WO2010000012A1

WO2010000012A1 - Method for preventing transport of a neurotropic virus

Info

Publication number: WO2010000012A1
Application number: PCT/AU2008/001403
Authority: WO
Inventors: Anthony Lawrence Cunningham; Russell John Diefenbach; April Mae Morton; Monica Miranda Saksena
Original assignee: The University Of Sydney; Sydney West Area Health Service
Priority date: 2008-07-03
Filing date: 2008-09-23
Publication date: 2010-01-07

Abstract

The present invention relates to viruses, particularly to cellular transport of neurotropic viruses and the viral and cellular proteins involved with cellular transport of neurotropic viruses.

Description

METHOD FOR PREVENTING TRANSPORT OF A NEUROTROPIC VIRUS

FIELD OF INVENTION

BACKGROUND OF THE INVENTION

The size and complexity of eukaiyotic cells effectively restricts free intracellular diffusion of molecules with a molecular mass greater than 500 000, requiring cells to have organised transport systems. In many cases, viruses utilise these intracellular transport mechanisms to complete their replication cycle (Greber and Way, 2006). Long distance intracellular movement of organelles and vesicles is driven by motor proteins which transport cargo along microtubules (MT). MTs are long cytoskeletal filaments with their fast growing plus-ends usually pointing towards the plasma membrane and in neuronal axons towards the nerve terminals. Intracellular transport is more important for viruses mat require access to the cell nucleus to replicate, such as the neurotropic herpesviruses and retroviruses. It has been estimated that a Herpes Simplex Virus (HSV) would take approximately 230 years to travel lcm through the cytoplasm by diffusion alone (Sodeik, 2000).

Herpesviruses are a large family of viruses which infect a wide range of organisms and include viruses in the α group (e.g. herpes simplex virus (HSV), Pseudorabies Virus (PrV)), the β group (e.g. Human Cytomegalovirus (HCMV)) and in the γ group (e.g. Epstein-Barr Virus (EBV)). HSV type I (HSV-I) is one of the most common human pathogens, infecting 40-80% of people worldwide. The her^fcδviruses are uniquely able to infect and remain latent within neurons. Although most clinical disease due to HSV-I is relatively mild, it can sometimes cause encephalitis in adults, or disseminated infection in neonates, both of which are frequently fetal if left untreated. Furthermore, mucocutaneous infection with HSV-I, especially in the genital region, causes significant morbidity, and reduced quality of life, for many people in the community. Genital herpes (HSV-I or HSV-2) is a common problem but more so in the developing world where co-infection with HIV leads to a two to four fold increase in HIV transmission (Cunningham and Dwyer, 2004).

The Herpes simplex virion has four components; an electron dense core containing the double-stranded DNA genome (152 kb), the capsid, the tegument and an outer envelope containing glycoprotein. ρUS9 is a 1OkDa type II membrane protein, although it is uncertain whether the protein is located in the tegument or envelope or both.

There remains limited knowledge of the mechanisms of transport of neurotropic viruses. Knowing how neurotropic viruses move in neurons and other cells and ultimately cause disease is important and is useful for the development of antiviral strategies.

SUMMARY OF THE INVENTION

The present inventors have identified that the viral protein pUS9 binds to the cellular protein kinesin-1. The binding of pUS9 to kinesin-1 is important for transport of a neurotropic virus in a cell.

Accordingly, the present invention provides a method of inhibiting transport of a neurotropic virus in a cell, the method comprising providing to tbe cell a compound which reduces binding of ρUS9 to kinesin-1. For, example, the compound may be a peptide or mimic thereof derived from pUS9 or apart of pUS9 which binds kinesin-1. In one embodiment, the peptide or mimic thereof is derived from residues 1 to

64 of pUS9. In another embodiment, the peptide or mimetic thereof is derived from residues 29 to 64 of pUS9.

In an embodiment, pUS9 comprises an amino acid sequence at least 80%, alternatively at least 85%, 90% or 95%, identical to SEQ ID NO:1. In another embodiment, the compound is a peptide or mimic derived from kiπesin-1 or a part of kinesin-1 which binds ppUS9. In one particular embodiment, the peptide or mimic thereof is derived from residues 814 to 963 of kinesin-1. In one embodiment, kinesin-1 comprises an amino acid sequence at least 80%, alternatively at least 85%, 90% or 95% identical to SEQ ID NO:2. While the methods of the invention may be used to inhibit the transport in a cell of any neurotropic virus, in one embodiment, the neurotropic virus is an alphaherpesvirus. For example, the alphahcrpesvirus may be selected from herpes simplex virus, varicella-zoster virus and pseudorabies virus. In one particular embodiment of the invention, the alphaherpesvirus is HSV-I. The peptide or mimic may be any suitable length as long as it functions to reduce binding of pUS9 to kinesin-1. For example, the peptide or mimic thereof may be 30 residues or Jess in length, 20 residues or less in length, or 15, 14, 13, 12, II or 10 residues or less in length.

The methods of the invention may be performed in any cell in which a neurotropic virus can be transported, In one embodiment, the cell is a neuron and the transport is neuronal transport In one particular embodiment, the neuronal transport is anterograde neuronal transport.

In an embodiment of the present invention, the method is performed in vivo in a subject by administering the compound to the subject In one embodiment, the cell or subject is mammalian, for example, the subject may be human.

In one embodiment, the compound is capable of reducing binding of pUS9 to kinesin-1, wherein pUS9 comprises the amino acid sequence of SBQ ID NO: 1.

In another embodiment, the compound is capable of reducing binding of pUS9 to kjnesjπ-l, wherein kinesin-1 comprises the amino acid sequence of SEQ ID NO:2. The present invention further provides a peptide or mimic thereof of 30 residues or less derived from pUS9 and which is capable of reducing binding of pUS9 to kinesin-1.

In one embodiment, pUS9 comprises an amino acid sequence at least 80% identical to SEQ ID NO: L In another embodiment, the peptide or mimic thereof is derived from residues 1 to 64 of pUS9. In yet another embodiment, the peptide or mimetic thereof is derived from residues 29 to 64 of pUS9.

The present invention also provides a peptide or mimic thereof of 30 residues or less derived from kinesin-1 and which is capable of reducing binding of pUS9 to kiπcsin-1.

In one embodiment, kinesin-1 comprises an amino acid sequence at least 80% identical to SEQ ΓD NO:2.

In another embodiment, the peptide or mimic thereof is derived from residues 814 to 963 ofkinesm-1. The present invention further provides a pharmaceutical composition comprising the peptide or mimic thereof of the invention. The compositions of the invention can be used, for example, to inhibit transport of a neurotropic virus in a cell.

The present invention further provides a method of screening for an inhibitor of neuronal transport of a neurotropic virus comprising determining whether a candidate compound reduces binding of pUS9 to kinesin-1.

In one embodiment, the compound is a peptide or mimic of 30 residues or less which is derived from amino acid residues 1 to 64 of pUS9.

In one particular embodiment, the peptide or mimic thereof is derived from a polypeptide comprising a sequence at least 80% identical to SEQ ID NO: 1. In one embodiment, the kinesin-1 comprises an amino acid sequence at least

80% identical to the amino acid sequence of SEQ ID NO:2. While the method of the invention may be used to screen for an inhibitor of neuronal transport for any neurotropic virus, in one embodiment me neurotropic virus is an alphaherpesvirus. The alphaherpesvirus may be selected from, for example, herpes simplex vims, varicella-zoster virus and pseudorabies vims. In one embodiment, the alphaherpesvirus is HSV-] .

By inhibiting transport of a neurotropic virus in a cell, the methods of the present invention may be used for the treatment or prevention of viral infection or reactivation. Accordingly, the present invention provides a method of treating or preventing infection with, or reactivation of, a neutrotropic virus in a subject, the method comprising administering to the subject a compound which reduces binding of pUS9 to kinesin-1.

In one embodiment, the present invention provides methods of treating or preventing infection with a neutrotropic virus in a subject

In a further embodiment, the present invention provides methods of treating or preventing reactivation of a neutrotropic virus in a subject.

In one particular embodiment, the present invention provides a method of treating reactivation of a neurotropic virus in a subject, the method comprising administering to the subject a compound which reduces binding of pUS9 to kinesin-1.

In one embodiment, the method comprises administering to the subject the peptide or mimic thereof of the invention or the pharmaceutical composition of the invention.

The present invention further provides use of the peptide or mimic thereof of the invention or the pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of infection or reactivation of a neutrotropic virus.

The present invention further provides use of the peptide or mimic thereof of the invention or the pharmaceutical composition of the invention as a medicament for the treatment or prevention of infection or reactivation of a neutrotropic virus.

The compounds, peptides or mimics of the present invention can be used in combination with additional agents. In one embodiment, the additional agent is a compound which reduces binding of pUSl l to kinesin-1. The USl I may be, for example, HSV-I USIl (NCBI Accession No. NPJM4674).

Accordingly, the present invention provides a method of inhibiting transport of a neurotropic virus in a cell, the method comprising providing to the cell a compound which reduces binding of pUS9 to kinesin-1 in combination with a compound which reduces binding of pUSl 1 to kinesin-1. As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Specific binding of His-tagged KIF5B 814-963 (heavy chain cargo-binding domain of human kinesin-1; detected with anti-His antibody) to both GST-tagged ppUS9 1-64 (cytoplasmic domain) and GST-tagged pUSll. No significant interaction was observed with GST only, GST-tagged cytoplasmic domain of pϋS8 (gE) or GST- tagged tegument protein pUL48. As an additional control, His-tagged pUSl 1 did not interact significantly with any of the GST-tagged proteins except with GST-tagged pUSl l.

Figure 2. Specific binding of kinesin-1 (detected with anti-KIF5B antibody) from HeLa lysates was the greatest with GST-ppUS9, although binding was observed with GST-pUS 11 and to a lesser extent GST-pUS8. Figure 3. SDS-PAGE of GST-tagged proteins stained with Coomassie blue to confirm the presence of the GST-tagged proteins.

Figure 4. Alignment of pUS9 protein from a number of alphaherpesviruses.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: I - Amino acid sequence of HSV-I pUS9. SEQ ID NO:2 - Amino acid sequence of human kinesin-1 (KIF5B)

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in virology, ceil culture, molecular, genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry). Unless otherwise indicated, the recombinant protein, cell culture, microbiological and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, 2^nd edn, Wiley (1988), J. Sambrook et al._t Molecular Cloning: A Laboratory Manual, 3"* edn, Cold Spring Harbour Laboratory Press (2001), TA. Brown (editor), Essential Molecular Biology: A Practica] Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and BD. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et cύ., (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present), and are incorporated herein by reference.

As used herein, the term "subject" refers to an animal, e.g., a mammal. In a preferred embodiment, the subject is human. Other preferred embodiments include companion animals such as cats and dogs, as well as livestock animals such as horses, cattle, sheep and pigs. The terms "inhibiting" or "inhibit" as used herein in relation to transport of a neurotropic virus in a cell refers to a reduced rate of transport of a virus and/or the transport of a reduced amount of virus in a cell comprising or having been provided with a compound of the invention when compared to a cell which does not comprise or has not been provided with a compound of the invention, hi one embodiment, transport of the virus in the cell is reduced by a measurable amount, for example by at least 10%, or at least 20%, or at least 30%, 40%, 50%, 60%, 70% or 80% or more, In another embodiment, the transport of the neurotropic virus in a cell is completely inhibited. The rate or amount of transport of a virus in the cell can be measured, for example, using the any suitable viral transport assays known to the skilled addressee, or using a viral transport assay as described herein.

By "reduces" or "reducing" binding is meant a decrease or inhibition in binding of pUS9 to kinesin-1 in the presence of a compound, for example a compound of the invention, when compared to binding of pUS9 to kinesin-1 in the absence of the compound, such as in a control sample. The degree of decrease or inhibition of binding will vary with the nature and quantity of the compound present, but will be evident e.g., as a detectable decrease in binding of pUS9 to kinesin-1; desirably a degree of decrease greater than 10%, 33%, 50%, 75%, 90%, 95% or 99% as compared to binding of pUS9 to kinesin-1 in the absence of the compound.

The phrase "specifically reduces binding" as used herein means that under particular conditions a compound binds to pUS9 or kinesin-1 and reduces or inhibits binding of pUS9 to kinesin-1, but the compound does not bind to a significant amount to other, for example, proteins or carbohydrates.

"Administering" as used herein is to be construed broadly and includes administering a compound of the invention to a subject as well as providing a compound of the invention to a cell. As used herein the terms "treating¹¹, "treat" or "treatment" include administering a therapeutically effective amount of a compound which reduces binding of pUS9 to kinesin-1 sufficient to reduce or eliminate at least one symptom of disease caused by infection with a neurotropic virus and/or by reactivation of a neurotropic virus.

The term "preventing" refers to protecting a subject that may be exposed to a neurotropic virus from developing at least one symptom resulting from infection and/or reactivation by the virus, or reducing the severity of a symptom of infection and/or reactivation in a subject.

Compounds that modulate pUS9 binding to kinesin

The Herpes simplex virion has four components; an electron dense core containing the double-stranded DNA genome (152 kb), the capsid, the tegument and an outer envelope containing glycoprotein. pUS9 is a 1OkDa type II envelope protein (NCBI Accession No. NP 044672; SEQ ID NO: 1), although it is uncertain whether the protein is located in the tegument or envelope or both. Despite pUS9 having been implicated in neuronal transport, the identification of the motor proteins with which it interacts in the host cell was considered to be a challenging task (LaVail et al., 2007).

The kinesin superfamily (KIF) of molecular motor proteins, which number 45 in mammalian cells, together with dyneiπ are responsible for microtubule-dependent transport of cargo in eukaryotic cells (Wozniak et al,, 2004; Hirokawa and Takemura, 2004; Miki et al., 2005). Kinesins are classified according to the position of the highly conserved motor domain (binds microtubules) which may be N-terminal (N-kinesin), middle (M-kinesin) or C-terminal (C-kinesin). The majority of kinesins are N-kinesins which transport cargo such as membranous vesicles, protein complexes, mRNA and viruses towards the plus or growing end of MTs. In the case of neurons, this corresponds to anterograde transport, that is away from the cell body towards the axon tip. The present inventors have found that pUS9 binds to the molecular motor protein kinesin-1 (KIF5B). Compounds that target and reduce the binding of pUS9 to ldnesin-1 and which inhibit the transport of neurotropic viruses in a cell may be useful as antiviral agents. Accordingly, in one embodiment the present invention provides a S method of inhibiting transport of a neurotropic virus in a cell, the method comprising providing to the cell a compound which reduces binding of pUS9 to kinesin-1. In one embodiment, the compound specifically reduces binding of pUS9 to kinesin-1

In one embodiment, the compound binds to the viral protein pUS9, for example the pUS9 protein of HSV-I. In one particular embodiment, the compound binds to the0 residues 1 to 64 in the cytoplasmic domain of pUS9.

Alternatively, the compound of the invention binds kinesin-1. In one embodiment, the compound of the invention binds to residues 814 to 963 of kinesin-1.

The compound may be any suitable compound which binds pUS9 or kinesin-1 and reduces binding of pUS9 to kinesin-1. For example, the compound may be a5 peptide or mimic thereof which is derived from pUS9 or kinesin-1. Alternatively, the compound may be an antibody which binds to pUS9 or kinesin-1.

Peptides and mimics thereof

In one embodiment, the compound of the invention which inhibits transport of a neurotropic virus in a cell includes a peptide or a mimic thereof derived from pUS9 or0 kinesin-1. In one embodiment, the compound is a peptide, which may be a naturally occurring peptide or fragment of a naturally occurring protein. In this way libraries of peptides may be made for screening in the methods of the invention.

In one embodiment, candidate compounds are peptides of from about 5 to about 30 amino acids, or from about 5 to about 20 amino acids, or from about 7 to about 155 amino acids. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each peptide consists of essentially random amino acids. Since generally these random peptides are chemically synthesized, they may incorporate any amino acid at any position. The synthetic process can be designed to0 generate randomized proteins, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate compounds.

In one embodiment, peptides are chemically or recombinantly synthesized as oligopeptides (approximately 10-25 amino acids in length) derived from a pUS9 or5 kinesin-1 sequence. Alternatively, pUS9 or kinesin-1 fragments are produced by digestion of native or recombinantly produced polypeptides by, for example, using a protease, e.g., trypsin, thermolysfai, chymotrypsin, or pepsin. Computer analysis (using commercially available software, e.g. MacVector, Omega, PCGene, Molecular Simulation, Inc.) is used to identify proteolytic cleavage sites. The proteolytic or synthetic fragments can comprise as many amino acid residues as are necessary to reduce binding of pUS9 to kinesin-1. Fragments may comprise 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more amino acids in length.

The terms "mimic", "mimetic" and "peptidomimetic" as used herein refer to a synthetic chemical compound, that has substantially the same structural and/or functional characteristics of the peptides, e.g., peptides of the invention derived from HSV pUS9 or from human kmesin-1. The mimic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural amino acid residues and partly non-natural analogs of amino acids. The mimic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimic's structure and/or activity. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

Table 1 - Exemplary substitutions.

As with peptides of the invention which are conservative variants, routine experimentation will determine whether a mimic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Peptide mimic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

A peptide may be characterized as a mimic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., ghrtaraldehyde, N-hydroxysuccinimide esters, bifunctional rnaleimides, N,N'- dicyclohexylcarbodiimide (DCC) or N,N¹-diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, but not limited to, ketomethylene (e.g.,

for -C(=O)— NH-), aminomethylene (CH₂-NIQ, ethylene, olefin (CH=CH), ether (CH₂-O), thioether (CH2-S), tetrazole (CN₄-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) In: Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY). A peptide can also be characterized as a mimic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature. The peptide or mimic thereof of the invention may be any length so long as it binds to kmesm-1 and blocks binding of pUS9 to kinesin-1. For example the peptide of the invention may be 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 or less residues in - I I - length, or even shorter, for example, the peptide or mimic thereof may be 10, 9, 8 or less residues in length. .

In one embodiment, numerous organic molecules may be assayed for their ability to modulate binding of pUS9 to kinesin-1. For example, within one S embodiment of the invention suitable organic molecules may be selected either from a chemical library, wherein chemicals are assayed individually, or from combinatorial chemical libraries where multiple compounds are assayed at once, then deconvoluted to determine and isolate the most active compounds.

Representative examples of such combinatorial chemical libraries include those0 described in, for example, U.S. Pat No. 5,463,564; WO 95/02566; WO 95/24186; WO

95/30642; WO 95/16918; WO 95/16712; U.S. Pat No. 5,288.514; WO 95/16209; WO

95/04277; U.S. Pat No. 5,506,337; WO 96/00148; Phillips and Wei, 1996; Ruhland et al., 1996; and Look el al, 1996.

Candidate compounds may be organic molecules, preferably small organic5 compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or0 heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

In one embodiment, the present invention involves screening small molecule5 chemodiversity represented within libraries of parent and fractionated natural product extracts, to detect bioactive compounds as potential candidates for further characterization.

In one embodiment of the present invention, the candidate compound is obtained from expression products of a gene library, a low molecular weight compound0 library (such as the low molecular weight compound library of ChemBridge Research

Laboratories), a cell extract, microorganism culture supernatant, bacterial cell components and the like.

Antibodies

The term "antibody" as used in this invention includes polyclonal antibodies,5 monoclonal antibodies, bispecific antibodies, diabodies, triabodies, heteroconjugate antibodies, chimeric antibodies including intact molecules as well as fragments thereof, such as Fab, F(ab')2, and Fv which are capable of binding the epitopic determinant, and other antibody-like molecules.

Antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab¹ fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody mat can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab)2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and

(5) Single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art (See for example,

Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,

New York (1988), incorporated herein by reference). (6) Single domain antibody, typically a variable heavy domain devoid of a light chain.

Polyclonal Antibodies

An antibody of the present invention may be a polyclonal antibody. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of the cells expressing the polypeptide and, if desired, an adjuvant Typically, the cells and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. Examples of adjuvants which may.be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by on& skilled in the art without undue experimentation.

Monoclonal Antibodies

The antibodies produced by the method of the invention may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with the cells expressing the polypeptide of the first species derived from the transgenic mammal to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide of the first species.

Generally, either peripheral blood lymphocytes (¹¹PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells arc used , if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, 3^rd edn, Academic Press, (1996)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfiised, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT- deficient cells. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the polypeptide of the first species. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RJPMI- 1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites hi a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in, for example, U.S. Pat No. 4,816,567.

Human and humanized antibodies The antibodies of the present invention may be humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab")₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. 1986; Riechmann et al. 1988; and Presta 1992).

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import¹¹ variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al. 1986; Riechmann et al. 1988; Verhoeyen et al. 1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR S residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. For example, one of the binding specificities may be0 for pUS9, the other one may be for kinesin-1 or any other antigen.

Methods for making bispecific antibodies are known in (he art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities. Because of the random assortment of immunoglobulin heavyS and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829 and in Traunecker (1991). 0 Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab")₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Various technique for making and isolating bispecific antibody fragments directly from recombinant cell5 culture have also been described.

Heteroconjugale Antibodies

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to0 unwanted cells (U.S. PaL No. 4,676,980), and for treatment of fflV infection (WO 91/00360; WO 92/200373; EP 03089). It is contemplated mat the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. Examples of suitable reagents for this purpose include iminothioJate and memyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. PaL No.4,676,980.

Protein-structure based design of binding inhibitors

The three-dimensional structure of a crystal of pUS9 and/or kinesin-1 (for example polypeptides comprising sequences such as provided in SEQ ID NO: 1 or SEQ ID NO:2), or a fragment thereof, can be used to identify compounds that reduce binding of pUS9 to kinesin-1 through the use of computer modelling using a docking program such as, for example, GRAM, DOCK, or AUTODOCK (Dunbrack et al., 1997). Alternatively, the three-dimensional structure of the proteins may be generated by computational modelling and the model structure used to dock potential inhibitors of binding. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of a candidate compound to the polypeptide. Generally the tighter the fit (e.g., the lower the steric hindrance, and/or the greater the attractive force) the more potent the potential antagonist of pUS9 binding to kinesin-1 will be since these properties are consistent with a tighter binding constant Furthermore, the more specificity in the design of a potential antagonist the more likely that it will not interfere with other proteins.

Initially a potential compound could be obtained, for example, by screening a random peptide library produced by a recombinant bacteriophage or a chemical library. A compound selected in this manner could be then be systematically modified by computer modeling programs until one or more promising potential compounds are identified.

Such computer modeling allows the selection of a finite number of rational chemical modifications, as opposed to the countless number of essentially random chemical modifications that could be made, and of which any one might lead to a useful agonist or antagonist. Each chemical modification requires additional chemical steps, which while being reasonable for the synthesis of a finite number of compounds, quickly becomes overwhelming if all possible modifications needed to be synthesized. Thus through the use of the three-dimensional structure and computer modeling, a large number of these compounds can be rapidly screened, and a few likely candidates can be determined without the laborious synthesis of untold numbers of compounds.

The prospective antagonist can be placed into the activity assay described herein to test its effect on the binding of pUS9 to kinesin-1.

For all of the screening assays described herein further refinements to the structure of the antagonist 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.

Viruses

The compounds and methods of the present invention can be used to inhibit the transport of a neurotropic virus, for example an alphaherpesvirus, in a cell. The compounds of the invention may be any compound suitable for administration or delivery to a cell and which can interfere with the binding of the structural protein pUS9 with a motor protein such as kinestn-1.

As used herein "neurotropic virus" refers to any virus which is capable of infecting neurons, including viruses which only transiently infect neurons. Neurotropic viruses include, but are not limited to viruses belonging to the herpesvirus family, for example herpes simplex virus type 1 (HSV-I), herpes simplex virus type 2 (HSV-2), varicella zoster virus (VZV), and the animal pathogens bovine herpesvirus type I (BHV-I), pseudorabies virus (PrV)₁ equine herpesvirus type I (EHY-I), and feline herpesvirus type I (FHV-I), as well as other neurotropic viruses such as rabies virus, West Nile Virus, and lyssavirus.

The alphaberpesvirus are of particular interest These viruses spread widely to produce infection of the skin, mucus membranes and nervous system, and establish a life-long latent infection of sensory nerve ganglia, from which later reactivation may occur. The alphaherpesviruses include herpes simplex virus 1 (HSV-I), herpes simplex virus 2 (HSV-2) equine herpesvirus 1 (EHV-I), EHV-3, EHV-4, bovine herpesvirus 1 (BHV-I), BHV-5, Rangiferine herpesvirus 1 (RanHV-1), Caprine herpesvirus 1 (CapHV-1), Cervid herpesvirus 1 (CerHV-1), feline herpesvirus 1 (FeHVl), pseudorabies virus (PrV), herpes simplex virus 1 (HSV-I), HSV-2, varicella zoster virus (VZV) and Asnine herpesvirus 1 (AHV-I).

Compositions and administration

In certain embodiments, the present invention provides compositions comprising a compound of the invention and a suitable carrier or excipient In one embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The compounds, for example peptides or mimics thereof, are incorporated into pharmaceutical compositions suitable for administration to a mammalian subject, e.g., a human. Such compositions typically comprise the "active" composition (e.g., the peptide or mimic derived from pUS9 or kinesin-1) and a "pharmaceutically acceptable carrier". As used hereinafter the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal), mucosal (e.g., oral, rectal, intranasal, buccal, vaginal, respiratory), enteral (e.g., orally, such as by tablets, capsules or drops, rectally) and transdermal (topical, e.g., epicutaneous, inhalational, intranasal, eyedrops, vaginal). Solutions or suspensions used for parenteral, intradermal, enteral or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor™ (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions is brought about by including hi the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound is incorporated with excipieπts and used in the form of tablets, troches, or capsules. Oral compositions are also prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as aiginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by mucosal or transdermal means. For mucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known hi the art, and include, for example, for mucosal administration, detergents, bile salts, and fusidic acid derivatives. Mucosal administration is accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds are also prepared in the form of suppositories (e.g, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydπdes, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials are also obtained commercially from, for example, Alza Corporation. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also used as pharmaceutically acceptable carriers. These are prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811 which is incorporated hereinafter by reference. It is advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used hereinafter refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

A pharmaceutically acceptable vehicle is understood to designate a compound or a combination of compounds entering into a pharmaceutical composition which does not cause side effects and which makes it possible, for example, to facilitate the administration of the active compound, to increase its life and/or its efficacy in the body, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be adapted by persons skilled in the art according to the nature and the mode of administration of the active compound chosen. Screening assays

One embodiment of the present invention relates to the use of pUS9 or kinesin-1 in a method for screening candidate compounds in vitro or in vivo for compounds that modulate the binding of pUS9 to kinesin-1 and which may be useful for inhibiting transport of a neurotropic virus in a cell.

In one embodiment, the method comprises:

(i) contacting kinesin-1 with pUS9 under conditions sufficient for pUS9 to bind to kinesin-1 to form a complex,

(ii) incubating the complex with a candidate compound, and (iii) monitoring for a reduction to binding of pUS9 to kinesin-1.

By a "candidate compound" is meant an agent to be evaluated for the ability to reduce pUS9 binding to kinesin-1. Candidate compounds may include, for example, peptides, polypeptides, mimics, mimetics, peptidomimetics, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules such as aptamers, peptide nucleic acid molecules, and components and derivatives thereof.

In certain embodiments, combinatorial libraries of potential inhibitors will be screened for an ability to bind to the protein sequence of pUS9 or kinesin-1 and modulate the ability of pUS9 to bind kjnesin-l.

Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., reducing binding, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

In one embodiment, high throughput screening methods involve providing a library containing a large number of candidate compounds. Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop etal., 1994).

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art Such combinatorial chemical libraries include, but are not limited to, peptide libraries, peptoids, encoded peptides, random bio-oϋgomers, nonpeptidal peptidomimetics, analogous organic syntheses of small compound libraries, nucleic acid libraries, peptide nucleic acid libraries, antibody libraries, carbohydrate libraries and small organic molecule libraries.

Compounds which bind to pUS9 or kinesiπ-1 may be identified and isolated by methods known to those of skill in the art Examples of methods that may be used to identify such binding compounds are the yeast-2-hybrid screening, phage display, affinity chromatography, expression cloning, Biacore systems, hnmunoprecipitation and GST pull downs coupled with mass spectroscopy.

The yeast-2-hybrid screening approach utilizes transcription activation to detect protein-protein interactions. Many transcription factors can be separated into two domains, a DNA binding domain and a transcriptional activation domain that are inactive when separated. When the two domains are brought into 'close proximity' their functional transcriptional activation activity is recreated.

The assays to identify modulators of pUS9 binding to kinesin-l may be amenable to high throughput screening. High throughput assays for the presence, absence, quantification, or other properties of particular protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins, while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/anubody binding.

In addition, high throughput screening systems are commercially available. These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detectors) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligaπd binding, and the like. Standard solid-phase ELISA assay formats are also useful for identifying antagonists of protein-protein interaction. In accordance with this embodiment, one of the binding partners, e.g pUS9, is immobilized on a solid matrix, such as, for example an array of polymeric pins or a glass support. Conveniently, the immobilized binding partner may be a fission polypeptide comprising, for example, Glutathione-S- transferase, wherein the GST moiety facilitates immobilization of the protein to the S solid phase support. The second binding partner (e.g. kinesin-1 or the KHC domain thereof) in solution is brought into physical relation with the immobilized protein to form a protein complex, which complex is then detected by methods known in the art Alternatively, Histidine-tagged protein complexes can be detected by their binding to nickel-NTA resin, or FLAG-labeled protein complexes detected by their binding to 10 FLAG M2 Affinity Gel. It will be apparent to the skilled person that the assay format described herein is amenable to high throughput screening of samples, such as, for example, using a microarray of bound peptides or fusion proteins.

EXAMPLES

Example 1. Binding of EKV-I structural proteins to kinesin-1 (KIFSB)

15 An w vitro pull-down assay was performed to test binding of HSV-I proteins to kinesin-1. The His-tag and the GST fusion constructs, were expressed, harvested and lysed as previously described (Diefenbach et al_^ 1998; 2002). Briefly, the GST-fusion tag constructs were expressed in E. coli strain BL21. Protein expression was induced with 0.1 DiM isopropyl-β-D-thiogalactoside (IPTG) for 3 h at 37⁰C except gpUS8(gE)

20 447-550 which was induced at 3O⁰C. The His-fusion tag constructs were expressed in E. coli strain BL21(DE3). Protein expression was induced with 1 mM IPTG for 3 h at 37⁰C. Conditions for addition of bacterial lysates containing GST-fusion proteins to GST-bind beads (Novagen), washing, subsequent addition of bacterial lysates containing His-tag fusion proteins before final washing and elution were essentially as

25 described previously (Diefenbach et ah, 2002). The only modification being incubation with GST-bind beads was 2 h at 4⁰C for both GST and His tagged proteins. GST- tagged proteins were then separated by 14% SDS-PAGE and immunoblotted for the presence of coeluting Ms-tag protein as previously described (Diefenbach et al., 2002; Vittoneef αf., 2005).

30 The results of the pulldown assay are shown in Figure 1. Immuπobiot with anti-

His tag antibody illustrates specific binding of His-tagged KIF5B 814-963 (heavy chain cargo-binding domain of human kinesin-1) to both GST-tagged pUS9 1-64 (cytoplasmic domain of the structural envelope protein pUS9 from HSV-I) and GST- tagged pUSl l (structural tegument protein of HSV-I). No significant interaction was observed with GST only, GST-tagged cytoplasmic domain of HSV-I gpUSS (glycoprotein E) or GST-tagged HSV-I tegument protein ptlL48.

As an additional control, His-tagged pUSl 1 did not interact significantly with any of the GST-tagged proteins except with GST-tagged pUSll. The interaction of pUS 11 with KBF5B has been previously described (Diefenbach et al. 2002) as has the self interaction of pUSl 1 (Vittone et al. 2005). In a related experiment using the same conditions as for the GST-pulldown assay above, specific binding of endogenous kinesin-1 (detected with aπti-KIF5B antibody (Niclas et al., 1994) from HeLa cell line lysates was the greatest with GST-pUS9 (Figure 2.) Coomassie staining confirmed the presence of GST-tagged proteins, eluted from GST-bind beads, in the pulldown assays

(Figure 3).

Example 2. Alignment of pUS9 from various alphaherpesvirnses

The cytoplasmic domain of pUS9 from several alphaherpesvirsuses were aligned using Lasergene version 7.0 MegAlign software from DNA star. The alignment indicates a number of highly conserved residues (Figure 4).

Example 3. Site-directed mutagenesis

To make Herpes virus mutants, the conserved residues of pUS9 are targeted for mutagenesis, initially to alanine, using a Quick change site-directed mutagensis kit (Stratagene) in the context of GST-pUS9. Example 4. Generation of nUS9 mutant virus

Residues critical for protein-protein interactions are mapped. Targeted mutagenesis of the HSV-I genome cloned as a bacterial artificial chromosome (BAC) is undertaken. This technique allows the maintenance and manipulation (such as mutagenesis) of large viral genomes (as a BAC) in Escherichia coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. In particular, mutations in pUS9 which specifically block binding of kinesiu-l, are introduced into the HSV-I BAC using galK selection (de Oliveira et al., 2008).

Example 5. Cell-based viral transport assay

Candidate compounds which reduce the binding of pUS9 to kinesin-1 are tested in an in vitro assay for their ability to inhibit the transport of neurotropic virus in a cell.

The adoption of two neuronal models - an SK-N-SH human neuroblastoma cell line, and dissociated rat dorsal root ganglionic (DRG) neurones - allows testing of peptide and small-molecule inhibitors of the KHC-pUS9 interaction for the inhibition of the anterograde transport of HSV. The SK-N-SH cell line is differentiated as previously described (Snyder et al., 2006), and is cultured and infects dissociated rat and human neurones, as has been have previously described (Miranda-Saksena et al., 2000). Comparisons of the kinetics of anterograde transport of the components of the herpes simplex virion in each biological system are examined by serial fixation, immunofluorescence and confocal microscopy. A commercial peptide carrier such as Chariot is used prior to infection with HSV-I to innoculate the minimal inhibitory pUSl 1 and KHC peptides into differentiated SK-N-SH cells or neuronal cultures.

For both SK-N-SH cells and rat/human DRGs, the efficacy of this approach is confirmed using transmission electron microscopy (TEM) and/or immuno-TEM, to determine whether viral capstds and associated tegument and not just individual viral proteins are being blocked for transport. This involves determining (i) the toxicity and specificity of peptide or small-molecule inhibitors, with trypan blue and fluorescein- dextran-amine staining to visualise live cells and neuronal projections, and (U) whether the transport of other known kinesin-I cellular cargo such as SNAP25 is impaired.

All publications discussed and/or referenced herein are incorporated herein m their entirety.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described.

The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

REFERENCES

Cunningham and Dwyer (2004) J HIV Ther, 9:9-13. de Oliveira et al (2008) J Virol, 82:4974-4990.

Diefenbach et al. (1998) Biochemistry, 41:14906-14915. Diefenbach et al. (2002) J Virol, 76:3282-3291.

Dunbrack et al. (1997) Folding and Design, 2: 27-42.

Gallop et al. (1994) J Med Chem, 37: 1233-1251.

Greber and Way (2006) Cell, 124:741-745.

Hirokawa and Takemura (2004) Exp Cell Res, 301:50-59. Jones et al (1986) Nature, 321:522-525.

Kohler and Milstein (1975) Nature, 256:495-497.

LaVail et al. (2007) Neuroscience, 146:974-985.

Look et al. (1996) Bioorg and Med Chem Letters, 6:707-712.

Miki et al. (2005) Trends Cell Biol, 15:467-476. Miranda-Sakseπa, et al. (2000) J Virol 74:1827-1839.

Munson and Pollard, (1980) Anal Biochem, 107:220.

Niclas etal. (1994) Neuron, 12:1059-1072

Phillips and Wei (1996) Tet Letters, 37:4887-4890.

Presta (1992) Curr Op Struct Biol, 2:593-59. Riechmann et al. (1988) Nature, 332:323-329.

Ruhland et al. (1996) J Amer Chem Soc, 111 :253-254.

Snyder et al (2006) J Virol, 80: 11165-11177.

Sodeik (2000) Trends Microbiol, 8:465-472.

Trauneckererα/. (199I)EMBO J, 10:3655-3659. Verhoeyen et al. (1988) Science, 239: 1534-1536.

Vittone et al. (2005) J Virol, 79:9566-9571.

Wozniak et al (2004) Traffic. 5:400-410.

Claims

CLAIMS;

1. A method of inhibiting transport of a neurotropic virus in a cell, the method comprising providing to the cell a compound which reduces binding of pUS9 to kinesin-1.

2. The method of claim 1, wherein the compound is a peptide or mimic thereof derived from pUS9 or a part of pUS9 which binds kinesin-1.

3. The method of claim 2, wherein the peptide or mimic thereof is derived from residues 1 to 64 of pUS9.

4. The method of any one of claims 1 to 3, wherein pUS9 comprises an amino acid sequence at least 80% identical to SEQ ID NO:1.

5. The method of claim 1, wherein the compound is a peptide or mimic derived from kinesin-1 or a part of kinesin-1 which binds pUS9.

6. The method of claim 5, wherein the peptide or mimic thereof is derived from residues 814 to 963 of kinesin-1.

7. The method of any one of claims 1 to 6, wherein kinesin-1 comprises an amino acid sequence at least 80% identical to SEQ ID NO:2.

8. The method of any one of claims 1 to 7, wherein the neurotropic virus is an alphaherpesvirus.

9. The method of claim 8, wherein the alphaherpesvirus is selected from herpes simplex virus, varicella-zoster virus and pseudorabies virus.

10. The method of claim 9, wherein the alphaherpesvirus is HSV- 1.

11. The method of any one of claims 2 to 10, wherein the peptide or mimic thereof is 30 residues or less in length.

12. The method of any one of claims 1 to 11, wherein the cell is a neuron and the transport is neuronal transport.

13. The method of claim 12, wherein the neuronal transport is anterograde neuronal 5 transport.

14. The method of any one of claims 1 to 13, wherein the method is performed in vivo in a subject by administering the compound to the subject

10 17. The method of any one of claims 1 to 16, wherein the cell or subject is mammalian.

18. The method of claim 17, wherein the cell or subject is human.

15 19. The method of any one of claims 1 to 18, wherein the compound is capable of reducing binding of p(JS9 to kinesin-l, wherein pUS9 comprises the amino acid sequence of SEQ ID NO:1.

20. The method of any one of claims 1 to 19, wherein the compound is capable of 20 reducing binding of ρUS9 to kinesin-1, wherein kinesin-1 comprises the amino acid sequence of SEQ ID NO:2.

21. A peptide or mimic thereof of 30 residues or less derived from pUS9 and which is capable of reducing binding of ρUS9 to kinesin-1.

25

22. The peptide or mimic thereof of claim 21, wherein pUS9 comprises an amino acid sequence at least 80% identical to SEQ ID NO:1.

23 The peptide or mimic thereof of claim 22, wherein the peptide or mimic thereof 30 is derived from residues 1 to 64 of pUS9.

24. A peptide or mimic thereof of 30 residues or less derived from kinesin-1 and which is capable of reducing binding of pUS9 to kinesin- 1.

35 25. The peptide or mimic thereof of claim 24, wherein kinesin-1 comprises an amino acid sequence at least 80% identical to SEQ ID NO:2. 26 The peptide or mimic thereof of claim 25, wherein the peptide or mimic thereof is derived from residues 814 to 963 of kinesin-1.

5 27. A pharmaceutical composition comprising the peptide or mimic thereof of any one of claims 21 to 26.

28. A method of screening for an inhibitor of neuronal transport of a neurotropic virus comprising determining whether a candidate compound reduces binding of pUS9

10 to kinesin-1.

29. The method of claim 28, -wherein the compound is a peptide or mimic of 30 residues or less which is derived from amino acid residues 1 to 64 of pUS9.

15 30. The method of claim 29, wherein the peptide or mimic thereof is derived from a polypeptide comprising a sequence at least 80% identical to SEQ ID NO: 1.

31. The method of any one of claims 28 to 30, wherein the kinesin-1 comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID

20 NO:2.

32. The method of any one of claims 28 to 31, wherein the neurotropic virus is an alphaherpesvirus.

25 33. The method of claim 32, wherein the alphaherpesvirus is selected from herpes simplex virus, varicella-zoster virus and pseudorabies virus.

34. The method of claim 33, wherein the alphaherpesvirus is HSV-I .

30 35. A method of treating or preventing infection with, or reactivation of, a neutrotropic virus in a subject, the method comprising administering to the subject a compound which reduces binding of pUS9 to kinesin-1.

36. The method of claim 35, comprising administering to the subject the peptide or 35 mimic thereof of any one of claims 21 to 26 or the pharmaceutical composition of claim 27. 37, Use of the peptide or mimic thereof of any one of claims 21 to 26 or the pharmaceutical composition of claim 27 in the manufacture of a medicament for the treatment or prevention of infection or reactivation of a neurotropic virus.

5

38. Use of the peptide or mimic thereof of any one of claims 21 to 26 or the pharmaceutical composition of claim 27 as a medicament for the treatment or prevention of infection or reactivation of a neurotropic virus.

10 39. A peptide or mimic thereof according to any one of claims 21 to 26 for the treatment or prevention of infection or reactivation of a neurotropic virus.

40. A composition according to claim 27 for the treatment or prevention of infection or reactivation of a neurotropic virus.

15

41. A peptide or mimic thereof according to any one of claims 21 to 26 for use as a medicament.

42. A composition according to claim 27 for use as a medicament 0

43. The steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.