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  Pharmaceutical Patents  

 

Title:  Druggable regions in the dengue virus envelope glycoprotein and methods of using the same
United States Patent:  7,524,624
Issued: 
April 28, 2009

Inventors:
 Modis; Yorgo (Brookline, MA), Harrison; Stephen C. (Cambridge, MA)
Assignee:
  Children's Medical Center Corporation (Boston, MA)
Appl. No.:
 11/257,777
Filed:
 October 24, 2005

 

Outsourcing Guide


Abstract

The present invention relates to novel druggable regions discovered in dengue virus envelope glycoprotein, or dengue virus E protein, which is a class II viral E protein. The present invention further relates to methods of using the druggable regions to screen potential candidate therapeutics for diseases caused by viruses having class II E proteins, e.g. viral fusion inhibitors.

Description of the Invention

A. General

We have determined the structures of the E protein in both its pre-fusion and post-fusion conformations.

The pre-fusion structure was determinned by solving the structure of a soluble fragment (residues 1-394) of the E protein from dengue virus type 2. This fragment contains all but about 45 residues of the E-protein ectodomain (FIG. 1A, see Original Patent). It resembles closely, in its dimeric structure and in the details of its protein fold, the E protein from tick-borne encephalitis (TBE) virus, studied previously. We have examined crystals grown in both the presence and the absence of the detergent n-octyl-.beta.-D-glucoside, .beta.-OG. The key difference between the two structures is a local rearrangement of the "k1" .beta.-hairpin (residues 268-280) and the concomitant opening up of a hydrophobic pocket, occupied by a molecule of .beta.-OG. Mutations affecting the pH threshold for fusion map to the hydrophobic pocket, which we propose is a hinge point in the fusion-activating conformational change. Detergent binding marks the k1 .beta.-hairpin and associated pocket as a potential target for viral fusion inhibitors. We have also discovered another region, the domain 1-3 region, which may serve as a target for viral fusion inhibitors.

The post-fusion structure of the soluble E ectodomain (sE) in its trimeric, post-fusion state reveals striking differences from the dimeric, pre-fusion form. The elongated trimer bears three "fusion loops" at one end, to insert into the host-cell membrane. Their structure allows us to model directly how they interact with a lipid bilayer. The protein folds back on itself, directing its C-terminus towards the fusion loop. We propose a fusion mechanism driven by essentially irreversible conformational changes in dengue virus E protein and facilitated by fusion-loop insertion into the outer bilayer leaflet. Specific features of the folded-back structure suggest strategies for inhibiting flavivirus entry, as well as druggable regions. The regions may serve as a target for viral fusion inhibitors and assays to discover such inhibitors.

Hence, we have discovered a variety of novel, structurally defined druggable regions which may present targets for a specific viral fusion inhibitor for dengue virus and other viruses having class II E protein. Because dengue virus type 2 E protein is strongly homologous to other dengue viral types, as well as other flavivirus E proteins and class II E proteins (Lindenbach and Rice, 2001, Rey, et al. 1995, Hahn, et al. 1998), these binding sites are likely present in those E proteins as well and may serve as targets for specific viral fusion inhibitors for those viruses.

Finally, we have also discovered that peptides corresponding to residues 396-429 and 413-447 (in the "stem" region) of dengue envelope protein (E) binds with fairly high affinity and specificity to the trimeric, post-fusion form of sE, the fragment of E spanning residues 1-395, which we crystallized first in the pre-fusion form and then in the post-fusion form. Inhibitor peptides derived from stem sequences may block completion of the conformational change by interacting with the relevant surfaces on the clustered domains II. Such inhibitors would interfere with the second stage of the conformational change. This peptide itself may serve as a specific viral fusion inhibitor, or may provide the basis from which to design improved specific viral fusion inhibitors.

C. Drug Discovery

C.1. Druggable Regions

Based in part on the structural information described in the Exemplification, we have identified novel druggable regions in dengue virus E protein. In one embodiment, the druggable region is comprised of the k1 hairpin or a portion thereof. In certain embodiments, the k1 hairpin may be comprised of at least one of residues 268-280 of a dengue virus E protein or the homologous residues in other class II E protein. In other embodiments, the druggable region or active site region may be comprised of the k1 hairpin and at least one of residues 47-54, 128-137, and 187-207.

In yet another embodiment, the druggable region may comprise the regions involved in the binding of residues 396-429 (the "stem" region of dengue envelope protein E) binds to the trimeric, post-fusion form of dengue virus E protein or other flavivirus E protein. In one embodiment, the druggable region is comprised of the stem region or a portion thereof. The stem region comprises residues 396-447, or fragments thereof, for example 396-429 and 413-447. In another embodiment, the druggable region is comprised of the channel in which the stem region binds. The channel is comprised of the residues at the trimer interface formed by domain II of each subunit in the trimer. Domain II consists of residues 52-132 and 193-280. A second region is the channel where the stem binds, formed by residues in domain II.

In another embodiment, the druggable region is comprised of the domain I-II region. In certain embodiments, the domain I-III region may be comprised of at least one of residues 38-40; 143-147; 294-296; and 354-365 of a dengue virus E protein or the homologous residues in other class II E protein. In other embodiments, the druggable region may be comprised of the domain I-domain III linker (residues 294-301).

In yet another embodiment, a druggable region is comprised of the fusion loop or a portion thereof.

Other regions of protein may in certain embodiments comprise a druggable region. For example, the hydrophobic core beneath the k1 hairpin or a portion thereof may comprise a druggable region. In another example, a druggable region may comprise domain II or a portion thereof. In still another example, a druggable region may comprise domain III or a portion thereof. In other examples, the pH-dependent hinge may serve as a druggable region. Further, a region or portion of a region of the E protein involved in trimerization, such as for example, the regions of domain II involved in trimerization, may present a druggable region. A region or a portion of a region involved in the stem fold back conformational change may comprise a druggable region, for example, such regions as the stem-domain II contact regions, the trimeric N terminal inner core, and C terminal outer layer surfaces on the clustered domains II, as well as the 53-residue stem. In certain embodiments, a druggable region may consist of the entire fragment of the E protein spanning residues 1-395.

In yet another aspect, the present invention is directed toward methods of identifying and designing modulators which bind with, interact with, or modulate the function or activity of an active or binding site of a dengue virus E protein or other class II E protein.

C.2. Modulators, Modulator Design and Screening Using the Subject Druggable Regions

In one aspect, the present invention provides methods of screening the subject druggable regions for potential modulators, as well as methods of designing such modulators. Modulators to polypeptides of the invention and other structurally related molecules, and complexes containing the same, may be identified and developed as set forth below and otherwise using techniques and methods known to those of skill in the art. The modulators of the invention may be employed, for instance, to inhibit and treat disease caused by a flavivirus or other virus having class II E protein, such as dengue fever, dengue hemorrhagic fever, tick-borne encephalitis, West Nile virus disease, yellow fever, Kyasanur Forest disease, louping ill, hepatitis C, Ross River virus disease, and O'nyong fever.

In one aspect, the present invention is directed towards a modulator that interacts with the subject druggable regions so as to reduce the activity of the dengue virus E protein or other class II E protein. Such modulators may in certain embodiments interact with a druggable region of the invention. In still another aspect, the present invention is directed toward a modulator that is a fragment of (or homolog of such fragment or mimetic of such fragment) the druggable region of a dengue virus E protein or other viral class II E protein and competes with that druggable region. Modulators of any of the above-described druggable regions may be used alone or in complementary approaches to treat dengue viral or other viral infections.

In certain embodiments, a modulator interacts with the k1 hairpin so as to preclude it from moving, thereby modulating the activity of the dengue virus E protein or other flavivirus E protein. In another aspect, the present invention is directed towards a modulator that interacts with the stem region or the channel so as to preclude them from interacting, thereby modulating the activity of the dengue virus E protein or other flavivirus E protein. Such modulators may be, as described above, derived from either the stem region or the channel, and compete with the stem region or channel for binding. In still other embodiments, a modulator of class II E protein activity interacts with the domain I-III region. The modulator may also preclude the movement of the domain I-III region. In another aspect, the present invention is directed towards a modulator that interacts with the fusion loop so as to preclude it from moving, thereby modulating the activity of the dengue virus E protein or other E protein.

Further, the present invention is in part directed toward an inhibitor that comprises SEQ ID NO: 3 or SEQ ID NO: 4, as well as fragments, homologs, variants, orthologs, and peptidomimetics thereof. Further, the present invention is directed towards an inhibitor that interacts with the relevant surfaces on the clustered domains II, so that completion of the conformational change is inhibited and thereby inhibiting the activity of the dengue virus E protein or other E protein. The present invention is also directed towards an inhibitor that interacts with the pocket beneath the k1 hairpin to infere with the first stage of the conformational change, thereby modulating the activity of the dengue virus E protein or other E protein. Such inhibitors may be used in complementary approaches to treat dengue viral or other viral infections.

A variety of methods for inhibiting the growth or infectivity of flaviviruses using the modulators are contemplated by the present invention. For example, exemplary methods involve contacting a flavivirus with a modulator thought or shown to be effective against such pathogen.

For example, in one aspect, the present invention contemplates a method for treating a patient suffering from an infection of dengue fever or other flavivirus comprising administering to the patient an amount of a modulator effective to modulate the expression and/or activity of a dengue virus E protein or other class II E protein. In certain instances, the animal is a human or a livestock animal such as a cow, pig, goat or sheep. The present invention further contemplates a method for treating a subject suffering from a flavivirus-related, alphavirus-related, or hepatitis-related disease or disorder, comprising administering to an animal having the condition a therapeutically effective amount of a molecule identified using one of the methods of the present invention.

In another embodiment, modulators of a dengue virus E protein or other class II E protein, or biological complexes containing them, may be used in the manufacture of a medicament for any number of uses, including, for example, treating any disease or other treatable condition of a patient (including humans and animals), and particularly a disease caused by a flavivirus or other virus having class II E protein, such as, for example, one of the following: dengue fever, dengue hemorrhagic fever, tick-borne encephalitis, West Nile virus disease, yellow fever, Kyasanur Forest disease, louping ill, hepatitis C, Ross River virus disease, and O'nyong fever. (a) Modulator Design

A number of techniques can be used to screen, identify, select and design chemical entities capable of associating with a dengue virus E protein or other class II E protein, structurally homologous molecules, and other molecules. Knowledge of the structure for a dengue virus E protein or other class II E protein, determined in accordance with the methods described herein, permits the design and/or identification of molecules and/or other modulators which have a shape complementary to the conformation of a dengue virus E protein or other class II E protein, or more particularly, a druggable region thereof. It is understood that such techniques and methods may use, in addition to the exact structural coordinates and other information for a dengue virus E protein or other class II E protein, structural equivalents thereof described above (including, for example, those structural coordinates that are derived from the structural coordinates of amino acids contained in a druggable region as described above).

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. In certain instances, it is desirable to use chemical entities exhibiting a wide range of structural and functional diversity, such as compounds exhibiting different shapes (e.g., flat aromatic rings(s), puckered aliphatic rings(s), straight and branched chain aliphatics with single, double, or triple bonds) and diverse functional groups (e.g., carboxylic acids, esters, ethers, amines, aldehydes, ketones, and various heterocyclic rings).

In one aspect, the method of drug design generally includes computationally evaluating the potential of a selected chemical entity to associate with any of the molecules or complexes of the present invention (or portions thereof). For example, this method may include the steps of (a) employing computational means to perform a fitting operation between the selected chemical entity and a druggable region of the molecule or complex; and (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the druggable region.

A chemical entity may be examined either through visual inspection or through the use of computer modeling using a docking program such as GRAM, DOCK, or AUTODOCK (Dunbrack et al., Folding & Design, 2:27-42 (1997)). This procedure can include computer fitting of chemical entities to a target to ascertain how well the shape and the chemical structure of each chemical entity will complement or interfere with the structure of a dengue virus E protein or other class II E protein (Bugg et al., Scientific American, Dec.: 92-98 (1993); West et al., TIPS, 16:67-74 (1995)). Computer programs may also be employed to estimate the attraction, repulsion, and steric hindrance of the chemical entity to a druggable region, for example. Generally, the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the chemical entity will be because these properties are consistent with a tighter binding constant. Furthermore, the more specificity in the design of a chemical entity the more likely that the chemical entity will not interfere with related proteins, which may minimize potential side-effects due to unwanted interactions.

A variety of computational methods for molecular design, in which the steric and electronic properties of druggable regions are used to guide the design of chemical entities, are known: Cohen et al. (1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol. 161: 269-288; DesJarlais (1988) J. Med. Cam. 31: 722-729; Bartlett et al. (1989) Spec. Publ., Roy. Soc. Chem. 78: 182-196; Goodford et al. (1985) J. Med. Cam. 28: 849-857; and Desjarlais et al. J. Med. Cam. 29: 2149-2153. Directed methods generally fall into two categories: (1) design by analogy in which 3-D structures of known chemical entities (such as from a crystallographic database) are docked to the druggable region and scored for goodness-of-fit; and (2) de novo design, in which the chemical entity is constructed piece-wise in the druggable region. The chemical entity may be screened as part of a library or a database of molecules. Databases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall). Computer programs such as CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions.

Chemical entities may be tested for their capacity to fit spatially with a druggable region or other portion of a target protein. As used herein, the term "fits spatially" means that the three-dimensional structure of the chemical entity is accommodated geometrically by a druggable region. A favorable geometric fit occurs when the surface area of the chemical entity is in close proximity with the surface area of the druggable region without forming unfavorable interactions. A favorable complementary interaction occurs where the chemical entity interacts by hydrophobic, aromatic, ionic, dipolar, or hydrogen donating and accepting forces. Unfavorable interactions may be steric hindrance between atoms in the chemical entity and atoms in the druggable region.

If a model of the present invention is a computer model, the chemical entities may be positioned in a druggable region through computational docking. If, on the other hand, the model of the present invention is a structural model, the chemical entities may be positioned in the druggable region by, for example, manual docking. As used herein the term "docking" refers to a process of placing a chemical entity in close proximity with a druggable region, or a process of finding low energy conformations of a chemical entity/druggable region complex.

In an illustrative embodiment, the design of potential modulator begins from the general perspective of shape complimentary for the druggable region of a dengue virus E protein or other class II E protein, and a search algorithm is employed which is capable of scanning a database of small molecules of known three-dimensional structure for chemical entities which fit geometrically with the target druggable region. Most algorithms of this type provide a method for finding a wide assortment of chemical entities that are complementary to the shape of a druggable region of a dengue virus E protein or other class II E protein. Each of a set of chemical entities from a particular data-base, such as the Cambridge Crystallographic Data Bank (CCDB) (Allen et al. (1973) J. Chem. Doc. 13: 119), is individually docked to the druggable region of a dengue virus E protein or other class II E protein in a number of geometrically permissible orientations with use of a docking algorithm. In certain embodiments, a set of computer algorithms called DOCK, can be used to characterize the shape of invaginations and grooves that form the active sites and recognition surfaces of the druggable region (Kuntz et al. (1982) J. Mol. Biol. 161: 269-288). The program can also search a database of small molecules for templates whose shapes are complementary to particular binding sites of a dengue virus E protein or other class II E protein (DesJarlais et al. (1988) J Med Chem 31: 722-729).

The orientations are evaluated for goodness-of-fit and the best are kept for further examination using molecular mechanics programs, such as AMBER or CHARMM. Such algorithms have previously proven successful in finding a variety of chemical entities that are complementary in shape to a druggable region.

Goodford (1985, J Med Chem 28:849-857) and Boobbyer et al. (1989, J Med Chem 32:1083-1094) have produced a computer program (GRID) which seeks to determine regions of high affinity for different chemical groups (termed probes) of the druggable region. GRID hence provides a tool for suggesting modifications to known chemical entities that might enhance binding. It may be anticipated that some of the sites discerned by GRID as regions of high affinity correspond to "pharmacophoric patterns" determined inferentially from a series of known ligands. As used herein, a "pharmacophoric pattern" is a geometric arrangement of features of chemical entities that is believed to be important for binding. Attempts have been made to use pharmacophoric patterns as a search screen for novel ligands (Jakes et al. (1987) J Mol Graph 5:41-48; Brint et al. (1987) J Graph 5:49-56; Jakes et al. (1986) J Mol Graph 4:12-20).

Yet a further embodiment of the present invention utilizes a computer algorithm such as CLIX which searches such databases as CCDB for chemical entities which can be oriented with the druggable region in a way that is both sterically acceptable and has a high likelihood of achieving favorable chemical interactions between the chemical entity and the surrounding amino acid residues. The method is based on characterizing the region in terms of an ensemble of favorable binding positions for different chemical groups and then searching for orientations of the chemical entities that cause maximum spatial coincidence of individual candidate chemical groups with members of the ensemble. The algorithmic details of CLIX is described in Lawrence et al. (1992) Proteins 12:3141.

In this way, the efficiency with which a chemical entity may bind to or interfere with a druggable region may be tested and optimized by computational evaluation. For example, for a favorable association with a druggable region, a chemical entity must preferably demonstrate a relatively small difference in energy between its bound and fine states (i.e., a small deformation energy of binding). Thus, certain, more desirable chemical entities will be designed with a deformation energy of binding of not greater than about 10 kcal/mole, and more preferably, not greater than 7 kcal/mole. Chemical entities may interact with a druggable region 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 chemical entity binds to the target.

In this way, the present invention provides computer-assisted methods for identifying or designing a potential modulator of the activity of a dengue virus E protein or other class II E protein including: supplying a computer modeling application with a set of structure coordinates of a molecule or complex, the molecule or complex including at least a portion of a druggable region from a dengue virus E protein or other class II E protein; supplying the computer modeling application with a set of structure coordinates of a chemical entity; and determining whether the chemical entity is expected to bind to the molecule or complex, wherein binding to the molecule or complex is indicative of potential modulation of the activity of a dengue virus E protein or other class II E protein.

In another aspect, the present invention provides a computer-assisted method for identifying or designing a potential modulator to a dengue virus E protein or other class II E protein, supplying a computer modeling application with a set of structure coordinates of a molecule or complex, the molecule or complex including at least a portion of a druggable region of a dengue virus E protein or other class II E protein; supplying the computer modeling application with a set of structure coordinates for a chemical entity; evaluating the potential binding interactions between the chemical entity and active site of the molecule or molecular complex; structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity, and determining whether the modified chemical entity is expected to bind to the molecule or complex, wherein binding to the molecule or complex is indicative of potential modulation of the dengue virus E protein or other class II E protein.

In one embodiment, a potential modulator can be obtained by screening a peptide or other compound or chemical library (Scott and Smith, Science, 249:386-390 (1990); Cwirla et al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990); Devlin et al., Science, 249:404-406 (1990)). A potential modulator selected in this manner could then be systematically modified by computer modeling programs until one or more promising potential drugs are identified. Such analysis has been shown to be effective in the development of HIV protease modulators (Lam et al., Science 263:380-384 (1994); Wlodawer et al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in Drug Discovery and Design 1:23-48 (1993); Erickson, Perspectives in Drug Discovery and Design 1:109-128 (1993)). Alternatively a potential modulator may be selected from a library of chemicals such as those that can be licensed from third parties, such as chemical and pharmaceutical companies. A third alternative is to synthesize the potential modulator de novo.

For example, in certain embodiments, the present invention provides a method for making a potential modulator for a dengue virus E protein or other class II E protein, the method including synthesizing a chemical entity or a molecule containing the chemical entity to yield a potential modulator of a dengue virus E protein or other class II E protein, the chemical entity having been identified during a computer-assisted process including supplying a computer modeling application with a set of structure coordinates of a molecule or complex, the molecule or complex including at least one druggable region from a dengue virus E protein or other class II E protein; supplying the computer modeling application with a set of structure coordinates of a chemical entity; and determining whether the chemical entity is expected to bind to the molecule or complex at the active site, wherein binding to the molecule or complex is indicative of potential modulation. This method may further include the steps of evaluating the potential binding interactions between the chemical entity and the active site of the molecule or molecular complex and structurally modifying the chemical entity to yield a set of structure coordinates for a modified chemical entity, which steps may be repeated one or more times.

Once a potential modulator is identified, it can then be tested in any standard assay for the macromolecule depending of course on the macromolecule, including in high throughput assays. Further refinements to the structure of the modulator will generally be necessary and can be made by the successive iterations of any and/or all of the steps provided by the particular screening assay, in particular further structural analysis by e.g., .sup.15N NMR relaxation rate determinations or x-ray crystallography with the modulator bound to a dengue virus E protein or other class II E protein. These studies may be performed in conjunction with biochemical assays.

Once identified, a potential modulator may be used as a model structure, and analogs to the compound can be obtained. The analogs are then screened for their ability to bind to a dengue virus E protein or other class II E protein. An analog of the potential modulator might be chosen as a modulator when it binds to a dengue virus E protein or other class II E protein with a higher binding affinity than the predecessor modulator.

In a related approach, iterative drug design is used to identify modulators of a target protein. Iterative drug design is a method for optimizing associations between a protein and a modulator by determining and evaluating the three dimensional structures of successive sets of protein/modulator complexes. In iterative drug design, crystals of a series of protein/modulator 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 modulators of each complex. For example, this approach may be accomplished by selecting modulators with modulatory activity, obtaining crystals of this new protein/modulator complex, solving the three dimensional structure of the complex, and comparing the associations between the new protein/modulator complex and previously solved protein/modulator complexes. By observing how changes in the modulator affected the protein/modulator associations, these associations may be optimized.

In addition to designing and/or identifying a chemical entity to associate with a druggable region, as described above, the same techniques and methods may be used to design and/or identify chemical entities that either associate, or do not associate, with affinity regions, selectivity regions or undesired regions of protein targets. By such methods, selectivity for one or a few targets, or alternatively for multiple targets, from the same species or from multiple species, can be achieved.

For example, a chemical entity may be designed and/or identified for which the binding energy for one druggable region, e.g., an affinity region or selectivity region, is more favorable than that for another region, e.g., an undesired region, by about 20%, 30%, 50% to about 60% or more. It may be the case that the difference is observed between (a) more than two regions, (b) between different regions (selectivity, affinity or undesirable) from the same target, (c) between regions of different targets, (d) between regions of homologs from different species, or (e) between other combinations. Alternatively, the comparison may be made by reference to the Kd, usually the apparent Kd, of said chemical entity with the two or more regions in question.

In another aspect, prospective modulators are screened for binding to two nearby druggable regions on a target protein. For example, a modulator that binds a first region of a target polypeptide does not bind a second nearby region. Binding to the second region can be determined by monitoring changes in a different set of amide chemical shifts in either the original screen or a second screen conducted in the presence of a modulator (or potential modulator) for the first region. From an analysis of the chemical shift changes, the approximate location of a potential modulator for the second region is identified. Optimization of the second modulator for binding to the region is then carried out by screening structurally related compounds (e.g., analogs as described above). When modulators for the first region and the second region are identified, their location and orientation in the ternary complex can be determined experimentally. On the basis of this structural information, a linked compound, e.g., a consolidated modulator, is synthesized in which the modulator for the first region and the modulator for the second region are linked. In certain embodiments, the two modulators are covalently linked to form a consolidated modulator. This consolidated modulator may be tested to determine if it has a higher binding affinity for the target than either of the two individual modulators. A consolidated modulator is selected as a modulator when it has a higher binding affinity for the target than either of the two modulators. Larger consolidated modulators can be constructed in an analogous manner, e.g., linking three modulators which bind to three nearby regions on the target to form a multilinked consolidated modulator that has an even higher affinity for the target than the linked modulator. In this example, it is assumed that is desirable to have the modulator bind to all the druggable regions. However, it may be the case that binding to certain of the druggable regions is not desirable, so that the same techniques may be used to identify modulators and consolidated modulators that show increased specificity based on binding to at least one but not all druggable regions of a target.

The present invention provides a number of methods that use drug design as described above. For example, in one aspect, the present invention contemplates a method for designing a candidate compound for screening for modulators of a dengue virus E protein or other class II E protein, the method comprising: (a) determining the three dimensional structure of a crystallized dengue virus E protein or other class II E protein or a fragment thereof; and (b) designing a candidate modulator based on the three dimensional structure of the crystallized polypeptide or fragment.

In another aspect, the present invention contemplates a method for identifying a potential modulator of a dengue virus E protein or other class II E protein, the method comprising: (a) providing the three-dimensional coordinates of a dengue virus E protein or other class II E protein or a fragment thereof; (b) identifying a druggable region of the polypeptide or fragment; and (c) selecting from a database at least one compound that comprises three dimensional coordinates which indicate that the compound may bind the druggable region; (d) wherein the selected compound is a potential modulator of a dengue virus E protein or other class II E protein.

In another aspect, the present invention contemplates a method for identifying a potential modulator of a molecule comprising a druggable region similar to that of an E protein k1 hairpin, the method comprising: (a) using the atomic coordinates of amino acid residues from a druggable region, such as an E protein k1 hairpin, or a fragment thereof, .+-. a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 .ANG., to generate a three-dimensional structure of a molecule comprising an E protein k1 hairpin-like druggable region; (b) employing the three dimensional structure to design or select the potential modulator; (c) synthesizing the modulator; and (d) contacting the modulator with the molecule to determine the ability of the modulator to interact with the molecule.

In another aspect, the present invention contemplates an apparatus for determining whether a compound is a potential modulator of a dengue virus E protein or other class II E protein, the apparatus comprising: (a) a memory that comprises: (i) the three dimensional coordinates and identities of the atoms of a dengue virus E protein or other class II E protein or a fragment thereof that form a druggable site, such as for example, an E protein k1 hairpin; and (ii) executable instructions; and (b) a processor that is capable of executing instructions to: (i) receive three-dimensional structural information for a candidate compound; (ii) determine if the three-dimensional structure of the candidate compound is complementary to the structure of the interior of the druggable site; and (iii) output the results of the determination.

In another aspect, the present invention contemplates a method for designing a potential compound for the prevention or treatment of a flavivirus related disease or disorder, the method comprising: (a) providing the three dimensional structure of a crystallized dengue virus E protein or other class II E protein, or a fragment thereof; (b) synthesizing a potential compound for the prevention or treatment of flavivirus related disease or disorder based on the three dimensional structure of the crystallized polypeptide or fragment; (c) contacting a dengue virus E protein or other class II E protein with the potential compound; and (d) assaying the activity of a dengue virus E protein or other class II E protein, wherein a change in the activity of the polypeptide indicates that the compound may be useful for prevention or treatment of a flavivirus related disease or disorder.

In another aspect, the present invention contemplates a method for designing a potential compound for the prevention or treatment of flavivirus related disease or disorder, the method comprising: (a) providing structural information of a druggable region derived from NMR spectroscopy of a dengue virus E protein or other class II E protein, or a fragment thereof; (b) synthesizing a potential compound for the prevention or treatment of flavivirus related disease or disorder based on the structural information; (c) contacting a dengue virus E protein or other class II E protein or a flavivirus with the potential compound; and (d) assaying the activity of a dengue virus E protein or other class II E protein, wherein a change in the activity of the polypeptide indicates that the compound may be useful for prevention or treatment of a flavivirus related disease or disorder. (b) Modulator Libraries

The synthesis and screening of combinatorial libraries is a validated strategy for the identification and study of organic molecules of interest. According to the present invention, the synthesis of libraries containing molecules bind, interact with, or modulate the activity/function of a subject druggable region may be performed using established combinatorial methods for solution phase, solid phase, or a combination of solution phase and solid phase synthesis techniques. The synthesis of combinatorial libraries is well known in the art and has been reviewed (see, e.g., "Combinatorial Chemistry", Chemical and Engineering News, Feb. 24, 1997, p. 43; Thompson et al., Chem. Rev. (1996) 96:555). Many libraries are commercially available. One of ordinary skill in the art will realize that the choice of method for any particular embodiment will depend upon the specific number of molecules to be synthesized, the specific reaction chemistry, and the availability of specific instrumentation, such as robotic instrumentation for the preparation and analysis of the inventive libraries. In certain embodiments, the reactions to be performed to generate the libraries are selected for their ability to proceed in high yield, and in a stereoselective and regioselective fashion, if applicable.

In one aspect of the present invention, the inventive libraries are generated using a solution phase technique. Traditional advantages of solution phase techniques for the synthesis of combinatorial libraries include the availability of a much wider range of reactions, and the relative ease with which products may be characterized, and ready identification of library members, as discussed below. For example, in certain embodiments, for the generation of a solution phase combinatorial library, a parallel synthesis technique is utilized, in which all of the products are assembled separately in their own reaction vessels. In a particular parallel synthesis procedure, a microtitre plate containing n rows and m columns of tiny wells which are capable of holding a few milliliters of the solvent in which the reaction will occur, is utilized. It is possible to then use n variants of reactant A, such as a ligand, and m variants of reactant B, such as a second ligand, to obtain n.times.m variants, in n.times.m wells. One of ordinary skill in the art will realize that this particular procedure is most useful when smaller libraries are desired, and the specific wells may provide a ready means to identify the library members in a particular well.

In other embodiments of the present invention, a solid phase synthesis technique is utilized. Solid phase techniques allow reactions to be driven to completion because excess reagents may be utilized and the unreacted reagent washed away. Solid phase synthesis also allows the use a technique called "split and pool", in addition to the parallel synthesis technique, developed by Furka. See, e.g., Furka et al., Abstr. 14th Int. Congr. Biochem., (Prague, Czechoslovakia) (1988) 5:47; Furka et al., Int. J. Pept. Protein Res. (1991) 37:487; Sebestyen et al., Bioorg. Med. Chem. Lett. (1993) 3:413. In this technique, a mixture of related molecules may be made in the same reaction vessel, thus substantially reducing the number of containers required for the synthesis of very large libraries, such as those containing as many as or more than one million library members. As an example, the solid support with the starting material attached may be divided into n vessels, where n represents the number species of reagent A to be reacted with the such starting material. After reaction, the contents from n vessels are combined and then split into m vessels, where m represents the number of species of reagent B to be reacted with the now modified starting materials. This procedure is repeated until the desired number of reagents is reacted with the starting materials to yield the inventive library.

The use of solid phase techniques in the present invention may also include the use of a specific encoding technique. Specific encoding techniques have been reviewed by Czarnik in Current Opinion in Chemical Biology (1997) 1:60. One of ordinary skill in the art will also realize that if smaller solid phase libraries are generated in specific reaction wells, such as 96 well plates, or on plastic pins, the reaction history of these library members may also be identified by their spatial coordinates in the particular plate, and thus are spatially encoded. In other embodiments, an encoding technique involves the use of a particular "identifying agent" attached to the solid support, which enables the determination of the structure of a specific library member without reference to its spatial coordinates. Examples of such encoding techniques include, but are not limited to, spatial encoding techniques, graphical encoding techniques, including the "tea bag" method, chemical encoding methods, and spectrophotometric encoding methods. One of ordinary skill in the art will realize that the particular encoding method to be used in the present invention must be selected based upon the number of library members desired, and the reaction chemistry employed.

In certain embodiments, molecules of the present invention may be prepared using solid support chemistry known in the art. For example, polypeptides having up to twenty amino acids or more may be generated using standard solid phase technology on commercially available equipment (such as Advanced Chemtech multiple organic synthesizers). In certain embodiments, a starting material or later reactant may be attached to the solid phase, through a linking unit, or directly, and subsequently used in the synthesis of desired molecules. The choice of linkage will depend upon the reactivity of the molecules and the solid support units and the stability of these linkages. Direct attachment to the solid support via a linker molecule may be useful if it is desired not to detach the library member from the solid support. For example, for direct on-bead analysis of biological activity, a stronger interaction between the library member and the solid support may be desirable. Alternatively, the use of a linking reagent may be useful if more facile cleavage of the inventive library members from the solid support is desired.

In regard to automation of the present subject methods, a variety of instrumentation may be used to allow for the facile and efficient preparation of chemical libraries of the present invention, and methods of assaying members of such libraries. In general, automation, as used in reference to the synthesis and preparation of the subject chemical libraries, involves having instrumentation complete one or more of the operative steps that must be repeated a multitude of times because a library instead of a single molecule is being prepared. Examples of automation include, without limitation, having instrumentation complete the addition of reagents, the mixing and reaction of them, filtering of reaction mixtures, washing of solids with solvents, removal and addition of solvents, and the like. Automation may be applied to any steps in a reaction scheme, including those to prepare, purify and assay molecules for use in the compositions of the present invention.

There is a range of automation possible. For example, the synthesis of the subject libraries may be wholly automated or only partially automated. If wholly automated, the subject library may be prepared by the instrumentation without any human intervention after initiating the synthetic process, other than refilling reagent bottles or monitoring or programming the instrumentation as necessary. Although synthesis of a subject library may be wholly automated, it may be necessary for there to be human intervention for purification, identification, or the like of the library members.

In contrast, partial automation of the synthesis of a subject library involves some robotic assistance with the physical steps of the reaction schema that gives rise to the library, such as mixing, stirring, filtering and the like, but still requires some human intervention other than just refilling reagent bottles or monitoring or programming the instrumentation. This type of robotic automation is distinguished from assistance provided by convention organic synthetic and biological techniques because in partial automation, instrumentation still completes one or more of the steps of any schema that is required to be completed a multitude of times because a library of molecules is being prepared.

In certain embodiments, the subject library may be prepared in multiple reaction vessels (e.g., microtitre plates and the like), and the identity of particular members of the library may be determined by the location of each vessel. In other embodiments, the subject library may be synthesized in solution, and by the use of deconvolution techniques, the identity of particular members may be determined.

In one aspect of the invention, the subject screening method may be carried out utilizing immobilized libraries. In certain embodiments, the immobilized library will have the ability to bind to a microorganism as described above. The choice of a suitable support will be routine to the skilled artisan. Important criteria may include that the reactivity of the support not interfere with the reactions required to prepare the library. Insoluble polymeric supports include functionalized polymers based on polystyrene, polystyrene/divinylbenzene copolymers, and the like, including any of the particles described in section 4.3. It will be understood that the polymeric support may be coated, grafted or otherwise bonded to other solid supports.

In another embodiment, the polymeric support may be provided by reversibly soluble polymers. Such polymeric supports include functionalized polymers based on polyvinyl alcohol or polyethylene glycol (PEG). A soluble support may be made insoluble (e.g., may be made to precipitate) by addition of a suitable inert nonsolvent. One advantage of reactions performed using soluble polymeric supports is that reactions in solution may be more rapid, higher yielding, and more complete than reactions that are performed on insoluble polymeric supports.

Once the synthesis of either a desired solution phase or solid support bound template has been completed, the template is then available for further reaction to yield the desired solution phase or solid support bound structure. The use of solid support bound templates enables the use of more rapid split and pool techniques.

Characterization of the library members may be performed using standard analytical techniques, such as mass spectrometry, Nuclear Magnetic Resonance Spectroscopy, including 195Pt and 1H NMR, chromatography (e.g, liquid etc.) and infra-red spectroscopy. One of ordinary skill in the art will realize that the selection of a particular analytical technique will depend upon whether the inventive library members are in the solution phase or on the solid phase. In addition to such characterization, the library member may be synthesized separately to allow for more ready identification. (c) In Vitro Assays

Any form of dengue virus E protein or other class II E protein, e.g. a full-length polypeptide or a fragment comprising the target druggable region, may be used to assess the activity of candidate small molecules and other modulators in in vitro assays. In one embodiment of such an assay, agents are identified which modulate the biological activity of a druggable region, the protein-protein interaction of interest or formation of a protein complex involving a subject druggable region. In another embodiment of such an assay, agents are identified which bind or interact with subject druggable region. In certain embodiments, the test agent is a small organic molecule. The candidate agents may be selected, for example, from the following classes of compounds: detergents, proteins, peptides, peptidomimetics, small molecules, cytokines, or hormones. In some embodiments, the candidate therapeutics may be in a library of compounds. These libraries may be generated using combinatorial synthetic methods as described above. In certain embodiments of the present invention, the ability of said candidate therapeutics to bind a target gene or gene product may be evaluated by an in vitro assay. In either embodiments, discussed in the next section, the binding assay may also be in vivo.

The invention also provides a method of screening multiple compounds to identify those which modulate the action of polypeptides of the invention, or polynucleotides encoding the same. The method of screening may involve high-throughput techniques. For example, to screen for modulators, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, a whole cell or tissue, or even a whole organism comprising a dengue virus E protein or other class II E protein and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a modulator of a dengue virus E protein or other class II E protein. The ability of the candidate molecule to modulate a dengue virus E protein or other class II E protein is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Detection of the rate or level of production of product from substrate may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to calorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in a nucleic acid of the invention or polypeptide activity, and binding assays known in the art.

Another example of an assay for a modulator of a dengue virus E protein or other class II E protein is a competitive assay that combines a dengue virus E protein or other class II E protein and a potential modulator with molecules that bind to a dengue virus E protein or other class II E protein, recombinant molecules that bind to a dengue virus E protein or other class II E protein, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. Polypeptides of the invention can be labeled, such as by radioactivity or a colorimetric compound, such that the number of molecules of a dengue virus E protein or other class II E protein bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential modulator.

A number of methods for identifying a molecule which modulates the activity of a polypeptide are known in the art. For example, in one such method, a dengue virus E protein or other class II E protein is contacted with a test compound, and the activity of the dengue virus E protein or other class II E protein in the presence of the test compound is determined, wherein a change in the activity of the dengue virus E protein or other class II E protein is indicative that the test compound modulates the activity of the dengue virus E protein or other class II E protein. In certain instances, the test compound agonizes the activity of the dengue virus E protein or other class II E protein, and in other instances, the test compound antagonizes the activity of the dengue virus E protein or other class II E protein.

In another example, a compound which modulates dengue virus E protein or other class II E protein dependent growth or infectivity of flavivirus may be identified by (a) contacting a dengue virus E protein or other class II E protein with a test compound; and (b) determining the activity of the polypeptide in the presence of the test compound, wherein a change in the activity of the polypeptide is indicative that the test compound may modulate the growth or infectivity of flavivirus.

In certain of the subject assays, to evaluate the results using the subject compositions, comparisons may be made to known molecules, such as one with a known binding affinity for the target. For example, a known molecule and a new molecule of interest may be assayed. The result of the assay for the subject complex will be of a type and of a magnitude that may be compared to result for the known molecule. To the extent that the subject complex exhibits a type of response in the assay that is quantifiably different from that of the known molecule then the result for such complex in the assay would be deemed a positive or negative result. In certain assays, the magnitude of the response may be expressed as a percentage response with the known molecule result, e.g. 100% of the known result if they are the same.

As those skilled in the art will understand, based on the present description, binding assays may be used to detect agents that bind a polypeptide. Cell-free assays may be used to identify molecules that are capable of interacting with a polypeptide. In a preferred embodiment, cell-free assays for identifying such molecules are comprised essentially of a reaction mixture containing a target and a test molecule or a library of test molecules. A test molecule may be, e.g., a derivative of a known binding partner of the target, e.g., a biologically inactive peptide, or a small molecule. Agents to be tested for their ability to bind may be produced, for example, by bacteria, yeast or other organisms (e.g. natural products), produced chemically (e.g. small molecules, including peptidomimetics), or produced recombinantly. In certain embodiments, the test molecule is selected from the group consisting of lipids, carbohydrates, peptides, peptidomimetics, peptide-nucleic acids (PNAs), proteins, small molecules, natural products, aptamers and oligonucleotides. In other embodiments of the invention, the binding assays are not cell-free. In a preferred embodiment, such assays for identifying molecules that bind a target comprise a reaction mixture containing a target microorganism and a test molecule or a library of test molecules.

In many candidate screening programs which test libraries of molecules and natural extracts, high throughput assays are desirable in order to maximize the number of molecules surveyed in a given period of time. Assays of the present invention which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins or with lysates, are often preferred as "primary" screens in that they may be generated to permit rapid development and relatively easy detection of binding between a target and a test molecule. Moreover, the effects of cellular toxicity and/or bioavailability of the test molecule may be generally ignored in the in vitro system, the assay instead being focused primarily on the ability of the molecule to bind the target. Accordingly, potential binding molecules may be detected in a cell-free assay generated by constitution of functional interactions of interest in a cell lysate. In an alternate format, the assay may be derived as a reconstituted protein mixture which, as described below, offers a number of benefits over lysate-based assays.

In one aspect, the present invention provides assays that may be used to screen for molecules that bind E protein druggable regions. In an exemplary binding assay, the molecule of interest is contacted with a mixture generated from target cell surface polypeptides. Detection and quantification of expected binding from to a target polypeptide provides a means for determining the molecule's efficacy at binding the target. The efficacy of the molecule may be assessed by generating dose response curves from data obtained using various concentrations of the test molecule. Moreover, a control assay may also be performed to provide a baseline for comparison. In the control assay, the formation of complexes is quantitated in the absence of the test molecule.

Complex formation between a molecule and a target E protein or microorganism containing a class II E protein may be detected by a variety of techniques, many of which are effectively described above. For instance, modulation in the formation of complexes may be quantitated using, for example, detectably labeled proteins (e.g. radiolabeled, fluorescently labeled, or enzymatically labeled), by immunoassay, or by chromatographic detection.

Accordingly, one exemplary screening assay of the present invention includes the steps of contacting a class II E protein or functional fragment thereof with a test molecule or library of test molecules and detecting the formation of complexes. For detection purposes, for example, the molecule may be labeled with a specific marker and the test molecule or library of test molecules labeled with a different marker. Interaction of a test molecule with a polypeptide or fragment thereof may then be detected by determining the level of the two labels after an incubation step and a washing step. The presence of two labels after the washing step is indicative of an interaction. Such an assay may also be modified to work with a whole target cell.

An interaction between a class II E protein target and a molecule may also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants. In one embodiment, a library of test molecules may be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the target is then flowed continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occurred. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.

In a preferred embodiment, it will be desirable to immobilize the target to facilitate separation of complexes from uncomplexed forms, as well as to accommodate automation of the assay. Binding of polypeptide to a test molecule may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein may be provided which adds a domain that allows the target to be bound to a matrix. For example, glutathione-S-transferase/polypeptide (GST/polypeptide) fusion proteins may be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with a labeled test molecule (e.g., S.sup.35 labeled, P.sup.33 labeled, and the like, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated. Alternatively, the complexes may be dissociated from the matrix, separated by SDS-PAGE, and the level of polypeptide or binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples. The above techniques could also be modified in which the test molecule is immobilized, and the labeled target is incubated with the immobilized test molecules. In one embodiment of the invention, the test molecules are immobilized, optionally via a linker, to a particle of the invention, e.g. to create the ultimate composition.

Other techniques for immobilizing targets or molecules on matrices may be used in the subject assays. For instance, a target or molecule may be immobilized utilizing conjugation of biotin and streptavidin. For instance, biotinylated polypeptide molecules may be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with a target or molecule may be derivatized to the wells of the plate, and the target or molecule trapped in the wells by antibody conjugation. As above, preparations of test molecules are incubated in the polypeptide presenting wells of the plate, and the amount of complex trapped in the well may be quantitated. Exemplary methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the complex, or which are reactive with one of the complex components; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with a target or molecule, either intrinsic or extrinsic activity. In an instance of the latter, the enzyme may be chemically conjugated or provided as a fusion protein with the target or molecule. To illustrate, a target polypeptide may be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in a complex with a molecule may be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-1-napthol. Likewise, a fusion protein comprising the polypeptide and glutathione-S-transferase may be provided, and complex formation quantitated by detecting the GST activity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes that rely on immunodetection for quantitating one of the components trapped in a complex, antibodies against a component, such as anti-polypeptide antibodies, may be used. Alternatively, the component to be detected in the complex may be "epitope tagged" in the form of a fusion protein which includes, in addition to the polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources). For instance, the GST fusion proteins described above may also be used for quantification of binding using antibodies against the GST moiety. Other useful epitope tags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem 266:21150-21157) which includes a 10-residue sequence from c-myc, as well as the pFLAG system (International Biotechnologies, Inc.) or the pEZZ-protein A system (Pharmacia, N.J.).

In certain in vitro embodiments of the present assay, the solution containing the target comprises a reconstituted protein mixture of at least semi-purified proteins. By semi-purified, it is meant that the components utilized in the reconstituted mixture have been previously separated from other cellular or viral proteins. For instance, in contrast to cell lysates, a target protein is present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity. In certain embodiments of the subject method, the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins (such as of cellular or viral origin) which might interfere with or otherwise alter the ability to measure binding activity. In one embodiment, the use of reconstituted protein mixtures allows more careful control of the target:molecule interaction conditions.

In still other embodiments of the present invention, variations of viral fusion or viral infectivity assays may be utilized in order to determine the ability of a test molecule to prevent a virus expressing type II E protein from binding to, fusing with, or infecting cells. If fusion, binding, or infecting is prevented, then the molecule or composition may be useful as a therapeutic agent.

All of the screening methods may be accomplished by using a variety of assay formats. In light of the present disclosure, those not expressly described herein will nevertheless be known and comprehended by one of ordinary skill in the art. Assay formats which approximate such conditions as formation of protein complexes or protein-nucleic acid complexes, and enzymatic activity may be generated in many different forms, as those skilled in the art will appreciate based on the present description and include but are not limited to assays based on cell-free systems, e.g. purified proteins or cell lysates, as well as cell-based assays which utilize intact cells. Assaying binding resulting from a given target:molecule interaction may be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. Any of the assays may be provided in kit format and may be automated. Many of the following particularized assays rely on general principles, such as blockage or prevention of fusion, that may apply to other particular assays. (d) In Vivo Assays

Animal models of viral infection and/or disease may be used as an in vivo assay for evaluating the effectiveness of a potential drug target in treating or preventing flavivirus related diseases or disorders. A number of suitable animal models are described briefly below, however, these models are only examples and modifications, or completely different animal models, may be used in accord with the methods of the invention. Animal models may be developed by methods known in the art, for example, by infecting an animal with dengue fever or another flavivirus, or by genetically engineering an animal to be predisposed to such infection (see, e.g., Wu, S.-J. L. et al. Evaluation of the severe combined immunodeficient (SCID) mouse as an animal model for dengue viral infection. Am. J. Trop. Med. Hyg. 52, 468-476 (1995)).

Further, viral infectivity assays may be used as in vivo assays to assess the effectiveness of a potential drug target in treating or preventing flavivirus related diseases or disorders. For example, the plaque assays described in Diamond et al (2000) J Virol 74:4957-4966 may be used to assess by analyzing virion production whether an agent may modulate infectivity of Dengue virus. Other assays, such as competitive, asymmetric reverse transcriptase-mediated PCR (RT-PCR) assays and flow cytometric assays that measure viral antigen, also described in Diamond, et al, may be used to assess the effectiveness of a potential drug target.

Still further, further, cell-cell fusion assays may be used as in vivo assays to assess the effectiveness of a potential drug target in treating or preventing flavivirus related diseases or disorders. For example, a cell-cell fusion assay in which the cell membrane fusion activity of dengue virus may be analyzed is described in Despres et al (1993) Virology 196:209-219.

A variety of other in vivo models are available and may be used when appropriate for specific pathogens or specific test agents.

It is also relevant to note that the species of animal used for an infection model, and the specific genetic make-up of that animal, may contribute to the effective evaluation of the effects of a particular test agent. For example, immuno-incompetent animals may, in some instances, be preferable to immuno-competent animals. For example, the action of a competent immune system may, to some degree, mask the effects of the test agent as compared to a similar infection in an immuno-incompetent animal. In addition, many opportunistic infections, in fact, occur in immuno-compromised patients, so modeling an infection in a similar immunological environment is appropriate.

E. Pharmaceutical Compositions

Pharmaceutical compositions of this invention include any modulator identified according to the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The term "pharmaceutically acceptable carrier" refers to 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.

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. 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 modulators described herein are useful for the prevention and treatment of disease and conditions, including diseases and conditions mediated by pathogenic species of origin for the polypeptides of the invention. 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). Alternatively, such preparations contain from about 20% to about 80% active compound.

G. Kits

The present invention provides kits for treating dengue fever and other flaviviral infections. For example, a kit may comprise compositions comprising compounds identified herein as modulators of dengue virus E protein or other class II E protein. The compositions may be pharmaceutical compositions comprising a pharmaceutically acceptable excipient. In other embodiments involving kits, this invention contemplates a kit including compositions of the present invention, and optionally instructions for their use. Kit components may be packaged for either manual or partially or wholly automated practice of the foregoing methods. Such kits may have a variety of uses, including, for example, imaging, diagnosis, therapy, and other applications.

H. Further Characterization of Dengue Virus E Protein or Other Flavivirus E Protein Druggable Regions and Complexes of the Same
 

Claim 1 of 18 Claims

1. A method for identifying a candidate therapeutic for a disease caused by a virus having class II E protein, comprising contacting a dengue virus class II E protein with a compound, wherein binding of said compound indicates a candidate therapeutic, wherein the dengue virus class II E protein consists of an amino acid sequence at least 20 amino acids but not greater than 394 amino acids in length having at least 85% identity along the length to the amino acid sequence of SEQ ID NO: 1 or 2, wherein amino acid residue 101 is Trp, amino acid residue 107 is Leu and amino acid residue 108 is Phe when numbered in accordance with SEQ ID NO: 1 or 2.

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