This invention relates to a method of inhibiting the generation of infectious viral progeny of pathogenic viruses and to a method for obtaining the tools necessary to carry out the method of the invention.

A well-known means of preventing viral disease is by vaccination. In addition, other viral diseases are controlled by pharmacological agents that act against the virus. However there remains a need for other means by which to control the unwanted effects of a viral infection.

Accordingly this invention relates to a method of controlling the unwanted effects of a viral infection by inhibiting the generation of infectious viral progeny of pathogenic viruses and to the a method for determining a pharmacological inhibitor that interrupts a cellular pathway recruited by a viral nucleic acid element or its encoded viral protein as essential for the generation of infectious viral progeny.

Figure IA is a schematic representation of the replication machinery of any host cells for the multiplication of its genetic material and for the production of infectious progeny.

Figure IB is a schematic representation of the current process centering on targeting an element of the virus itself.

Figure 1C is a schematic representation of the process of the instant invention wherein viral replication is limited.

Figure 2 presents an overview of the structure of the HTV-I genome, identifies the retroviral sequences that encode the tat protein and the rev protein, and specified the relative position of the retroviral nucleotide sequences to which they bind, TAR and RRE, respectively and also shows the relative amounts of CMV- vs. HTV-encoded luciferase activities.

A virus relies on and recruits the replication machinery of any host cells for the multiplication of its genetic material and for the production of infectious progeny (Figure 1 A). Irrespective of whether a virus encodes its genetic material as DNA or RNA, this is the basic principle of any viral infection afflicting any living organism, whether unicellular or multicellular, whether vertebrate or non-vertebrate, whether plant or insect or animal or human.

Current antiviral drugs and treatment strategies, including those involving vaccination, center on targeting an element of the virus itself, generally a viral protein or enzyme or a viral nucleic acid (Figure 1 B). This classical approach, while proven to be highly practical and efficient, has inherent limitations and disadvantages. These are particularly severe in the case of viruses that can undergo rapid mutations. The selective pressure exerted by a classical antiviral drug, which targets and incapacitates an element of the virus itself, hands that virus the initiative to develop escape mutations and resistant strains, often by simple point mutation and often after the administration of the first dose of a classical antiviral drug.

This invention relates to an alternative strategy of limiting viral multiplication is to deny the invading virus the use of the infected host cell's replication machinery (Figure 1 C). The most extreme realization of this strategy consists in the altruistic suicide of infected cells, or apoptosis. To save other cells of an organism from the spread of infection by viral progeny, any cell is genetically preprogrammed to initiate and execute self-destruction once viral takeover of its replication machinery, i.e. viral infection, is evident. Viruses therefore have developed an array of anti-apoptotic proteins that keep an infected cell alive and force it to continue functioning, at the pleasure of the virus, as a virally controlled production facility for infectious viral progeny.

Since a cell's replication machinery consists of a multitude of pathways, suppression of those, or of the single one, critical for the multiplication of a given virus provides a rational alternative to the total destruction of the entire replication machinery in the apocalypse of apoptosis. In principle, suppression of a pathway critical for the multiplication of a given virus can be achieved by a small molecule that is able to interfere with and abrogate the virus / host cell interactions which recruit the infected host cell's replication machinery for viral multiplication. Such a small molecule inhibitor denies the host cell's replication machinery to the invading virus without targeting, or physically contacting, any viral macromolecule (Figure 1 C). Interacting only with cellular elements and thus 'invisible' to the invading virus, such drugs convert the internal milieu of the host cell from permissive for viral multiplication into one that is non-permissive and that does not support infectivity. Since the virus itself is never in contact with, nor the target of, an antiviral drug of this non- classical type, the selection of resistant strains and their multiplication requires the comprehensive revision of the evolutionarily established viral interaction with the cell's

replication machinery. Whereas resistance against compounds that target viral molecules and physically contact them is known to emerge rapidly, e.g. by single-point mutation in the viral genome to abolish the single-point impact of the classical antiviral agents (CAAs), such one- step genetic adaptation is ineffective in the case of non-classical antiviral agents (NCAAs). To overcome the pharmacological denial of the host cell's critical replication machinery elements, the invading virus must rewire its entire interaction pattern, or interactome, with the host cell and thus, throw out its very blueprint that had been optimized and customized through eons of evolution and billions of viral generations.

The viral recruitment of a critical path in the host cell's replication machinery involves the direct or indirect interaction of viral macromolecules, including viral nucleic acid sequences and virally encoded proteins, with nucleic acids and proteins of the host cell. These recruitment interactions are, at the ultimate molecular level, determined by specific sequences within the genome of the invading virus. For a given virus, detailed information on these specific viral nucleotide sequences, including the viral proteins they encode, and the specific cellular partner(s) they recruit for viral multiplication is, however, sparse or entirely lacking, and so is the knowledge of small molecules that can disrupt and incapacitate this recruitment process for a given virus by denying participation of the cellular molecules critical for the multiplication of this virus.

The diagram summarizing the mode-of-action of NCAAs (Figure 1 C) can, for a given virus, be put to use for therapeutic purposes. The diagram can also be employed for molecule identification in two distinct ways:

The first one consists in using a know viral nucleotide sequence, which causes viral dependence on a particular critical element of the cell's replication machinery, as a tool to identify a specific inhibitor from a library of small molecules that blocks the function of this critical cellular element and thus, leaves unmet the viral requirement for and reliance on it.

The second one consists in using a known compound, which incapacitates the virus- specific critical element of the cell's replication machinery, as a tool to identify a nucleotide sequence within the genome of the invading virus that encodes the viral requirement for and reliance on this critical element.

Accordingly, the instant invention provides a method i) to identify small molecules that interfere with and abrogate the function of specific viral nucleotide sequences essential for viral multiplication in cells by direct or indirect recruitment of host cell proteins; and ii) to

establish the existence and sequence of such viral nucleic acid segments even if their precise mode of interaction with their specific cellular protein partner, or with several partners, remains obscure.

More particularly, one embodiment of this invention relates to a method of inhibiting the generation of infectious viral progeny which comprises interrupting a cellular pathway essential for infectious progeny generation by contacting a virally infected cell with a pharmacological inhibitor of said cellular pathway, incapacitating said cellular pathway and obtaining a suppressive effect on the generation of infectious progeny. The virally infected cell occurs outside or inside a living organism. The infectious viral progeny is derived from a virus pathogenic for a living organism.

Another embodiment of the invention relates to a method of identifying viral nucleic acid elements, or their encoded viral proteins, that interrupt a cellular pathway for the generation of infectious viral progeny which method comprises contacting a pharmacological inhibitor of said pathway in an expression system with said viral nucleic acid elements or proteins; and assessing the suppressive effect on said viral nucleic acid elements, viral proteins or infectious viral progeny. The viral nucleic acid elements are derived from the genome of a virus that is pathogenic for a living organism. The viral nucleic acid elements are derived from viruses whose genome is encoded as DNA or RNA. More particularly, the viruses are lentiviridae. Li a more particular embodiment, the lentiviridae are selected from the group of HIV, SIV or FIV.

Yet another embodiment of the invention relates to a method of identifying a pharmacological inhibitor that interrupts a cellular pathway recruited by a viral nucleic acid element or its encoded viral protein as essential for the generation of infectious viral progeny which comprises contacting said viral nucleic acid element or its encoded viral protein in an expression system with an individual compound of a library of compounds and assessing the suppressive effect of said compound on said viral nucleic acid elements, viral proteins or infectious viral progeny. Particularly, the viral nucleic acid element is derived from the genome of a virus that is pathogenic for a living organism. The viral nucleic acid element is derived from viruses whose genome is encoded as DNA or RNA. In one embodiment of the method of the invention, the viruses are lentiviridae. More particularly, the lentiviridae are selected from the group of HTV, SIV or FIV.

An example of the embodiments of this invention is provided for the case of human immunodeficiency virus type 1 (HIV-I).

For its multiplication, HIV-I critically relies on two elements encoded in its genome: the tat-TAR system, to enhance the transcription into retroviral mRNAs of its own genome once it has been incorporated into the host cell; and the rev-RRE system to enhance the translation of retroviral mRNAs and the packaging into infective virions of the mRNA species that represents the HTV-I genome. Retroviral sequences encode the tat protein and the rev protein, and represent the nucleotide sequences to which they bind, TAR and RRE, respectively. When tat is bound to TAR, as well as when rev is bound to RRE, either one of them recruits and requires specific cellular partners that are indispensable for the biological function of the tat-TAR complex and the rev-RRE complex in promoting and enhancing multiplication of HTV-I and generation of infectious progeny.

In the case of the rev-RRE complex, several cellular proteins are assembled at or subsequently interacting with this complex, and are essential for its activity. Among those proteins is the eukaryotic translation initiation factor 5A (eTF5A), which exists in two isoforms, eTF5Al and eIF5A2. Each eTF5A protein molecule must be hydroxylated by the enzyme deoxyhypusine hydroxylase (DOHH) at its single deoxyhypusine residue, a lysine- derived intermediate that occurs in position X within an -GIy-X- Y-GIy- motif and is formed by the enzyme deoxyhypusine synthase (DOHS). DOHS and DOHH generate, in this sequential two-step process, the eIF5 A-specific residue hypusine that determines the function ofeIF5A.

DOHH, which catalyzes the decisive and final product-forming step, is a non-heme metalloprotein that consumes atmospheric oxygen and can be classified among the 2- oxoacid-utilizing dioxygenases. The catalytic pathway of these enzymes, including DOHH, is known at the subatomic level, and the pertinent orbital interactions and rehybridizations have been established to a degree that allows for the knowledge-guided design and the knowledge- directed synthesis of small molecule inhibitors.

If eTF5A cannot be hydroxylated, e.g. due to pharmacological inhibition of DOHS or DOHH or due to a point mutation of the lysine precursor, then eIF5A is inactive. Experimental mutations within eIF5A of HTV-infected cells are known to block retroviral multiplication. This genetically established, functionally essential interrelation of the rev- RRE complex with hypusine-containing eTF5A suggests that pharmacological inhibition of

eIF5A hydroxylation , i.e. suppression of DOHH activity in HIV-infected cells, should likewise abrogate, directly or indirectly, the biological function of the rev-RRE complex. We have established earlier that DOHH inhibitors, such as the drug deferiprone, indeed suppress HTV-I replication (United States Patent 5,849,587). However, it has not been established that this susceptibility is imparted by a topographically defined segment, and thus by a particular nucleic acid sequence, within the genomic organization of HTV-I itself.

To establish the genetic basis for this susceptibility, the DOHH inhibitors deferiprone and ciclopirox were used to assess their effect on HTV-I constructs designed to express a virally encoded reporter protein, luciferase. The effect of each compound was studied on two non-infections representative constructs. One construct, molecular clone pNL4- 3-Luc E ^" , contains the firefly luciferase as part of the viral we/ gene within an otherwise complete viral genome (except for a deletion in the env gene to render the virus non-infectious). The other construct, pLTR-Luc, contains the firefly luciferase directly next to the LTR segment with the TAR element that optimizes retroviral transcription, without any other viral nucleic acid segments (see Figure 2). 293T cells were used as expression system for both constructs. As a virological expression control, the cells were co-transfected with a commercially available cytomegalovirus (CMV)-encoded Renilla luciferase construct, pCMV-Renilla.

Only if a nucleic acid construct contains the viral nucleic acid segments that require involvement of functional eTF5A and thus of DOHH for their translation, should the expression of virally encoded luciferase be sensitive to DOHH inhibition, and in consequence become suppressed by DOHH inhibitors. In the absence of such sensitivity-imparting viral nucleic acid segments, the expression of the virally encoded luciferase should be totally insensitive to DOHH inhibition, and should proceed even in the presence of any compound that blocks DOHH activity and thus, the availability of bioactive eIF5A.

The activity of CMV-encoded luciferase (pCMV-Renilla construct) was as expected unaffected by the presence of DOHH inhibitors deferiprone and ciclopirox. This is^opnsistent with the fact that CMV does not recruit an eIF5A-involving and thus hypusine-, i.e. DOHH- dependent critical pathway of the host cell's replication machinery for expression of the proteins it encodes. This finding also indicates that in this expression system, neither DOHH inhibitor affected CMV-encoded luciferase expression in a global or non-specific manner. Subsequent results were therefore determined as the relative expression of the CMV- vs. the HTV-encoded luciferase activities.

In contrast to the molecular CMV clone, the molecular clone of HIV-I that carries the luciferase gene (pNL4- 3 -Luc E ^" construct) was sensitive to both DOHH inhibitors. However, each DOHH inhibitor failed to have any suppressive effect if luciferase expression occurred from the HIV construct that lacks all HIV sequences except for the LTR segment with the TAR element (pLTR-Luc). Thus, the suppressive activity of the DOHH inhibitors deferiprone and ciclopirox on virally encoded protein expression, though mediated via an eIF5A-involving critical pathway in the replication machinery of the cell, is categorically dependent on a specific topographically defined nucleic acid segment within the genomic organization of HIV-I itself (Figure 2). Absence of this segment, exemplified by the severely truncated version of the HIV genome in the pLTR-Luc construct, causes absence of the suppressive effect and renders the previously effective inhibitors ineffective.

The judicious integration of viral constructs expressing a reporter molecule, but engineered to have specific deletions, point mutations, insertions, etc. in their genomic sequence, with the testing of specific small molecules that target the cellular protein partners on which a particular virus relies on for its multiplication, provides the basis for high- throughput assays able to identify antiviral drug candidates (drug discovery) as well as identify the precise viral nucleotide sequences (gene discovery) to whom they deny availability of cellular partner protein(s). Knowledge of the latter, which may vary from cell type to cell type or even during the life cycle of a given cell, is not required.

The drug discovery application of this invention can be utilized for the identification of small molecules that are suitable to treat, by topical or systemic pharmacological means, infections by viruses that cause diseases and cancers in plants, animals, and man.

The gene discovery application of this invention can be utilized for the identification of nulceic acid sequences and motifs that are suitable to treat, by means of nucleic acid interference (RNAi) or nucleic acid vaccination, infections by viruses that cause diseases and cancers in plants, animals, and man.

The pharmacological inhibitors of the present invention can be used that interrupt a cellular pathway for the generation of infectious viral progeny. Such infectious viral progeny are generated by viruses that include, but are not limited to, the lentiviruses pathogenic for humans and animals, in particular the human, bovine, feline, and simian immunodeficiency viruses, the equine infectious anemia virus, the caprine arthritis-encephalitis virus, and the visna virus.

These inhibitors can be administered topically or systemically. More particularly, such administration can be orally; parenterally, i.e. by subcutaneous, intravascular, or intramuscular injection; intraperitoneally; intrathecally; or by topical application, e.g. to skin or eyes, or by application to the mucous membranes of the nose, throat, bronchial tree, or rectum, etc. They may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form such as tablets, capsules, powders, solutions, suspensions, or emulsions. The dosage of the active compound depends on the species of animal or plant, the weight, age, and mode of administration.

The pharmaceutical products of the present invention are prepared by dissolving, mixing, granulating, or tablet-coating processes which are known per se.

For oral administration, the pharmacological inhibitors, ie the active compounds or their physiologically tolerated derivatives such as salts, esters, or amides, are mixed with the additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and are converted by customary methods into a suitable form for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic, or oily suspensions, or aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders like acacia, cornstarch, gelatin, or with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant like stearic acid or magnesium stearate. Examples of suitable oily vehicles or solvents are vegetable or animal oils, such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules.

For parenteral administration (subcutaneous, intravascular, or intramuscular injection), the active compounds or their physiologically tolerated derivatives such as salts, esters, or amides, are converted into a solution, suspension, or emulsion, if desired, with the substances customary and suitable for this purpose, such as solubilizers or other auxiliaries. Examples are: sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions.

For use as aerosols, the active compounds or their physiologically tolerated derivatives such as salts, esters, or amides, may be dissolved or suspended in a physiologically acceptable liquid and packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. The agents which block intracellular hypusine formation, in accordance with the present invention, may also be administered from a non- pressurized container such as a nebulizer or atomizer.

For topical administration to external or internal body surfaces, e.g., in the form of creams, gels, or drops, etc., the active compounds or their physiologically tolerated derivatives such as, salts, esters, or amides, are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.

Materials and Methods

Deferiprone and ciclopirox were obtained from commercial suppliers and were of the highest purity available. The pCMV-Renilla construct was purchased. The HTV-I molecular pNL4- 3-Luc E- , in which part of the nef gene is replaced by the firefly luciferase gene and the env gene is mutated to abrogate viral packaging, was described by B. K. Chen et al. (Journal of Virology 68, 654-660,1994; hereby incorporated by reference) and generously provided by Dr. David Baltimore. The pLTR-Luc plasmid, which contains the firefly luciferase gene, and its expression using co-transfection with pRSV-Tat, were described by M. Hoque et al. (Molecular and Cellular Biology 23, 1688-1702, 2003; hereby incorporated by reference). Transfection in 293T cells, grown in Dulbecco's Modified Eagle's medium with 10% fetal bovine serum, and dual luciferase assays were performed as described by M. Hoque et al. (Molecular and Cellular Biology 23, 1688-1702, 2003; hereby incorporated by reference). Briefly, 293T cells were co-transfected with 500 ng pNL4-3-Luc E ^" , or with 100 ng of pLTR- Luc and 20 ng of pRSV-Tat, together with 100 ng ofpCMV-Renilla. Buffer (control), 30 μM ciclopirox, or 250 μM deferiprone were added during transfection, and cells were harvested at 12 hours post-transfection for dual luciferase assay. Results were expressed as the ratio of firefly luciferase to Renilla luciferase, normalized to the controls, ± standard deviations.

Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made by those skilled in the art without departing from the spirit and scope of the invention that is defined by the following claims.