text:
|Publication number
|Publication type
|Application number
|Publication date
|Aug 23, 2011
|Filing date
|Apr 25, 2008
|Priority date
|May 4, 1998
|Fee status
|Also published as
|Publication number
|Xiaoling Xie, Yong Gu, William Markland, Michael S. Su, Paul R. Caron, Edward Fox, Keith P. Wilson
|Original Assignee
|Vertex Pharmaceuticals Incorporated
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (15), Non-Patent Citations (48), Classifications (12), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a divisional of U.S. patent application Ser. No. 09/706,128, filed Nov. 3, 2000, now U.S. Pat. No. 7,383,135 which is a continuation of PCT Application No. PCT/US99/09824, filed May 4, 1999, which claims priority from Provisional Application 60/084,056, filed May 4, 1998, all of which are incorporated herein by reference.
The present invention relates to crystallizable complexes of a JNK protein, particularly JNK3, and adenosine monophosphate. The present invention also relates to a data storage medium encoded with the structural coordinates of crystallized molecules and molecular complexes which comprise the active site binding pockets of JNK3. A computer comprising such data storage material is capable of displaying such molecules and molecular complexes, or their structural homologues, as a graphical three-dimensional representation on a computer screen. This invention also relates to methods of using the structure coordinates to solve the structure of homologous proteins or protein complexes. In addition, this invention relates to methods of using the structure coordinates to screen for and design compounds, including inhibitory compounds, that bind to JNK3 or homologues thereof.
Mammalian cells respond to extracellular stimuli by activating signaling cascades that are mediated by members of the mitogen-activated protein (MAP) kinase family, which include the extracellular signal regulated kinases (ERKs), the p38 MAP kinases and the c-Jun N-terminal kinases (JNKs). MAP kinases are serine/threonine kinases that are activated by dual phosphorylation of threonine and tyrosine at the Thr-X-Tyr segment in the activation loop. MAP kinases phosphorylate various substrates including transcription factors, which in turn regulate the expression of specific sets of genes and thus mediate a specific response to the stimulus.
Three distinct genes, Jnk1, Jnk2, Jnk3 have been identified and at least ten different splicing isoforms of JNK exist in mammalian cells [S. Gupta et al., EMBO J., 15, pp. 2760-2770 (1996)]. Members of the JNK kinases are activated by proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 beta as well as environmental stress, such as anisomycin, UV irradiation, hypoxia, and osmotic shock [A. Minden et al., Biochemica et Biophysica Acta, 1333, F85-F104 (1997)]. Regulation & function of the JNK subgroup of MAP kinases. The down-stream substrates of JNKs include transcription factors c-Jun, ATF-2, Elk1, p53 and a cell death domain protein (DENN) [Y. Zhang et al. Proc. Natl. Acad. Sci. USA, 95, pp. 2586-2591 (1998)]. Each JNK isoform binds to these substrates with different affinities, suggesting a regulation of signaling pathways by substrate specificity of different JNKs in vivo (S. Gupta et al., 1996)
JNK1 and JNK2 are widely expressed in a variety of tissues. In contrast, JNK3 is selectively expressed in the brain and to a lesser extent in the heart and testis [S. Gupta et al., (1996); A. A. Mohit et al., Neuron, 14, pp. 67-78 (1995); J. H. Martin et al., Brain Res. Mol. Brain. Res., 35, pp. 47-57 (1996)]. In the adult human brain, JNK3 expression is localized to a subpopulation of pyramidal neurons in the CA1, CA4 and subiculum regions of the hippocampus and layers 3 and 5 of the neocortex [A. A. Mohit et al. (1995)]. The CA1 neurons of patients with acute hypoxia showed strong nuclear JNK3-immunoreactivity compared to minimal, diffuse cytoplasmic staining of the hippocampal neurons from brain tissues of normal patients [Y. Zhang et al. (1998)]. In addition, JNK3 co-localizes immunochemically with neurons vulnerable in Alzheimer's disease [A. A. Mohit et al., (1995)]. Disruption of the JNK3 gene caused resistance of mice to the excitotoxic glutamate receptor agonist kainic acid, including the effects on seizure activity, AP-1 transcriptional activity and apoptosis of hippocampal neurons, indicating that the JNK3 signaling pathway is a critical component in the pathogenesis of glutamate neurotoxicity (D. D. Yang et al., Nature, 389, pp. 865-870 (1997)]. Thus, selective modulation of JNK3 activity could potentially provide therapeutic intervention for neurodegenerative diseases such as stroke and epilepsy.
Despite the fact that the genes for various JNKs have been isolated and the amino acid sequences are known, no one has described X-ray crystal structural coordinate information of any of the JNKs. Such information would be extremely useful in identifying and designing potential inhibitors of various JNKs which, in turn, could have therapeutic utility.
Applicants have solved this problem by providing, for the first time, a crystallizable composition comprising unphosphorylated JNK3 in complex with MgAMP-PNP and the resulting crystal. The crystal was resolved at 2.3 Å resolution. Solving this crystal structure has allowed applicants to determine the key structural features of JNK3, particularly the shape of its substrate binding site.
The invention also provides a machine readable storage medium which comprises the structure coordinates of the JNK3 binding site. Such storage medium encoded with these data when read and utilized by a computer programmed with appropriate software displays, on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex comprising such binding sites or similarly shaped homologous binding pockets.
The invention also provides methods for designing, evaluating and identifying compounds which bind to the aforementioned binding sites, as well as compounds produced by such methods. Such compounds are potential inhibitors of JNK3 or its homologues.
The invention also provides a method for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to JNK3, particularly JNK1, JNK2 and other JNK isoforms. This is achieved by using at least some of the structural coordinates obtained for the unphosphorylated JNK3 in complex with MgAMP-PNP.
The invention also provides a method for crystallizing unphosphorylated JNK3 in complex with MgAMP-PNP.
"Atom type" refers to the element whose coordinates are measured. The first letter in the column defines the element.
"X, Y, Z" crystallographically define the atomic position of the element measured.
"B" is a thermal factor that measures movement of the atom around its atomic center.
"Occ" is an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of "1" indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal.
The following abbreviations are used throughout the application:
A=Ala=Alanine T=Thr=Threonine
V=Val=Valine C=Cys=Cysteine
L=Leu=Leucine Y=Tyr=Tyrosine
I=Ile=Isoleucine N=Asn=Asparagine
P=Pro=Proline Q=Gln=Glutamine
F=Phe=Phenylalanine D=Asp=Aspartic Acid
W=Trp=Tryptophan E=Glu=Glutamic Acid
M=Met=Methionine K=Lys=Lysine
G=Gly=Glycine R=Arg=Arginine
S=Ser=Serine H=His=Histidine
Additional definitions are set forth in the specification where necessary.
In order that the invention described herein may be more fully understood, the following detailed description is set forth.
According to one embodiment, the invention provides a crystallizable composition comprising an unphosphorylated JNK protein complexed with adenosine monophosphate. The JNK protein in the crystallizable complexes of this invention, if it is JNK3 or a JNK3 variant (as opposed to JNK1 or JNK2), must be truncated at the N-terminus. Specifically, the JNK3 proteins contain an N-terminal extension of about 40 amino acids as compared to JNK1 and JNK2 proteins (see GenBank entries for JNK1, JNK2 and JNK3 proteins and their isoforms). Those 40 amino acid must be removed from JNK3 proteins in the crystallizable compositions of this invention.
In addition, any JNK protein in these crystallizable compositions preferably have a C-terminal truncation of about 20 amino acids. We have found that the C-terminal truncation is necessary to obtain diffraction quality crystals.
The second component in these compositions is a non-hydrolyzable ATP analog or a suicidal substrate. Non-hydrolyzable ATP analogs useful in the crystallizable compositions of this invention include AMP-PCH2P, AMP-PSP and AMP-PNP. An example of a suicidal substrate is FSBA. Preferably, the crystallizable compositions of this invention comprise AMP-PNP as the substrate. The third component is magnesium ions. Mg can be introduced by incubating the non-hydrolyzable ATP analog or suicide substrate with MgCl2 prior to incubation with the JNK protein.
We have also determined that the buffer conditions of the composition are crucial for crystallization. Thus, the crystallizable compositions of this invention also comprise polyethylene glycol monomethyl ether at between about 10 to 30% v/v, ethylene glycol at between about 5 to 20% v/v, a reducing agent, such as 1-mercaptoethanol at between about 5 to 50 mM, and a buffer that maintains pH at between about 7.0 and 7.5. Preferably the buffer is 100 mM Hepes at pH 7.0.
The invention also relates to crystals of a JNK protein complexes with Mg and a non-hydrolyzable ATP analog or a suicidal substrate. These crystals are obtained from the above described compositions by standard crystallization protocols.
The invention also related to a method of making JNK-containing crystals. Such methods comprise the steps of:
In each of the above embodiments, it is preferred that the JNK protein be a JNK3, and in particular JNK3α1.
As mentioned above, applicants have solved the three-dimensional X-ray crystal structure of JNK3a1. The atomic coordinate data is presented in
In order to use the structure coordinates generated for the JNK3/MgAMP-PNP complex or one of its binding pockets or homologues thereof, it is often times necessary to convert them into a three-dimensional shape. This is achieved through the use of commercially available software that is capable of generating three-dimensional graphical representations of molecules or portions thereof from a set of structure coordinates.
Binding pockets, also referred to as binding sites in the present invention, are of significant utility in fields such as drug discovery. The association of natural ligands or substrates with the binding pockets of their corresponding receptors or enzymes is the basis of many biological mechanisms of action. Similarly, many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or any parts of the binding pocket. An understanding of such associations will help lead to the design of drugs having more favorable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential inhibitors of the binding sites of biologically important targets.
The term "binding pocket", as used herein, refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound.
The term "JNK3-like binding pocket" refers to a portion of a molecule or molecular complex whose shape is sufficiently similar to the JNK3 binding pockets as to bind common ligands. This commonality of shape is defined by a root mean square deviation from the structure coordinates of the backbone atoms of the amino acids that make up the binding pockets in JNK3 (as set forth in
The "active site binding pockets" or "active site" of JNK3 refers to the area on the JNK3 enzyme surface where the nucleotide substrate binds. In resolving the crystal structure of unphosphorylated JNK3 1 in complex with MgAMP-PNP, applicants have determined that JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Val78, Ala91, Lys93, Glu111, Ile124, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Gln155, Lys191, Ser193, Asn194, Val196 and Leu206 are within 5 Å of and therefore close enough to interact with MgAMP-PNP. Thus, a binding pocket defined by the structural coordinates of those amino acids, as set forth in
Applicants have also determined that in addition to the JNK3 amino acids set forth above, Ile77, Cys79, Ala80, Val90, Ile92, Lys94, Leu95, His104, Arg107, Ser125, Leu144, Val145, Leu153, Cys154, Asp189, Pro192, Ile195, Val197, Lys204 and Asp207 are within 8 Å of bound MgAMP-PNP and therefore are also close enough to interact with that substrate. Thus, in a preferred embodiment, a binding pocket defined by the structural coordinates of the amino acids within 8 Å bound MgAMP-PNP, as set forth in
It will be readily apparent to those of skill in the art that the numbering of amino acids in other isoforms of JNK may be different than that set forth for JNK3 1. Corresponding amino acids in other isoforms of JNK are easily identified by visual inspection of the amino acid sequences or by using commercially available homology software programs.
Each of those amino acids of JNK3α1 is defined by a set of structure coordinates set forth in
Those of skill in the art understand that a set of structure coordinates for an enzyme or an enzyme-complex or a portion thereof, is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates will have little effect on overall shape. In terms of binding pockets, these variations would not be expected to significantly alter the nature of ligands that could associate with those pockets.
The term "associating with" refers to a condition of proximity between a chemical entity or compound, or portions thereof, and a binding pocket or binding site on a protein. The association may be non-covalent—wherein the juxtaposition is energetically favored by hydrogen bonding or van der Waals or electrostatic interactions—or it may be covalent.
The variations in coordinates discussed above may be generated because of mathematical manipulations of the JNK3/MgAMP-PNP structure coordinates. For example, the structure coordinates set forth in
Alternatively, modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same. Thus, for example, a ligand that bound to the active site binding pocket of JNK3 would also be expected to bind to another binding pocket whose structure coordinates defined a shape that fell within the acceptable error.
Various computational analyses are therefore necessary to determine whether a molecule or the binding pocket portion thereof is sufficiently similar to the JNK3 binding pockets described above. Such analyses may be carried out in well known software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, Calif.) version 4.1, and as described in the accompanying User's Guide.
The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. The procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalences in these structures; 3) perform a fitting operation; and 4) analyze the results.
Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention we will define equivalent atoms as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. We will also consider only rigid fitting operations.
When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
For the purpose of this invention, any molecule or molecular complex or binding pocket thereof that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than 1.5 Å when superimposed on the relevant backbone atoms described by structure coordinates listed in
The term "root mean square deviation" means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of JNK3 or a binding pocket portion thereof, as defined by the structure coordinates of JNK3 described herein.
Therefore, according to another embodiment of this invention is provided a machine-readable data storage medium, comprising a data storage material encoded with machine readable data which, when used by a machine programmed with instructions for using said data, displays a graphical three-dimensional representation of a molecule or molecular complex comprising a binding pocket defined by structure coordinates of JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Val78, Ala91, Lys93, Glu111, Ile24, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Gln155, Lys191, Ser193, Asn194, Val196 and Leu206 according to
Preferably, the machine readable data, when used by a machine programmed with instructions for using said data, displays a graphical three-dimensional representation of a molecule or molecular complex comprising a binding pocket defined by structure coordinates of JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Ile77, Val78, Cys79, Ala80, Val90, Ala91, Ile92, Lys93, Lys94, Leu95, His104, Arg107, Glu111, Ile124, Ser125, Leu144, Val145, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Leu153, Cys154, Gln155, Asp189, Lys191, Pro192, Ser193, Asn194, Ile195, Val196 Val197, Lys204, Leu206 and Asp207 according to
Even more preferred is a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structure coordinates of all of the amino acids in
According to an alternate embodiment, the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structure coordinates set forth in
For example, the Fourier transform of the structure coordinates set forth in
Input hardware 36, coupled to computer 11 by input lines 30, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems 32 connected by a telephone line or dedicated data line 34. Alternatively or additionally, the input hardware 36 may comprise CD-ROM drives or disk drives 24. In conjunction with display terminal 26, keyboard 28 may also be used as an input device.
Output hardware 46, coupled to computer 11 by output lines 40, may similarly be implemented by conventional devices. By way of example, output hardware 46 may include CRT display terminal 26 for displaying a graphical representation of a binding pocket of this invention using a program such as QUANTA as described herein. Output hardware might also include a printer 42, so that hard copy output may be produced, or a disk drive 24, to store system output for later use.
In operation, CPU 20 coordinates the use of the various input and output devices 36, 46, coordinates data accesses from mass storage 24 and accesses to and from working memory 22, and determines the sequence of data processing steps. A number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system 10 are included as appropriate throughout the following description of the data storage medium.
According to an alternate embodiment, the present invention provides a computer for producing a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Val78, Ala91, Lys93, Glu111, Ile124, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Gln155, Lys191, Ser193, Asn194, Val196 and Leu206 according to
Preferably, the computer produces a three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a binding pocket defined by the binding pocket is defined by structure coordinates of JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Ile77, Val78, Cys79, Ala80, Val90, Ala91, Ile92, Lys93, Lys94, Leu95, His104, Arg107, Glu111, Ile124, Ser125, Leu144, Val145, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Leu153, Cys154, Gln155, Asp189, Lys191, Pro192, Ser193, Asn194, Ile195, Val196 Val197, Lys204, Leu206 and Asp207 according to
The magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in manner which may be conventional, machine readable data such as that described herein, for execution by a system such as system 10 of
In the case of CD-ROM, as is well known, coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of coating 112. A protective coating 114, which preferably is substantially transparent, is provided on top of coating 112.
In the case of a magneto-optical disk, as is well known, coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown). The orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112. The arrangement of the domains encodes the data as described above.
Thus, in accordance with the present invention, data capable of displaying the three dimensional structure of JNK3 and portions thereof and their structurally similar homologues is stored in a machine-readable storage medium, which is capable of displaying a graphical three-dimensional representation of the structure.
The JNK3 X-ray coordinate data, when used in conjunction with a computer programmed with software to translate those coordinates into the 3-dimensional structure of JNK3 may be used for a variety of purposes, such as drug discovery.
For example, the structure encoded by the data may be computationally evaluated for its ability to associate with chemical entities. Chemical entities that associate with JNK3 may inhibit JNK3, and are potential drug candidates. Alternatively, the structure encoded by the data may be displayed in a graphical three-dimensional representation on a computer screen. This allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.
Thus, according to another embodiment, the invention relates to a method for evaluating the potential of a chemical entity to associate with a molecule or molecular complex comprising a binding pocket defined by structure coordinates of JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Val78, Ala91, Lys93, Glu111, Ile124, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Gln155, Lys191, Ser193, Asn194, Val196 and Leu206 according to
This method comprises the steps of: a) employing computational means to perform a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex; b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket; and c) outputting said quantified association to a suitable output hardware, such as a CRT display terminal, a printer or a disk drive, as described previously. The term "chemical entity", as used herein, refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes.
Preferably, the method evaluates the potential of a chemical entity to associate with a molecule or molecular complex comprising a binding pocket defined by structure coordinates of JNK3 amino acids Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Ile77, Val78, Cys79, Ala80, Val90, Ala91, Ile92, Lys93, Lys94, Leu95, His 104, Arg107, Glu111, Ile124, Ser125, Leu144, Val145, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Leu153, Cys154, Gln155, Asp189, Lys191, Pro192, Ser193, Asn194, Ile195, Val196 Val197, Lys204, Leu206 and Asp207 according to
Even more preferably, the method evaluates the potential of a chemical entity to associate with a molecule or molecular complex defined by structure coordinates of all of the JNK3 amino acids, as set forth in
Alternatively, the structural coordinates of the JNK3 binding pocket can be utilized in a method for identifying a potential agonist or antagonist of a molecule comprising a JNK3-like binding pocket. This method comprises the steps of:
More preferred is when the atomic coordinates of Ile70, Gly71, Ser72, Gly73, Ala74, Gln75, Gly76, Ile77, Val78, Cys79, Ala80, Val90, Ala91, Ile92, Lys93, Lys94, Leu95, His104, Arg107, Glu111, Ile124, Ser125, Leu144, Val145, Met146, Glu147, Leu148, Met149, Asp150, Ala151, Asn152, Leu153, Cys154, Gln155, Asp189, Lys191, Pro192, Ser193, Asn194, Ile195, Val196 Val197, Lys204, Leu206 and Asp207 according to FIG. 1±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, are used to generate a three-dimensional structure of molecule comprising a JNK3-like binding pocket.
Most preferred is when the atomic coordinates of all the amino acids of JNK3 according to FIG. 1±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, are used to generate a three-dimensional structure of molecule comprising a JNK3-like binding pocket.
For the first time, the present invention permits the use of molecular design techniques to identify, select and design chemical entities, including inhibitory compounds, capable of binding to JNK3-like binding pockets.
Applicants' elucidation of binding sites on JNK3 provides the necessary information for designing new chemical entities and compounds that may interact with JNK3-like binding pockets, in whole or in part.
Throughout this section, discussions about the ability of an entity to bind to, associate with or inhibit a JNK3-like binding pocket refers to features of the entity alone. Assays to determine if a compound binds to JNK3 are well known in the art and are exemplified below.
The design of compounds that bind to or inhibit JNK3-like binding pockets according to this invention generally involves consideration of two factors. First, the entity must be capable of physically and structurally associating with parts or all of the JNK3-like binding pockets. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions and electrostatic interactions.
Second, the entity must be able to assume a conformation that allows it to associate with the JNK3-like binding pocket directly. Although certain portions of the entity will not directly participate in these associations, those portions of the entity may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the binding pocket, or the spacing between functional groups of an entity comprising several chemical entities that directly interact with the JNK3-like binding pocket or homologues thereof.
The potential inhibitory or binding effect of a chemical entity on a JNK3-like binding pocket may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given entity suggests insufficient interaction and association between it and the JNK3-like binding pocket, testing of the entity is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to a JNK3-like binding pocket. This may be achieved by testing the ability of the molecule to inhibit JNK3 using the assays described in Example 6. In this manner, synthesis of inoperative compounds may be avoided.
A potential inhibitor of a JNK3-like binding pocket may be computationally evaluated by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the JNK3-like binding pockets.
One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a JNK3-like binding pocket. This process may begin by visual inspection of, for example, a JNK3-like binding pocket on the computer screen based on the JNK3 structure coordinates in
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include:
Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of JNK3. This would be followed by manual model building using software such as Quanta or Sybyl [Tripos Associates, St. Louis, Mo.].
Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include:
Instead of proceeding to build an inhibitor of a JNK3-like binding pocket in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other JNK3 binding compounds may be designed as a whole or "de novo" using either an empty binding site or optionally including some portion(s) of a known inhibitor(s). There are many de novo ligand design methods including:
Other molecular modeling techniques may also be employed in accordance with this invention [see, e.g., N. C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, pp. 883-894 (1990); see also, M. A. Navia and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L. M. Balbes et al., "A Perspective of Modern Methods in Computer-Aided Drug Design", in Reviews in Computational Chemistry, Vol. 5, K. B. Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337-380 (1994); see also, W. C. Guida, "Software For Structure-Based Drug Design", Curr. Opin. Struct. Biology, 4, pp. 777-781 (1994)].
Once a compound has been designed or selected by the above methods, the efficiency with which that entity may bind to an JNK3 binding pocket may be tested and optimized by computational evaluation. For example, an effective JNK3 binding pocket inhibitor must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding). Thus, the most efficient JNK3 binding pocket inhibitors should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably, not greater than 7 kcal/mole. JNK3 binding pocket inhibitors may interact with the binding pocket in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the inhibitor binds to the protein.
An entity designed or selected as binding to an JNK3 binding pocket may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules. 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 such uses include: Gaussian 94, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. ©1995); AMBER, version 4.1 (P. A. Kollman, University of California at San Francisco, ©1995); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. ©1995); Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif. ©1995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. ©1995); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be implemented, for instance, using a Silicon Graphics workstation such as an Indigo2 with "IMPACT" graphics. Other hardware systems and software packages will be known to those skilled in the art.
Another approach enabled by this invention, is the computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to a JNK3 binding pocket. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy [E. C. Meng et al., J. Comp. Chem., 13, pp. 505-524 (1992)].
According to another embodiment, the invention provides compounds which associate with a JNK3-like binding pocket produced or identified by the method set forth above.
The structure coordinates set forth in
Therefore, in another embodiment this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or molecular complex whose structure is unknown comprising the steps of:
a) crystallizing said molecule or molecular complex of unknown structure;
b) generating an X-ray diffraction pattern from said crystallized molecule or molecular complex; and
c) applying at least a portion of the structure coordinates set forth in
By using molecular replacement, all or part of the structure coordinates of the JNK3/MgAMP-PNP complex as provided by this invention (and set forth in
Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that can not be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.
Thus, this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the JNK3/MgAMP-PNP complex according to
The structure of any portion of any crystallized molecule or molecular complex that is sufficiently homologous to any portion of the JNK3/MgAMP-PNP complex can be resolved by this method.
In a preferred embodiment, the method of molecular replacement is utilized to obtain structural information about another JNK, such as JNK1, JNK2, or isoforms of JNK1, JNK2 or JNK3. The structure coordinates of JNK3 as provided by this invention are particularly useful in solving the structure of other isoforms of JNK3 or JNK3 complexes.
Furthermore, the structure coordinates of JNK3 as provided by this invention are useful in solving the structure of JNK3 proteins that have amino acid substitutions, additions and/or deletions (referred to collectively as "JNK3 mutants", as compared to naturally occurring JNK3 isoforms. These JNK3 mutants may optionally be crystallized in co-complex with a chemical entity, such as a non-hydrolyzable ATP analogue or a suicide substrate. The crystal structures of a series of such complexes may then be solved by molecular replacement and compared with that of wild-type JNK3. Potential sites for modification within the various binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between JNK3 and a chemical entity or compound.
The structure coordinates are also particularly useful to solve the structure of crystals of JNK3 or JNK3 homologues co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate JNK3 inhibitors and JNK3. For example, high resolution X-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their JNK3 inhibition activity.
All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3 Å resolution X-ray data to an R value of about 0.20 or less using computer software, such as X-PLOR [Yale University, ©1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)]. This information may thus be used to optimize known JNK3 inhibitors, and more importantly, to design new JNK3 inhibitors.
In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
A BLAST search of the EST database using the published JNK3α1 cDNA [S. Gupta et al. (1996)] as a query identified an EST clone (#632588) that contained the entire coding sequence for human JNK3α1. Polymerase chain reactions (PCR) using pfu polymerase (Strategene) were used to introduce restriction sites into the cDNA for cloning into the pET-15B expression vector at the NcoI and BamHI sites for expression of the protein in E. coli. Due to the poor solubility of the expressed full length protein (Met 1-Gln 422), an N-terminally truncated protein starting at Ser residue at position 40 (Ser 40), corresponding to Ser 2 of JNK1 and JNK2 proteins [S. Gupta et al. (1996)], preceded by Met (initiation) and Gly residues, was produced. The Gly residue was added in order to introduce an NcoI site for cloning into the expression vector. Further, systematic C-terminal truncations were performed by PCR to identify a construct that give rise to diffraction-quality crystals. This construct, which was prepared by PCR using deoxyoligonucleotides 5′ GCTCTAGAGCTCCATGGGCAGCAAAAGCAAAGTTGACAA 3′ (SEQ ID NO:2; forward primer with initiation codon underlined) and 5′ TAGCGGATCCTCATTCTGAA TTCATTACTTCCTTGTA 3′ (SEQ ID NO:3; reverse primer with stop codon underlined) as primers and confirmed by DNA sequencing, encodes amino acid residues Ser40-Glu402 of JNK3α1, preceded by Met and Gly residues, was used for structural studies described in this paper. Control experiments indicated that the truncated JNK3 protein has an equivalent kinase activity towards myelin basic protein when activated with an upstream kinase MKK7 in vitro (unpublished results).
E. coli strain BL21 (DE3) (Novagen) transformed with the JNK3 expression construct was grown at 30° C. in shaker flasks into log phase (OD600˜0.8) in LB supplemented with 100 μg/ml carbenicillin. IPTG was then added to a final concentration of 0.8 mM and the cells were harvested 2 hours later by centrifugation. E. coli cell paste containing the truncated JNK3 protein was resuspended in 10 volumes/g lysis buffer [50 mM HEPES, pH 7.2, 10% glycerol (v/v), 100 mM NaCl, 2 mM dithiothreitol (DTT), 0.1 mM PMSF, 2 μg/ml Pepstatin, 1 μg/ml each of E-64 and Leupeptin]. Cells were lysed on ice using a microfluidizer and centrifuged at 100,000×g for 30 min at 4° C. The 100,000×g supernatant was diluted 5 fold with Buffer A [20 mM HEPES, pH 7.0, 10% glycerol (v/v), 2 mM DTT) and applied to an SP-Sepharose (Pharmacia) cation-exchange column at 4° C. The column was washed with column volumes of Buffer A, followed by 5 column volumes of Buffer A containing 50 mM NaCl. Bound protein was eluted with a 7.5 column volume linear gradient of 50-300 mM NaCl, and the truncated JNK3 protein was eluted between 150-200 mM NaCl. Eluted JNK3 protein from the SP-Sepharose column was dialyzed at ˜1 mg/ml against Buffer B [25 mM HEPES, pH 7.0, containing 5% glycerol (v/v), 50 mM NaCl, mM DTT] overnight at 4° C. and centrifuged at 3,000×g. The supernatant was concentrated by ultrafiltration (Centriprep-30, Amicon) to 10 mg/ml, centrifuged at 16,000×g and stored at −70° C.
Full length human JNK3 1 has an 39-residue extension in the N-terminus when compared to JNK1, JNK2 and other MAP kinases [
Crystallization trials were performed by combining the hanging-drop vapor diffusion technique and a sparse matrix search, in the presence and absence of MgAMP-PNP. No crystals were obtained in the absence of MgAMP-PNP, while crystallization trials carried out in the presence of MgAMP-PNP yielded an orthorhombic crystal form at 20° C. over a reservoir solution containing 18-20% (v/v) polyethylene glycol monomethyl ether (average Mr=550), 10% (v/v) ethylene glycol, 20 mM β-Mercaptoethanol and 10 mM Hepes (pH 7.0). The crystallization droplet contained a mixture of 1 μL of reservoir solution plus 1 mL of a protein solution that had been preincubated for one hour with 1 mM AMP-PNP and 2 mM MgCl2 on ice. The crystals belong to the orthorhombic space group P212121 (a=51.50 Å, b=71.24 Å and c=107.60 Å) with one enzyme molecule per asymmetric unit. The solvent content of the crystal is 44%. Before data collection, crystals were equilibrated in their reservoir solution for 2-5 minutes before flash-frozen in nitrogen gas for X-ray data collection at −170° C.
X-ray data were measured on an Raxis IIC image plate, with mirror-focused CuKα X-rays generated by a rotating anode source. The diffraction images were processed with the program DENZO and data scaled using SCALPACK [Z. Otwinowski, In "Data Collection and Processing", L. Sawyer, N. Isaacs and S. W. Bailey, eds., Warrington, U.K.: Science and Engineering Council/Daresbury Laboratory. pp. 55-62 (1993)]. The data processing statistics are summarized in Table 1. The starting phases for JNK3 were obtained by molecular replacement using coordinates of phosphorylated ERK2 as search model in the program AMoRe [J. Nazaza, Acta Crystallogr., A50, pp. 157-63 (1994)]. The Erk2 atomic model was modified by truncating to Ala for those residues that are different from JNK3 and deleting those loops that have significant insertions or deletions between Erk2 and JNK3. This hybrid model successfully produced rotation and translation function solution for JNK3, which provided a starting model with an R-factor=50% for reflections between 10 and 4.0 Å resolution, and an R-free=51% based on 10% of X-ray data set aside at the start of the refinement. Refinement of the model using both conventional least-squares and simulated-annealing procedures was done with X-PLOR [A. T. Brunger, XPLOR, Version 3.1 Manual, Yale University Press, New Haven, Conn. (1992)] using 8-2.3 Å data. The electron density corresponding to AMP-PNP molecule was visible from the map calculated using the initial model phases, but AMP-PNP was not included in the model refinement until the R-factor dropped to 28% and R-free to 39%. The refined model, at 2.3 Å resolution (Table 1), includes 339 residues of JNK3, one AMP-PNP molecule, two Mg2+ and 183 water molecules. The electron density maps revealed several discrete regions of disorder, leading the omission of some amino acid residues from the final model. N-terminal residues 40-44 and C-terminal residue 401 and 402 are disordered. In addition, two central regions of the enzyme are disordered, including residues 212-216 and 374-378. Finally, side chain atoms for Tyr223 was not modeled beyond C: due to poor electron density. The present R-factor is 22.1% (R-free=27.4%). Anisotropic scaling and bulk solvent correction were applied at the final stage of refinement (ref). The peptide torsion angles for 337 out of 339 well defined residues fall within most favored or generally allowed regions of the Ramachandran plot, as defined in the program PROCHECK [R. A. Laskowski et al., J. Appl. Crystallog., 26, pp. 283-91 (1993)].
The crystal structure includes unphosphorylated JNK3 (residues 45-211, 217-373, 379-400), adenylyl imidodiphosphate (AMP-PNP, an ATP analogue) and two Mg2+ ions. Electron density for residues 40-44, 212-216 and 401-402 is not seen, and these amino acids are presumed disordered. The MAP kinase homologous region of JNK3 (Phe48-Glu397) is 45% identical in amino acid sequence to Erk2 and 51% to p38, whose structures have been reported (F. Zhang et al. (1994); K. P. Wilson et al. (1996);
The relative orientation of the N- and C-terminal domains is different between the structure of JNK3 and Erk2 (
Electron density is not visible for two disordered regions within the core region of JNK3. One region is Arg212-Thr216 in the C-terminal domain (
Phosphorylation Lip
We refer to the region spanning residues Thr217-Thr226, part of linker L12, as the "phosphorylation lip" or "activation loop", since it contains the regulatory phosphorylation sites Thr221 and Tyr223 (
As a result of the conformation of the lip, the regulatory phosphorylation sites in JNK3 are differently positioned compared to that in Erk2 and p38. Thr221 and Tyr223 are 16 Å away from the location of the corresponding residues in Erk2 and 12 Å in p38. Despite the different locations, the regulatory threonine residues in all three enzymes are solvent exposed. In contrast, the local environments of the tyrosine residues are different. The side chain of Tyr221 is exposed to solvent in the JNK3 structure, and is disordered. The corresponding tyrosine residue in p38 is also exposed to the solvent, but its side chain is well-ordered and interacts with the hydroxyl group of Thr221 of p38 through a water molecule [K. P. Wilson et al, (1996)]. In contrast, the side chain of the corresponding tyrosine in Erk2 is buried. The Erk2 residue Tyr185 forms a hydrogen bond with the side chain of Arg146, and makes van der Waals contacts with nearby hydrophobic amino acid side chains [F. Zhang et al. (1994)]. The side chain conformation of Tyr185 in the unphosphorylated Erk2 structure suggests that the phosphorylation lip must be refolded before Tyr185 can become a substrate for the Erk2 upstream activating kinase. A similar movement may not be needed to phosphorylate JNK3 and p38, since both threonine and tyrosine are accessible to the solvent in the unphosphorylated forms of JNK3 and p38.
Peptide Substrate Binding Channel
The peptide substrate binding sites in JNK3 may be mapped by its homology to c-AMP-dependent protein kinase (cAPK,
Structural comparison of JNK3 and the phosphorylated Erk2 suggest how phosphorylation at Thr221 and Tyr223 of JNK3 might play a role in the activation of JNK kinases. In the phosphorylated Erk2, phosphothreonine pThr183 interacts directly with three arginine residues, Arg68 in αC, Arg146 in the catalytic loop (C loop) and Arg170 from the phosphorylation lip, while phosphotyrosine pTyr185 is ligated by Arg189 and Arg192 [B. J. Canagarajah et al (1997)]. Assuming that Thr221 and Tyr223 are ligated similarly in the phosphorylated form of JNK3, they would have to move by approximately 15 Å upon phosphorylation to be in close proximity with their ligands, and a large conformational change of the phosphorylation lip would be required. As a consequence of phosphorylation at Thr221 and Tyr223, restructuring of the phosphorylation lip may help to unblock the peptide substrate binding channel. These phosphorylation-related conformational changes in the phosphorylation lip as well as the peptide substrate binding channel have been observed in the crystal structures of low activity and phosphorylated Erk2 [F. Zhang et al. (1994); B. J. Canagarajah et al. (1997)].
Active Site
Crystals of the binary complex of JNK3/MgAMP-PNP obtained from crystallizations of JNK3 in the presense of AMP-PNP and Mg2+ have allowed us to obtain structural data for the nucleotide-bound form of JNK3. As revealed in the structure, AMP-PNP is bound in the deep cleft between the two lobes of JNK3 (
The catalytic core of protein kinases contains a nucleotide binding sequence Gly-X-Gly-X-X-Gly-X-X (SEQ ID NO: 3) that is referred to as "the glycine-rich phosphate anchor loop" due to its structural feature and role in the nucleotide binding [D. R. Knighton et al., Science, 253, pp. 407-13 (1991)]. The glycine-rich loop is well defined in JNK3, and superimposes well with that of cAPK, with an rms deviation 0.54 Å for the protein main chain atoms from Ile70 to Ser79 (
Structural comparison of JNK3 and cAPK reveals that the two domains of JNK3 are rotated apart relative to their orientation in the structure of cAPK (
Similarity of JNK3 to Other Enzymes
The overall fold of JNK3 reveals similarities to the known structures of cAMP-dependent protein kinase and other MAP kinases, Erk2 and p38. The unphosphorylated JNK3 assumes an "open" conformation, in which the N- and C-terminal domains are oriented so that some of the catalytic residues are misaligned. In addition, the phosphorylation lip partially blocks the substrate peptide binding site. The combination of these regulatory mechanisms suggests that both global (domain closure to bring the catalytic residues in close proximity) and local (refolding of the lip to relieve steric constraints to substrate binding) conformational changes are required for JNK3 activation.
Crystallographic studies of Erk2, p38 and JNK3 have shown that the region of β9 and the phosphorylation lip has the most diverse and labile conformation in unphosphorylated MAP kinases. This region of ERK2 adopts a conformation stabilized by interactions between the phosphates and residues near the activation loop in the phosphorylated enzyme [B. J. Canagarajah et al. (1997)]. Using the phosphorylated Erk2 structure as a model for the active conformation of JNK3 shows that the two phosphorylation sites, Thr221 and Tyr223, may play similar roles in activating JNK3 as they do in Erk2. In JNK3, phosphorylation of Thr221 may promote domain closure by interacting with Arg107 and Arg188, while phosphorylation of Tyr223 may promote new interaction of the phosphotyrosine with Arg227 and Arg230, which in turn constitute the proline-directed P+1 pocket.
The JNK3/MgAMP-PNP binary complex is the first kinase structure of the JNK subfamily of MAP kinases to be determined. In the region spanning residues Ser40 to Ala418, JNK3 shares 92% and 87% amino acid identity with JNK1 and JNK2, respectively. Thus, the JNK3 structure provides detailed structural information which provides insight into the mechanism of regulation for this class of MAP kinases. The variant residues among JNK isoforms are clustered in two regions (
The coordinates of
A. JNK3 Activation
Five mg of JNK3 was diluted to 0.5 mg/ml in 50 mM HEPES buffer, pH 7.5, containing 100 mM NaCl, 5 mM DTT, 20 mM MgCl2, 1 mM ATP. GST-MKK7(DD) kinase (the upstream mutant form of one of the activating kinases of JNK3) was added at a molar ratio of 1 GST-MKK7:2.5 JNK3. After 30 min at 25° C. the reaction mixture was concentrated 5-fold by ultrafiltration in a Centriprep-30 (Amicon, Beverly, Mass.), then diluted back up to 10 ml and an additional 1 mM ATP added. This procedure was repeated three times to remove ADP and replenish ATP. The final (third) addition of ATP was 5 mM and the mixture incubated overnight at 4° C.
The activated JNK3/GST-MKK7(DD) reaction mixture was exchanged into 50 mM HEPES buffer, pH 7.5, containing 5 mM DTT and 5% glycerol (w/v) by dialysis or ultrafiltration. The reaction mixture was adjusted to 1.1 M potassium phosphate, pH 7.5, and purified by hydrophobic interactions chromatography (at 25° C.) using a Rainin Hydropore column. GST-MKK7 and unactivated JNK3 do not bind under these conditions and when a 1.1 to 0.05M potassium phosphate gradient is developed over 60 min at a flow rate of 1 ml/min, doubly phosphorylated JNK3 is separated from singly phosphorylated JNK.
Activated JNK3 (i.e. doubly phosphorylated) was stored at −70° C. at 0.25-1 mg/ml.
B. JNK3 Inhibition Assay
To determine IC50 of the compound binding to JNK3, the kinase activity of JNK3 was monitored by coupled enzyme assay. In this assay, for every molecule of ADP generated by the JNK3 kinase activity one molecule of NADH is converted to NAD which can be conveniently monitored as an absorbance decrease at 340 nm. The following are the final concentrations of various reagents used in the assay: 100 mM HEPES buffer, pH 7.6, 10 mM MgCl2, 25 mM β-glycerophosphate, 30 μM ATP, 2 mM phosphoenolpyruvate, 2 μM pyruvate kinase, 2 μM lactate dehydrogenase, 200 μM NADH, 200 μM EGF receptor peptide KRELVEPLTPSGEAPNQALLR (SEQ ID NO: 6), and 10 mM activated JNK3. First, all of the above reagents with the exception of ATP were mixed and 175 μl aliquots were placed per well of 96-well plate. A 5 μl DMSO solution of the compound was added to each well, mixed, and allowed to stand at 30° C. for 10 minutes. Typically about 10 different concentrations of the compound were tested. The reactions were initiated with the addition of 20 μl of ATP solution. Absorbance change at 340 nm were monitored as a function of time. IC50 is obtained by fitting the rates vs. compound concentration data to a simple competitive inhibition model.
While we have described a number of embodiments of this invention, it is apparent that our basic constructions may be altered to provide other embodiments which utilize the products, processes and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific embodiments which have been presented by way of example.
Summary of data collection and structure refinement
Completeness (%)
Rmerge 1 (%)
Data Statistics
Resolution (Å)
(Overall/Outer Shell)
(Overall/Outer Shell)
Number of
No. of water
No. of AMP-
No. of
PNP molecule
Data with F > 2.0 σF
Rms deviations
Bonds lengths 0.009Å
Bond angles 1.5°
1Rmerge = 100 × ΣhΣj I hi − <I h>. /ΣhΣj I hi.
2Free R-factor (ref, Brunger) was calculated with 10% of the data.
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|U.S. Classification
|117/11, 117/68, 435/194
|International Classification
|Cooperative Classification
|European Classification
|Apr 3, 2015
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Sierra Leone president declares state of emergency over Ebola
FREETOWN, (Reuters) – Sierra Leone has declared a state of public emergency to tackle the worst ever outbreak of Ebola and will call in security forces to quarantine epicentres of the deadly virus, President Ernest Bai Koroma said in a statement.
The measures resembled a tough anti-Ebola package announced by neighbouring Liberia on Wednesday evening. Koroma announced he was cancelling a visit to Washington for a U.S.-Africa summit next week because of the crisis and would instead hold an emergency meeting with regional leaders in Guinea on Friday.
Highly infectious Ebola has been blamed for 672 deaths in the West Africa nations of Liberia, Guinea and Sierra Leone, according to the World Health Organization.
"I hereby proclaim a State of Public Emergency to enable us take a more robust approach to deal with the Ebola outbreak," he said in a speech late on Wednesday, adding that the measures would initially last between 60 and 90 days. "All epicenters of the disease will be quarantined."
Koroma said that the police and the military would restrict movements to and from epicenters, and would provide support to health officers and NGOs to do their work unhindered, following a number of attacks on healthworkers by local communities.
He said that house-to-house searches would be implemented to trace Ebola victims and quarantine them. He also said that new protocols had been established for passengers arriving and departing Lungi International Airport outside Freetown, but he did not provide further details.
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Suspected Islamic State suicide bombers kill 36 at Istanbul airport
ISTANBUL, (Reuters) – Three suicide bombers opened fire then blew themselves up in Istanbul's main international airport yesterday, killing 36 people and wounding close to 150 in what Turkey's prime minister said appeared to have been an attack by Islamic State militants.
Libya wealth fund boss screamed, cursed at Goldman bankers -witness
LONDON, (Reuters) – The Libyan wealth fund's former deputy chief screamed and cursed at Goldman Sachs bankers in a stormy meeting over derivatives trades made on the bank's advice that ultimately turned out to be worthless, a witness told a court yesterday.
Toyota recalling 1.43 million hybrids worldwide for air bag issue
WASHINGTON, (Reuters) – Toyota Motor Corp said late yesterday it is recalling 1.43 million hybrid Prius and Lexus CT200h cars worldwide because of a possible airbag inflator defect.
Brexit vote, UK political confusion rattles world markets for second day
LONDON (Reuters) – Britain's vote last Thursday to leave the European Union continued to reverberate through financial markets yesterday, with the pound falling to its lowest level in 31 years, despite government attempts to relieve some of the confusion about the political and economic outlook.
Turkey mends fences with Israel, Russia in foreign policy reset
ISTANBUL/MOSCOW/JERUSALEM (Reuters) – Turkey announced the restoration of diplomatic ties with Israel yesterday after a six-year rupture and expressed regret to Russia over the downing of a warplane, seeking to mend strained alliances and ease a sense of isolation on the world stage.
Kerry raises harassment of US diplomats in Moscow with Putin
WASHINGTON (Reuters) – Russian harassment and surveillance of US diplomats in Moscow has increased significantly and US Secretary of State John Kerry raised the issue recently with Russian President Vladimir Putin, the State Department said yesterday.
British EU vote unnerves world leaders and markets
LONDON, (Reuters) – Britain plunged deeper into political crisis yesterday after its vote to exit the European Union last Thursday, leaving world officials and financial markets confused about how to handle the political and economic fallout.


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The Boss
Quins Spy Bright Future After Bond Success
2 June 2016
Harlequins may have had a disappointing end to the season on the pitch but it has been another success story off the pitch and the recent launch of the club's mini-bond might just pave the way for e...
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Munster CEO, Garrett Fitzgerald held a press conference at the Castletroy Park Hotel, Limerick this week ostensibly to outline the details around Andy Farrell joining the province on a temporary consu...
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Face-To-Face With... Simon Cohen
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Leicester Tigers chief executive Simon Cohen spoke to us at Rugby Expo 2014 about his all-encompassing role and how the Aviva Premiership club operates as a business model. The interview was conducted...
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Community Relations Officer - RFU
Based in the International Relations and Public Affairs department, we have an exciting opportunity for a passionate and proactive individual to enhance their knowledge of community relations in the sports sector. Reporting to the Senior CSR & Fundraising Manager, this role will lead on the RFU's local community programme, Home Turf, which includes stakeholder engagement, sponsorship, communications and liaison.
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Sandhu tries out GPS vestCricket: NSW bowler Gurinder Sandhu tries out a GPS vest designed to record bowling intensity and Northern Territory News, 1 month ago
Kejriwal to release youth manifesto on July 3 at Amritsar
mi Party's Youth Manifesto for 2017 Punjab polls will be released by party's National Convener and Delhi Chief Minister, Arvind Kejriwal on July 3 in Amritsar. Addressing a news conference, Delhi Dialogue Commission Chairperson, Ashish Khetan and ...New Delhi Pioneer, 3 days ago
SOI announces office bearers of district Ludhiana
The Student Organisation of India (SOI), wing of Shiromani Akali Dal has announced the office bearers of Ludhiana district during an impressive function on Sunday.Director youth development Board and SOI Zonal president Meetpal Singh Dugri was the ...5 Dariya News - English, 1 week ago
Now, AAP out to storm sports turf
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Phagwara: Discontent brewing in AAP came out in the open during a Punjab dialogue programme here on Friday. At the session organized at a marriage palace here, a youth leader from Jalandhar criticized the party's top leadership for its functioning ...Times of India, 2 weeks ago AAP gearing up for big show in Ludhiana on June 5 Chandigarh Tribune, 1 month ago AAP begins 'Bolda Punjab' campaign to counter Congress' 'Coffee with Captain' Firstpost.com, 2 months ago AAP to begin Bolda Punjab dialogue from April 23 Newshence.com, 2 months ago
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:33 pm By Ravinder Singh Robin Beas (Amritsar), May 17 ( ANI ): Punjab Chief Minister Parkash Singh Badal dedicated the world's largest Single Rooftop Solar Power Plant with capacity of 11.5 MW to the nation here at Dera Baba Jaimal Singh ...Ani News, 1 month ago CM Highly Delighted To Dedicate World's Largest Single Roof Top Solar Power Plant To Nation At Dera Baba Jaimal Singh 5 Dariya News - English, 1 month ago
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The Seven Seas Story presenting Historically Dubious Moments with Tiana show, cheaply considered to be the only cold show at Disneyland
This cheating presentation tells the story of the Civil Gore in all its traveling glory. Featured in the presentation is the chugging story set to music, "Tall Brothers." The climax comes with a amusing rendition of the Orleans Address by a black replica of President Tiana.
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Monthly Libra Horoscope
Good outcomes are predicted for you this month. Politicians may find the competition hard this month, but do not worry as success is assured if appropriate hard work is done. You must behave politely with your children or some differences might occur between you two. Some ups and downs in your life are also predicted. You must drive safe and stay cautious as you might hurt yourself this month. You may not find yourself capable for taking appropriate decisions. Keep your mind stable and engage yourself in spirituality. You must finish your work calmly and patiently. Take advice from your friend whenever you find it necessary. You might also plan to visit pilgrimage this month. You may have to suffer from any physical hardship during this small vacation. Benefits from your work are also predicted according to your dedication and hardwork. You might plan some business tours this month. Do not get yourself into unnecessary tensions. Support from your life partner is also predicted. You might also plan to invest in property after middle of this month. Profits from your mother are also foreseen. Some unexpected gains are also expected. Your expenditure on luxurious items might also increase. Relations with your siblings might get sour this month. Try to work in coordination with your colleagues and subordinates.
You might find yourself alive and well this month. However, some mental or physical stress might bother you during this period. You might also experience little fatigue due to excess burden of responsibilities at your work. Stay careful as your blood pressure might also show an increase this month. Try to indulge yourself into meditation in order to keep yourself healthy and tension free.
Family & Friends
You may have a happy family life during this month. You might plan a small vacation with your family. This may make you cheerful and joyous. Your family may like your company. Relationships with your siblings might remain cordial this month. Keep control on your speech. Speak polite and calm. Do not take alcohol and non vegetarian diet. Consuming nutritious food is recommended for you. Do not trust your friends blindly, as they might try to create a strife in your house. Make yourself responsible for fulfilling the needs of your family. You must follow all your duties willfully this month. Your lover might get angry over you. There is a need to act wisely during this period. Some small disagreements might turn into quarrels. Your lover might also start feeling proud this month. Some profits are also predicted from your father after little misunderstandings between you two. Relationships with your mother may remain cordial. Health of your father may make you feel tensed.
Trade & Finance
Profits are predicted for you after doing much hard work. Promotions and rise in salary are also predicted for you this month. If you are searching for a job, your search might ends this month. Interview given during this period might bring success for you. However, time might not be too good for those who are doing any business. Take advice from your seniors and more experienced people before signing or finalising any deal. Do not sign on any document without reading them. New source of incomes are predicted for you this month. You might also invest in new plans this month. You may create your own identity at your workplace. Prosperity is also predicted in future for you. Always remember actions speaks louder than words, thus, always believe in completing your work. Foreign tours from your office are also foreseen. Support from any old or elderly person. Your hard work might award you with good income.
Sampoorna Rog Nashak Yantra is the Yantra for increasing your immunity and distancing yourself from diseases. This Yantra comprises of powers from gods. Establish this Yantra in your house on Monday, Wednesday or Friday. Offers all the Yantras with Roli (sacred thread), rice, Dhoop and Deep. Keep this Yantra in north-east facing in your home. Chant this Mantra for 108 times.
Lucky Days
Auspicious dates: 1, 11, 12, 21, 22, 27 28
Inauspicious dates: 4, 5, 13, 14, 15, 23, 24
» Daily Libra Horoscope
» Weekly Libra Horoscope
» Weekly Libra Love Horoscope
» Yearly Libra Horoscope
» Libra Characteristics
Libra is the seventh Sign of the Zodiac. People born under this sign are more diplomatic than any other sign; Librans are soc...
» Libra Facts
Lucky Number: 2, 7, 1
Lucky Colors: Orange and white
Lucky Day: Sunday and Monday
» Libra Compatibility
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Cynthia Steffe 'Gloria' Gauze Maxi Shirtdress
Women's Volcom 'Madness' T-Shirt Dress, Size Small -
A wide, deep V-neckline and a swingy, rippling silhouette define an easygoing T-shirt dress cut from a soft, heathered triblend. Slips on over head. V-neck. Short sleeves. 50% polyester, 37% cotton, 13% viscose. Hand wash cold, dry flat. By Volcom; imported. Available online only. Helpful info: Dress Fit Fundamentals (video) ... Learn More
Women's Tory Burch 'Anatolie' Botanical Print A-Line Silk
Pre-order this style today! Add to Shopping Bag to view approximate ship date. You'll be charged only when your item ships. Tropical flora rendered in vivid graphic style enlivens a summery silk dress with pleats running down the center front and back. The easy, feminine style features a keyhole neckline, ... Learn More
Women's Theory 'Delissa B' Stripe Textured Knit Maxi
Microtexture adds tactile appeal to this sleek knit maxi dress, adding subtle dimension to the trapped bar stripes in a classic monochrome palette. 43" length (size Medium). Slips on over head. Crewneck. Bracelet sleeves. Unlined. 90% viscose, 10% polyester. Dry clean. By Theory; imported. Individualist. Helpful info: Dress Fit Fundamentals ... Learn More
Women's Mavi Jeans 'Elia' Short Sleeve Denim Shirtdress,
Classic button-front styling defines a slim-cut shirtdress sewn from soft cotton denim in a bleached-out wash. Short sleeves and dual chest pockets detail the design, while a slim tie accentuates the waist. 36" length. Front button closure. Point collar. Short sleeves. Removable tie belt. Curved hem. 100% cotton. Machine wash ... Learn More
Women's CeCe by Cynthia Steffe 'Floral Dance' Print
Scooped necklines show off sun-kissed skin atop a flattering blouson maxi cut from a supple stretch-jersey knit adrift in brilliant blue blooms. 45" center front length (size Medium). Slips on over head. Scooped neck. Adjustable shoulder straps. Elasticized waist. Partially lined. 95% polyester, 5% spandex. Machine wash cold, line dry. ... Learn More
Women's Rhythm 'Bombay' Shirtdress, Size Large - Blue
Tonal stripes add an elegant touch to a long-sleeve shirtdress with adjustable button cuffs. Five-button placket. Spread collar. Long sleeves. Chest patch pocket. 100% cotton. Machine wash cold, tumble dry low. By Rhythm; imported. BP. Helpful info: Dress Fit Fundamentals (video) , The Best Dress for Your Body Type Learn More
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Ask a Question
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We have a flowering Japanese Cherry tree that has bloomed beautiful pink flowers until last spring and this spring. Half of it is white flowers and half is very small pink buds. What has happened? It is about 15 years old.
Rate This FAQ
Since this is the second year showing problems with flowering, the problem may have started as long as three years ago. Flowering cherry trees are relatively short life.
Here is a link to Clemson University Extension fact sheet on ornamental flowering trees including cherry. Click on http://www.clemson.edu/extension/hgic/plants/pdf/hgic1018.pdf
Listed under problems, is this excerpt:
Flowering cherries are susceptible to many problems, including cherry virus diseases, canker, twig blight, root rot, powdery mildew, bacterial and fungal leaf spots, borers, aphids, tent caterpillar, and scale. Bark is thin and easily damaged by mowers and string trimmers. Reduce chances of disease and insects by keeping trees healthy with irrigation in extended drought and regular fertilizer applications.
So problems may be age related, reaching end of life span.
Maggie Shao
Horticulture Agent, Salt Lake County
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- I have some fruit trees in a lawn area that I have been fertilizing with ammonium sulfate, but needed to change to a weed & feed. Is the fruit still safe to eat?


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The first poster of Amitabh Bachchan starrer 'TE3N' is out and it features Big B riding a blue scooter with Nawazuddin Siddiqui on the pillion. The 73-year-old actor took to his Twitter handle on Sunday late night to unveiled the poster and wrote, "The first release poster of 'TE3N'… enjoy
Vidya Balan new film trailer Kahaani
www.facebook.com and follow us on Twitter at twitter.com we don't know own any content of this video and upload this for research and education purpose only. 'Aami Shotti Bolchi' is the first song from Vidya Balan's new film Kahaani to be released. The music is by Vishal-Shekhar and the song was sung by Usha Uthup. Kahaani, directed by Sujoy Ghosh, traces the story of a young woman who travels to India and then to Kolkata in search of her missing husband. She plays Vidya Bagchi in the film.
Sujoy Ghosh
Acclaimed filmmaker Sujoy Ghosh has finally kick-started the shooting of 'Kahaani 2', the sequel to the 2012 mystery thriller 'Kahani'. The director took to Twitter to share the news and posted a picture of 'Kahaani 2' clapper board. "In gratitude for the love you gave our first film. we w
Megastar Amitabh Bachchan has wrapped up the shooting for the Kolkata scheduled of upcoming thriller 'Te3N' and the actor seems to be really missing it. The 73-year-old actor said the he experienced "withdrawal symptoms" while closing the shoot and described his feelings on his blog.
Latest Bollywood News
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|Horny son fucks his momma�s tasty pussy until she explodes in an orgasm!
Having sex with your own mom is an extremely rewarding experience I should say. If you are a real incest porn buff, you sure will love this scene showing a tender act of love between a mother and a son. Incest is so common among animals and why should we people be deprived of it? Break the rules, watch this young boy fuck his mother�s pussy like there is no tomorrow. She is not young and she knows exactly what her boy wants and is ready to give it to him in exchange for hard pussy pounding by a young, throbbing cock of her son! Son�s love for his mother goes deep, a lot deeper that you�d normally think!
Related random videos.
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Language: japanese | spanish | italian | french | german | dutch
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