants have produced crystals containing S. aureus EF-P which are suitable for X-ray crystallographic analysis. The three-dimensional structure of S. aureus EF-P was elucidated using high resolution X-ray crystallography. Preferably, the crystals have the orthorhombic space group symmetry P2 ₁ 2 ₁ 2 ₁ . More preferably, the crystal comprises unit cells that are rectangular in shape, each unit cell having a dimension a,
b and c, where a is about 25Å to about 50Å and b is about 35Å
To about 60Å and c is about 85Å to about 110Å; α = β = γ
= 90 °. The crystallized enzyme is a monomer and has one molecule in the asymmetric unit.

S. aureus EF-P purified at a concentration of about 1 mg / ml to about 50 mg / ml
Is, for example, about 0% to about 50% by weight of polyethylene glycol (PEG).
And having a number average molecular weight of between about 200 and about 20,000), containing from 0 to about 20% by weight of DMSO and buffered to a pH of about 3.5 to about 5.5. -Can be crystallized using the hanging drop method. Two
It is preferred to use a buffer having a pKa between 0.5 and 6.5. In a particularly preferred embodiment of the method, the buffer comprises about 10 mM to about 300 mM sodium acetate. Variations in buffer and buffer pH and other additives such as PEG will be apparent to those of skill in the art and can result in similar crystals.

In the present invention, a is about 25Å to about 50Å and b is about 35Å to about 60Å.
With dimensions a, b and c in which c is about 85Å to about 110Å; α
Further included are S. aureus EF-P crystals or S. aureus EF-P / ligand crystals that are isomorphic to S. aureus EF-P crystals characterized by a unit cell with = β = γ = 90 °.

X-Ray Crystallographic Analysis Recombinant S. aureus EF-P crystals (FIG. 1) were obtained from a crystallization screening solution containing 100 mM sodium acetate, pH 4.6 and 4% PEG 4000. Recombinant S. aureus EF-P used for crystallization contains a 6-residue polyhistidine tag at the C-terminus to facilitate purification of the recombinant protein. The improved conditions resulted in ideal crystal growth at pH 5.2-5.4. EF-P incorporating selenomethionine was prepared by down-regulation of methionine, since there was no cognate structure available for displacement of the molecule (TE Benson et al. Nat. Struct. Biol 2: 6
44-53 (1995); GD Van Duyne et al., J. Mol. Biol; 229: 105-24 (1993)).
. The selenomethionine EF-P was crystallized in the same manner as the natural protein, and these crystals were diffracted by a synchrotron (Advance Photon Source, Argonne, IL) to a resolution of 1.9Å. The crystal has a cell constant a = 37.5Å, b = 47.3Å, c = 9.
It belongs to the orthorhombic space group P2 ₁ 2 ₁ 2 ₁ having 7.5 Å and α = β = γ = 90 °.
The Patterson map of anomalous and dispersive differences (Fig. 2) revealed two of the four potential selenium sites, while the third site uses the cross difference Fourier method. Turned out through. The fourth site could not be localized by either method due to the multiple conformations of methionine as subsequently observed in the electron density map. Solvent flat (
As a result of maximum likelihood phasing with solvent-flattening, the electron density maps were separated but could be explained (Fig. 3). The structure is 29.0%
Refined to 25.6% R-factor with free R-factor of. Details of structure determination and refinement are given in Tables 3 and 4.

[0025]

[Table 3]

[0026]

[Table 4] Each construct amino acid of S. aureus EF-P was defined by the set of structural coordinates described in Figure 4. The term "structural coordinates" refers to Cartesian coordinates derived from a mathematical formula related to the pattern obtained on the diffraction of a monochromatic beam of X-rays by the atoms (dispersion center) of the S. aureus EF-P complex in crystalline form. . The diffraction data was used to calculate an electron density map of the repeating units of the crystal. . Then, using the electron density map, S. aureus
Establishes the position of individual atoms in the EF-P protein or protein / ligand complex.

Subtle changes in structural coordinates can be created by mathematically manipulating S. aureus EF-P structural coordinates. For example, the structural coordinates described in FIG. 4 are manipulated by crystallographic exchange of structural coordinates, division of structural coordinates, integer addition or subtraction from a set of structural coordinates, transformation of structural coordinates or any combination of the above. did it. Alternatively, modifications in the crystal structure due to amino acid mutations, additions, substitutions and / or deletions, or other changes in any of the components that make up the crystal may also give rise to variations in the structural coordinates. Such slight deformations in individual coordinates will have little effect on the overall shape. If such deformation is within an acceptable standard error compared to the original coordinates, the resulting three-dimensional shape is considered to be structurally equivalent. Structural equivalence is described in more detail below.

It should be noted that slight modifications in individual structural coordinates of S. aureus EF-P are not expected to significantly alter the properties of chemical groups such as ligands that can associate with the binding surface. In this context, the phrase “associated with” refers to the condition of proximity between a chemical group or portion thereof and a S. aureus EF-P molecule or portion thereof. The association may be non-covalent, where the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces or electrostatic interactions, or it may be covalent.

Thus, for example, bound to the binding surface of S. aureus EF-P or S. aureus
A ligand that interfered with the binding surface of EF-P would also be expected to bind or interfere with another binding surface that defines a shape whose structure coordinates are within acceptable error.

The amino acid numbering in the other isoforms of S. aureus EF-P is E
It will be readily apparent to those skilled in the art that it may differ from that of S. aureus EF-P expressed in E. coli.

The structural elongation factor P, which has an effect on function, is mainly composed of β-strands organized into three distinct domains (FIGS. 5 and 6). Domain 1 contains four antiparallel β-strands and a single turn of the α-helix. Both domains 2 and 3 contain putative oligonucleotides-
An antiparallel β-barrel that resembles the folding of an oligosaccharide bond (A. G.
. Murzin, EMBO. J .; 12: 861-67 (1993)). Further, domain 2 comprises a single turn of the 3 ₁₀ helix between strands β7 and beta 8. Cα from domains 2 and 3
Superposition produced a rms deviation of 1.55Å (Fig. 7). There are some unconserved loop regions (including the absence of a true helix region in domain 3),
The general folding is maintained. This fold was observed in many other proteins, some of which were IF1 (M. Sette et al., EMBO. J .; 16: 1436-4.
3 (1997)), CspA (W. Jiang et al., J. Biol. Chem .; 272: 196-202 (1997).
); H. Schindelin et al., Proc. Natl. Acad. Sci. USA; 91: 5119-23 (1994
)) And EF-Tu (P. Nissen et al., Science; 270: 1464-72 (1995)). This suggests that domains 2 and 3 of EF-P may play a role in interacting with RNA-possibly either tRNA or ribosomal RNA. Other structures that utilize this putative oligonucleotide-oligosaccharide bond fold are chains 1 and 2, chains 3 and alpha helices, or chains 4 and 5.
Shows specific interactions with their respective ligands with a loop between (AG
Murzin, EMBO. J .; 12: 861-67 (1993)). Identification of these residues within the two β-barrels for EF-P reveals potential sites for interaction with RNA (Figure 8). Based on evidence from related structures, the residues for S. aureus EF-P that can be associated with oligonucleotide binding include residue 7 from domain 2.
7-80, 99-105 and 117-120 and residue 1 from domain 3
49-150, 164-169 and 177-181. These residues correspond to the loops of chain 1 and chain 2 and chain 3 in the model beta barrel oligonucleotide binding folding described in AG Murzin, EMBO. J .; 12: 861-67 (1993).
To the loop of helix 1 and the loop between strands 4 and 5.

A notable feature of EF-P is its polarity of surface charge shown in the surface depiction (FIG. 9). Such polarities in proteins likely to interact with RNA indicate that the positively charged face of such polar proteins (FIG. 9a) will interact with negatively charged oligonucleotides. In addition, this surface delineation reveals that EF-P resembles the shape and dimension of tRNA (FIG. 10) (MA Rould et al., Science 2).
46: 1135-42 (1989)). This similarity in shape to tRNA can provide a hypothesis about the mechanism of action of EF-P. In the model of translation, three distinct sites on the large subunit of the ribosome-a P site for a tRNA containing an extended aminoacyl chain, an A site for an incoming tRNA and a deacylated t.
An E site for RNA has been proposed. During the first step of translation, the E site will be unoccupied because there is still no deacylated tRNA. One possibility is that EF-P could bind at the E site during translation initiation and act as a mimic on the deacylated tRNA. This function may have functioned to bring the ribosome into a more active early conformation that enhances the synthesis of the first peptide bond.

Comparison of EF-Ps to Related Structures Two related structures have been elucidated from archebacteria (M. jannaschii and P. aerophilum), which are more closely related to the human protein, eIF-5A (KK).
Kim et al., Proc. Natl. Acad. Sci. US A; 95: 10419-24 (1998); TS Peat
Et al., Structure; 6: 1207-14 (1998)). These archaeal proteins are
Although containing homology to domains 1 and 2 of EF-P, domain 3 is surprisingly absent (Figure 11). Although it is not clear why domain 3 is absent in the archaeal and eukaryotic sequences, but is present in E. coli and S. aureus EF-P,
The similarity between domain 3 or domain 2 and the oligonucleotide binding folds suggests that the interaction with rRNA or tRNA may be slightly different for E. coli and S. aureus. The superposition of domain 1 from S. aureus and M. jannaschii gives an rmsd for a C _α atom of 2.02Å, the superposition of domain 2 of these two structures is for a C _α atom of 3.02Å The rmsd
Was given. The superposition of domain 1 from S. aureus and P. aerophilum is 3.
Given the rmsd for a C _α atom of 29Å, the domain 2 of these two structures
Superposition gave a rmsd for a C _α atom of 4.25Å. Although there are good agreements individually for each domain, the relative orientation of domains 1 and 2 is
It is quite flexible as shown in FIGS. EIF from M. jannaschii
A second crystal form of 5A has been solved, which revealed some flexibility in the relative orientation of domains 1 and 2 (KK Kim et al., Proc. Natl. Acad. Sci.
US A; 95: 10419-24 (1998); TS Peat et al., Structure; 6: 1207-14 (19
98)). Since the inventors only clarified one crystal, this linker is S. aureu
s It is unclear how flexible EF-P is.

The structures of S. aureus, M. jannaschii and P. aerophilum are such that the highly conserved motif within domain 1-xKxGKGxA (which in yeast and humans (also called deoxyhypusine) N ^ε- (4- Aminobutyl) lysine) was identified as the site for post-translational modification of the second lysine). This modification is essential for cell survival in yeast (J. Schnier et al., Mol. Cell. Biol; 11: 3105-14.
(1991)), and it was shown to occur only in eIF5A by the enzyme deoxyhypusine synthase (DI Liao et al., Structure; 6: 23-32 (1998)). The position of this conserved loop in EF-P lies between β2 and β3 and is K3 of S. aureus EF-P.
3 is a conserved residue, which is modified in the eukaryotic EF-P homolog. This revealed lysine is shown in Figure 14 and projects out of this loop. In bacteria, no K33 amino acid hypusination of the cognate EF-P was observed, and in fact, an electron density analysis for this residue in S. aureus EF-P revealed that
It does not reveal any evidence of post-translational modifications in this recombinant protein. EF-P from archaea
Similar studies of electron density on homologues did not reveal any modifications (KK Kim et al., Proc. Natl. Acad. Sci. US A; 95: 10419-24 (1998); T.
S. Peat et al., Structure; 6: 1207-14 (1998)). Mass spectrometry of S. aureus EF-P samples (both methionine and selenomethionine) did not reveal any additional mass that might be explained by the post-translational modification. S. aureus EF-P is EF
Since it was purified from the -P overexpressing strain, it is possible that the modification pathway was overwhelmed and the post-translational modification could not be performed sufficiently. Also, there is no direct evidence that hypusination occurs in bacteria, which suggests that another type of modification is S. aureus.
It may be included in EF-P.

Binding Surfaces and Other Structural Features Applicants' invention, for the first time, provided information on the shape and structure of the putative oligonucleotide binding surface of S. aureus EF-P.

Bonding surfaces have considerable utility in areas such as drug discovery. The association of natural ligands or substrates with their corresponding receptor or enzyme binding surface is the basis of many biological mechanisms of action. Similarly, many drugs exert their biological effects through the association of receptors and enzymes from the binding surface. Such association can occur with all or any portion of the binding surface. An understanding of such associations will help lead to drug designs with more favorable association with their targets, and then improved biological effects. Therefore, this information is valuable for designing potential inhibitors of S. aureus EF-P-like binding surfaces, as described in more detail below.

By “molecular complex” is meant a protein that is in a covalent or non-covalent association with a chemical moiety. The term "binding surface" as used herein refers to a region of a molecule or molecular complex that, as a result of its shape, advantageously associates with another chemical group.

The amino acid component of the S. aureus EF-P oligonucleotide binding surface as defined herein as well as its selected constituent atoms are located in three dimensions according to the structural coordinates listed in FIG. In one embodiment, the structural coordinates of the S. aureus EF-P binding surface include the structural coordinates of all atoms in the constituent amino acids; in another embodiment, the structural coordinates of the S. aureus EF-P binding surface. Includes the structural coordinates of only the skeletal atoms of the constituent atoms.

A particular chemical group can be attached to an amino acid surface residue of any of S. aureus EF-P as listed in Table 1. Preferably, the surface residue comprising the binding surface comprises amino acid K33. More preferably, the surface residues comprising the binding surface include those residues located within 4Å, preferably within 7Å, more preferably within 10Å and most preferably within 15Å of K33 as listed in Table 2. .

The term “S. aureus EF-P-like binding surface” refers to at least the binding surface of S. aureus EF-P such that its shape is expected to bind a common or structurally related ligand. A portion of a molecule or molecular complex that is sufficiently similar to a portion. A structurally equivalent bond surface is at most about 1.9Å (as shown in Figure 4) S. aureus
It is defined by the root mean square deviation from the structural coordinates of the backbone atoms of the amino acids that make up the binding surface on EF-P. How this calculation is obtained will be described later.

Accordingly, the present invention, as defined by the set of structural coordinates above, provides a S. a.
A molecule or molecular complex comprising a ureus EF-P oligonucleotide binding surface or a S. aureus EF-P-like binding surface is provided.

The three-dimensional arrangement X-ray structural coordinates define a unique arrangement of points in space. Those skilled in the art will appreciate that the set of structural coordinates for a protein or protein / ligand complex or a portion thereof defines the set of relative points, which in turn defines the three-dimensional arrangement. Similar or identical arrangements may be defined by a completely different set of coordinates, provided that the distances and angles between the coordinates remain substantially the same. In addition, the measurable placement of the points can be defined by increasing or decreasing the distance between the coordinates by a scalar factor while keeping the angles substantially the same.

Thus, the present invention provides a measurable three-dimensional arrangement of points derived from the structural coordinates of at least a portion of the molecule or molecular complex of S. aureus EF-P shown in FIG. 4, as well as the structural equivalents described below. It includes various arrangements. Preferably, the three-dimensional arrangement is S. aureu
s Points derived from structural coordinates that represent the positions of multiple amino acids that define the EF-P binding surface are included. In one embodiment, the measurable three-dimensional arrangement has structural coordinates that represent the positions of the plurality of amino acids that define the S. aureus EF-P binding surface, preferably the backbone atoms of those amino acids listed in Table 1. The points derived from are included. In another embodiment, the measurable three-dimensional configuration includes S. aureus EF-P binding surfaces, preferably side chains and backbone atoms (hydrogen) of a plurality of amino acids defining those amino acids listed in Table 1. Points other than) are included that are derived from the structural coordinates that represent the position. Alternatively, the measurable three-dimensional arrangement of the points is determined by the amino acid backbone atom within 4 Å, preferably within 7 Å, more preferably within 10 Å and most preferably within 15 Å of Lys33 shown in Table 2, and optionally the side chain. Derived from the structural coordinates that represent an atom (other than hydrogen).

Similarly, the invention also includes three-dimensional arrangements of points derived from molecules or molecular complexes that are structurally homologous to S. aureus EF-P, as well as structurally equivalent arrangements. Structurally homologous molecules or molecular complexes are defined below. Advantageously, structurally homologous molecules can be identified using the structural coordinates of S. aureus EF-P according to the method of the invention (Figure 4).

The arrangement of points in space derived from the structural coordinates according to the invention can be visualized, for example, as a holographic image, a stereogram, a model or a computer-depicted image, and thus the invention also relates to such an image, figure or model. Include.

Structurally Equivalent Crystal Structures Using various computer analyses, the molecule or its binding surface moieties are defined by their three-dimensional structure into S. aureus EF-P or all or part of its binding surface. Alternatively, it may be determined whether or not it is "structurally equivalent." Such analysis is
UANTA (Molecular Simulations Inc., San Diego, CA) version 4.1
Modern software applications, such as the Molecular Similarity application, can be performed as described in the accompanying User Guide.

The Molecular Similarity application allows comparison between different structures, different conformations of the same structure, and different parts of the same structure. The process used in Molecular Similarity to compare structures is divided into four steps: (1) loading the structures to be compared; (2) atomic equivalence (at) in these structures.
om equivalence); (3) perform fitting operation; then (4) analyze the results.

Each structure is identified by a name. One structure was identified as the target (ie, fixed structure); all remaining structures are working structures (ie, moving structures). QUANT
For the purposes of the present invention, the equivalent atoms are the protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared, since the atom equivalents in A are defined by user input. ). For this purpose a conserved residue is defined as a set of atoms with the same interatomic connectivity. Consider only exact fitting operations.

When using the exact fitting method, the working structure is transposed and rotated to obtain the best fit with the target structure. The fitting operation uses an algorithm that computes the optimal transitions and rotations that fit the moving structure such that the root mean squared difference of the fits over a particular pair of equivalent atoms has an absolute minimum. This number, obtained in Angstroms, is reported by QUANTA.

For the purposes of the present invention, when superposed on a suitable skeletal atom described by the reference structural coordinates set forth in FIG.
), Any molecule or molecular complex or their binding surface, or any portion thereof having a root mean square deviation of), is considered to be "structurally equivalent" to the reference molecule. That is, the crystal structures of these portions of the two molecules are substantially the same, within acceptable error. Particularly preferred structurally equivalent molecules or molecular complexes are those defined by the full set of structural coordinates of Figure 4 less than about 1.9 Å from the conserved backbone atoms of these amino acids ± root mean square deviation. More preferably, the root mean square deviation is less than about 1.0Å.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations. It is a way of expressing deviations or variables from trends or goals. For purposes of the present invention, "root mean square deviation" refers to S. aureus EF-P as described herein.
Defines the variables in the protein backbone from the S. aureus EF-P backbone or its binding surface moieties, as defined by the structural coordinates of

Equipment A readable storage medium for all or part of the S. aureus EF-P or S. aureus EF-P / ligand complex, for structurally homologous molecules as defined below, or as defined above. The conversion of structural coordinates for the structural equivalence of any of these molecules or molecular complexes into a three-dimensional graphic representation of the molecule or complex can be conveniently accomplished through the use of commercially available software.

Thus, the present invention further provides a device-readable storage medium comprising device-readable data-encoded data storage material, which is a device programmed with instructions for using the data. Can be used to display a graphical three-dimensional depiction of any of the molecules or molecular complexes of the invention described above. In a preferred embodiment, the device-readable data storage medium includes a data storage material that encodes device-readable data, which, when used with a device programmed with instructions for using the data, A graphical three-dimensional depiction of a molecule or molecular complex that includes all or a portion of the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface defined in. In another preferred embodiment, the device-readable data storage medium is a molecule or molecule defined by the root mean square deviation from the skeletal atoms of the amino acids of the structural coordinates of all amino acids ± about 1.9Å or less in FIG. A graphic three-dimensional depiction of the complex can be displayed.

In another embodiment, the instrument-readable data storage medium comprises a first set of instrument-readable data-encoding data storage materials including a Fourier transform of the structural coordinates set forth in FIG. This is combined with the second set of instrument readable data containing the X-ray diffraction pattern of the molecule or molecular complex when using the instrument programmed instructions for using the second instrument. At least a portion of the structural coordinates corresponding to the readable data may be determined.

For example, a system for reading storage media includes a central processing unit (“CPU”), such as RAM (random access memory) or “core”, all interconnected by a conventional bidirectional system bus. Working memory, which can be memory, (one or more disk drives or CD-RO
Mass storage memory (such as M drive), one or more cathode ray tubes (“C
RT ”) display terminal, liquid crystal display (“ LCD ”)), electric fluorescent display, vacuum fluorescent display, field emission display (“ F ”)
ED "), plasma display, projection panel, etc.), one or more user input devices (eg, keyboard, microphone, mouse, touch screen, etc.), one or more input lines, and one or more output lines. Computers including are included. The system can be a single computer or networked to other systems (eg, computers, hosts, servers, etc.) (eg, local area networks, wide area networks, intranets, extranets or the Internet). The system also includes additional computer controls such as consumer electronics and appliances.

Input hardware can be coupled to the computer by input lines and implemented in a variety of ways. The device readable data of the present invention can be entered through the use of a modem or multiple modems connected by telephone lines or dedicated data lines. Alternatively or in addition, the input hardware may include a CD-ROM drive or disk drive. A keyboard can be used as an input device in contacting the display terminal.

The output hardware can be connected to a computer by an input line and can be executed by an ordinary device. For example, the output hardware includes a display device for displaying a graphical depiction of the bonding surface of the invention using a program such as QUANTA as described herein. Also, output hardware may include a printer, or disk drive, so that hardcopy output may be created to store system output for later use.

In operation, the CPU coordinates the use of various input and output devices,
Corresponds to data access from bulk storage and to and from working memory and determines the sequence of data processing steps. Multiple programs may be used to process the instrument readable data of the present invention. Such programs are discussed with reference to the drug discovery calculation methods as described herein. Specific references to hardware system components are included where appropriate throughout the following description of data storage media.

Instrument-readable storage media useful in the present invention include, but are not limited to, magnetic devices, electronic devices, optical devices, and combinations thereof. Examples of such data storage media include, but are not limited to, hard disk devices, CD devices, digital video disk devices, flexible disk devices, movable hard disk devices, magneto-optical disk devices, magnetic tape devices, flash memory devices. ,
Includes bubble memory devices, holographic devices and any other peripheral devices. These devices include the necessary hardware (eg, drives, controllers, power supplies, etc.) as well as any necessary media (eg, disks, flash cards, etc.) that allow the storage of data.

Structurally Homologous Molecules, Molecular Complexes, and Crystal Structures The structural coordinates listed in FIG. 4 can be used to help obtain structural information about another crystallized molecule or molecular complex. The methods of the present invention can determine at least a portion of the three-dimensional structure of a molecule or molecular complex that contains one or more structural features similar to those of S. aureus EF-P. These molecules are referred to herein as "structurally homologous" to S. aureus EF-P. Similar structural features include, for example, regions of amino acid identity, conserved active or binding site motifs,
And similarly arranged secondary structure elements such as α-helices and β-sheets. If desired, structural homology may be used to align the residues of two amino acid sequences so that the amino acids in each sequence remain in their proper order, but with identical amino acids along the length of those sequences. , By determining the gaps that allow either or both sequences in creating an alignment to optimize the number of identical amino acids. Preferably Ta
tusova et al., FEMS Microbiol. Lett. 174: 247-250 (1999), ht
The amino acid sequences of two are compared using the BLASTp program, version 2.0.9 of the BLAST2 search algorithm, available at tp: //www.ncbi.nlm.nih.gov/gorf/bl2.html. Preferably, matrix = BLOSUM62; open gap penalty value = 11, extension gap penalty value = 1, gap x Dropoff value = 50
, All BLAS including expected number threshold = 10, word size = 3 and filter ON
Use the default values for the T2 search parameters. Structural similarity is referred to as "identity" in a comparison of two amino acid sequences using the BLAST search algorithm. Preferably, the structurally homologous molecules are amino acid sequences of S. aureus EF-P (
A protein having an amino acid sharing at least 65% identity with SEQ ID NO: 1). More preferably, the protein structurally homologous to S. aureus EF-P includes S. a
At least one contiguous stretch of at least 50 amino acids that shares at least 80% amino acid sequence identity with a similar portion of ureus EF-P is included. Methods for generating structural information about structurally homologous molecules or molecular complexes are well known and include, for example, molecular replacement techniques.

Accordingly, in another embodiment, the invention provides: (a) crystallizing a molecule or molecular complex of unknown structure; (b) X-rays from the crystallized molecule or molecular complex. Generating a diffraction pattern; and (c) applying at least a portion of the structural coordinates shown in FIG. 4 to the X-ray diffraction pattern to create a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown. Provided is a method for obtaining structural information about a molecule or a molecular complex whose structure is unknown by utilizing molecular substitution, which includes steps.

By using molecular replacement, all or part of the structural coordinates of the S. aureus EF-P or S. aureus EF-P / ligand complex provided by the invention (eg, as listed in FIG. 4). Can be used to determine the structure of crystallized molecules or molecular complexes whose structure is unknown, more quickly and efficiently than attempts to determine such information from scratch.

Molecular replacement provides an accurate estimate of phase for unknown structures. Phase is a factor in the equation used to solve crystal structures that cannot be directly determined. Obtaining accurate values for the phase by methods other than molecular replacement is a time consuming process involving iterative cycles of approximation and refinement, and significantly impedes crystal structure elucidation. However, when the crystal structure of a protein containing at least structurally homologous portions is elucidated, it provides a satisfactory estimate of the phase of the unknown phase from the known structure.

Thus, the method provides for the unknown molecular or molecular complex to maximize the observed X-ray diffraction pattern of a crystal of the unknown molecular or molecular complex of its structure. S. aureus EF-P or S. a according to Fig. 4 in the unit cell of the crystal of
Orienting or locating relevant parts of the ureus EF-P / ligand complex to create a preliminary model of a molecule or molecular complex whose structural coordinates are unknown. The phase is then calculated from this model and combined with the observed X-ray diffraction pattern amplitude to create an electron density map of the structure whose coordinates are unknown. It can then be subjected to any of the well known model building and structure refinement techniques to obtain the final precise structure of an unknown crystallized molecule or molecular complex (E. Lattman, "Use of the Rotation and
Translation Functions "in Meth. Enzymol., 115, pp. 55-77 (1985); M. G
. Rossman, "The Molecular Replacement Method", Int. Sci. Rev. Ser., No.
. 13, Gordon & Breach, New York (1972)).

Structural information for a portion of any crystallized molecule or molecular complex that is sufficiently structurally homologous to a portion of S. aureus EF-P may be elucidated by this method. In addition to the molecules that share one or more structural features with S. aureus EF-P, it has the same biological activity as S. aureus EF-P, such as catalytic activity, substrate specificity or ligand binding activity. The molecule with may also be sufficiently structurally homologous to S. aureus EF-P and the structural coordinates of S. aureus EF-P may be used to solve its crystal structure.

In a preferred embodiment, the method of molecular replacement is utilized to obtain structural information about a molecule or molecular complex, wherein the molecule or molecular complex is at least one S. aur.
Contains subunits or homologues of eus EF-P. The "subunit" of S. aureus EF-P refers to S. aureu truncated at the N-terminus or C-terminus, or both.
s EF-P molecule. In the context of the present invention, a "homolog" of S. aureus EF-P means
Substitution of one or more amino acids in the amino acid sequence of S. aureus EF-P,
It contains deletions, additions or rearrangements but is or reasonably expected to show at least part of the third (three-dimensional) structure of S. aureus EF-P when folded into its native conformation. Protein. For example, a structurally homologous molecule may comprise a deletion or addition of one or more contiguous or non-contiguous amino acids such as loops or domains. Structurally homologous molecules also include acetylation, hydroxylation, methylation, amidation and side-chain modifications, including carbohydrate or lipid groups, cofactor attachment, backbone modifications and N- and C-terminal modifications. A "modified" S that is chemically or enzymatically derivatized with one or more constituent amino acids, including S.
Also includes aureus EF-P.

Heavy atom derivatives of S. aureus EF-P are also included as S. aureus EF-P homologues.
The term "heavy atom derivative" refers to a derivative of S. aureus EF-P made by chemically modifying a crystal of S. aureus EF-P. In practice, the crystals are immersed in a solution containing a heavy metal atom salt or an organometallic compound capable of diffusing through the crystals and binding to the surface of the protein, such as lead chloride, gold thiomalate, thimerosal or uracil acetate. The position (s) of the bound heavy metal atom (s) can be determined by X-ray diffraction analysis of the soaked crystals. This information is then used to generate the topological information used to build the three-dimensional structure of the protein (TL Blundell and NL Johnson,
Protein Crystallography, Academic Press (1976)).

Since S. aureus EF-P was expected to crystallize in more than one crystal form, the structural coordinates of S. aureus EF-P provided by the present invention are S. aureus EF-P or S. aureus EF-P. aureus
It is particularly useful for elucidating other crystal forms of the EF-P / substrate complex.

The structural coordinates of S. aureus EF-P provided by the present invention are particularly useful for elucidating the structure of S. aureus EF-P mutants. Mutants can be prepared, for example, by expressing a cDNA of S. aureus EF-P that has been previously modified in its coding sequence by oligonucleotide site-directed mutagenesis. Mutants were also engineered by site-specific incorporation of unnatural amino acids into the EF-P protein using the general biosynthetic method of CJ Noren et al., Science, 244: 182-188 (1989). Can be generated. In this method, the codon encoding the amino acid of interest in wild type S. aureus EF-P is replaced with the "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis. The sublesser tRNA directed against this codon is then aminoacylated with the desired unnatural amino acid in vitro. Then the aminoacylated tR
NA was added to the translation system in vitro to yield mutant S. aureus EF-P with its site-specific incorporated unnatural amino acid.

Selenocysteine or selenomethionine are S. cerevisiae in auxotrophic E. coli strains.
Expression of a cDNA encoding aureus EF-P results in wild-type or mutant S.
Can be embedded in aureus EF-P (WA Hendrickson et al., EMBO J., 9 (5): 166)
5-1672 (1990)). In this way, wild-type or mutated S. aure
us EF-P cDNA is depleted in native cystine and / or methionine (or both) but enriched in selenocysteine and / or selenomethionine (or both) host organisms on growth media. Can be expressed in. Alternatively, selenomethionine analogs can be prepared by downregulated methionine biosynthesis (TE Benson et al., Nat. Struct. Biol., 2: 644-53 (1995); GD.
Van Duyne et al., J. Mol. Biol. 229: 105-24 (1993)).

The structural coordinates of S. aureus EF-P in FIG. 4 show S. aureus EF-P co-complexing with various chemical groups.
It is also particularly useful for elucidating the crystal structure of aureus EF-P, S. aureus EF-P mutants or S. aureus EF-P homologues. With this approach, candidate S. aureu
The optimal site for interaction between chemical groups, including s EF-P inhibitors and S. aureus EF-P, can be determined. Potential sites for modification within the various binding sites of the molecule may also be identified. This information provides additional tools to determine the most efficient binding interactions, such as, for example, the highly hydrophobic interactions between S. aureus EF-P and chemical groups. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvents can determine where each type of solvent molecule resides. Small molecules that bind tightly to these sites can then be designed and synthesized and tested for their S. aureus EF-P inhibitory activity.

All the complexes mentioned above can be examined using the well-known X-ray diffraction technique, X-PLOR (Yale University, 81992, Molecular Simulati
distributed by ons, Inc .; eg, Blundell and Johnson, supra; Meth. Enzymol.
., Vol. 114 and 115, edited by HW Wyckoff et al., Academic Press (1985)) using computer software to obtain R values of about 0.20 or less and to obtain 1.5-3Å resolution X-ray data. Can be refined. Therefore, this information can be used to optimize known S. aureus EF-P inhibitors,
And, more importantly, new S. aureus EF-P inhibitors can be designed.

The present invention provides a unique tertiary order defined by a set of points defined by the structural coordinates of a molecule or molecular complex structurally homologous to S. aureus EF-P determined using the method of the invention. It also includes a magnetic storage medium containing an original configuration, a structurally equivalent configuration, and a set of such structural coordinates. Further, the invention includes structurally homologous molecules identified using the methods of the invention.

Homology Modeling Homology modeling can be used to construct or refine computer models of S. aureus EF-P homologs without crystallizing the homolog. First, S. a
A preliminary model of the ureus EF-P homologue is obtained by sequence alignment with S. aureus EF-P, secondary structure prediction, screening of structural libraries, or any combination of these techniques. Computer software can be used to perform sequence alignments and secondary structure predictions. Structural inconsistencies, eg, structural fragments around insertions and deletions, can be modeled by screening structural libraries for peptides of the desired length and proper conformation. For side chain conformation prediction, side chain rotamer libraries may be utilized. If the S. aureus EF-P homolog has been crystallized, the final homology model can be used to elucidate the crystal structure of the homolog by the molecular substitutions described above. Next, the preliminary model is subjected to energy minimization to obtain an energy minimization model. The energy minimization model can include regions where stereochemical constraints are disturbed, in which case such regions are remodeled to obtain the final homology model. The homology model is localized according to the results of molecular replacement and subjected to further refinement including molecular dynamics calculations.

Rational Drug Design Computer-engineered techniques are used to screen, identify, screen, and / or design chemical moieties or structurally homologous molecules that can associate with S. aureus EF-P. You can The knowledge of the structural coordinates for S. aureus EF-P indicates that S. a
The design and / or identification of synthetic and / or other molecules having a shape complementary to the conformation of the ureus EF-P binding site is acceptable. In particular, computational techniques can be used to identify or design chemical moieties, such as inhibitors, agonists and antagonists, that associate with the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface. The inhibitor can bind to all or part of the active site of S. aureus EF-P and can be a competitive, non-competitive or non-competitive inhibitor; or a dimer by binding to the interface between two monomers. Can interfere with the conversion. For example, an inhibitor that binds to the binding surface of S. aureus EF-P can interfere with the binding of S. aureus EF-P to ribosomal proteins or ribosomal RNA during translation. Once identified and screened for biological activity,
These inhibitors / agonists / antagonists are used therapeutically or prophylactically to block S. aureus EF-P activity, thus inhibiting bacterial growth and causing cell death. Also, structure-activity data for analogs of ligands that bind or interfere with S. aureus EF-P or S. aureus EF-P-like binding surfaces can be obtained by computer manipulation.

As used herein, the phrase “chemical group” refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes. The chemical moieties determined to associate with S. aureus EF-P are potential drug candidates.

Thus, a device capable of displaying a graphical three-dimensional depiction of the structure of S. aureus EF-P or a structurally homologous molecule or portion thereof identified herein-a readable storage medium. The data stored in can be advantageously used for drug discovery. The structural coordinates of the chemical groups are used to create a three-dimensional image that can be computationally fitted to the three-dimensional image of S. aureus EF-P or a structurally homologous molecule. The three-dimensional molecular structure encoded by the data in the magnetic storage medium can then be computationally evaluated for its ability to associate with a chemical moiety. Displaying the molecular structure encoded by the data in a graphical three-dimensional representation on a computer screen also allows the protein structure to be visually inspected for potential association with chemical moieties.

One embodiment of a method of drug design includes S. aureus EF-P or a structurally homologous molecule, especially S. aureus EF-P binding surface or S. aureus EF-P-like binding surface. , Assessing potential associations with known chemical groups. Thus, methods of drug design include computationally assessing the potential of selected chemical moieties for association with any of the aforementioned molecules or molecular complexes. In this method, (a) a computer operation means is used to perform a fitting operation between the selected chemical atomic group and the binding surface of the molecule or the molecular complex; and then (
b) analyzing the results of the fitting operation to quantify the association between the chemical moieties and the binding surface.

In another embodiment, methods of drug design include computer-aided design of chemical moieties associated with S. aureus EF-P, its homologs, or portions thereof. Chemical groups can be designed in a step-wise fashion, one fragment at a time, or as a whole or "de novo".

In order to be a valuable drug candidate, a chemical moiety identified or designed according to the method is structurally associated with at least a portion of a S. aureus EF-P or S. aureus EF-P-like binding surface. Therefore, it must be able to sterically or energetically predict a conformation that allows it to associate with S. aureus EF-P or S. aureus EF-P-like binding surfaces. Noncovalent molecular interactions important for this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. Conformations to consider include the overall three-dimensional structure and orientation of the chemical group with respect to the binding surface, and the groups of groups that interact directly with the S. aureus EF-P-like binding surface or its homologues. Includes spacing between various functional groups.

Optionally, the potential attachment of the chemical moiety to the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface is determined by the actual synthesis and testing of the chemical moiety.
Analyze using computer modeling technology. If these computational experiments suggest insufficient interaction and association between it and the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface, testing of the group is unnecessary. Is. However, if computer modeling shows strong interactions, the molecule is synthesized and tested for its ability to bind or interfere with the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface. obtain. The compound is actually S. aureus
Binding assays to determine if EF-P binds can also be performed and are well known in the art. The binding assay uses a very wide range of analyses, including microcalorimetry, circular dichroism spectroscopy, capillary zone electrophoresis, nuclear magnetic resonance, fluorescence emission and combinations thereof, to determine kinetic or Thermodynamic methods can be used.

One of ordinary skill in the art would use one of several methods to obtain S. aureus EF-P or S. aureus EF-P.
Chemical groups or fragments can be screened for their ability to associate with an aureus EF-P-like binding surface. This process is, for example, S. aureus EF-P in FIG.
S. aureus EF-P or S. on a computer screen based on structural coordinates or other coordinates that define similar shapes created from an instrument-readable storage medium.
It can be initiated by visual inspection of the aureus EF-P-like binding surface. The selected fragments or chemical groups can then be located or docked in various orientations within the binding surface. Docking can be done using software such as QUANTA and SYBYL, followed by standard molecular mechanics force fields such as CHARMM and AMBER and energy minimization and molecular dynamics.

Professional computer programs may also assist in the process of selecting fragments or chemical groups. Examples include GRID (PJ Goodford, J.
Med. Chem. 28: 849-857 (1985); available from Oxford University, Oxford, UK); MCSS (A. Miranker et al., Proteins: Struct. Funct. Gen., 11: 29-34.
(1991); available from Molecular Simulations, San Diego, CA); AUTOD
OCK (DS Goodsell et al., Proteins: Struct. Funct. Genet. 8: 195-202 (1
990); available from Scripps Research Institute, La Jolla, CA); and D
OCK (ID Kuntz et al., J. Mol. Biol. 161: 269-288 (1982); University o
f available from California, San Francisco, CA).

Once the appropriate chemical groups or fragments have been selected, they can be assembled into a single compound or complex. Assembly can be done first by visually examining the relationship of the fragments to each other in a three-dimensional image displayed on a computer screen with respect to the structural coordinates of S. aureus EF-P. Following this, QUANTA or SYBYL (Tripos Associates, St. Louis, MO)
Manual model building will be performed using software such as.

Programs useful in aiding one of skill in the art in associating individual chemical groups or fragments include, but are not limited to, CAVEAT (P.
A. Bartlett et al., In "Molecular Recognition in Chemical and Biological Pro
blems ", Special Publ., Royal Chem. Soc., 78: 182-196 (1989); G. Lauri
Et al., J. Comput. Aided Mol. Des. 8: 51-66 (1994)); University of Califo
Available from rnia, Berkeley, CA); ISIS (MDL Information Systems, San
Available from Leandro, CA; YC Martin, J. Med. Chem. 35: 2145-2154 (199
2) and HOOK (MB Eisen et al., Proteins: Struc., Funct., Gene.
t. 19: 199-221 (1994); available from Molecular Simulations, San Diego, CA).

S. aureus EF-P binding compounds are designed “de novo” with an empty binding site or optionally with some portion (s) of known inhibitor (s). You can LUDI (H.-J. Bohm, J. Comp. Aid. Mo
lec. Design. 6: 61-78 (1992); Molecular Simulations Inc., San Diego, C
A.); LEGEND (Y. Nishibata et al., Tetrahedron, 47: 8985 (19
91); Available from Molecular Simulations Inc., San Diego, CA); LeapFrog
(Available from Tripos Associates, St. Louis, MO); and SPROUT (V.
Gillet et al., J. Comput. Aided Mol. Design 7: 127-153 (1993); University.
There are many de novo-ligand design methods, including available from Leeds, UK).

Once compounds have been designed or selected by the methods described above, the computational efficiency of the ability of the groups to bind to the S. aureus EF-P binding surface or the S. aureus EF-P-like binding surface has been evaluated. Can be tested and optimized by. For example, valid
The S. aureus EF-P or S. aureus EF-P-like binding surface inhibitor is preferably
It must exhibit a relatively small difference in energy between its bound and free states (ie, a small deformation energy of the bond). Therefore, the most efficient S. aureus EF-P or S. aureus EF-P-like binding surface inhibitors should preferably be designed with binding deformation energies of about 10 kcal / mol or less; more preferably 7 kcal / mol or less. Is. S. aureus EF-P or S. aureus EF-P-like binding surface inhibitors can interact with the binding surface in more than one conformations that are similar in total binding energy. In these cases, the binding deformation energy is considered to be the difference between the energy of the free radical and the average energy of the conformation observed when the inhibitor binds to the protein.

An atomic group designed or selected to bind to a S. aureus EF-P binding surface or a S. aureus EF-P-like binding surface further comprises a target enzyme and surrounding water molecule, preferably in its bound state. It can be computer-optimized to lack repulsive electrostatic interactions with. Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.

Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for this purpose are: Gaussian 94, revision C (MJ Frisch, Gaussian, Inc., Pit
tsburgh, PA C1995); AMBER, version 4.1 (PA Kollman, University
of California at San Francisco, C 1995); QUANTA / CHARMM (M
olecular Simulations, Inc., San Diego, CA C 1995); Insight II / Discover
(Molecular Simulations, Inc., San Diego, CA C 1995); DelPhi (Molecular
Simulations, Inc., San Diego, CA C 1995); and AMSOL (Quantum Ch
emistry Program Exchange, Indiana University) is included. These programs include, for example, "IMPACT" graphics and Silicon Graphic such as Indigo ^2.
s workstation. Other hardware systems and software packages will be known to those skilled in the art.

Another approach encompassed by the present invention is S. aureus EF-P or S. aur
eus EF-P-like binding surface is computationally screened a small molecule database for chemical groups or compounds that can bind in whole or in part. In this screen, the quality of the fitting of such groups to the binding site can be judged either by shape complementarity or putative interaction energies (EC Meng et al., J. Comp. Chem. 13: 505-524 (1992). ).

The present invention also allows for the development of chemical groups that can isomerize to short-lived reaction intermediates in the chemical reaction of substrates or other compounds that bind to or bind to S. aureus EF-P. A time-dependent analysis of structural changes in S. aureus EF-P during its interaction with other molecules is performed. The reaction intermediate of S. aureus EF-P is S. aureus EF.
It can be estimated from the reaction product in the co-complex with -P. Such information is known
Designed improved analogs of S. aureus EF-P inhibitors, or S. aureus EF-P
And is useful in designing a new class of inhibitors based on the reaction intermediates of inhibitor co-complexes. It is a S. aureu that has both high properties and stability.
s Provides a new route for designing EF-P inhibitors.

Yet another approach to rational drug design is to explore the S. aureus EF-P crystals of the invention with molecules containing a variety of different functional groups to identify candidate S. aureus EF-P inhibitors. It involves determining the optimal site of interaction with the protein. For example, high resolution X-ray diffraction data collected from crystals soaked in or co-crystallized with other molecules allows for determination of which type of solvent molecule does not leave. Molecules that bind tightly to these sites can then be further modified and synthesized and tested for their herpes protease inhibitor activity (J. T.
ravis, Science, 262: 1374 (1993)).

In a related approach, iterative drug design is used to identify inhibitors of S. aureus EF-P activity. Iterative drug design is a method of optimizing the association between proteins and compounds by determining and assessing the three-dimensional structure of a continuous set of protein / compound complexes. In iterative drug design, crystals of a series of protein / compound complexes are obtained, and then the three-dimensional structure of each complex is solved. Such an approach provides insights into the association between the protein and compound of each complex. It selects compounds for their inhibitory activity and obtains crystals of this new protein / compound complex,
This is done by elucidating the three-dimensional structure of the complex and then comparing the association between the new protein / compound complex and the previously elucidated protein / compound complex. By observing how changes in compounds affected protein / compound associations, these associations can be optimized.

The compound identified or designed as a result of any of these methods can be obtained (or synthesized) and then its biological activity, eg, S. aureu.
s Can be tested for inhibition of EF-P activity.

Pharmaceutical Composition The pharmaceutical composition of the present invention comprises an inhibitor of S. aureus EF-P activity identified according to the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle. . The term "pharmaceutically acceptable carrier" refers to carrier (s) "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to its recipient. The pH of the formulation, if desired, is adjusted with pharmaceutically acceptable acids, bases or buffers to improve the stability of the formulated compound or its delivery form.

Also included in the invention are methods of making and using such pharmaceutical compositions. The pharmaceutical composition of the present invention is administered orally, parenterally, by inhalation spray, topically, rectally,
Administration may be intranasal, buccal, vaginal or via an implanted container. Oral administration or administration by injection is preferred. The term parenteral as used herein includes subcutaneous,
Intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques are included.

About 0.01 to about 100 mg / kg body weight / day, preferably about 0.5 to about 7
A dose level of 5 mg / kg body weight / day of the S. aureus EF-P inhibitor compound described herein is useful for the prevention and treatment of S. aureus EF-P mediated diseases. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 5 times per day, or alternatively, as a continuous infusion. Such administration may be used as a chronic or acute therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form is
It will vary depending on the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Preferably, such preparations contain from about 20% to about 80% active compound.

In order that the invention may be more fully understood, the following examples are set forth. These examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0099]   Example   Example 1: Analysis of the structure of S. aureus EF-P   EF-P expression and selenomethionine uptake   Escherichia coli construct M15 (pREP4) (pQE-6 expressing S. aureus EF-P
0 EF-P) was obtained from Human Genome Sciences. Derived from S. aureus EF-P
Various genes and polypeptides are published in WO 0012678. EF-
For the preparation of selenomethionine analogs of P, the construct was a source of carbon and nitrogen.
And NH as_FourGrow in minimal salt medium M9 with Cl. Next
Thus, endogenous methionine biosynthesis was paired with growth medium just prior to IPTG induction of EF-P synthesis.
And added selenomethionine in excess (T. E. Benson et al., Nat. Str.
uct. Biol 2: 644-53 (1995); G. D. Van Duyne et al., J. Mol. Biol; 229: 105-
24 (1993)). The basic M9 medium formulation is Na in 1 L of deionized water.₂HPO_Four, 6 g; KH₂PO _Four , 3 g; NH_FourCl, 1.0 g; and NaCl, 0.5 g. pH to 7.4 with concentrated KOH
The prepared medium was sterilized by autoclaving. Prior to inoculation, sterilize by filtration as follows:
Solution: 1 M MgSO_Four, 1.0 ml; 1 M CaCl₂, 0.1 ml; Trace metal salt solution, 0.1
ml, 10 mM thiamine, 1.0 ml; and 20% glucose, 20 ml to the basal medium.
Added Trace amount of metal salt solution is MgCl per liter of deionized water.₂ 6H₂0,39.
44 g; MnSO_Four H₂O, 5.58 g; FeSO_Four 7H₂O, 1.11 g; Na₂MoO_Four 2H₂O, 0.48 g; CaCl₂ , 0.33 g; NaCl, 0.12 g; and ascorbic acid, 1.0 g. Filter sterilized
Ampicillin and kanamycin, respectively, to a final concentration of 100 μg / ml and
And 30 μg / ml were added to the medium.

The fermentation was prepared in a 100 ml volume of M9 medium contained in a 500 ml wide neck flask. Inoculate the medium with a 0.1 ml aliquot of the storage medium, 200 rpm
They were grown at 37 ° C for 18-20 hours at a shaking rate of. Seed cultures were obtained by centrifugation and then resuspended in an equal volume of M9 medium. The resuspended seed was used to inoculate the expression fermentation at a rate of 3%. For expression, cultures were grown under identical conditions to an A ₆₀₀ of 0.6. In this respect, methionine biosynthesis is 100 μg / m 2 each.
L-lysine, L-threonine and L-phenylalanine to a final concentration of 1 and L-leucine, L-isoleucine and L to a final concentration of 50 μg / ml each.
-Down-regulated by addition of valine. D, L-selenomethionine is 100μ
Simultaneously added to a final concentration of g / ml. After 15 to 20 minutes, protein expression was 1 m
It was induced by the addition of IPTG (isopropyl thio-β-D-galactosidase, Gibco BRL) up to M. The growth of the culture is further 3 up to an A _{600 of} 2.0-2.2
I continued for hours. Cells were then obtained by centrifugation and frozen at -80 ° C. Under these conditions, the average yield of cell paste was 4.0 g / l, about 20 mg / l.
L selenomethionine EF-P was produced.

Purification of selenomethionine EF-P Purification of E. coli cell paste obtained from 1 liter of induced cell culture was carried out using 25 ml of buffer A (300 mM NaCl and 10% glycerin containing pH 7. 6 in 50 mM Tris). 10 mg DNase I
And one Complet ^™ Protase Inhibitor Tablet was added to the suspension.
The cells were then lysed by passing the suspension 3 times through a French Press (Spectronic Instruments, Rochester, NY) at a pressure of 100 PSI using a refrigerated 40 ml high pressure cell. Soluble cytosol is 60 at 4 ° C
Prepared from lysates by ultracentrifugation at 100,000 xg for minutes. Benzonase (25 μl) was added to the cytosol to remove any residual polynucleotide. The clarified cytosol was HR10 / 1 loaded with Ni-NTA Superflow resin (Qiagen, Chatsworth, CA) previously equilibrated in buffer A.
0 FPLC column (Pharmacia Biotech, Piscataway, NJ). All column chromatography was performed on the AKTA 100 Explorer biochromatography system at a flow rate of 1 ml / min. After injection, the column was rinsed with buffer A until the absorbance of the column eluate at 280 nm was less than 0.1 AUFS. The material was then eluted from the column by a linear gradient of buffer B (500 mM imidazole in buffer A). The gradient consisted of a linear gradient of 0-8% buffer B for 20 minutes, 8% buffer B of the segment for 10 minutes, then 8-100% buffer B over 20 minutes. The column was washed with 100% buffer B for 30 minutes. 4 ml fractions were collected starting at the beginning of the gradient. Selected fractions were analyzed by SDS-PAGE for purity before making the final pool for crystallographic studies.

S. aureus EF-P was prepared and purified in the same manner as for selenomethionine EF-P.

Protein Crystallization The finally purified EF-P protein sample contained 10 mM Tris (5 mM DT, pH 7.6).
T was added for selenomethionine EF-P) and 1 for crystallization experiments
Concentrated to 2 mg / ml. The initial crystals of EF-P are crystal screening solution-Hampton screen 1, condition 37, 0.1M sodium acetate, pH 4.6, 8%.
PEG 4000 from a standard library. The optimization of crystallization conditions is
Performed with EF-P at a concentration of 12 mg / ml. The crystals have a pH of 5.2-5.4 of 0.
Grows best with 1 M sodium acetate and 1-4% PEG 4000. The crystals were grown by evaporative diffusion in hanging drops by addition of protein to a 1: 1 starting ratio of well solution. Protein precipitation was always observed after addition of the well solution to the protein solution, since the pH of the crystallization buffer was at or very close to the pI of the protein. During the process of equilibrium, the protein is redissolved, eventually
Crystallized for a period of 5 days. Subsequently, the EF-P crystal sits
It was determined that there was no well solution added to the protein drop and that equilibrium occurred throughout the overnight evaporation phase (this method avoided the early precipitation event before crystal formation). It was E during the early stages of this project
Due to lack of access to the coordinates for any of the IF5A structures, selenomethionine EF-P is overexpressed by down-regulating methionine biosynthesis (TE Benson et al., Nat. Struct. Biol 2: 644- 53 (1995); GD Van Du
yne et al., J. Mol. Biol; 229: 105-24 (1993)), immobilized metal affinity chromatography (see above). Purified selenomethionine EF-P was maintained in 50 mM Tris and 5 mM DTT, pH 7.8. Crystals of selenomethionine EF-P grew similarly to methionine EF-P. Crystals grown under these conditions were diffracted by a synchrotron against 1.9Å resolution.

DATA COLLECTION AND STRUCTURE DETERMINATION Multiple wavelength anomalous diffusion data are available for beamline ID-1 for sodium acetate crystal form.
Advanced Photon Source at Argonne National Laboratory, Argonne, IL
It is a collection in. The complete data set consists of three different wavelengths-one remote low energy wavelength (1.0332Å), one wavelength at the refraction point of the selenium X-ray fluorescence spectrum (0.997746Å), which would have the largest dispersive difference. One wavelength (0.99796) at the peak of the X-ray fluorescence spectrum where the anomalous difference would be maximum
17Å). Each of these individual data sets was separately indexed and consolidated (see Table 1 for consolidated statistics). The dataset is the program SCALEIT (Collaborative Computationa) in the CCP4 program suite.
l Project No. 4, Acta crys. D 50: 760-63 (1994)). The Patterson map initially revealed only two of the four selenium sites. The solution of the Patterson function was complicated by the presence of a cross vector between the two top selenium sites on the Harker section. The Cross Difference Fourier Map revealed a weaker third selenium site, which actually corresponded to the position of selenomethionine in the EF-P structure.

Heavy atom refinement and phase calculation are based on MLPHARE (Z. Otwinowski, in Isomorph
ous Replacement and Anomalous Scattering 80-86 (W. Wolf et al. SERC Daresb
ury Laboratory, Warrington, 1991), followed by DM (KD
Solvent flattening in Cowtan & P. Main, Acta Cryst .; D49: 148-57 (1993)) gave heavily separated electron density maps. At this point in the structural solution, after exploring some alternative space groups, heavy atom refinement and topological calculations are performed using SH
ARP SHARP (E. La Fortelle et al., A Maximum-Likelihood Heavy-Atom Parameter
Refinement and Phasing Program for the MIR and MAD Methods, P. Bourne &
K. Watenpaugh, eds., Crystallographic Computing 7 (1997)). The phase calculation in SHARP is done by the program SOLOMON (Collaborative Computatio
nal Project N4, Acta Cryst .; D50: 760-63 (1994)) and then obtained a significantly improved electron density map. At this stage in structure elucidation, M. j
Coordinates for eIF5A from annaschii became available, which greatly assisted the process of model building. Model building is done by the program CHAIN (JS Sack, J. Mol. G
raph; 6: 224-25 (1988)) and LORE (BC Finzel, Meth. Enzymol .; 2
77: 230-42 (1997)). Refinement is refinement (JS Jiang & A
. T. Brunger, J. Mol. Biol; 243: 100-15 (1994)) in bulk solvent (
bulk solvent) XPLOR98 (AT Brunger, X-PLOR version 3.1:
A system for X-ray Crystallography and NMR, New Haven: Yale Univ. Press
(1992)). The progress of refinement was monitored by the reduction of both R and free R factors. The stereochemistry of the model is PROCHECK (RA Laskowski et al., J.
ournal of Applied Crystallography; 26: 283-91 (1993)), which revealed that there were no residues in the region that did not allow the Ramachandran plot. 3 and 10 show SETOR (SV Evans, J. Mol.
Graphics; 11: 134-38 (1993)), and
Was produced in MOLSCRIPT (P. Kraulis, J. Appl. Cryst .; 24: 946-50 (1991)).

[Sequence list]

[Brief description of drawings]

FIG. 1 shows crystals of S. aureus elongation factor P grown in 0.1 M sodium acetate, pH 5.4, 1% PEG 4000. The width of the crystal is about 0.350 mm.

FIG. 2 shows a Patterson map of anomalous differences for selenomethionine EF-P at the reflection point at the selenium K-edge (λ = 0.979746Å, 10-1.9Å resolution). a) x Harker section, b) y Harker section and c) z Harker section.

Figure 3, a) the calculated final 2F _O -F _C Map for multiple anomalous dispersion map after solvent flattening calculated and b) 1.9 Å resolution for 2.3Å 3 shows an electron density map for EF-P for residues 73-77 for.

FIG. 4 lists the atomic structure coordinates of S. aureus EF-P derived from the crystals of the complex by X-ray diffraction. The following abbreviations are used in FIG. “Atom” refers to the element whose coordinates are measured. The second column defines the atomic number in the structure. The letters in the third column define the element. Columns 4 and 5 define the amino acids and amino acid numbers in the structure. "X, Y, Z" defines the atomic position of the measured element crystallographically. "Occ" is an occupancy factor that indicates a fragment of a molecule in which each atom occupies a position specified by coordinates. A value of "1" indicates that each atom has the same conformation, ie the same position, in all molecules of the crystal. "B" is a thermal factor that measures the motion of an atom about its atomic center.

FIG. 5: a) Ribbon representation of the X-ray crystal structure of S. aureus EF-P, b) S. aureus according to the amino acid sequence of recombinant EF-P (SEQ ID NO: 1 including His ₆ tag). The structure and sequence of EF-P is shown, with the start of each domain indicated by an arrow. Modulated residues are underlined.

[Figure 6]   FIG. 6 shows the secondary structure diagram for S. aureus EF P.

FIG. 7 shows the conserved second and third structures between domains 2 and 3 of EF-P. Two drawings (a and b) of superposition of domain 2 and domain 3 from S. aureus elongation factor P are shown.

[Figure 8]   FIG. 8 shows two stereoscopic views (a and b) of EF-P.

FIG. 9 shows a surface depiction of S. aureus EF-P. Surface charge density of elongation factor P (
Alternative drawings (a and b) of 180 °, separately) are shown.

FIG. 10. EF-P and tRNA ^gln from E. coli (MA Rould et al., Scien.
Figure 24 shows a superposition of the hypotheses of tRNA from ce 246: 1135-42 (1989), PDB access code 1 gtr). a) EF-P is oriented in domain 3 of the anticodon axis and domain 1 of the acceptor axis. b) Another possible orientation of EF-P with domain 3 of the acceptor axis and domain 1 of the anticodon axis.

FIG. 11 shows a structural comparison and sequence alignment of EF-P homologues. a) S. a
Three analyzed structures from ureus, b) Methanococcus jannaschii and c) Pyrobaculum aerophilum are shown. d) Four EF-P homologues: EF from E. coli
-P (SEQ ID NO: 2), eIF5A from Methanococcus jannaschii (SEQ ID NO: 3)
, IF5A from Pyrobaculum aerophilum (SEQ ID NO: 4) and eIF5 from human
Sequence alignment of recombinant S. aureus EF-P with a (SEQ ID NO: 5) (SEQ ID NO: 1, which contains a His ₆ tag that is not among the compared sequences). Identical residues are shaded or boxed.

Figure 12: eI from S. aureus EF-P (dark) and Methanococcus jannaschii.
F5A (bright) overlay is shown. a) Domain 1 alignment (S. aureus EF-P,
From residues 2-15, 19-33, 34-43 and 49-65 and M. jannasc
Residues 10-23, 27-41, 43-52, 58-74 from hii). b) Alignment of domain 2 (residues 66-95 and 99-128 from S. aureus EF-P and M.ja.
residues 75-103 and 104-132 from nnaschii).

FIG. 13. eIF from S. aureus EF-P (dark) and Pyrobaculum aerophilum.
5A (bright) overlay is shown. a) Alignment of domain 1 (residues 2-15, 19-33, 34-43 and 49-65 from S. aureus EF-P and P. aerophilu
Residues 11-24, 28-42, 44-53 and 59-75 from m). b) Alignment of domain 2 (residues 66-95 and 99-128 from S. aureus EF-P and P.
Residues 76-105 and 109-139 from aerophilum).

FIG. 14 shows certain residues of interest in S. aureus EF-P. Lys33 is a proposed site for post-translational modification based on the hypusine modification found in EF-P homologues of eukaryotic systems.

FIG. 15 lists structure factors and a number of anomalous dispersion phases for the crystal structure of S. aureus EF-P (SEQ ID NO: 1). “INDE” means the index h of the lattice plane,
k and I (columns 2, 3 and 4 respectively). "FOBS" refers to the structure factor of the observed reflection. "SIGMA" is the standard deviation for observations.
“PHAS” refers to the phase used for observation. "FOM" means fig
A figure of figure of merit.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12P 1/00 C12P 1/00 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD) , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, C , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (38)

[Claims] 1. A S. aureus EF-P or S. aureus EF-P
At least a portion of the binding surface, wherein the binding surface comprises amino acids selected from the surface residues listed in Table 1, the binding surface being represented by the structural coordinates listed in FIG. Amino acids Val29, Lys30, Pro31,
A molecule or molecular complex defined by a set of points having a root mean square deviation of less than about 1.9Å from the points representing the skeletal atoms of Gly32, Lys33, Gly34, Ser35 and Ala36. 2. The binding surface is less than about 1.9Å from the point representing the backbone atom of the amino acid within about 15Å of Lys33 represented by the structural coordinates listed in Table 2 and listed in FIG. 2. The molecule or molecular complex of claim 1, defined by a set of points having a root mean square deviation. 3. Structurally homologous to the S. aureus EF-P molecule or molecular complex, wherein the S. aureus EF-P molecule or molecular complex is structurally coordinated as listed in FIG. A molecule or molecular complex characterized by being represented by: 4. At least a part of said point has amino acids Val29, Lys.
30, Pro31, Gly32, Lys33, Gly34, Ser35 and A
Measurable three-dimensional arrangement of points, characterized in that they are derived from the structural coordinates of at least a portion of the S. aureus EF-P molecule or molecular complex listed in FIG. 4, including la36. 5. Substantially all of said points are S. aureus EF-P listed in FIG.
The measurable three-dimensional arrangement of points according to claim 4, which is derived from structural coordinates of a molecule or a molecular complex. 6. At least a part of the points derived from the S. aureus EF-P structural coordinates represents at least the position of the backbone atom of the amino acid defining the binding surface of S. aureus EF-P or S. aureus EF-P. A measurable three-dimensional arrangement of points according to claim 4, characterized in that said binding surface comprises the amino acids listed in Table 1. 7. The measurable three-dimensional arrangement of points according to claim 4, wherein said binding surface comprises amino acids within about 15Å of Lys33 listed in Table 2. 8. The measurable three-dimensional arrangement of points according to claim 4, which is displayed as a holographic image, a three-dimensional view, a model or a computer display image. 9. An EF-P or EF structurally homologous to a S. aureus EF-P molecule or molecular complex and comprising amino acids selected from the surface residues listed in Table 1.
-In the structural coordinates of at least part of a molecule or molecular complex containing a P-like binding surface,
A measurable three-dimensional arrangement of points, characterized in that at least a portion of the points originates. 10. The measurable three-dimensional arrangement of points according to claim 9, which is displayed as a holographic image, a three-dimensional view, a model or a computer display image. 11. An S. aureus E containing (i) an amino acid selected from the surface residues listed in Table 1 when using an instrument programmed with instructions using said data.
FP or S. aureus EF-P-like binding surface comprising at least a portion of said binding surface, wherein said binding surface is represented by the amino acid Val29, Ly represented by the structural coordinates listed in FIG.
a molecule or molecular complex defined by a set of points having a root mean square deviation of less than about 1.9Å from the point representing the s30, Pro31, Gly32, Lys33, Gly34, Ser35 or Ala36 skeletal atom; and (ii) a table S. aureu containing amino acids within approximately 15 Å of Lys33 listed in 2.
s root-mean-square deviation of less than about 1.9Å from a point that comprises at least a portion of the EF-P molecule, the EF-P molecule representing the backbone atom of the amino acid represented by the structural coordinates listed in FIG. A molecule or molecular complex defined by a set of points having; and (iii) structurally homologous to the S. aureus EF-P molecule or molecule complex, wherein the S. aureus EF-P molecule is Or encrypted device-readable data capable of displaying a graphic three-dimensional representation of at least one molecule or molecular complex selected from the group consisting of molecules or molecular complexes whose molecular complex is represented by the structural coordinates listed in FIG. A device-readable data storage medium comprising the data storage material described above. 12. When combined with a second set of device-readable data, a device programmed with instructions for using the first set of data and the second set of data provides a second set of device-readable data. At least a portion of the corresponding structural coordinates can be determined, where the first set of data is the S. aureus listed in FIG.
A first set of instrument-readable data comprising a Fourier transform of at least a portion of the structural coordinates for EF-P; the second set of data comprises an X-ray diffraction pattern of a molecule or molecular complex of unknown structure. A device-readable data storage medium that contains data storage materials that are encrypted data. 13. Crystallizing the molecule or molecular complex; creating an X-ray diffraction pattern from the crystallized molecule or molecular complex; at least a portion of the structural coordinates set forth in FIG. Structural information about a molecule or molecular complex of unknown structure characterized by creating a three-dimensional electron density map of at least a portion of the molecule or molecular complex of unknown structure when applied to the pattern. How to get. 14. The S. aureus EF-P homologue amino acid sequence and the S. aureus EF-P molecule amino acid sequence are aligned and the S. aureus EF-P homologue sequence is shown in FIG. Incorporation into the model of S. aureus EF-P derived from coordinates to obtain a preliminary model of the S. aureus EF-P homologue; subjecting the preliminary model to energy minimization to obtain an energy minimization model; stereochemical constraint Re-model the domain of the energy minimization model that violates S. a
A method for homology modeling S. aureus EF-P homologues, characterized by obtaining a final model of the ureus EF-P homologue. 15. A computer modeling application is provided with a set of structural coordinates of a molecule or molecular complex, wherein the molecule or molecular complex is S. aureus EF-P or S. aureus EF-P. A binding surface comprising at least a portion of the binding surface, the binding surface comprising amino acid surface residues listed in Table 1; providing a computer modeling application with a set of structural coordinates of the chemical moiety; Is an inhibitor expected to bind to or interfere with the molecule or molecular complex, where:
Binding to or interfering with the molecule or molecular complex is an indicator of potential inhibition of S. aureus EF-P activity.
A computer-assisted method for identifying inhibitors of eus EF-P activity. 16. The binding surface comprises amino acids selected from the surface residues listed in Table 1, wherein the binding surface is represented by the structural coordinates listed in FIG. 4, amino acids Val29, Lys30, Pro31, Gly32, Lys33, Gl
16. The method of claim 15, further defined by a set of points having a root mean square deviation of less than about 1.9Å from the points representing the skeletal atoms of y34, Ser35 and Ala36. 17. A fitting operation between the chemical atomic group and the binding surface of the molecule or molecular complex is carried out, and subsequently, the result of the fitting operation is analyzed by a computer to analyze the chemical atomic group and the chemical atomic group. Is the inhibitor expected to bind to or interfere with the molecule or molecular complex, characterized by quantifying the association with the binding surface? 16. The method according to claim 15, which determines whether or not. 18. The method according to claim 15, further comprising screening a library of chemical groups. 19. A computer modeling application for S. a.
ureus EF-P or S. aureus EF-P providing a set of structural coordinates of a molecule or molecular complex comprising at least a portion of a binding surface, the binding surface comprising amino acid surface residues listed in Table 1; Providing a set of structural coordinates of chemical groups to a computer modeling application; assessing potential binding interactions between the chemical groups and the binding surface of the molecule or molecular complex; Structurally modified to obtain a set of structural coordinates of a modified chemical group; the modified chemical group then binds to or interferes with the molecule or molecular complex Was determined to be an expected inhibitor, where binding or interference with the molecule or molecular complex was determined by S. aureu.
s A computer-aided method for designing inhibitors of S. aureus EF-P activity, characterized in that it is an indicator of potential inhibition of EF-P activity. 20. The binding surface comprises amino acids selected from the surface residues listed in Table 1, wherein the binding surface is represented by the structural coordinates listed in FIG. 4, the amino acids Val29, Lys30, Pro31, Gly32, Lys33, Gl
20. The method of claim 19, wherein the method is defined by a set of points having a root mean square deviation of less than about 1.9 Å from the points representing the skeletal atoms of y34, Ser35 and Ala36. 21. A fitting operation is performed between the chemical atomic group and the binding surface of the molecule or molecular complex, and subsequently, the result of the fitting operation is analyzed by computer operation to analyze the chemical atomic group and the chemical atomic group. Inhibitors expected to bind or interfere with said molecule or molecular complex by said modified chemical group, characterized by quantifying association with a binding surface 20. The method of claim 19, wherein determining whether or not 22. The method of claim 19, wherein the set of structural coordinates for the chemical group is obtained from a chemical fragment library. 23. The computer modeling application includes a S. a.
ureus EF-P or S. aureus EF-P-like binding surface provides a set of structural coordinates of a molecule or molecular complex comprising at least a portion of the binding surface, wherein the binding surface comprises amino acid surface residues listed in Table 1. Computationally constructing a chemical group represented by a set of structural coordinates; then expected that the chemical group binds to or interferes with the molecule or molecular complex Determined that the binding or interference with the molecule or molecular complex is an indicator of potential inhibition of S. aureus EF-P activity. Computer aided method for designing inhibitors of de novo S. aureus EF-P activity. 24. The binding surface comprises amino acids selected from the surface residues listed in Table 1, wherein the binding surface is represented by the structural coordinates listed in FIG. 4, the amino acids Val29, Lys30, Pro31, Gly32, Lys33, Gl
24. The method of claim 23, defined by a set of points having a root mean square deviation of less than about 1.9 Å from the points representing the skeletal atoms of y34, Ser35 and Ala36. 25. A fitting operation between a chemical atomic group and the binding surface of the molecule or molecular complex is performed, and subsequently, the result of the fitting operation is analyzed by a computer to analyze the chemical atomic group and the bond. Whether the chemical group is an inhibitor expected to bind to or interfere with the molecule or molecular complex, characterized by quantifying the association with the surface 24. The method of claim 23, wherein 26. A chemical group is chemically or enzymatically synthesized to produce S. aureus.
Obtaining an inhibitor of EF-P activity and providing a set of structural coordinates of a molecule or molecular complex for computer modeling applications, which molecule or molecular complex is S. aureus EF-P or S. aureus EF-P. Providing a set of structural coordinates of chemical groups to a computer modeling application; the chemical groups then bind to the molecule or molecular complex at the binding surface, or To determine whether it is expected to interfere with the molecule or molecular complex and whether binding or interference with the molecule or molecular complex is an indication of potential inhibition of S. aureus EF-P activity. A method of making an inhibitor of the characterized S. aureus EF-P activity. 27. A chemical group is chemically or enzymatically synthesized to give S. aureus.
Designed during a computer-assisted process in which an inhibitor of EF-P activity is obtained and the chemical group supplies a set of structural coordinates of a molecule or molecular complex to a computer modeling application, the molecule or molecular complex is a. aureus EF-P or S. aureus EF-P containing at least a portion of a binding surface; providing a set of structural coordinates for chemical groups to a computer modeling application;
Assessing potential binding interactions between the chemical moiety and the binding surface of the molecule or molecular complex; structurally modifying the chemical moiety to provide structural coordinates for the modified chemical moiety. The chemical group binds to or interferes with the molecule or molecular complex at the binding surface, where it binds to the molecule or molecular complex. Alternatively, a method of making an inhibitor of S. aureus EF-P activity, characterized in that interference is an indicator of potential inhibition of S. aureus EF-P activity. 28. A chemical group is chemically or enzymatically synthesized to obtain S. aureus.
Designed during a computer-assisted process in which an inhibitor of EF-P activity is obtained and the chemical group supplies a set of structural coordinates of a molecule or molecular complex to a computer modeling application, the molecule or molecular complex is A. aureus EF-P or S. aureus EF-P-like binding surface; a chemical group represented by a set of structural coordinates is computationally constructed; Determine whether the inhibitor is expected to bind to or interfere with the molecule or molecular complex, where binding or interference with the molecule or molecular complex is S. A method for producing an inhibitor of S. aureus EF-P activity, which is an indicator of potential inhibition of aureus EF-P activity. 29. An inhibitor of S. aureus EF-P activity identified, designed or produced by the method of any one of claims 15, 19, 23, 26, 27 or 28. 30. A composition comprising an inhibitor of S. aureus EF-P activity identified, designed or produced by the method of any one of claims 15, 19, 23, 26, 27 or 28. 31. An inhibitor of S. aureus EF-P activity or a salt thereof identified or designed by the method according to any one of claims 15, 19, 23, 26, 27 or 28, and a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising a carrier. 32. Purified S. aureus EF-P is prepared at a concentration of about 1 mg / ml to about 50 mg / ml; then about 0% to about 50% polyethylene glycol, 0 to about 20% by weight. S. aureu from a solution containing DMSO and buffered to a pH of about 3.5 to about 5.5.
s A method of crystallizing S. aureus EF-P molecule or molecular complex, which comprises crystallizing EF-P. 33. A crystal of S. aureus EF-P. 34. The crystal of claim 33 having orthorhombic space group symmetry P2 ₁ 2 ₁ 2 ₁ . 35. A has a dimension of a, b and c in which a is about 25Å to about 50Å, b is about 35Å to about 60Å and c is about 85Å to about 110Å; and α = β 34. The crystal according to claim 33, comprising a unit cell in which γ = 90 °. 36. A crystal according to claim 33, comprising atoms arranged in a spatial relationship represented by the structural coordinates listed in FIG. 37. The crystal of claim 33 having the S. aureus EF-P amino acid sequence SEQ ID NO: 1. 38. The crystal of claim 33 having the S. aureus EF-P amino acid sequence SEQ ID NO: 1, except that at least one methionine is replaced with a selenomethionine.