# Patent

## Title
Method for crystallizing human beta secretase in complex with an inhibitor

## Abstract

An inhibitor bound form of human beta secretase, also known as memapsin 2 and BACE, particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system has been crystallized, and the three dimensional x-ray crystal structure has been solved to 3.2 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of human beta secretase activity.

## Background
RELATED APPLICATIONS
This is a continuation of U.S. application Ser. No. 10/144,441 filed May 10, 2002 (now abandoned), which is a continuation-in-part of U.S. application Ser. No. 10/027,277, filed Dec. 21, 2001 (now abandoned), which is a continuation of U.S. application Ser. No. 09/808,262, filed Mar. 14, 2001 (now abandoned), which is a continuation-in-part of U.S. application Ser. No. 09/747,420, filed Dec. 23, 2000 (now abandoned), which are incorporated herein by reference in their entireties.


FIELD OF THE INVENTION
This invention relates to the crystallization and structure determination of beta secretase, also known as memapsin 2 and BACE, from human (Homo sapiens), particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system.
BACKGROUND
Alzheimer's disease (AD) causes progressive dementia with consequent formation of amyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. The disease occurs in both genetic and sporadic forms whose clinical course and pathological features are quite similar. Three genes have been discovered to date which, when mutated, cause an autosomal dominant form of Alzheimer's disease. These encode the amyloid protein precursor (APP) and two related proteins, presenilin-1 (PS1) and presenilin-2 (PS2), which, as their names suggest, are structurally and functionally related. Mutations in any of the three proteins have been observed to enhance proteolytic processing of APP via an intracellular pathway that produces amyloid beta peptide (Aβ peptide, or sometimes here as Abeta), a 40-42 amino acid long peptide that is the primary component of amyloid plaque in AD.
Dysregulation of intracellular pathways for proteolytic processing may be central to the pathophysiology of AD. In the case of plaque formation, mutations in APP, PS1 or PS2 consistently alter the proteolytic processing of APP so as to enhance formation of Aβ 1-42, a form of the Aβ peptide which seems to be particularly amyloidogenic, and thus very important in AD. Different forms of APP range in size from 695-770 amino acids, localize to the cell surface, and have a single C-terminal transmembrane domain. The Abeta peptide is derived from a region of APP adjacent to and containing a portion of the transmembrane domain. Normally, processing of APP at the α-secretase site cleaves the midregion of the Aβ sequence adjacent to the membrane and releases the soluble, extracellular domain of APP from the cell surface. This α-secretase APP processing creates soluble APP-α, which is normal and not thought to contribute to AD. Pathological processing of APP at the β- and γ-secretase sites, which are located N-terminal and C-terminal to the a-secretase site, respectively, produces a very different result than processing at the a site. Sequential processing at the β- and γ-secretase sites releases the Aβ peptide, a peptide possibly very important in AD pathogenesis. Processing at the β- and γ-secretase sites can occur in both the endoplasmic reticulum (in neurons) and in the endosomal/lysosomal pathway after reinternalization of cell surface APP (in all cells). Despite intense efforts, for 10 years or more, to identify the enzymes responsible for processing APP at the β and γ sites, to produce the Aβ peptide, those proteases remained unknown until recently.
The identification and characterization of the β secretase enzyme, termed Aspartyl Protease 2 (Asp2) has been established. In addition, the X-ray crystal structure of human beta secretase in complex with a peptide inhibitor was solved and published Hong et al., Science 290: 150-53 (2000) from protein expressed in E. coli that contained no covalent sugar (glycosylation) at any of the four putative glycosylation sites within the enzyme.
SUMMARY OF THE INVENTION
This invention relates to the crystallization and structure determination of beta secretase, also known as memapsin 2 and BACE, from human (Homo sapiens), particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system.
In one aspect, the present invention provides a method for crystallizing a human beta secretase molecule or molecular complex. The method involves crystallizing a human beta secretase molecule or molecular complex by preparing purified human beta secretase in the presence of an inhibitor and crystallizing human beta secretase from a solution having a pH of at most about 6.0.
In another aspect, the present invention provides crystalline forms of a human beta secretase molecule. In one embodiment, the present invention provides a crystal of beta secretase having the trigonal space group symmetry P3221. In another embodiment, a crystal of human beta secretase is provided having the trigonal space group symmetry P3221 with unit cell dimensions of a, b, and c, wherein a is about 77 Å to about 147 Å, b is about 77 Å to about 147 Å, and c is about 77 Å to about 147 Å; and α=β=90°, and γ=120°. Preferably, the crystal has unit cell dimensions of a=112.0 Å, b=112 Å, c=110 Å, α=β=90°, γ=120°.
In another aspect, the present invention provides a method of producing human beta secretase, the method including expressing the human beta secretase in a mammalian cell line.
In another aspect, the present invention provides a method of producing human beta secretase, the method including expressing the human beta secretase in an insect cell line.
Abbreviations
The following abbreviations are used throughout this disclosure:

    Alzheimer's disease (AD)
    Amyloid beta peptide (Aβ peptide or Abeta)
    Amyloid protein precursor (APP)
    Aspartyl protease 2 (Asp2)
    Baculovirus expression system (BVES)
    Beta secretase (memapsin 2, BACE)
    Chinese hamster ovary (CHO)
    Dimethyl sulfoxide (DMSO)
    Multiple anomalous dispersion (MAD)
    Presenilin-1 (PS1)
    Presenilin-2 (PS2)
    Polyethylene glycol (PEG)


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













BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is an illustration of the chemical structure of an inhibitor used in co-crystallization experiments.
FIG. 2 is the synthetic peptide Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine, SEQ ID NO:3, PNU-292593E), used for affinity purification of beta secretase.
FIG. 3 is an electron density map for the inhibitor illustrated in FIG. 1 at 3.2 Å.
FIG. 4 is a stereo view of a Cα trace of human beta secretase (black line) in the presence of the inhibitor illustrated in FIG. 1. The general location of the inhibitor trace (gray line) is indicated by an arrow.
FIG. 5 is a stereo view of Cα traces of human beta secretase as a superposition of a data set from the present invention (black line) with a data set from Hong et al., Science 290:150-53 (2000) (gray line). The r.m.s. deviation on Cα between the illustrated data sets was 0.43 Å.
FIG. 6 depicts a stereo view of the active site of human beta secretase (illustrated with light gray carbons, dark gray oxygens, and black nitrogens) with the inhibitor illustrated in FIG. 1. The general location of the inhibitor is indicated by an arrow (illustrated with light gray carbons, dark gray oxygens, and black nitrogens). The iodine atom is circled and the two fluorine atoms are indicated by a “●” symbol.
FIG. 7 depicts the sequence (SEQ ID NO:1) of residues for recombinant human beta secretase present in the X-ray structure.



DETAILED DESCRIPTION OF THE INVENTION
Crystalline Form(s) and Method of Making
The three-dimensional structure of human beta secretase was solved using x-ray crystallography to 3.2 Å resolution. Accordingly, the invention includes a human beta secretase crystal and/or a crystal with human beta secretase co-crystallized with a ligand, such as an inhibitor. Preferably, the crystal has trigonal space group symmetry P3221. More preferably, the crystal includes hexagonal shaped unit cells, each unit cell having dimensions a=l 112.0±35 Å, b=112±35 Å, c=110±35 Å, α=β=90°, γ120. The crystallized enzyme is a monomer with a single monomer in the asymmetric unit.
According to the present invention, human beta secretase can be isolated from a variety of cell lines, for example, the mammalian cell line CHO-K1 or an insect cell line.
A “molecular complex” means a protein in covalent or non-covalent association with a chemical entity. In a preferred embodiment, molecular complexes of purified human beta secretase at a concentration of about 1 mg/ml to about 80 mg/ml may be crystallized in the presence of an inhibitor at a concentration from about 0.1 to about 10 mM, for example, by using a streak seeding procedure from a solution including about 5% by weight to about 50% by weight PEG or PEG-MME (PEG monomethyl ether), PEG-DME (PEG dimethyl ether), or polyoxyalkylenepolyamines (e.g., materials available under the trade designation JEFFAMINE from Huntsman Corp., Salt Lake City, Utah) (preferably having a number average molecular weight between about 200 and about 20,000), preferably, a salt (more preferably about 0.001 M to about 0.5 M salt), and about 0% by weight to about 20% by weight organic solvent (such as DMSO). Exemplary salts include sodium chloride, ammonium sulfate, magnesium sulfate, lithium sulfate, or combinations thereof.
The solution has a pH of at most about 6.0, preferably at most about 5.8, more preferably at most about 5.6, even more preferably at most about 5.5, and most preferably at most about 4.7. The lower pH limit is not believed to be critical, but certain embodiments provide crystallization of beta secretase at a pH of at least about 3.5, and more preferably at least about 4.0. Although the method of crystallizing beta secretase is only exemplified herein for protein derived from CHO cells and Baculovirus cells, the method will also work for crystallizing beta secretase derived from other sources (e.g., E. coli).
Use of a buffer having a pKa of about 3 to about 6 is preferred. A particularly preferred buffer is about 10 mM to about 200 mM sodium acetate. Variation in buffer and buffer pH as well as other additives such as PEG or PEG-MME (PEG monomethyl ether), PEG-DME (PEG dimethyl ether), or polyoxyalkylenepolyamines (e.g., materials available under the trade designation JEFFAMINE from Huntsman Corp., Salt Lake City, Utah) is apparent to those skilled in the art and may result in similar crystals.
The invention further includes an human beta secretase crystal that is isomorphous with an human beta secretase crystal characterized by a unit cell having dimensions of a, b, and c; wherein a is about 77 Å to about 147 Å, b is about 77 Å to about 147 Å, and c is about 75 Å to about 145 Å; and α=β=90°, and γ=120°.
X-Ray Crystallographic Analysis
Each of the constituent amino acids of human beta secretase is defined by a set of structure coordinates. The term “structure coordinates” refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of an human beta secretase complex in crystal form. The diffraction data are used to calculate an electron density map of the repeating unit of the crystal. The electron density maps are then used to establish the positions of the individual atoms of the human beta secretase protein or protein/ligand complex.
Slight variations in structure coordinates can be generated by mathematically manipulating the human beta secretase or human beta secretase/ligand structure coordinates. For example, structure coordinates could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above. 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 yield variations in structure coordinates. Such slight variations in the individual coordinates will have little effect on overall shape. If such variations are within an acceptable standard error as 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 variations in individual structure coordinates of the human beta secretase would not be expected to significantly alter the nature of chemical entities such as ligands that could associate with the inhibitor binding pockets. In this context, the phrase “associating with” refers to a condition of proximity between a chemical entity, or portions thereof, and an human beta secretase molecule or portions thereof. The association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent.
Thus, for example, a ligand that bound to a inhibitor binding pocket of human beta secretase would also be expected to bind to or interfere with another inhibitor binding pocket whose structure coordinates define a shape that falls within the acceptable error.
It will be readily apparent to those of skill in the art that the numbering of amino acids in other isoforms of human beta secretase may be different than that of human beta secretase expressed in CHO or HKE 293 cells.
Active Site and Other Structural Features
Applicants' invention provides information about the shape and structure of the inhibitor binding pocket of human beta secretase in the presence of an inhibitor. The secondary structure of the human beta secretase monomer includes two domains consistent with a typical aspartic protease fold.
Binding pockets 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 helps lead to the design of drugs having more favorable associations with their target, and thus improved biological effects. Therefore, this information is valuable in designing potential inhibitors of beta secretase-like inhibitor binding pockets, as discussed in more detail below.
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. Thus, a binding pocket may include or consist of features such as cavities, surfaces, or interfaces between domains. Chemical entities that may associate with a binding pocket include, but are not limited to, cofactors, substrates, inhibitors, agonists, and antagonists.
The amino acid constituents of an human beta secretase inhibitor binding pocket as defined herein are positioned in three dimensions. In one aspect, the structure coordinates defining a inhibitor binding pocket of human beta secretase include structure coordinates of all atoms in the constituent amino acids; in another aspect, the structure coordinates of a inhibitor binding pocket include structure coordinates of just the backbone atoms of the constituent atoms.
The inhibitor binding pocket of human beta secretase preferably includes the amino acids listed in Table 1, more preferably the amino acids listed in Table 2, and most preferably the amino acids listed in Table 3. Alternatively, the inhibitor binding pocket of human beta secretase may be defined by those amino acids whose backbone atoms are situated within about 4 Å, more preferably within about 7 Å, most preferably within about 10 Å, of one or more constituent atoms of a bound substrate or inhibitor. In yet another alternative, the inhibitor binding pocket may be defined by those amino acids whose backbone atoms are situated within a sphere centered on the coordinates representing the alpha carbon atom of residue Thr 231, the sphere having a radius of about 15 Å, preferably about 20 Å, and more preferably about 25 Å.
The term “beta secretase-like inhibitor binding pocket” refers to a portion of a molecule or molecular complex whose shape is sufficiently similar to at least a portion of a inhibitor binding pocket of human beta secretase as to be expected to bind related structural analogues. A structurally equivalent inhibitor binding pocket is defined by a root mean square deviation from the structure coordinates of the backbone atoms of the amino acids that make up inhibitor binding pockets in human beta secretase of at most about 0.35 Å. How this calculation is obtained is described below.
Accordingly, the invention provides molecules or molecular complexes including an human beta secretase inhibitor binding pocket or beta secretase-like inhibitor binding pocket, as defined by the sets of structure coordinates described above.







TABLE 1





Residues with 4 Å of inhibitor binding site.




















GLY 11
SER 35
PHE 108
ASP 228



GLY 13
PRO 70
ILE 110
GLY 230



LEU 30
TYR 71
TRP 115
THR 231



ASP 32
THR 72
ILE 126
THR 232



GLY 34
GLN 73
TYR 198
ARG 235

















TABLE 2





Residues with 7 Å of inhibitor binding site.




















SER 10
ASN 37
PHE 109
GLY 230



GLY 11
VAL 69
ILE 110
THR 231



GLN 12
PRO 70
TRP 115
THR 232



GLY 13
TYR 71
ILE 118
ASN 233



TYR 14
THR 72
ILE 126
ARG 235



LEU 30
GLN 73
ALA 127
SER 325



VAL 31
GLY 74
ARG 128
THR 329



ASP 32
LYS 75
TYR 198
VAL 332



THR 33
TRP 76
LYS 224
ALA 335



GLY 34
ASP 106
ILE 226



SER 35
LYS 107
ASP 228



SER 36
PHE 108
SER 229

















TABLE 3





Residues with 10 Å of inhibitor binding site.




















ARG 7
TYR 71
LEU 121
THR 232



GLY 8
THR 72
ALA 122
ASN 233



LYS 9
GLN 73
TYR 123
LEU 234



SER 10
GLY 74
ALA 124
ARG 235



GLY 11
LYS 75
GLU 125
ARG 307



GLN 12
TRP 76
ILE 126
PHE 322



GLY 13
GLU 77
ALA 127
ALA 323



TYR 14
ILE 102
ARG 128
ILE 324



TYR 15
SER 105
PRO 129
SER 325



ILE 29
ASP 106
LEU 154
GLN 326



LEU 30
LYS 107
TRP 197
SER 327



VAL 31
PHE 108
TYR 198
SER 328



ASP 32
PHE 109
TYR 199
THR 329



THR 33
ILE 110
ASP 223
GLY 330



GLY 34
ASN 111
LYS 224
THR 331



SER 35
SER 113
SER 225
VAL 332



SER 36
TRP 115
ILE 226
MET 333



ASN 37
GLU 116
VAL 227
GLY 334



ALA 39
GLY 117
ASP 228
ALA 335



TYR 68
ILE 118
SER 229
VAL 336



VAL 69
LEU 119
GLY 230
MET 338



PRO 70
GLY 120
THR 231
GLU 339










Three-Dimensional Configurations

X-ray structure coordinates define a unique configuration of points in space. Those of skill in the art understand that a set of structure coordinates for protein or an protein/ligand complex, or a portion thereof, define a relative set of points that, in turn, define a configuration in three dimensions. A similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remain essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.
The present invention thus includes the scalable three-dimensional configuration of points derived from the structure coordinates of at least a portion of an human beta secretase molecule or molecular complex, as well as structurally equivalent configurations, as described below. Preferably, the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of a plurality of the amino acids defining an human beta secretase inhibitor binding pocket.
In one embodiment, the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations the backbone atoms of a plurality of amino acids defining the human beta secretase inhibitor binding pocket, preferably the amino acids listed in Table 1, more preferably the amino acids listed in Table 2, and most preferably the amino acids listed in Table 3. Alternatively, the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of the side chain and the backbone atoms (other than hydrogens) of a plurality of the amino acids defining the human beta secretase inhibitor binding pocket, preferably the amino acids listed in Table 1, more preferably the amino acids listed in Table 2, and most preferably the amino acids listed in Table 3.
Likewise, the invention also includes the scalable three-dimensional configuration of points derived from structure coordinates of molecules or molecular complexes that are structurally homologous to beta secretase, as well as structurally equivalent configurations. Structurally homologous molecules or molecular complexes are defined below. Advantageously, structurally homologous molecules can be identified using the structure coordinates of human beta secretase according to a method of the invention.
The configurations of points in space derived from structure coordinates according to the invention can be visualized as, for example, a holographic image, a stereodiagram, a model or a computer-displayed image, and the invention thus includes such images, diagrams or models.
Structurally Equivalent Crystal Structures
Various computational analyses can be used to determine whether a molecule or a inhibitor binding pocket portion thereof is “structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of human beta secretase or its inhibitor binding pockets. Such analyses may be carried out in current 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 equivalent atoms are defined as protein backbone atoms (N, Cα, C, and 0) for all conserved residues between the two structures being compared. A conserved residue is defined as a residue which is structurally or functionally equivalent. Only rigid fitting operations are considered.
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 inhibitor binding pocket thereof, or any portion thereof, that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than about 0.35 Å, when superimposed on the relevant backbone atoms is considered “structurally equivalent” to the reference molecule. That is to say, the crystal structures of those portions of the two molecules are substantially identical, within acceptable error. Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structure coordinates ± a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 0.35 Å. More preferably, the root mean square deviation is less than about 0.2 Å. Another embodiment of this invention is a molecular complex for those amino acids listed in Table 1, ± a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 0.35 Å, preferably less than about 0.2 Å. Still another embodiment of this invention is a molecular complex for those amino acids listed in Table 2, ± a root mean square deviation from the conserved backbone atoms of those amino acids of not more than 0.35 Å, preferably less than about 0.2 Å.
The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations. 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 human beta secretase or a inhibitor binding pocket portion thereof, as defined by the structure coordinates of human beta secretase described herein.
Machine Readable Storage Media
Transformation of the structure coordinates for all or a portion of human beta secretase or the human beta secretase/ligand complex or one of its inhibitor binding pockets, for structurally homologous molecules as defined below, or for the structural equivalents of any of these molecules or molecular complexes as defined above, into three-dimensional graphical representations of the molecule or complex can be conveniently achieved through the use of commercially-available software.
The invention thus further provides a machine-readable storage medium including a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of any of the molecule or molecular complexes of this invention that have been described above. In a preferred embodiment, the machine-readable data storage medium includes a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex including all or any parts of an human beta secretase inhibitor binding pocket or an beta secretase-like inhibitor binding pocket, as defined above. In another preferred embodiment, the machine-readable data storage medium includes a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex ± a root mean square deviation from the backbone atoms of said amino acids of not more than 0.43 Å.
In an alternative embodiment, the machine-readable data storage medium includes a data storage material encoded with a first set of machine readable data which includes the Fourier transform of structure coordinates, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data including the x-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.
For example, a system for reading a data storage medium may include a computer including a central processing unit (“CPU”), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), electroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, track balls, touch pads, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus. The system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.). The system may also include additional computer controlled devices such as consumer electronics and appliances.
Input hardware may be coupled to the computer by input lines and 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 connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may include CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.
Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices. By way of example, the output hardware may include a display device 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, so that hard copy output may be produced, or a disk drive, to store system output for later use.
In operation, a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, 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. References to components of the hardware system are included as appropriate throughout the following description of the data storage medium.
Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof. Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device. It should be understood that these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data.
Structurally Homologous Molecules, Molecular Complexes, and Crystal Structures
Structure coordinates can be used to aid in obtaining structural information about another crystallized molecule or molecular complex. The method of the invention allows determination of at least a portion of the three-dimensional structure of molecules or molecular complexes which contain one or two more structural features that are similar to structural features of human beta secretase. These molecules are referred to herein as “structurally homologous” to human beta secretase. Similar structural features can include, for example, regions of amino acid identity, conserved active site or binding site motifs, and similarly arranged secondary structural elements (e.g., α helices and β sheets. Optionally, structural homology is determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. Preferably, two amino acid sequences are compared using the Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as described by Tatusova et al.,FEMS Microbiol Lett 174, 247-50 (1999) Preferably, the default values for all BLAST 2 search parameters are used, including matrix =BLOSUM62; open gap penalty =11, extension gap penalty =1, gap x—dropoff =50, expect =10, wordsize =3, and filter on. In the comparison of two amino acid sequences using the BLAST search algorithm, structural similarity is referred to as “identity.” Preferably, a structurally homologous molecule is a protein that has an amino acid sequence sharing at least 65% identity with a native or recombinant amino acid sequence of human beta secretase (for example, SEQ ID NO:1). More preferably, a protein that is structurally homologous to human beta secretase includes at least one contiguous stretch of at least 50 amino acids that shares at least 80% amino acid sequence identity with the analogous portion of the native or recombinant human beta secretase (for example, SEQ ID NO:1). Methods for generating structural information about the structurally homologous molecule or molecular complex are well-known and include, for example, molecular replacement techniques.
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 including the steps of:
(a) crystallizing the 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 to the x-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
By using molecular replacement, all or part of the structure coordinates of human beta secretase or the human beta secretase/ligand complex as provided by this invention can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.
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 cannot 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 structurally 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 human beta secretase or the human beta secretase/inhibitor complex within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed x-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed x-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (Lattman, “Use of the Rotation and Translation Functions,” in Meth. Enzymol. 115, pp. 55-77 (1985); M. G. Rossman, ed., The Molecular Replacement Method—A Collection of Papers on the Use of Non-Crystallographic Symmetry, Intl. Sci. Rev. Ser. No. 13, Gordon & Breach, New York (1972)).
Structural information about a portion of any crystallized molecule or molecular complex that is sufficiently structurally homologous to a portion of human beta secretase can be resolved by this method. In addition to a molecule that shares one or more structural features with human beta secretase as described above, a molecule that has similar bioactivity, such as the same catalytic activity, substrate specificity or ligand binding activity as human beta secretase, may also be sufficiently structurally homologous to human beta secretase to permit use of the structure coordinates of human beta secretase 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 includes at least one human beta secretase subunit or homolog. A “subunit” of human beta secretase is an human beta secretase molecule that has been truncated at the N-terminus or the C-terminus, or both. In the context of the present invention, a “homolog” of human beta secretase is a protein that contains one or more amino acid substitutions, deletions, additions, or rearrangements with respect to the amino acid sequence of human beta secretase (SEQ ID NO:1), but that, when folded into its native conformation, exhibits or is reasonably expected to exhibit at least a portion of the tertiary (three-dimensional) structure of human beta secretase. For example, structurally homologous molecules can contain deletions or additions of one or more contiguous or noncontiguous amino acids, such as a loop or a domain. Structurally homologous molecules also include “modified” human beta secretase molecules that have been chemically or enzymatically derivatized at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
A heavy atom derivative of human beta secretase is also included as an human beta secretase homolog. The term “heavy atom derivative” refers to derivatives of human beta secretase produced by chemically modifying a crystal of human beta secretase. In practice, a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thiomersal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein. The location(s) of the bound heavy metal atom(s) can be determined by x-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the protein (Blundell et al., Protein Crystallography, Academic Press (1976)).
Because human beta secretase can crystallize in more than one crystal form, the structure coordinates of human beta secretase as provided by this invention are particularly useful in solving the structure of other crystal forms of human beta secretase or human beta secretase complexes.
The structure coordinates of human beta secretase as provided by this invention are particularly useful in solving the structure of human beta secretase mutants. Mutants may be prepared, for example, by expression of human beta secretase cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis. Mutants may also be generated by site-specific incorporation of unnatural amino acids into beta secretase proteins using the general biosynthetic method of Noren et al., Science 244:182-88 (1989). In this method, the codon encoding the amino acid of interest in wild-type human beta secretase is replaced by a “blank” nonsense codon, TAG, using oligonucleotide-directed mutagenesis. A suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid. The aminoacylated tRNA is then added to an in vitro translation system to yield a mutant human beta secretase with the site-specific incorporated unnatural amino acid.
Selenocysteine or selenomethionine may be incorporated into wild-type or mutant human beta secretase by expression of human beta secretase-encoding cDNAs in auxotrophic E. coli strains (Hendrickson et al., EMBO J 9:1665-72 (1990)). In this method, the wild-type or mutagenized human beta secretase cDNA may be expressed in a host organism on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both). Alternatively, selenomethionine analogues may be prepared by down regulation methionine biosynthesis. (Benson et al., Nat. Struct. Biol. 2:644-53 (1995); Van Duyne et al., J. Mol. Biol. 229:105-24 (1993)).
The structure coordinates of human beta secretase are also particularly useful to solve the structure of crystals of human beta secretase, human beta secretase mutants or human beta secretase homologs co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate human beta secretase inhibitors and human beta secretase. Potential sites for modification within the various binding site of the molecule can also be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between human beta secretase and a chemical entity. 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 human beta secretase inhibition activity.
All of the complexes referred to above may be studied using well-known x-ray diffraction techniques and may be refined versus 1.5-3.5 Å resolution x-ray data to an R value of about 0.30 or less using computer software, such as X-PLOR (Yale University, 81992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., Vol. 114 & 115, H. W. Wyckoffet al., eds., Academic Press (1985)). This information may thus be used to optimize known human beta secretase inhibitors, and more importantly, to design new human beta secretase inhibitors.
The invention also includes the unique three-dimensional configuration defined by a set of points defined by the structure coordinates for a molecule or molecular complex structurally homologous to human beta secretase as determined using the method of the present invention, structurally equivalent configurations, and magnetic storage media including such set of structure coordinates.
Further, the invention includes structurally homologous molecules as identified using the method of the invention.
Homology Modeling
Using homology modeling, a computer model of an human beta secretase homolog can be built or refined without crystallizing the homolog. First, a preliminary model of the human beta secretase homolog is created by sequence alignment with human beta secretase, secondary structure prediction, the screening of structural libraries, or any combination of those techniques. Computational software may be used to carry out the sequence alignments and the secondary structure predictions. Structural incoherences, e.g., structural fragments around insertions and deletions, can be modeled by screening a structural library for peptides of the desired length and with a suitable conformation. For prediction of the side chain conformation, a side chain rotamer library may be employed. If the human beta secretase homolog has been crystallized, the final homology model can be used to solve the crystal structure of the homolog by molecular replacement, as described above. Next, the preliminary model is subjected to energy minimization to yield an energy minimized model. The energy minimized model may contain regions where stereochemistry restraints are violated, in which case such regions are remodeled to obtain a final homology model. The homology model is positioned according to the results of molecular replacement, and subjected to further refinement including molecular dynamics calculations.
Rational Drug Design
Computational techniques can be used to screen, identify, select and/or design chemical entities capable of associating with human beta secretase or structurally homologous molecules. Knowledge of the structure coordinates for human beta secretase permits the design and/or identification of synthetic compounds and/or other molecules which have a shape complementary to the conformation of the human beta secretase binding site. In particular, computational techniques can be used to identify or design chemical entities, such as inhibitors, agonists and antagonists, that associate with an human beta secretase inhibitor binding pocket or an beta secretase-like inhibitor binding pocket. Inhibitors may bind to or interfere with all or a portion of an active site of human beta secretase, and can be competitive, non-competitive, or uncompetitive inhibitors; or interfere with dimerization by binding at the interface between the two monomers. Once identified and screened for biological activity, these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block human beta secretase activity and, thus, prevent the onset and/or further progression of Alzheimer's disease. Structure-activity data for analogues of ligands that bind to or interfere with human beta secretase or beta secretase-like inhibitor binding pockets can also be obtained computationally.
The term “chemical entity,” as used herein, refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes. Chemical entities that are determined to associate with human beta secretase are potential drug candidates.
Data stored in a machine-readable storage medium that is capable of displaying a graphical three-dimensional representation of the structure of human beta secretase or a structurally homologous molecule, as identified herein, or portions thereof may thus be advantageously used for drug discovery. The structure coordinates of the chemical entity are used to generate a three-dimensional image that can be computationally fit to the three-dimensional image of human beta secretase or a structurally homologous molecule. The three-dimensional molecular structure encoded by the data in the data storage medium can then be computationally evaluated for its ability to associate with chemical entities. When the molecular structures encoded by the data is displayed in a graphical three-dimensional representation on a computer screen, the protein structure can also be visually inspected for potential association with chemical entities.
One embodiment of the method of drug design involves evaluating the potential association of a known chemical entity with human beta secretase or a structurally homologous molecule, particularly with an human beta secretase inhibitor binding pocket or beta secretase-like inhibitor binding pocket. The method of drug design thus includes computationally evaluating the potential of a selected chemical entity to associate with any of the molecules or molecular complexes set forth above. This method includes the steps of: (a) employing computational means to perform a fitting operation between the selected chemical entity and a inhibitor binding pocket or a pocket nearby the inhibitor binding pocket of the molecule or molecular complex; and (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the inhibitor binding pocket.
In another embodiment, the method of drug design involves computer-assisted design of chemical entities that associate with human beta secretase, its homologs, or portions thereof. Chemical entities can be designed in a step-wise fashion, one fragment at a time, or may be designed as a whole or “de novo.”
To be a viable drug candidate, the chemical entity identified or designed according to the method must be capable of structurally associating with at least part of an human beta secretase or beta secretase-like inhibitor binding pockets, and must be able, sterically and energetically, to assume a conformation that allows it to associate with the human beta secretase or beta secretase-like inhibitor binding pocket. Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions. Conformational considerations include the overall three-dimensional structure and orientation of the chemical entity in relation to the inhibitor binding pocket, and the spacing between various functional groups of an entity that directly interact with the beta secretase-like inhibitor binding pocket or homologs thereof.
Optionally, the potential binding of a chemical entity to an human beta secretase or beta secretase-like inhibitor binding pocket is analyzed using computer modeling techniques prior to the actual synthesis and testing of the chemical entity. If these computational experiments suggest insufficient interaction and association between it and the human beta secretase or beta secretase-like inhibitor 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 or interfere with an human beta secretase or beta secretase-like inhibitor binding pocket. Binding assays to determine if a compound actually interferes with human beta secretase can also be performed and are well known in the art. Binding assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof.
One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with an human beta secretase or beta secretase-like inhibitor binding pocket. This process may begin by visual inspection of, for example, an human beta secretase or beta secretase-like inhibitor binding pocket on the computer screen based on the human beta secretase structure coordinates or other coordinates which define a similar shape generated from the machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the inhibitor binding pocket. Docking may be accomplished using software such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER.
Specialized computer programs may also assist in the process of selecting fragments or chemical entities. Examples include GRID (Goodford, J. Med. Chem. 28:849-57 (1985); available from Oxford University, Oxford, UK); MCSS (Miranker et al., Proteins: Struct. Funct. Gen. 11:29-34 (1991); available from Molecular Simulations, San Diego, Calif.); AUTODOCK (Goodsell et al.,. Proteins: Struct. Funct. Genet. 8:195-202 (1990); available from Scripps Research Institute, La Jolla, Calif.); and DOCK (Kuntz et al., J. Mol. Biol. 161:269-88 (1982); available from University of California, San Francisco, Calif.).
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 human beta secretase. 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, without limitation, CAVEAT (Bartlett et al., in “Molecular Recognition: Chemical and Biological Problems,” Special Publ., Royal Chem. Soc., 78:182-96 (1989); Lauri et al., J. Comput. Aided Mol. Des. 8:51-66 (1994); available from the University of California, Berkeley, Calif.); 3D database systems such as ISIS (available from MDL Information Systems, San Leandro, Calif.; reviewed in Martin, J. Med. Chem. 35:2145-54 (1992)); and HOOK (Eisen et al., Proteins: Struc., Funct., Genet. 19:199-221 (1994); available from Molecular Simulations, San Diego, Calif.).
Human beta secretase binding compounds may be designed “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, without limitation, LUDI (Böhm, J. Comp. Aid. Molec. Design. 6:61-78 (1992); available from Molecular Simulations Inc., San Diego, Calif.); LEGEND (Nishibata et al., Tetrahedron, 47:8985 (1991); available from Molecular Simulations Inc., San Diego, Calif.); LeapFrog (available from Tripos Associates, St. Louis, Mo.); and SPROUT (Gillet et al., J. Comput. Aided Mol. Design 7:127-53 (1993); available from the University of Leeds, UK).
Once a compound has been designed or selected by the above methods, the efficiency with which that entity may bind to or interfere with an human beta secretase or beta secretase-like inhibitor binding pocket may be tested and optimized by computational evaluation. For example, an effective human beta secretase or beta secretase-like inhibitor 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 human beta secretase or beta secretase-like inhibitor 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. human beta secretase or beta secretase-like inhibitor binding pocket inhibitors may interact with the inhibitor 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 or interfering with an human beta secretase or beta secretase-like inhibitor 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. 81995); AMBER, version 4.1 (P. A. Kollman, University of California at San Francisco, 81995); QUANTA/CHARMM (Molecular Simulations, Inc., San Diego, Calif. 81995); Insight II/Discover (Molecular Simulations, Inc., San Diego, Calif. 81995); DelPhi (Molecular Simulations, Inc., San Diego, Calif. 81995); 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 encompassed 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 human beta secretase or beta secretase-like inhibitor 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. 13:505-24 (1992)).
This invention also enables the development of chemical entities that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that interferes with or with human beta secretase. Time-dependent analysis of structural changes in human beta secretase during its interaction with other molecules is carried out. The reaction intermediates of human beta secretase can also be deduced from the reaction product in co-complex with human beta secretase. Such information is useful to design improved analogues of known human beta secretase inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the human beta secretase and inhibitor co-complex. This provides a novel route for designing human beta secretase inhibitors with both high specificity and stability.
Yet another approach to rational drug design involves probing the human beta secretase crystal of the invention with molecules including a variety of different functional groups to determine optimal sites for interaction between candidate human beta secretase inhibitors and the protein. For example, high resolution x-ray diffraction data collected from crystals soaked in or co-crystallized with other molecules allows the determination of where each type of solvent molecule sticks. Molecules that bind tightly to those sites can then be further modified and synthesized and tested for their beta secretase inhibitor activity (Travis, Science 262:1374 (1993)).
In a related approach, iterative drug design is used to identify inhibitors of human beta secretase. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes. In iterative drug design, crystals of a series of protein/compound complexes are obtained and then the three-dimensional structures of each complex is solved. Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
A compound that is identified or designed as a result of any of these methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of beta secretase activity.
Pharmaceutical Compositions (Inhibitors)
Pharmaceutical compositions of this invention include an inhibitor of human beta secretase activity identified according to the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The term “pharmaceutically acceptable carrier” refers to a carrier(s) that is “acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof. Optionally, the pH of the formulation is adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form.
Methods of making and using such pharmaceutical compositions are also included in the invention. The pharmaceutical compositions of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or administration by injection is preferred. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.
Dosage levels of between about 0.01 and about 100 mg/kg body weight per day, preferably between about 0.5 and about 75 mg/kg body weight per day of the human beta secretase inhibitory compounds described herein are useful for the prevention and treatment of human beta secretase mediated disease. 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 can be used as a chronic or acute therapy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon 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 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.
EXAMPLES
Crystallization and Structure Determination of Human Beta Secretase
Expression, Purification, and Crystallization
Expression and Purification of Beta Secretase from HEK 293 Cells.
The expression plasmid=pcDNA3.1/myc/his (neomycin) (Invitrogen) contains beta secretase extending from Met [−21] to Ser [432] with a myc tag followed by a hexahistidine tag [EQKLISEEDLNMHTEHHHHHH*] (SEQ ID NO:2) at the C-terminus. Following transfection in HEK293 cells, stable cells were selected using 0.8 mg/ml G418. A stable clone of transfected HEK293 cells that secretes human beta-secretase was expanded in static, monolayer cell culture. Confluent cultures were detached by shaking and a plurality of plastic, 225 cm2 T-flasks were each inoculated with a suspension of 1-5×106 cells in 100 ml of High-Glucose Dulbecco's Modified Eagle medium that was supplemented with 5% fetal bovine serum and 500 micrograms/ml G418. These cell cultures were incubated in a humidified. 37° C. incubator gassed with 95% air and 5% C02. Once the cells reached confluence the growth medium in each flask was removed and replaced with 100 ml fresh medium. The conditioned, culture medium supernatant was harvested aseptically and replaced by fresh medium every 48-72 hours. The harvested medium was pooled, centrifuged at 1000×g to remove cell debris, and was stored in plastic bottles at 4° C. Cell monolayers were maintained in semi-continuous culture for several weeks until the cells either began to die or to detach from the culture flasks. The cells were then resuspended and used to inoculate a fresh set of production flasks.
For purification, the medium was concentrated approximately 10-fold using a tangential flow concentrator equipped with a 30,000 molecular weight cutoff cartridge. Solid ammonium sulfate was then slowly added with stirring to the concentrate at 4° C. to a final value of 40% saturation (242 g/L). After stirring at 4° C. for 30 minutes, the suspension was clarified by centrifugation (16,000×g, 60 minutes) and the supernatant taken for further analysis. The 40% ammonium sulfate supernatant was adjusted to 80% saturation by slow addition of solid ammonium sulfate with stirring at 4° C. (281 g/L). After stirring for 30 minutes at 4° C., the insoluble material was collected by centrifugation as indicated above and the 40-80% ammonium sulfate pellet taken for further analysis.
The 40-80% ammonium sulfate pellet was dissolved in 25 mM Tris-HCl (8.5)10.5 M NaCIIIO mM imidazole ( 1/10 the original volume) and applied to a 12.5 ml column containing Ni+-NTA Fast Flow resin previously equilibrated in the same buffer. Following sample application, the column was washed with 10 column volumes of loading buffer and then eluted with 25 mM Tris-HCl (8.5)/0.5 M NaCl/50 mM imidazole. The material eluting in 50 mM imidazole was pooled, concentrated approximately 10-fold using a YM 30 membrane (30,000 MWCO), and then dialyzed against 10 mM HEPES-Na (8.0) using 50,000 molecular weight cutoff tubing. For affinity purification, the synthetic peptide Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine, SEQ ID NO:3, PNU-292593E, the structure being illustrated in FIG. 2) was synthesized and coupled to sulfolink resin (Pierce Chemical Company) as recommended by the manufacture. The dialyzed material from above was adjusted to 0.1 M NaOAc (4.5) by addition of 1/10 volume of 1.0 M NaOAc (4.5) and immediately applied to the PNU-292593E/sulfolink column (6 ml containing 1.0 mg PNU-292593/ml of resin) that had been previously equilibrated in 25 mM NaOAc (4.5). Following sample application, the column was washed with 10 column volumes of 25 mM NaOAc (4.5) and then eluted with 50 mM NaBO3 (8.5). N-terminal sequence analysis of the affinity purified material revealed an equimolar mixture of pro- and processed human beta-secretase beginning at Thr1 and Glu25 respectively. The final protein concentration was determined by amino acid analysis assuming a 60 kDa glycoprotein.
Production of Recombinant Human β-Secretase in Insect sf9 Cells and CHO-K1 Cells. The coding sequence was engineered to delete the terminal transmembrane and cytoplasmic domain and introduce a C-terminal hexahistidine tag using the polymerase chain reaction. The 5′ sense oligonucleotide primer [CGCTTTGGATCCGTGGACAACCTGAGGGGCAA] (SEQ ID NO:4) was designed to incorporate a BamHI site for ease in subcloning and Kozak consensus sequence around the initiator methionine for optimal translation initiation. The 3′ antisense primer [CGCTTTGGTACCCTATGACTCATCTGTCTGTGGAATGTTG] (SEQ ID NO:5) incorporated a hexahistidine tag and translation termination codon just upstream of the predicted transmembrane domain (Ser432) and a NotI restriction site for cloning. The PCR was performed on the plasmid template pcDNA3.hygroAsp2R for 15 cycles [94° C., 30 sec., 65° C., 30 sec., 72° C., 30 sec] using Pwo I polymerase (Roche Biochemicals, Indianapolis, Ind.) as outlined by the manufacturer. The PCR product was digested to completion with BamHI and NotI and ligated into the BamHI and NotI sites of the Baculovirus transfer vector pVL1393 (PharMingen, San Diego, Calif.). A portion of the ligation was used to transform competent E. coli DH5α cells and recombinant clones were selected on ampicillin. Individual clones containing the proper cDNA inserts were identified by PCR. Plasmid DNA from clone (pVL1393/Hu_Asp-2LΔTM(His)6) was prepared by alkaline lysis and banding in CsCl. The integrity of the insert was confirmed by complete DNA sequencing. For CHO-K1 cell expression, plasmid pVL1393/Hu_Asp-2LΔTM(His)6 was digested with BamHI and NotI and the resulting fragment subcloned into the mammalian expression vector pcDNA3.1 (hygro) as described above to yield pcDNA3.1 (hygro)/Hu_Asp-2LΔTM(His)6).
For expression, CHO-K1 cells (50% confluent) were transfected with cationic liposome/pcDNA3.1 (hygro)/Hu_Asp-2LΔTM(His)6 complexes in α-MEM medium containing 10% FBS overnight. Selection was performed in the same medium containing 0.5 mg/L hygromycin B for seven days and surviving cells were cloned by limiting dilution. Eight cell lines were screened for soluble β-secretase by Western blot analysis using a polyclonal rabbit antiserum specific for human β-secretase (UP-191). Conditioned medium from each clonal cell line was concentrated by Ni+-NTA chromatography and the histidine-tagged polypeptide eluted with buffer containing 50 mM imidazole. Aliquots of the latter fraction were displayed on a PVDF membrane and recombinant soluble human β-secretase was visualized using UP-191 antiserum and alkaline phosphatase conjugated goat antirabbit second antibody. Based on these results, clone #4 showed the highest expression level and was used for all subsequent experiments.
For Baculovirus expression (BVES), recombinant virus containing the coding sequence of soluble β-secretase was isolated following recombination of the plasmid transfer vector pVL1393/Hu_Asp-2LΔTM(His)6 in sf9 cells using standard methods. Individual virus isolates were used to infect sf9 cells and expression of the desired polypeptide was quantified by Western blot analysis of the conditioned medium collected 69 hr post infection as described above. The recombinant virus directing the synthesis of the highest level of β-secretase was scaled-up for protein production.
For production of soluble β-secretase in insect cells, sf9 cells were infected with recombinant Baculovirus at a multiplicity of infection of 1.0 in serum free medium and conditioned medium harvested 69 hours post-infection. For CHO cells, the production cell line expressing secreted soluble human β-secretase was expanded in either roller bottles or in a packed-bed bioreactor in medium containing 0.5 fetal bovine serum. Conditioned medium was collected and stored at 4° C. until processing.
Purification of Recombinant Human β-Secretase from BVES or CHO-K1 Cells. For purification from either source, the medium was concentrated approximately 10-fold using a tangential flow concentrator equip with a 30,000 molecular weight cutoff cartridge. Solid ammonium sulfate was then slowly added with stirring to the concentrate at 4° C. to a final value of 40% saturation (242 g/L). After stirring at 4° C. for 30 minutes, the suspension was clarified by centrifugation (16,000×g, 60 minutes) and the supernatant taken for further analysis. The 40% ammonium sulfate supernatant was adjusted to 80% saturation by slow addition of solid ammonium sulfate with stirring at 4° C. (281 g/L). After stirring for 30 minutes at 4° C., the insoluble material was collected by centrifugation as indicated above and the 40-80% ammonium sulfate pellet taken for further analysis.
The 40-80% ammonium sulfate pellet was dissolved in 25 mM Tris-HCl (8.5)/0.5 M NaCl/10 mM imidazole ( 1/10 the original volume) and applied to a 12.5 ml column containing Ni+-NTA Fast Flow resin previously equilibrated in the same buffer. Following sample application, the column was washed with 10 column volumes of loading buffer and then eluted with 25 mM Tris-HCl (8.5)/0.5 M NaCl/50 mM imidazole. The material eluting in 50 mM imidazole was pooled, concentrated approximately 10-fold using a YM 30 membrane (30,000 MWCO), and then dialyzed against 10 mM HEPES-Na (8.0) using 50,000 molecular weight cutoff tubing. For affinity purification, the synthetic peptide Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys (where Sta=statine, SEQ ID NO:3, PNU-292593E) was synthesized and coupled to sulfolink resin (Pierce Chemical Company) as recommended by the manufacture. The dialyzed material from above was adjusted to 0.1 M NaOAc (4.5) by addition of 1/10 volume of 1.0 M NaOAc (4.5) and immediately applied to the PNU-292593E/sulfolink column (6 ml containing 1.0 mg PNU-292593/ml of resin) that had been previously equilibrated in 25 mM NaOAc (4.5). Following sample application, the column was washed with 10 column volumes of 25 mM NaOAc (4.5) and then eluted with 50 mM NaBO3 (8.5). N-terminal sequence analysis of the affinity purified material revealed an equimolar mixture of pro- and processed human β-secretase beginning at Thr1 and Glu25, respectively. The final protein concentration was determined by amino acid analysis assuming a 52 kDa glycoprotein for insect cells and a 60 kDa glycoprotein for CHO cells, respectively.
Protein Crystallization. Based on observations of the initial screening effort, fresh protein derived from CHO cells was concentrated to 42 mg/m1 and mixed with a new inhibitor (FIG. 1) so that the final concentration of the mix was 40 mg/ml beta secretase, and 2 mM of the inhibitor, in 20 mM Hepes pH 7.8, 10% DMSO. This preparation was screened with Hampton Screen 1 (Hampton Research, Laguna Nigel, CA.) and Wizard Screen 1 (Emerald Biostructures, Bainbridge Island, WA) at room temperature (20° C.). 500-μL well volumes were used. A 1:1 ratio of protein-compound mix to the well solution was used in a hanging drop format to complete the screen. After 10 days, but less than 18 days later crystals were observed in Wizard Screen 1, Condition No. 45 (20% PEG 3000, 0.1 M NaOAc pH 4.5). Optimization and seeding efforts around this condition provided crystals that grew in 17-20% PEG 3000, 0.1 M Na Acetate pH 4.5. Seeding was done utilizing a cat whisker which was touched to a drop containing microcrystals and stepwise diluted by streaking through one row of the optimization tray. Cross-seeding efforts provided crystals of HEK 293 cell derived protein (38 mg/ml in 20 mM Hepes pH 7.8 , 50 mM NaCl, 10% DMSO, 2 mM of the inhibitor from CHO cell derived seed stock. Macroseeding by moving small crystals with a loop from the target drop to a fresh drop containing 17-20% PEG3000, 0.1 M Na Acetate pH 4.5 also resulted in crystals that doubled or more in size, usually with a shower of microcrystals also. Crystals were obtained in hanging drop or sitting drop methods by seeding. Crystals obtained from streak seeding attempts were frozen in a cryoprotectant solution based on the mother liquor plus 20% Ethylene Glycol. The crystals were then soaked incrementally through 5% intervals of the cryoprotectant in 3 to 5-minute intervals. Crystals have also been grown in the presence of 10% glycerol or 10% ethylene glycol to facilitate stabilization into cryogenic solutions. In these cases, the crystals were soaked incrementally through 5% intervals of the cryoprotectant in 3 to 5-minute intervals. The use of other cryo agents would be apparent to one of skill in the art.
Fresh Asp2 protein derived from Baculovirus cells (BVES) was concentrated to 18.6 mg/mL and mixed with the inhibitor illustrated in FIG. 1, so that the final concentration of the mix was 18 mg/mL beta secretase and 2 mM of the inhibitor illustrated in FIG. 1 in 20 mM Hepes pH 7.8 with 5% DMSO. Observations of bow CHO derived Asp2 crystallized led to the following crystallization effort for BVES Asp2. A PEG range from 17-22% PEG 3000, 0.1M sodium acetate pH 4.5, with none, 10% DMSO, or 10% glycerol was used in 1000 μL wells. Sample drops were streak seeded at set up, with 10−1 seed dilution of CHO seed stocks as described above. A crystal measuring 1.0×0.1×0.1 mm3 was obtained in 17% PEG 3000, 0.1M sodium acetate pH 4.5 with 10% DMSO, and 3 days post set up. Crystals were incrementally soaked into a cryoprotectant solution containing a final concentration of 20% DMSO in 5 minute intervals.
X-Ray Diffraction Characterization for Beta Secretase Expressed in CHO Cells
All data collection was carried out at the Advanced Photon Source (Argonne, IL) at beamline 17-ID. The crystals diffracted to 3.2 Å using synchrotron radiation. Crystals were of the space group P3221 with cell constants a=112 ±35 Å, b=112 ±20 Å, c=110 ±20 A, α=β=90°, γ=120°. The Matthews coefficient for these crystals assuming that there is one molecule in the asymmetric unit is 3.5 ÅDa with 65% solvent. The structure determination (see below) revealed the presence of electron density in the active site appropriate for the inhibitor.
Molecular Replacement for Beta Secretase Expressed in CHO Cells
A molecular replacement solution was determined using AMORE (Navaza, Acta Cryst., D50:157-63 (1994); Collaborative Computational Project N4, Acta Cryst. D50:760-3 (1994)) by utilizing a previously published model of human beta secretase, 1FKN, (Hong et al., Science 290: 150-53 (2000)) made available from the Protein Data Bank. Using the 1FKN model, the initial rotation solution gave a single strong peak of 9.7 σ with the next strongest peak appearing at 4.0 σ. The final determination of the space group (P321, P3121, or P3221) was determined experimentally by testing translation searches in each space group. A translation search in the correct space group, P3221, resulted in a correlation coefficient of 55.1 with an R-factor of 39.9% to 4 Å resolution.







TABLE 4







Data collection statistics for structure of Human Beta Secretase (data


collected at λ 1.0000 Å at APS, 17-ID)










Resolution Range
Rsym







  20-6.81
0.048



6.81-5.44
0.095



5.44-4.77
0.112



4.77-4.33
0.113



4.33-4.03
0.181



4.03-3.79
0.224



3.79-3.60
0.248



3.60-3.45
0.288



3.45-3.31
0.321



3.31-3.20
0.324



All reflections
0.098










Model Building and Refinement for Beta Secretase Expressed in CHO Cells

Further rigid body refinement of the model in CNX (Molecular Simulations, Inc) followed by minimization and group b-factor refinement gave an R-factor of 35.1% and a Free R-factor of 37.7% to 3.2 Å. During each cycle of refinement a bulk solvent correction was incorporated (Jiang et al., J. Mol. Biol. 243:100-15 (1994)). Progress of the refinement was monitored by a decrease in both the R-factor and Free R-factor.
At this point, inspection of the electron density map within the active site revealed electron density that was unaccounted for by the protein model and consistent with the shape of the inhibitor shown in FIG. 1 that was present in the crystallization conditions. Model building was done using the program CHAIN (Sack,Journal of Molecular Graphics 6: 224-225 (1988)) and LORE (Finzel, Meth. Enzymol. 277:230-42 (1997)). Modest rebuilding of the model into the 3.2 Å low resolution map afforded the opportunity for further cycles of refinement giving improvement of the R-factor to 31.6% and a Free R-factor of 35.7%. Finally, the inhibitor was included in the refinement giving to give the current R-factor of 29.9% and a Free R-factor of 34.9%.
Inspection of the electron density throughout the molecule indicates that all three disulfide bonds are intact (Cys155-Cys359,Cys217-Cys382, and Cys269-Cys 319). In addition, the electron density near Asn92, Ans162, and Asn293, indicates the presence of glycosylation. Only the glycosylation at Asn111 is disordered enough not to be visible in the electron density map. Residues 158-170 and 311 to 317 were disordered in the electron density and therefore have been omitted from the model.







TABLE 5







Refinement Statistics for structure of Human Beta Secretase











R-factor
Free R-factor
No. of reflections


20-3.2 Å F ≧ 2σ
0.2991
0.3483
9883









r.m.s deviation from ideal
Bonds (Å)
Angles(°)


geometry
0.012
1.7















Number
Average 




of atoms
B-factor















Protein
2880
65.24



Waters
41
74.89



Total
2921
65.38










X-ray Diffraction Characterization for Baculovirus Produced Human Beta Secretase Crystals

All data collection was carried out at the Advanced Photon Source (Argonne, IL) at beamline 17-ID. The crystals diffracted to 2.7 Åusing synchrotron radiation. Crystals were of the space group P3221 with cell constants a=99.4 ±35 Å, b=99.4 ±35 Å, c=117 ±35 Å, α=β=90°, γ=120°. The Matthews coefficient for these crystals assuming that there is one molecule in the asymmetric unit is 2.9 Å/Da with 58% solvent. The structure determination (see below) revealed the presence of electron density in the active site appropriate for the inhibitor.
Molecular Replacement for Baculovirus Produced Human Beta Secretase Crystals
A molecular replacement solution was determined using AMORE (Navaza 1994; Collaborative Computational Project N4, Acta Cryst. D50:760-3 (1994)) by utilizing the structure of human beta secretase produced from CHO cells. Using the CHO beta secretase model, the initial rotation solution gave a strong peak of 10.2σ with the strongest peak appearing at 8.0σ. The space group was P3221. A translation search in the correct space group, P3221, resulted in a correlation of 57.8 with an R-Factor of 41.5% to 4 Å resolution.







TABLE 6







Data collection statistics for structure of Human Beta Secretase


expressed in a Baculovirus expression system (data collected at λ 1.0000


Å at APS, 17-ID)










Resolution Range
Rsym







99.0-5.81
0.045



5.81-4.61
0.076



4.61-4.03
0.093



4.03-3.66
0.117



3.66-3.40
0.139



3.40-3.19
0.176



3.19-3.03
0.149



3.03-2.90
0.174



2.90-2.79
0.169



2.79-2.69
0.273



All reflections
0.073










Model Building and Refinement for Baculovirus Produced Human Beta Secretase Crystals

Further rigid body refinement of the model in CNX (Molecular Simulations, Inc) followed by minimization refinement gave an R-factor of 36.4% to 2.7 Å. During each cycle of refinement a bulk solvent correction was incorporated (Jiang et al., J. Mol. Biol. 243:100-15 (1994)).
At this point, inspection of the electron density map within the active site revealed electron density that was unaccounted for by the protein model and consistent with the shape of the inhibitor illustrated in FIG. 1, that was present in the crystallization conditions. Model building was done using the program CHAIN (Sack, Journal of Molecular Graphics 6:224-25 (1988)) and LORE (Finzel, Meth. Enzymol. 277:230-42 (1997)).
The complete disclosure of all patents, patent applications including provisional applications, and publications, and electronically available material (e.g., GenBank amino acid and nucleotide sequence submissions) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described; many variations will be apparent to one skilled in the art and are intended to be included within the invention defined by the claims.
SEQUENCE LISTING FREE TEXT


    SEQ ID NO:1 Residues for recombinant human beta secretase present in the X-ray structure
    SEQ ID NO:2 Peptide
    SEQ ID NO:3 Synthetic peptide
    SEQ ID NO:4 Primer
    SEQ ID NO:5 Primer

## Claims

1. A method of crystallizing a complex of a purified human beta secretase polypeptide and a ligand, wherein the purified human beta secretase polypeptide comprises the amino acid sequence of SEQ ID NO:1 and the ligand has the formula N1-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-iodobenzylamino)butan-2yl)-5-methyl-N3,N3-dipropylisophthalamide, the method comprising combining equal volumes of (a) an aqueous solution of 40 mg/mL of the purified human beta secretase polypeptide and 2mM of the ligand, and (b) a solution of 17% PEG 3000, 0.1 M sodium acetate pH 4.5 at 20 degrees C. and crystallizing a complex of the purified human beta secretase polypeptide and the ligand.

2. The method of claim 1 wherein solution (b) further includes 10% glycerol or 10% ethylene glycol.

3. A method of crystallizing a complex of a purified human beta secretase polypeptide and a ligand, wherein the purified human beta secretase polypeptide comprises the amino acid sequence of SEQ ID NO:1 and the ligand has the formula N1-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-iodobenzylamino)butan-2-yl)-5-methyl-N3,N3-dipropylisophthalamide, the method comprising combining equal volumes of (a) an aqueous solution of 18 mg/mL of the purified human beta secretase polypeptide and 2mM of the ligand, and (b) a solution of 17% PEG 3000, 0.1 M sodium acetate pH 4.5, and 10% dimethylsulfoxide at 20 degrees C. and crystallizing a complex of the purified human beta secretase polypeptide and the ligand.

4. The method of claim 3 wherein solution (b) further includes 10% glycerol or 10% ethylene glycol.

5. A method for crystallizing a complex of a purified human beta secretase polypeptide and a ligand, wherein the purified human beta secretase polypeptide comprises the amino acid sequence of SEQ ID NO:1, except at least one methionine of SEQ ID NO:1 is replaced with selenomethionine, and the ligand has the formula N1-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-iodobenzylamino)butan-2-yl)-5-methyl-N3,N3-dipropylisophthalamide, the method comprising: preparing the purified human beta secretase in the presence of the ligand;
and crystallizing a complex of the purified human beta secretase polypeptide and the ligand from a solution comprising the purified human beta secretase and the ligand, wherein the solution has a pH of at most about 5.8, and wherein the crystal of the complex of the purified human beta secretase polypeptide and the ligand has the trigonal space group symmetry P3221 and unit cell constants of a=112±20 Å, b=112±20 Å, c=110±20 Å, α=β=90 and Υ=120°.

6. A method for crystallizing a complex of a purified human beta secretase polypeptide and a ligand, wherein the purified human beta secretase polypeptide comprises the amino acid sequence of SEQ ID NO:1 and the ligand has the formula N1-((2S,3R)-1-(3,5-difluorophenyl)-3-hydroxy-4-(3-iodobenzylamino)butan-2-yl)-5-methyl-N3,N3-dipropylisophthalamide, the method comprising: preparing the purified human beta secretase in the presence of the ligand; and crystallizing a complex of the purified human beta secretase polypeptide and the ligand from a solution comprising the purified human beta secretase and the ligand, wherein the solution has a pH of at most about 5.8, and wherein the crystal of the complex of the purified human beta secretase polypeptide and the ligand has the trigonal space group symmetry P3221 and unit cell constants of a=112±20 Å, b=112±20 Å, c=110±20 Å, α=β=90° and Υ=120°.

7. The method of claim 6, wherein the solution has a pH of at most 6.0.

8. The method of claim 6, wherein the solution has a pH of at most about 5.6.

9. The method of claim 6, wherein the solution has a pH of about 3.5 to about 5.5.

10. The method of claim 6, wherein the solution has a pH of about 4.0 to about 4.7.

11. The method of claim 6, wherein the solution comprises about 5% by weight to about 50% by weight of polyethylene glycol (PEG) 3000.

12. The method of claim 6, wherein the solution comprises sodium acetate.

13. The method of claim 12 wherein the concentration of sodium acetate is about 0.001 M to about 0.5 M.

14. The method of claim 6, wherein the solution has a pH of 4.5 and the crystallizing step is by hanging drop method at 20 degrees centigrade, and the method further comprises the step of isolating a crystal suitable for x-ray diffraction studies following the crystallizing step.