WO2007002441A1

WO2007002441A1 - Methods of use for non-atp competitive tyrosine kinase inhibitors to treat pathogenic infection

Info

Publication number: WO2007002441A1
Application number: PCT/US2006/024539
Authority: WO
Inventors: Daniel Kalman; William Gerard Bornmann
Original assignee: Emory University
Priority date: 2005-06-24
Filing date: 2006-06-23
Publication date: 2007-01-04

Abstract

Compositions and methods are provided for using non-ATP competitive tyrosine kinase inhibitors to treat pathogenic infection. In particular, methods for using non-ATP competitive inhibitors such as amino-substituted (E)-2,6-dialkoxystyryl 4-substituted- benzylsulfones, particularly ON012380, to treat pathogenic infection are provided. Infections to be treated according to the present invention include, particularly, those caused by microbial pathogens such as bacteria and viruses.

Description

METHODS OF USE FOR NON-ATP COMPETITIVE TYROSINE KINASE INHIBITORS TO TREAT PATHOGENIC INFECTION

5 FIELD OF THE INVENTION

The invention relates to compositions and methods for using non-ATP competitive tyrosine kinase inhibitors to treat pathogenic infection associated with or caused by host-cell interactions involving tyrosine kinases. In particular, the present invention relates to the use of non-ATP competitive inhibitors of the Abl-family tyrosine 10 kinase inhibitors to treat infection from microbial pathogens such as bacteria and viruses,

BACKGROUND OF THE INVENTION

The last several decades have witnessed an onslaught of deadly pathogens around the globe. A broad array of human pathogens exists, including various microbes such as

15 bacteria, protozoa, viruses, algae, and fungi. The innate capacity to respond to selective pressures has driven the evolution of microbes and enabled them to adapt to complex and variable environments. It is perhaps no surprise, then, that infectious microbes have readily evolved mechanisms to evade our attempts to destroy them with synthetic or natural anti-microbial compounds.

20 The fact that microbes develop resistance at a rate that far exceeds development of new therapeutics arguably poses the single most serious public health threat in this century in both developing and developed nations. There is no denying that antimicrobial strategies have met with spectacular success over the last century. For example, antibacterial and antiviral drugs directed at targets within the pathogen have

25 been used to save countless lives. But it is becoming increasingly evident that such success is not sustainable. To counter these drugs, bacteria and viral pathogens have evolved sophisticated mechanisms to inactivate these compounds. Examples include the pan-drug resistant strains of Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aerginosa, and Mycobacterium tuberculosis (TB) among bacteria and

30 human immunodeficiency virus (HIV) among viruses.

More worrisome still is the lack of effort on the part of pharmaceutical companies (big or small) to pursue development of new antimicrobials. Efforts to develop new antibiotics by the pharmaceutical industry by large-scale screens of chemical libraries that inhibit growth have largely failed, and new tetracycline and sulfanilamide analogs will likely engender resistance and will quickly be rendered useless. The resistance problem is compounded further by indiscriminate and inappropriate use of antibiotics and antiviral compounds without compliance measures or public health policies to reduce disease burden. With the astounding costs of clinical trials (e.g., approximately $400M to bring new tetracyclines to the market for an expected revenue of $100M), the failure to control generic sales, and the capacity to generate substantial revenues from medications for chronic illnesses there is little if any financial incentive for big pharmaceutical companies to even develop new antibiotics, and small biotechnology companies simply do not have the resources. Even with the current level of effort there is cause for concern. Of the new drugs under development, most, if not all, will likely engender resistance quickly upon release (e.g., folate biosynthesis inhibitor Icalprim). The search for novel antiviral compounds has been somewhat more successful and largely motivated by the HIV pandemic, but drugs have been developed principally against viral targets, and mutation rates among viruses still outpaces new development. One positive development has been vaccines, which are promising for some bacterial and viral illnesses. But vaccines are not successful in all cases (e.g., in young children), and adequate resources have not been made available to implement large-scale vaccination programs.

Recently, certain tyrosine kinase inhibitors such as STI-571 (also called imatinib mesylate or Gleevec^®) have been shown to be useful in the treatment of bacterial and viral infections (see, e.g., Reeves et al. (2005) Nature Med. 11 : 731-739; PCT App. Pub. No. WO2005072826). Such tyrosine kinase inhibitors are ATP competitive, meaning that they exhibit their effects through binding to the ATP-binding sites of tyrosine kinases (Marsilje etal. (2000) Bioorg. Med. Chem. Lett. 10:477-481; Milkiewicz et al. (2000) Bioorg. Med. Chem. Lett. 10:483-486; Gumireddy et al. (2005) Proc. Nat. Acad. ScL, 102:1992-1997). However, it is known that some individuals carry mutations in the genes encoding certain tyrosine kinases that disrupt the binding of such compounds to the ATP-binding sites of tyrosine kinases (Gumireddy et al (2005) Proc. Nat. Acad. Sci, 102:1992-1997). There is therefore a need to develop new compounds and methods effective for the prevention and treatment of pathogenic infection that are independent of compounds that bind to ATP-binding sites of tyrosine kinases. SUMMARY OF THE INVENTION

Compositions and methods for treating pathogenic infection are provided. Compositions of the invention comprise compounds that are non-ATP competitive inhibitors of tyrosine kinases involved in pathogen-host cell interactions that are associated with or cause pathogenic infection. The invention relates to the use of non- ATP competitive inhibitors of Ableson (AbI) family tyrosine kinases such as amino- substituted (E)-2,6-dialkoxystyryl 4-substituted-benzylsulfones, in particular ONO 12380, or pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof. The methods of the invention comprise administering the compositions described above in therapeutically effective amounts to a patient in need thereof for treating infection by a broad array of pathogens, including microbial pathogens such as bacteria, protozoa, viruses, algae, and fungi. In particular, the invention relates to the use of these compositions to treat disease associated with bacterial and viral pathogens including pathogenic Escherichia coli (enteropathogenic Escherichia coli (EPEC), enterohemmorhagic Escherichia coli (EHEC), uropathogenic Escherichia coli (UPEC), and enteroinvasive Escherichia coli (EIEC)), Helicobacter pylori, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Mycobacterium tuberculosis (mTB), Pox viruses (including Vaccinia monkeypox and variola viruses), polyoma viruses (including JC and BK viruses), Herpes viruses, cytomegalovirus (CMV), and human immunodeficiency viruses (for example, HIV-I), and Pseudomonas aeruginosa. The compositions may be administered by any means of administration as long as a therapeutically effective amount for the treatment of pathogenic infection is delivered.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows quantification of extracellular enveloped virus (EEV) from supernatants of vaccinia (strain IHD-J)-infected BSC-40 cells or BSC-40 cells treated with 10 μM of a drug that was either an ATP competitive or a non-ATP competitive tyrosine kinase inhibitor. Drugs used were PD166326 (PD), AMN-107 (AMN), BMS- 354825 (BMS)₅ and ON012380 (ON).

Figure 2 shows a comparison of the ability of PD166326, STI-571, BMS354825, AMN107, and ON012380 to inhibit plaque formation by variola using BSC-40 monolayers. The presence of "comets" associated with the major plaques in the control cells are due to EEV released from those plaques. The non-ATP competitive inhibitor ON012380 blocked formation of EEV comets compared to controls.

Figure 3 shows a comparison of the ability of PD166326, STI-571, BMS354825, AMN107, and ON012380 to inhibit plaque formation by variola using BSC-40 monolayers that were overlayed with CMC agar to restrict formation of EEV comets. Plaque size is then used as an indicator of actin motility, which mediates spread of virus to an apposing uninfected cell. The non-ATP competitive inhibitor ONO 12380 reduced plaque size to "pinpoints" indicating that the inhibitor blocked Src and AbI family kinases associated with actin motility. Figure 4 shows a comparison of the ability of ATP competitive and non-ATP competitive tyrosine kinase inhibitors to inhibit polyoma virus (PyV) infection. Figures A and B show that infection of 3T3 cells lacking either AbI or Arg kinase produced lower levels of the viral T proteins (Large T, Middle T and Small T) compared to matched 3T3 cells derived from wild-type animals (negative and positive lanes, respectively). Figure A shows that gleevec substantially reduced early viral protein expression in 3T3 cells. Figure 4B shows that BMS-354825, AMN- 107, and ONO 12380 each reduced PyV replication, as measured by production of viral T proteins. Figure 4C shows that no viral T proteins were detectable in 3T3 cells derived from mice deficient in both AbI and Arg. Note that F4 cross-reacts with actin, the ~40 kD band running just ahead of Middle T. The band immediately below the actin band in the infected samples is a LT degradation product.

Figure 5 shows results from a fluorescence assay to detect PyV-infected cells. Primary macrophages or 3T3 cells were infected with PyV or left uninfected, and were treated with either Gleevec or ONO 12380 (10 μM) before and during infection. Figure 5 A shows that T antigen was evident in infected but not uninfected cells, effects that were observed in both primary macrophages and 3T3 fibroblast cell lines. Quantitation of these data is shown in Figure 5B and Figure 5C. To quantitate data, the number of T- antigen positive cells was scored as a fraction of the number of DAPI+ nuclei in at least 15 images, and averaged. Figure 5B shows that the production of T antigen was blocked by both Gleevec and ON012380, and Figure 5C shows that none was evident in Abl^"A /Arg^"A cells.

Figure 6 shows results from the addition of 10 μM ON0123802 hrs after PyV infection of 3T3 cells compared to control. Results demonstrate that administratin of ONO 12380 reduces the number of PyV infected cells. The minus sign on the left designates results from the control condition.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of compounds that are non-ATP competitive inhibitors of tyrosine kinases involved in pathogen-host cell interactions that are associated with or cause pathogenic infection. In particular, the present invention relates to the use of non-ATP competitive tyrosine kinase inhibitors to treat or prevent diseases associated with infection from microbial pathogens, including bacterial and viral pathogens such as Escherichia coli, Helicobacter pylori, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, Mycobacterium tuberculosis (TB), Pox viruses (including Vaccinia monkeypox and variola viruses), polyoma viruses (including JC and BK viruses), Herpes viruses, cytomegalovirus (CMV), and human immunodeficiency viruses (for example, HIV-I), and Pseudomonas aeruginosa. Particularly, non-ATP competitive tyrosine kinase inhibitors for use in the present invention include non-ATP competitive inhibitors of Ableson (AbI) family tyrosine kinases such as amino-substituted (E)-2,6-dialkoxystyryl 4-substituted-benzylsulfones, particularly ONO 12380, or pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof. The non-ATP competitive tyrosine kinase inhibitors described herein can be used in the methods of the invention to treat or prevent any pathogenic infection that is associated with or caused by tyrosine kinase-mediated host-pathogen interactions, particularly microbial infection, and more particularly viral and bacterial infection. Without being bound by theory, it is believed that the non-ATP competitive tyrosine kinase inhibitors described herein target host cells and interfere with cellular mechanisms that allow for the interaction of these host cells with pathogens and in so doing prevent the pathogenic effects caused by the pathogen. Because cellular mechanisms regulating pathogen-host interactions are remarkably conserved, it is believed that the non-ATP competitive tyrosine kinase inhibitors described herein can be applied to combat infection by a wide range of pathogens. Such pathogens include various microbes such as bacteria, protozoa, viruses, algae, and fungi. In a preferred embodiment of the present invention, the pathogens are bacteria and viruses. Advantageously, the therapeutic approach described herein targets the host, rather than the pathogen as is seen with antibiotics, and therefore decreases the likelihood of the development of pathogen drug resistance. In one embodiment, the present invention relates to the use of non-ATP competitive tyrosine kinase inhibitors to treat or prevent bacterial infections. Such infections include those caused by members of the following genera and species: Agrobacterium tumefaciens, Aquaspirillum, Bacillus, Bacteroides, Bordetella pertussis, Borrelia burgdorferi, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacteriurn diptheriae, Coxiella burnetii, Deinococcus radiodurans, Enterococcus, Escherichia, Francisella tularemsis, Geobacillus, Haemophilus influenzae, Helicobacter pylori, Lactobacillus, Listeria monocytogenes, Mycobacterium, Mycoplasma, Neisseria meningitidis, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Streptomyces coelicolor, Vibro, and Yersinia. In a preferred embodiment, such infections include those caused by Escherichia coli, Helicobacter pylori, Listeria monocytogenes, Salmonella typhimurium, Shigella flexneri, and Mycobacterium tuberculosis (TB). In an other embodiment, such infections include those caused by pathogenic and/or diarrheagenic Escherichia coli strains, including enteropathogenic Escherichia coli (EPEC), enterohemmorhagic Escherichia coli (EHEC), uropathogenic Escherichia coli (UPEC), and enteroinvasive Escherichia coli (EIEC).

In another embodiment, the present invention relates to the use of non-ATP competitive tyrosine kinase inhibitors to treat viral infections. Such infections include those caused by members of the following virus families: Adenoviridae, Arenaviridae, Astroviridae, Bacteriophages, Baculoviridae, Bunyaviridae, Calciviridae; Coronaviridae, Deltavirus, Filoviridae, Flaviviridae, Geminiviridae, Hepadnaviridae, Herpesviridae, Nodaviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Phycodnaviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, Tobamoviridae, and Toqaviridae. In a preferred embodiment, such infections include those caused by Pox viruses (including Vaccinia monkeypox and variola viruses), polyoma viruses (including JC and BK viruses), Herpes viruses, cytomegalovirus (CMV), and human immunodeficiency viruses (for example, HIV-I), and Pseudomonas aeruginosa. In one embodiment, non-ATP competitive tyrosine kinase inhibitors are administered to make vaccines more effective. For example, it is well known that immunization of neonates with live viruses does not contribute to acquired immunity because maternal antibodies neutralize the vaccine (Bot and Bona (2002) Microbes Infect. 4:511). In one embodiment, administration of a non-ATP competitive tyrosine kinase inhibitor of the present invention allows for safe administration of higher doses of virus to overcome antibody response and permit acquisition of cellular immunity. In another embodiment, non-ATP competitive tyrosine kinase inhibitors of the present invention facilitate immune clearance of the pathogen. For some chronic viruses (e.g., HIV and polyoma), high viral loads have been found to compromise T cell function (Welsh (2001) J. Exp. Med. 193:F19). Thus, lowering the viral burden could permit recovery of T cell function and thereby facilitate clearance. In another embodiment, non-ATP competitive tyrosine kinase inhibitors of the present invention permit immunocompromised individuals to be vaccinated.

The non-ATP competitive tyrosine kinase inhibitors of the present invention are for administration in a living subject or patient, including a human being or an animal such as a laboratory monkey or mouse.

Non-ATP Competitive Tyrosine Kinase Inhibitor Compounds for Use in the Methods of the Invention

Protein kinases are enzymes that modify other proteins via a phosphorylation step that removes a phosphate group from ATP and covalently attaches it to one of three amino acids that have a free hydroxyl group. Protein kinases may be roughly divided into those that phosphorylate tyrosine residues (tyrosine kinases), those that phosphorylate serine and threonine residues (serine/threonine kinases), and those that phosphorylate all three. In addition, a variety of subclasses of protein kinases are known. For example, a number of families of tyrosine kinases are currently recognized, including AbI, Fes/Fer, Syk/Zap70, Jak, Tec, Fak, Ack, Src, and Csk (see, e.g., Neet & Hunter (1996) Genes Cells 1:147-169; Robinson et al. (2000) Oncogene 19:5548-5557).

Certain inhibitors of tyrosine kinases such as STI-571 (also called imatinib mesylate or Gleevec^®) have recently been shown to be useful in the treatment of bacterial and viral infections (see, e.g., Reeves et al. (2005) Nature Med. 11:731-739; PCT App. Pub. No. WO2005072826). Such tyrosine kinase inhibitors are ATP competitive, meaning that they exhibit their effects through binding to the ATP -binding sites of tyrosine kinases (Marsilje et al. (2000) Bioorg. Med. Chem. Lett. 10:477-481; Milkiewicz et al. (2000) Bioorg. Med. Chem. Lett. 10:483-486; Gumireddy et al. (2005) Proc. Nat. Acad. ScL, 102:1992-1997). However, it is known that some individuals carry mutations in the genes encoding certain tyrosine kinases that disrupt the binding of such compounds to the ATP-binding sites of tyrosine kinases (Gumireddy et al. (2005) Proc. Nat. Acad. ScL, 102:1992-1997). For example, in cancer patients treated with STI-571, some patients exhibit or develop resistance to treatment due to mutations in the oncogene encoding the BCR-ABL tyrosine kinase that introduce amino acid substitutions which interfere with the ability of STI-571 to bind to the BCR-AbI protein (Gumireddy et al. (2005) Proc. Nat. Acad. ScL, 102:1992-1997).

The present invention is directed to methods for preventing or treating pathogenic infection through the administration of inhibitory compounds that target activity of protein kinases in a non-ATP competitive manner. Such compounds do not compete with ATP to inhibit the tyrosine kinase but do compete with its substrates. Non-ATP competitive tyrosine kinase inhibitors are particularly useful in the treatment of individuals resistant to treatment with such ATP competitive tyrosine kinase inhibitors as STI-571. Compounds that may be used in the methods of the present invention include non-ATP competitive inhibitors of several tyrosine kinase families, including, but not limited to, members of the AbI and Src families of tyrosine kinases. Thus, in one embodiment of the present invention, a non-ATP competitive tyrosine kinase inhibitor is used to treat pathogenic infection that inhibits at least members of the AbI family of tyrosine kinases, including c-Abl and c-Arg. It is recognized, however, that compounds which cross-react with multiple protein kinases (such as both AbI and Src tyrosine kinases) or compounds that bind to protein kinases other than AbI and Src tyrosine kinases may be used in the methods of the present invention.

In one embodiment of the present invention, a method for preventing or treating a bacterial infection or a viral infection is provided comprising administering a therapeutically effective amount of the non-ATP competitive tyrosine kinase inhibitor ON012380, having the following structure:

ONO 12380 has been shown to inhibit the BCR-AbI tyrosine kinase and is known to inhibit both Abl-family and Src-family tyrosine kinases (Gumireddy et al. (2005) Proc. Nat. Acad. ScL, 102:1992-1997). ON012380 is greater than 10 fold more potent than the ATP competitive tyrosine kinase inhibitor STI-571 and exhibits low in vivo toxicity, with mice able to tolerate doses of approximately 300 mg/kg. Thus in one embodiment of the present invention, a method for preventing or treating a bacterial infection or a viral infection is provided comprising administering a therapeutically effective amount of a non-ATP competitive inhibitor of the BCR-AbI tyrosine kinase, where the non-ATP competitive inhibitor of the BCR-AbI tyrosine kinase is ON012380. In another embodiment, a method for preventing or treating a bacterial infection or a viral infection is provided comprising administering a therapeutically effective amount of a non-ATP competitive inhibitor of the a member of either the Abl-family or Src-family of tyrosine kinases, where the non-ATP competitive inhibitor is ON012380.

Other non-ATP competitive inhibitors of the BCR-AbI tyrosine kinase include amino-substituted (E)-2,6-dialkoxystyryl 4-substituted-benzylsulfones. Accordingly, in another embodiment of the present invention, a method for preventing or treating a bacterial infection or a viral infection is provided, comprising administering a therapeutically effective amount of an amino-substituted (E)-2,6-dialkoxystyryl 4- substituted-benzylsulfone. Amino-substituted (E)-2,6-dialkoxystyryl 4-substituted- benzylsulfones for use in the present application include, for example, compounds as disclosed in PCT Patent App. Publication No. WO 03/072062 and U.S. Patent App. Pub. No. 20050130942, herein incorporated by reference in their entireties. Specifically, the present invention encompasses compounds of Formula I

wherein: X is selected from the group consisting of (i) and (ii) below :

X₁ is selected from the group consisting of (i), (ii) and (iii) below:

wherein Xi is optionally protected with one or more chemical protecting groups; g is 0 or 1; each M is a bivalent connecting group independently selected from the group consisting of -(C-C₆) alkylene-, -(CH₂)_a-V-(CH₂)_b-, -(CH₂)d-W-(CH₂)_e-, and -Z-; each y is independently selected from the group consisting of 0 and 1; each V is independently selected from the group consisting of arylene, heteroarylene, -C(=O)-, -C(=S)-, -S(=O)-, -SO₂-, -C(=O))-; -C(=O)(Ci- C₆)perfluoroalkylene-, -C(=O)NR₄-, -C(=S)NR₄-, and-SO₂NR₄-; each W is independently selected from the group consisting Of -NR₄-, -O-, and -S-

each a is independently selected from the group consisting of O, 1, 2 and 3; each b is independently selected from the group consisting of 0, 1, 2 and 3; each d is independently selected from the group consisting of 1, 2 and 3; each e is independently selected from the group consisting of 0, 1, 2 and 3;

wherein the absolute stereochemistry of -Z- is D or L or a mixture of D and L; each R_a is independently selected from the group consisting of -H, -(C₁- C₆)alkyl, -(CH₂)₃-NH-C(NH₂)(=NH), -CH₂C(=O)NH₂, -CH₂COOH, -CH₂SH, -(CH₂)₂C(=O)-NH₂, -(CH₂)₂COOH, -CH₂-(2-imidazolyl), -CH(CH₃)-CH₂-CH₃, -CH₂CH(CH₃)₂, -(CH₂)₄-NH₂,

-(CH₂)₂-S-CH₃, phenyl, CH₂-phenyl,-CH₂-OH, -CH(OH)-CH₃,-CH₂-(3-indolyl), -CH₂-(4- hydroxyphenyl), -CH(CH₃)₂, and -CH₂-CH₃; and includes compounds wherein R_a and Ri combine to form a 5-, 6-, or 7-membered heterocyclic ring; each Ri is independently selected from the group consisting of -H, unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₅, - C(=O)N(R₄)₂, -CR₄R₆R₇, -C(=NH)-N(R₄)₂, -(C₁ -C₆)perfluoroalkyl, -CF₂Cl, - P(=O)(OR₄)₂, -OP(=O)(OR₄)₂ and a monovalent peptidyl moiety with a molecular weight of less than 1000; provided that when y is 0 and R₁ is -CO₂R₅, R₅ is not -H; each R₂ is independently selected from the group consisting of -H, -(C₁ -C₆) alkyl, and aryl(Ci-C₃)alkyl, wherein -R₂ and -(M)y-Ri may optionally be linked covalently to form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle; each R₃ is independently selected from -(Ci-C6)alkyl; each R₄ is independently selected from the group consisting of -H, and -(C₁ -C₆) alkyl; wherein: when R₄ and R] are bonded to the same nitrogen atom, Ri and R₄ may combine to form a heterocycle; and when two R₄ groups are geminally bonded to the same nitrogen, the two R₄ groups may combine to form a heterocycle; each R₅ is independently selected from the group consisting of -H, -(C₁ -C₆)alkyl and -(C₁ -C₆)acyl; each R₆ is independently selected from the group consisting of -H, -(C₁ -C₆)alkyl, -CO₂R₅, -C(=O)R₇, -OR₅, -OC(=O)(CH₂)₂CO₂R₅, -SR₄, guanidino, -N(R₄)₂,-N⁺(R₄)₃, -N⁺ (CH₂CH₂OH)₃, phenyl, substituted phenyl, heterocyclic, substituted heterocyclic and halogen; each R₇ is independently selected from the group consisting of -H, -R_a, halogen, -(C₁ -C₆)alkyl, -N(R₄)₂ and heterocycles containing two nitrogen atoms; and

Q is selected from the group consisting of -H, -(C₁ -C₆)alkoxy, halogen, -(Ci- C6)alkyl and -N(R₄)₂; wherein the substituents for the substituted aryl and substituted heterocyclic groups comprising or included within Ri, R_a, R₂, R₆, and R₇, are independently selected from the group consisting of halogen, (Ci-C₆)alkyl, (Ci- C₆)alkoxy, -NO₂, -C≡N, -CO₂R₅, -C(=O)O(C,-C₃)alkyl, -OR₅, -(C₂-C₆)-OH, phosphonato, -N(R₄);,, -NHC(=O)(Ci-C₆)alkyl, sulfamyl, -OC(=O)(C,-C₃)alkyl, -0(C₂- C₆)-N((C,-C₆)alkyl)₂, and -CF₃; provided

(1) when Ri is a monovalent peptidyl moiety of molecular weight less than 1000 and V is -C (=O)-, -C(=S)-, -S(=O)-, or -SO₂-, and b is 0; then said peptidyl moiety is coupled to M through the amino terminus of the peptidyl moiety or through a side chain amino group to form an amide, thioamide, sulfonamide, or sulfonamide respectively;

(2) when R₁ is a monovalent peptidyl moiety of molecular weight less than 1000 and V is -C(=O)NR₃-, -SO₂NR₃-, or -NR₄-, and b is 0, then said peptidyl moiety is coupled to M through the carboxy terminus of the peptidyl moiety or through a sidechain carboxyl group to form an imide, sulfonimide, or carboxamide respectively; and (3) when Ri is a monovalent peptidyl moiety of molecular weight less than 1000 and W is -S- or -O-, and d is 0, then said peptidyl moiety is coupled to M through the carboxy terminus of the peptidyl moiety or through a sidechain carboxyl group to form a carbothioic acid ester or the carboxylic ester respectively; or a salt of such a compound.

In another embodiment, the present invention encompasses compounds of Formula I wherein: each V is independently selected from the group consisting of -C(=O)-, - C(=S)-, -S(=O)-, -SO₂-; -C(=O)NR₄-, -C(=S)NR₄-, and -SO₂NR₄-;

wherein the absolute stereochemistry of -Z- is either D or L each R₃ is independently selected from the group consisting of -H, -CH₃, -(CH₂)₃-

NH-C(NH₂)C=NH), -CH₂CC=O)NH₂, -CH₂COOH, -CH₂SH, -(CH₂)₂C(=O)-NH₂, - (CH₂)₂COOH, -CH₂-(2-imidazolyl), -CH(CH₃)-CH₂-CH₃, -CH₂CH(CH₃)₂, -(CH₂)₄-NH₂, - (CH₂)2-S-CH₃, phenyl, CH₂-phenyl, -CH₂-OH, -CH(OH)-CH₃, -CH₂-(3-indolyl), -CH₂- (4-hydroxyphenyl), -CH(CH₃)₂, and- CH₂-CH₃; and includes compounds wherein R₃ and Ri combine to form a 5-, 6-, or 7-membered heterocyclic ring; each R] is independently selected from the group consisting of -H, unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₅, - C(=O)N(R₄)₂, -CHR₆R₇, -C(=NH)-N(Rt)₂, and a monovalentpeptidyl moiety with a molecular weight of less than 1000; provided that when y is 0 and Ri is -CO₂R₅, R₅ is not -H; each R₆ is independently selected from the group consisting of -H, -(C₁ -C₆)alkyl, -CO₂R₅, -C(=O)R₇, -OH, -SR₄, -(Ci-C₃)alkoxy, -(Ci-C₃)alkylthio, guanidino, -N(R₄)₂, phenyl, substituted phenyl, heterocyclic, substituted heterocyclic and halogen; and each R₇ is independently selected from the group consisting of -H, halogen,-(C₁ -C₆)alkyl, N(R₄)₂, and heterocycles containing two nitrogen atoms; wherein the substituents for the substituted aryl and substituted heterocyclic groups comprising or included within R₁, R_a, R₂, R₆, and R₇, are independently selected from the group consisting of halogen, (C₁-C₆)alkyl, (Ci-C6)alkoxy, -NO₂, -C=N, CO₂R₅, -C(=O)O(Ci-C₃)alkyl, -OH, -(C₂-C₆)-OH, phosphonato, -N(R₄)₂, -NHC (=O)(C₁- C₆)alkyl, sulfamyl, -OC(=O) (C,-C₃)alkyl, -O(C₂-C₆)-N((Ci-C₆)alkyl)₂, and -CF₃.

In another embodiment, the present invention encompasses compounds of Formula I, wherein each V is independently selected from the group consisting of

In another embodiment, the present invention encompasses compounds of

Formula I, wherein each V is independently selected from the group consisting of

In another embodiment, the present invention encompasses compounds of Formula I, wherein Z has an L absolute configuration.

In another embodiment, the present invention encompasses compounds of Formula I and salts thereof that include:

(E)-2,4,6-trimethoxystyryl-3-[4-(4-methylpiperazin-l-yl]benzamido)-4-methoxy- benzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(acetoxyacetamido)-4-methoxy-benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(triethylammoniumacetamido)-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-[tri- (2-hydroxyethylammonium)acetamido]-4- methoxy-benzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(2-methyl-2-hydroxypropionamido)-4- methoxybenzyl-sulfone; (E)-2,4,6-trimethoxystyryl-3-(2-metliyl-2-acetoxypropionamido)-4- methoxybenzyl-sulfone;

(E)-2,4,6-trimethoxystyryl-3-(2-acetoxypropionamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(trifluoroacetamido)-4-methoxybenzylsulfone; (E)-2,4,6-trimethoxys1yryl-3-(trifluoromethanesulfonamido)-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-[3-(3-carboxypropanoyloxy)acetamido]-4-methoxy- benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(phosphonatoacetamido)-4-methoxybenzylsulfone, disodium salt;

(E)-2,4,6-trimethoxystyryl-3-(methylcarbamoyl)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(2,2-difluoromalonamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(pentafluoropropionamido)-4- methoxybenzylsulfone; (E)-2,4,6-trimethoxystyryl-3 -(methyl-2,2-difluoromalonamido-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-(2,2-difluoromalonamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(dimethylamino-α, α-difluoroacetamido)-4-methoxy- benzylsulfone; and (E)-2,4,6-trimethoxystyryl-3-(2, 2,3, 3, tetrafluorosuccinamido)-4- methoxybenzyl-sulfone.

In another embodiment, the present invention encompasses compounds of Formula I, wherein

X is

and y is 0; and

R₂ is -H.

In a sub-embodiment thereof, the present invention encompasses compounds of Formula II

wherein: g is 0 or 1; each R₂ is independently selected from the group consisting of -H, -(Ci-C6)alkyl, and aryl(Ci-C₃)alkyl, wherein -R₂ and -(M)_y-Ri may optionally be linked covalently to form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle; each R₃ is independently selected from -(C₁ -C₆)alkyl; each R₄ is independently selected from the group consisting of -H, and -(Q- C₆)alkyl;

Q is selected from the group consisting of -H, -(C₁ -C₆)alkoxy, halogen, -(Ci- C₆)alkyl, and -N(R₄)₂; and

Xi is selected from the group consisting of (i), (ii) and (iii) below:

wherein Xi is optionally protected with one or more chemical protecting groups;

Suitable protecting groups will be stable to reactions designed to derivatize the 3- amino group of Formula II. Subsequently, said protecting groups are optionally removed to regenerate the Xi.

In another sub-embodiment, the present invention encompasses compounds of Formula Ha

wherein X₂ is selected from the group consisting of -NO₂ and -NH₂, wherein said -NH₂ is optionally protected with a chemical protecting group.

In another sub-embodiment, the present invention encompasses compounds of Formula Ha wherein Q is -(C₁ -C₆)alkoxy.

In another sub-embodiment, the present invention encompasses compounds of Formula Ha wherein Q is -OCH₃.

In another sub-embodiment, the present invention encompasses compounds of Formula Ha wherein R₃ is -CH₃. One such compound is (E)-2,4,6-trimethoxystyryl-4- methoxy-3-amino-benzylsulfone.

In yet another embodiment, the present invention encompasses compounds of Formula I wherein X is;

and R₂ is -H, y is 0; and

Ri is selected from the group consisting of unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₃; -C(=O)N(R₄)₂,-CHR6R7, -C (=NH)-N(Rt)₂ and a monovalent peptidyl moiety with a molecular weight of less than 1000.

In yet another embodiment, the present invention encompasses compounds of Formula I wherein X is:

In a sub-embodiment thereof, the present invention encompasses compounds of Formula III

In another embodiment, the present invention encompasses compounds of Formula III and salts thereof that include:

(E)-2,4,6-trimethoxystyryl-3-(carboxyacetamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(3, 5-dinitrobenzamido)-4-methoxybenzyl-sulfone;

(E)-2,4₎6-trimethoxystyryl-3-(3,5-diaminobenzasmido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(chloroacetamido)-4-methoxybenzylsulfone_;

(E)-2, 4,6-trimethoxystyryl-3-[ (4-methylpiperazinyl) acetamido]-4- methoxybenzylsulfone_;

(E)-2,4,6-trimethoxystyryl-3-(beri2;amido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(4-nitrobenzamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(4-aminobenzamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(acetamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(dimethylaminoacetamido)-4-methoxybenzylsulfone_;

(E)-2,4,6-trimethoxystyryl-3-(hydroxyacetamido)-4-methoxy-benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(2-hydroxypropionamido)-4-methoxy- benzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(pyridinium-l-yl) acetamido-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-(ethylmalonamido)-4-methoxy-benzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(glutaramido)-4-methoxybenzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(methylsuccinamido)-4-methoxybenzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(succinamido)-4-methoxybenzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(3-chlorosuccinamido)-4-methoxybenzylsulfone; and (E)-2,4,6-trimethoxystyryl-3-(aminoacetamido)-4-methoxyben2ylsulfone. In yet another embodiment, the present invention encompasses compounds of Formula I wherein X is:

and y is 1 ; and M is -Z-. In a sub-embodiment thereof, the present invention encompasses compounds of

Formula IV and salts thereof

wherein: each R_a is independently selected from the group consisting of -H, -CH3, -(CH₂)₃- NH-C(NH₂)(=NH), -CH₂C(=O)NH₂,-CH₂COOH, -CH₂SH, -(CH₂)₂C(=O)-NH₂, - (CH₂)₂COOH, -CH₂-(2-imidazolyl), -CH(CH₃)-CH₂-CH₃, -CH₂CH(CH₃)₂, -(CH₂)₄-NH₂,- (CH₂)₂-S-CH₃, phenyl, CH₂-phenyl, -CH₂-OH, -CH(OH)-CH₃, -CH₂-(3-indolyl), -CH₂- (4-hydroxyphenyl), -CH(CH₃)₂, and -CH₂-CH₃; and includes compounds wherein R₃ and Ri combine to form a 5-, 6~, or 7-membered heterocyclic ring;

Heterocyclic rings formed by the combination of R₅ and R₁ include, for example, pyrrolidine, hydroxypyrrolidine, piperidine, homopiperidine and thiazolidine.

In another embodiment, the present invention encompasses compounds of Formula IV and salts thereof that include:

(E)-2,4,6-trimethoxystyryl-3-amino-4-methoxybenzylsulfone-L-lysineamide;

(E)-2,4,6-trimethoxystyryl-3-amino-4-methoxybenzylsulfone-L-serineamide; and

(E)-2,4,6-trimethoxystyryl-3-amino-4-methoxybenzylsulfone-D-serineamide.

and y is 1; and M is -(CH₂)_a-V-(CH₂)_b-; and V is -SO₂-.

In a sub-embodiment thereof, the present invention encompasses compounds of Formula V and salts thereof

In another embodiment, the present invention encompasses compounds of Formula V and salts thereof that include:

(E)-2,4,6-trimethoxystyryl-3-carboxymethylsulfamyl-4-methoxy- benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(4-methoxybenzenesulfamyl)-4-methoxy- benzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(2, 4-dinitτobenzenesulfamyl)-4-methoxy- benzylsulfone; and

(E)-2,4,6-trimethoxystyryl-3-(2,4-diamdinobenzenesulfamyl)-4-methoxy- benzylsulfone.

In yet another embodiment, the present invention encompasses compounds of

Formula I wherein X is:

and y is 0; and Ri is -C(=NH)-N(R₄)₂. In a sub-embodiment thereof, the present invention encompasses compounds of

Formula VI and salts thereof

One such compound is (E)-2,4,6-trimethoxystyryl-3-guanidino-4-methoxy- benzylsulfone, or a salt thereof. In yet another embodiment, the present invention encompasses compounds of

Formula I wherein X is:

and y is 1; and M is -(C₁ -C₆)alkylene-. In a sub-embodiment thereof, the present invention encompasses compounds of

Formula VII and salts thereof

Exemplary compounds of Formula VII include, for example:

(E)-2,4,6-trimethoxystyryl-3-(N-methylamino)-4-methoxybenzylsulfone; racemic-(E)-2,4,6-trimethoxystyryl-3-(l-carboxyethyl) amino-4-methoxy- ben2ylsulfone;

D-(E)-2,4,6-trimethoxystyryl-3-(l-carboxyethyl)amino-4-methoxybenzyl- sulfone_;

L-(E)-2,4,6-trimethoxystyryl-3 -( 1 -carboxyethyl)amino-4-methoxybenzyl-sulfone; and

(E)-2,4,6-trimethoxy-styryl-3-(carboxymethylamino)-4-methoxybenzyI-sulfone and salts thereof.

In another embodiment, the present invention encompasses compounds of Formula VIII and salts thereof

One such compound is (E)-2,4,6-trimethoxystyryl-3-(4-nitrophenylimino)-4 methoxybenzylsulfone or a salt thereof.

and y is 1; and M is -(CH₂)_a-V-(CH₂)_b-; and V is -C(=O)NR₄-. In a sub-embodiment thereof, the present invention encompasses compounds of Formula IX and salts thereof

An exemplary compound of Formula IX is (E)-2,4,6-trimethoxystyryl~3-ureido-4- methoxybenzylsulfone, or a salt thereof.

In another embodiment, the present invention encompasses compounds of Formula X and salts thereof

wherein: g is 0 or 1; each R₃ is independently selected from -(C₁-C₆)alkyl; each R₄ is independently selected from the group consisting of -H, and -(C ₁-C₆) alkyl;

Xi is selected from the group consisting of (i), (ii), and (iii) below:

g groups; tize the 3- y removed

ounds of

ionally

thoxy-3-

nds of

(i) and y is 0; Ri is -CHR₆R₇; R₆ is CO₂R₅ and R₇ is R_a;

In a sub-embodiment thereof, the present invention encompasses compounds of Formula XI and salts thereof

Exemplary compounds of Formula XI are (E)-2,4,6-trimethoxystyryϊ-3-(l- carboxyethyl)amino-4-methoxybenzylsulfone; and (E)-2,4,6-trimethoxystyryl-3 - carboxymethylamino-4-methoxybenzylsulfone; or salts thereof.

Preferred compounds are the sodium and potassium salts of (E)-2,4,6- trimethoxystyiyl-3-carboxymethylamino-4-methoxybenzylsulfone, particularly the sodium salt.

and y is 1; and M is -(C₁ -C₆)alkylene-;

In a sub-embodiment thereof, the present invention encompasses compounds of Formula XII and salts thereof

Exemplary compounds of Formula XII are:

(E)-2,4,6-trimethoxystyryl-3-(3-carboxypropylamino)-4-methoxybenzylsulfone; (E)-2,4,6-trimethoxystyryl-3-(2-carboxyethylamino)-4-methoxybenzyl-sulfone; or a salt of such a compound.

In another embodiment, the present invention encompasses compounds as described in Table 1.

Additional non-ATP competitive tyrosine kinase inhibitors for use within the methods of the present invention include hydroxynapthalene derivatives as disclosed and described in Marsilje et al. (2000) Bioorg. Med. Chem. Lett. 10:477-481 as well as hydroxyindole derivatives as disclosed and described in Milkiewicz et al. (2000) Bioorg. Med. Chem. Lett. 10:483-486. Assays and methods for the identification of further non- ATP competitive tyrosine kinase inhibitors for use within the methods of the present invention are known in the art (as disclosed and described in, for example, Marsilje et al. (2000) Bioorg. Med. Chem. Lett. 10:477-481; Milkiewicz et al. (2000) Bioorg. Med. Chem. Lett. 10:483-486; and Gumireddy et al. (2005) Proc. Nat. Acad. Sci, 102:1992- 1997). It is to be understood that the present invention encompasses the use not only of the specific compounds described above, but also any pharmaceutically acceptable salts, enantiomers, analogs, esters, amides, prodrugs, metabolites, or derivatives thereof.

Combination Therapies

In accordance with the methods of the present invention, the non-ATP competitive tyrosine kinase inhibitors described herein may be administered in combination with one another, for example, administering ONO 12380 with another amino-substituted (E)-2,6- dialkoxystyryl 4-substituted-benzylsulfone, or with other compounds, particularly antipathogenic compounds. Such antipathogenic compounds include conventional antimicrobials. In other embodiments, one or more of the non-ATP competitive tyrosine kinase inhibitors described herein can be used in combination with other compounds such as cidofovir, for example, in cases related to smallpox, wherein the combination of these agents would provide for lower dosages of cidofovir to be administered, thereby decreasing the toxicity effects of this nucleoside analogue antiviral compound. Where the non-ATP competitive tyrosine kinase inhibitors of the present invention are administered as part of a combination therapy to treat or prevent pathogenic infection, they may be administered concurrently or sequentially, in either order, with the additional compound(s). In one embodiment, the non-ATP competitive tyrosine kinase inhibitors described herein may be administered in combination with ATP competitive tyrosine kinase inhibitors. Without being bound by theory, combined administration of non-ATP and ATP competitive tyrosine kinase inhibitors provides superior results in the treatment or prevention of pathogenic infection compared to administration of either type of tyrosine kinase inhibitor alone due to their different mechanisms of action. Such combination of ATP and non-ATP competitive tyrosine kinase inhibitors also allow lower dosages of each agent to be administered, thereby decreasing any undesireable side effects associated with individual agents.

Thus, in one embodiment, a method for preventing or treating a bacterial infection or a viral infection is provided, comprising administering to a subject in need thereof a therapeutically effective amount of a non-ATP competitive tyrosine kinase inhibitor such as ONO 12380 in combination with a therapeutically effective amount of an ATP competitive tyrosine kinase inhibitor selected from the group consisting of: a) STI-571 (also called imatinib mesylate or Gleevec^®), designated chemically as 4-[(4-Methyl-l-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2- pyrimidinyl]amino]-phenyl]benzamide methanesulfonate, which has the following structure;

b) STI-X (a benzylated derivative of imatinib mesylate), which has the following structure;

c) N-phenyl-2-pyrimidine-amine derivatives as generally and specifically disclosed in U.S. Patent No. 5,521,184, herein incorporated by reference in its entirety, including, for example, N-phenyl-2-pyrimidine-amine derivatives of the following structural formula

wherein:

Ri is 4-pyrazinyl, 1 -methyl- lH-pyrrolyl, amino-, or amino-lower alkyl-substituted phenyl wherein the amino group in each case is free, alkylated, or acylated, lH-indolyl or IH- imidazolyl bonded at a five-membered ring carbon atom, or unsubstituted or lower alkyl- substituted pyridyl bonded at a ring carbon atom and unsubstituted or substituted at the nitrogen atom by oxygen, R₂, R₃, R₉, X, Y, n and Ri₀ are as defined in claim 1 therein; d) Pyrido[2,3-d]pyrimidines as described in Kraker et al. (2000) Biochem. Pharmacol. 60:885-898 and synthesized using methods adopted from Klutchko et al. (1998) J. Med. Chem. 41:3276-3292 and Boschelli et al. (1998) J. Med. Chem. 41:4365- 4377, herein incorporated by reference in their entireties, including, for example, compounds represented by the following structural formula;

e) BMS-354825, also called [N-(2-chloro-6-methylphenyl)-2-(6-(4-(2- hydroxyethyl)piperazin-l-yl)-2-methylpryimidin-4-ylamino)thiazole-5-carboxamide and which has the following structure;

f) cyclic compounds as generally and specifically disclosed in U.S. Patent Application No. 20040054186, herein incorporated by reference in its entirety, including, for example, compounds represented by the following structural formula;

wherein the individual substituents Q, Z, X], X₂, R₁, R₂, R₃, R₄, and R₅ have the meaning as set forth and explained therein; g) AMNl 07, also called N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2- hydroxyethyl)- 1 -piperazinyl] -2-methyl-4-pyrimidinyl] amino] -5 -thiazolecarboxamide and which has the following structure; and

h) Substituted pyrimidinylaminobenzamides as disclosed in PCT Publication No. WO 04/005281, herein incorporated by reference in its entirety, including, for example, compounds represented by the following structural formula

wherein the individual substituents R₁, R₂, and R₄ have the meaning as set forth and explained therein.

Pharmaceutical Compositions Because many tyrosine kinase inhibitors are already the subject of drug development or are in use to treat certain cancers, data has established that they are well tolerated in humans even for extended periods (months), and are not toxic. Such drugs can be ingested orally, are stable at room temperature, and are simple and inexpensive to manufacture. In one embodiment of the present invention, a method of treating or preventing pathogenic infection, particularly microbial infection, comprises administering to a living subject in need of such treatment an effective amount of a pharmaceutical composition suitable for administration to the living subject where the pharmaceutical composition comprises: (a) at least one non-ATP competitive tyrosine kinase inhibitor in an amount effective for augmenting an inhibitable response from a host cell of the living subject responsive to at least one pathogen, particularly a microbe; and (b) a pharmaceutically acceptable carrier suitable for administration to the living subject.

In another embodiment, the present invention also relates to pharmaceutical compositions suitable for administration to a living subject, comprising: (a) at least one non-ATP competitive tyrosine kinase inhibitor in an amount effective for augmenting an inhibitable response from a host cell of the living subject responsive to at least one bacteria; and (b) a pharmaceutically acceptable carrier suitable for administration to a living subject.

In another embodiment, the present invention also relates to pharmaceutical compositions suitable for administration to a living subject, comprising: (a) at least one non-ATP competitive tyrosine kinase inhibitor in an amount effective for augmenting an inhibitable response from a host cell of the living subject responsive to at least one virus; and (b) a pharmaceutically acceptable carrier suitable for administration to a living subject. The pharmaceutically acceptable carrier can be suitable for oral administration to the living subject, and the pharmaceutical composition is administered to the living subject orally. The pharmaceutically acceptable carrier can also be suitable for nasal administration to the living subject, and the pharmaceutical composition is administrated to the living subject nasally. Or the pharmaceutically acceptable carrier is suitable for rectal administration to the living subject, and the pharmaceutical composition is administrated to the living subject rectally. Moreover, the pharmaceutically acceptable carrier can be suitable for intravenous administration to the living subject, and the pharmaceutical composition is administrated to the living subject intravenously. Furthermore, the pharmaceutically acceptable carrier can be suitable for inoculative administration to the living subject, and the pharmaceutical composition is administrated to the living subject inoculatively. Additionally, the pharmaceutically acceptable carrier can be suitable for hypodermic administration to the living subject, and the pharmaceutical composition is administrated to the living subject hypodermically. Thus, depending upon the pathogenic infection to be treated or prevented, the pharmaceutical composition comprising a non-ATP competitive tyrosine kinase inhibitor described herein can be administered by any suitable route, including, but not limited to, orally, nasally, buccally, sublingually, intravenously, transmucosally, rectally, topically, transdermally, subcutaneously, by inhalation, or intrathecally. In particular, in another embodiment, these pharmaceutical compositions may be in the form of orally administrable suspensions, drinking solutions, or tablets; nasal sprays or nasal drops; or olegenous suspensions or suppositories.

When administered orally as a suspension, compositions of the present invention are prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. Components in the formulation of a mouthwash or rinse include antimicrobials, surfactants, cosurfactants, oils, water and other additives such as sweeteners/flavoring agents known in the art.

When administered by a drinking solution, the composition comprises one or more of the non-ATP competitive tyrosine kinase inhibitor compounds described herein dissolved in drinking liquid such as water, with appropriate pH adjustment, and with carrier. The compound dissolved in the drinking liquid is an amount sufficient to give a concentration in the bloodstream on the order of 1 nM and above, preferably in an effective amount that is effective in vivo. When administered nasally, these compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents known in the art (see, for example, Ansel et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Systems (7^th ed.).

Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the preparation of nasal dosage forms and some of these can be found in Remington 's Pharmaceutical Sciences (18th ed., Mack Publishing Company, Eaton, PA; 1990), a standard reference in the field. The choice of suitable carriers is highly dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, jelling agents, or buffering and other stabilizing and solubilizing agents may also be present.

The formulations of this invention may be varied to include: (1) other acids and bases to adjust the pH; (2) other tonicity-imparting agents such as sorbitol, glycerin, and dextrose; (3) other antimicrobial preservatives such as other parahydroxy benzoic acid esters, sorbate, benzoate, propionate, chlorbutanol, phenylethyl alcohol, benzalkonium chloride, and mercurials; (4) other viscosity imparting agents such as sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, polyvinyl alcohol and other gums; (5) suitable absorption enhancers; (6) stabilizing agents such as antioxidants, like bisulfate and ascorbate, metal chelating agents such as sodium edentate, and drug solubility enhancers such as polyethylene glycols.

The above nasal formulations can be administered as drops, sprays, or by any other intranasal dosage form. Optionally, the delivery system can be a unit dose delivery system. The volume of solution or suspension delivered per dose can be anywhere from 5 to 500 microliters, and preferably 5 to 200 microliters. Delivery systems for these various dosage forms can be dropper bottles, plastic squeeze units, atomizers, and the like in either unit dose or multiple dose packages. Lozenges can be prepared according to U.S. Patent No. 3,439,089, herein incorporated by reference for these purposes.

When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters, or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.

Dosage levels on the order of 1 mg/day or above may be useful in the treatment or prevention of pathogenic infections and related diseases within a host organism as noted herein above. In one embodiment of the present invention, a patient in need of treatment or prevention of pathogenic infection is administered a non-ATP competitive tyrosine kinase inhibitor described herein in an amount equal to or greater than about 1 mg/day, equal to or greater than about 5 mg/day, equal to or greater than about 10 mg/day, equal to or greater than about 20 mg/day, equal to or greater than about 30 mg/day, equal to or greater than about 40 mg/day, equal to or greater than about 50 mg/day, equal to or greater than about 60 mg/day, equal to or greater than about 70 mg/day, equal to or greater than about 80 mg/day, equal to or greater than about 90 mg/day, equal to or greater than about 100 mg/day, equal to or greater than about 110 mg/day, equal to or greater than about 120 mg/day, equal to or greater than about 130 mg/day, equal to or greater than about 140 mg/day, equal to or greater than about 150 mg/day, equal to or greater than about 160 mg/day, equal to or greater than about 170 mg/day, equal to or greater than about 180 mg/day, equal to or greater than about 190 mg/day, equal to or greater than about 200 mg/day, equal to or greater than about 210 mg/day, equal to or greater than about 220 mg/day, equal to or greater than about 230 mg/day, equal to or greater than about 240 mg/day, equal to or greater than about 250 mg/day, equal to or greater than about 260 mg/day, equal to or greater than about 270 mg/day, equal to or greater than about 280 mg/day, equal to or greater than about 290 mg/day, equal to or greater than about 300 mg/day, equal to or greater than about 310 mg/day, equal to or greater than about 320 mg/day, equal to or greater than about 330 mg/day, equal to or greater than about 340 mg/day, equal to or greater than about 350 mg/day, equal to or greater than about 360 mg/day, equal to or greater than about 370 mg/day, equal to or greater than about 380 mg/day, equal to or greater than about 390 mg/day, equal to or greater than about 400 mg/day, equal to or greater than about 410 mg/day, equal to or greater than about 420 mg/day, equal to or greater than about 430 mg/day, equal to or greater than about 440 mg/day, equal to or greater than about 450 mg/day, equal to or greater than about 460 mg/day, equal to or greater than about 470 mg/day, equal to or greater than about 480 mg/day, equal to or greater than about 490 mg/day, equal to or greater than about 500 mg/day, equal to or greater than about 510 mg/day, equal to or greater than about 520 mg/day, equal to or greater than about 530 mg/day, equal to or greater than about 540 mg/day, equal to or greater than about 550 mg/day, equal to or greater than about 560 mg/day, equal to or greater than about 570 mg/day, equal to or greater than about 580 mg/day, equal to or greater than about 590 mg/day, or equal to or greater than about 600 mg/day, for a patient having approximately 70 kg body weight. In some embodiments, the dose to be administered ranges from about 1 mg/day to about 1000 mg/day, including about 10 mg/day, 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day 100 mg/day, 125 mg/day, 150 mg/day, 175 mg/day, 200 mg/day, 225 mg/day, 250 mg/day, 275 mg/day, 300 mg/day, 350 mg/day, 400 mg/day, 450 mg/day, 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, and other such values between about 1 mg/day to about 1000 mg/day, for a patient having approximately 70 kg body weight. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific salt or other form employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

In one preferred regimen, such dosages can be administered to a subject in need thereof by either nasal spray or by oral lozenge. The effectiveness of using the pharmaceutical compositions of the present invention to treat or prevent a specific pathogenic infection, particularly microbial infection, may vary, for example, depending on the infectious agent, stage of infection, severity of infection, age, weight, and sex of the patient, and the like.

"Treatment" is herein defined as the application or administration of a non-ATP competitive tyrosine kinase inhibitor described herein to a subject, where the subject has a pathogenic infection as noted elsewhere herein, a symptom associated with a pathogenic infection, or a predisposition toward development of a pathogenic infection, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the pathogenic infection, any associated symptoms of the pathogenic infection, or the predisposition toward the development of the pathogenic infection. By "treatment" is also intended the application or administration of a pharmaceutical composition comprising a non-ATP competitive tyrosine kinase inhibitor described herein to a subject, where the subject has a pathogenic infection as noted elsewhere herein, a symptom associated with a pathogenic infection, or a predisposition toward development of a pathogenic infection, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the pathogenic infection, any associated symptoms of the pathogenic infection, or the predisposition toward the development of the pathogenic infection.

The non-ATP competitive tyrosine kinase inhibitors described herein are useful in treating or preventing pathogenic infections as noted herein above. Treatment or prevention of pathogenic infection in the manner set forth herein is particularly useful for transplant patients, for example, kidney transplant patients, where emergence of pathogens, particularly polyoma viruses, for example, JC and BK, and pathogenic infection can diminish function of the transplanted organ. In like manner, HIV infection can destroy oligodendrocytes in the brain, leading to AIDS-related dementia. Thus, in addition to treating or preventing pathogenic infections as noted elsewhere herein, the non-ATP competitive tyrosine kinase inhibitors described herein can be used to control secondary infection in HIV-positive and AIDS patients and in patients receiving transplants, for example, kidney transplants, and to control AIDS-related dementia. Further, the non-ATP competitive tyrosine kinase inhibitors can be used prophylactically to prevent spread of infectious virions, for example, associated with Vaccinia infections, in immunocompromised individuals, including HIV-positive and AIDS patients and in patients receiving transplants.

EXPERIMENTAL

The following section describes the use of non-ATP competitive tyrosine kinase inhibitors on the infection of host cells by pathogens, particularly bacterial and viral pathogens. Before describing such uses in more detail, it will be helpful to provide a basic description of various pathogens and host-pathogen interactions. Pathogenic E. coli, including enteropathogenic E. coli (EPEC) and enterohemmorhagic E. coli (EHEC), contaminate water and food supplies and cause infantile diarrhea. EPEC and EHEC are classified by NIAID as category B pathogens. In developing nations, EPEC causes sickness in some 20 million per year, killing 500,000 (Goosney et al. (2000) Annu. Rev. Cell Dev. Biol., 16: 173). EHEC, causative agent of "raw hamburger disease," contaminates food and is associated with diarrhea and an often fatal consequence, hemolytic-uremic syndrome. EHEC possess two Shiga toxins, which cause the symptoms associated with hemolytic-uremic syndrome (Perna et al (2001) Nature, 409(6819): 529-33). EPEC, EHEC, and Citrobacter (C.) rodentium (mouse EPEC) form actin-filled membrane protrusions or "pedestals" beneath themselves on the surface of epithelial cells (Knutton et al (1989) Lancet 2: 218; McDaniel et al (1997) MoL Microbiol, 23: 399). Pedestals prevent phagocytosis, allow colonization of the host, and are required for subsequent development of disease (Goosney et al. (1999) Infect. Immun., 67: 490; Jerse et al. (1990) Proc. Natl. Acad. Sd. USA, 87: 7839). The mechanisms by which pedestals form have been extensively investigated (Kalman et al. {1999) Nat. Cell Biol, 1: 389). The development of both pedestals and diarrhea are critically dependent on the activation of a host tyrosine kinase beneath the bacterium, which phosphorylates a bacterial protein secreted into the host cell called Tir (Kenny et al (1997) Cell, 91: 511; Kenny (1999) MoI. Microbiol, 31: 1229). Upon binding of the bacterial ligand intimin, a host signal transduction cascade is initiated that leads to pedestal formation.

The watershed event in EPEC pathogenesis is the phosphorylation of EPEC Tir (Kenny (1999) MoI Microbiol, 31: 1229). Once phosphorylated, EPEC Tir facilitates recruitment and activation of host cell proteins, including Nek, N-WASP, and Arp2/3 complex, that initiate actin polymerization to construct and brace the pedestal Kalman et al. (1999) Nat. Cell Biol, 1: 389; Lommel et al (2001) EMBO Rep., 2: 850; Gruenheid et al (2001) Nat. CellBiol, 3: 85619; Rohatgi et al (1999) Cell, 97: 221).

Vaccinia virus (W) and variola viruses are members of the Poxviridae family that are 95% identical in sequence (Esposito et al (1990) Poxviruses, in Fields Virology, D.M. Knipe, Editor, Raven Press: New York. p. 2336; Moss (1990) Poxviridae: The Viruses and Their Replication, in Fields Virology, D.M. Knipe, Editor. Raven Press: New York. p. 2336). W western reserve (WR) strain serves as a vaccinating agent for variola major, the cause of smallpox. W and variola enter mammalian cells, establish extranuclear replication "factories," and produce enveloped virions (Moss (1990)

Poxviridae: The Viruses and Their Replication, in Fields Virology, D.M. Knipe, Editor. Raven Press: New York. p. 2336). These virions travel to the cell surface using microtubule motors and transit into apposing cells by polymerizing actin (Ploubidou et al (2000) EMBO J., 19(15): p. 3932-44; Rietdorf et al (2001) Nat. CellBiol, 3(11): p. 992- 1000; Ward and Moss (2001) J. Virol, 75(23): p. 11651-63; Ward and Moss (2001) J. Virol, 75(10): p. 4802-13; Cudmore et al (1996) J. Cell Sd., 109 ( Pt 7): p. 1739-47; Cudmore et al (1997) Trends Microbiol, 5(4): p. 142-8). There the virions polymerize actin to propel themselves through the host cell cytoplasm and towards the plasma membrane, where they exit the cell and enter apposing cells. Formation of actin "comets" is considered critical for vaccinia to spread from cell to cell. For actin-based motility, vaccinia relies on the recruitment of host cell molecules to the surface of the particle, including tyrosine kinases. Ultimately, the host cell undergoes cytolysis thereby releasing additional infectious particles. Tyrosine and serine/threonine kinases are important for several aspects of viral infection. Actin-based motility depends on the activity of the host cell tyrosine kinases related to c-Src and AbI, and replication at least in part depends on a viral kinase, though the precise mechanism is less well understood (Frischknecht et al (1999) Nature 401(6756):926-929; Rempel et al (1992) J. Virol. 66(7):4413-4426; Traktman et al. (1995) J. Virol. 69(10):6581-6587; Traktman et al. (1989) J. Biol. Chem. 264(36):21458- 21461)

Upon entry of the pox virus into host cells, the virion moves to a juxtanuclear location where it replicates up to 10⁴ concatameric genomes (Moss (1990) Poxviridae: The Viruses and Their Replication, in Fields Virology, D.M. Knipe, Editor. Raven Press: New York, p. 2336). The concatamers ultimately form individual enveloped particles (called intracellular mature virions (IMVs), some of which are packaged in additional membranes to form intracellular enveloped virions (IEVs; Smith et al. (2003) Annu. Rev. Microbiol, pp. 323-342). Cytolysis releases IMVs from the cell. Prior to cytolysis, however, IEVs travel towards the host cell periphery via a kinesin/microtubule transport system (Carter et al (2003) J. Gen. Virol, pp. 2443-2458; Hollinshead et al (2001) J. Cell Biol, pp. 389-402; Rietdorf et al. (2001) Nat. Cell Biol, pp. 992-1000; Ward and Moss (2001) J. Virol, pp., 11651-11663).

To exit the cell, the IEV particle fuses with the plasma membrane of the host cell to form a cell-associated enveloped virus (CEV), leaving behind one of its two outer membranes (Smith et al (2003) Ann. Rev. Microbiol, pp., 323-342; Smith et al. (2002) J. Gen. Virol, pp. 2915-2931). CEVs either detatch directly, or initiate actin polymerization to propel the particle on an actin-fϊlled membrane protuberance towards an apposing cell and then detach (Smith et al (2003) Ann. Rev. Microbiol, pp., 323-342). Actin motility depends on AbI and Src family kinases whereas detachment of CEvs to form extraceullar enveloped virus (EEV) depends on AbI family kinases (Smith et al. (2003) Ann. Rev. Microbiol, pp., 323-342).

It is known that the protein encoded by the W A36R gene (called A36R), located in the membrane surrounding the CEV, is required for actin polymerization and virulence (Wolffe et al. (1998) Virology pp. 20-26; Parkinson and Smith (1994) Virology pp. 376- 390). The watershed event in actin polymerization and cell-to-cell spread is the phosphorylation of A36R tyrosine residues by a host cell tyrosine kinase (Newsome et al.

(2004) Science 306:124-128; Frischknecht et al. (1999) Nature 401(6756):926-929). There is a remarkable homology between the EPEC Tir protein decribed above and the W protein A36R, therefore using using similar but not identical host signalling factors as EPEC to polymerize actin and exit from the host cell (Frischknecht and Way (2001) Trends Cell Biol. l l(l):30-38).

Previous reports suggest that the mammalian tyrosine kinase c-Src localizes to virions (Frischknecht et al. (1999) Nature 401(6756):926-929). Moreover, the release of virions from microtubules and nucleation of actin to form actin tails depends on phosphorylation of A36R by Src or other kinases (Newsome et al. (2004) Science 306: 124-128; Frischknecht et al. (1999) Nature 401(6756):926-929; Kalman et al. (1999) Nat. Cell. Bio. 1:389-391). Once phosphorylated, A36R facilitates detachment of kinesin and recruitment and activation of host cell proteins, including Nek, Grb2, N-WASP, and the Arp2/3 complex, which initiate actin polymerization beneath the particle

(Frischknecht and Way (2001) Trends Cell Biol. 1 l(l):30-38; Moreau et al. (2000) Nat. Cell Biol, pp. 441-448; Scaplehorn et al. (2002) Curr. Biol., pp. 740-745). Indeed vaccinia uses mechanisms similar to those used by Shigella flexneri to propel itself through the host cytoplasm. For example, both Shigella and Vaccinia recruit and activate N-WASP and the Arp2/3 complex as a means of polymerizing actin (Frischknecht and Way (2001) Trends Cell Biol. l l(l):30-38).

Previous studies have demonstrated that tyrosine kinases are participants in motility, release, and pathogenic infection of Vaccinia virus (see, e.g., Reeves et al.

(2005) Nature Med. 11:731-739; PCT App. Pub. No. WO2005072826). In particular, Abl-family kinases, but not Src-family kinases, are required for efficient actin motility, and ATP-dependent tyrosine kinase inhibitors that inhibit Abl-family kinases, including pyrido[2,3-d]pyrimidine (PD) compounds, block actin motility. PD compounds and STI- 571 block release of infectious virions, and STI-571 reduces viral load in W-infected mice. In this regard, these results indicate that drugs such as PD and STI-571 are useful for the prevention or treatment of W infection. Because Vaccinia and variola viruses are similar, it is likely that these drugs would also have increased efficacy against variola infections in humans that cause smallpox.

Previous studies have also demonstrated that tyrosine kinase activity, in particular c-Abl activity, sufficient for pedestal formation initiated by EPEC or EHEC and of EPEC Tir phosphorylation (see, e.g., Swimm et al. (2004) MoI. Biol. Cell 15:3520-3529; PCT App. Pub. No. WO2005072826). These studies provided the first results identifying a role for tyrosine phosphorylation in EHEC pedestal formation and the first description of any tyrosine kinase sufficient for either EPEC or EHEC signalling. These results indicate that PD or related compounds are useful to treat or prevent EPEC and EHEC infections.

Experiment 1 — Effects of Non-ATP Competitive Tyrosine Kinase Inhibitors on Aspects of W and Variola Infection

This experiment is designed to study the efficacy of non-ATP competitive tyrosine kinase inhibitors such as ONO 12380 in reducing or minimizing pathogenicity in

W or Variola infected mice. C57 BL/6 mice are used for these studies. Mice are infected in a BSL2 facility to prevent infection of other mice.

ON012380 on W and variola infection. Intradermal inoculation of mice with

W has been proposed to model W vaccination in humans (Tscharke et al. (1999) J. Gen. Virol, 80: 2751-5; Tscharke et al (2002) J. Gen. Virol, 83: 1977-86). Using this model, it has been shown that intradermal inoculation on the ears of 6 week-old C57BL/6 mice with W strain WR produces 3 mm lesions within 8 days. The lesion disappears after about three weeks indicating that the animal has developed an immune response and cleared the infection. This model was developed based on experimental groups of 5, female, age matched 6 week old C57BL/6 mice infected with 10⁴ pfu intradermally on the ear, with lesion diameter measured daily over a three week time course. The present experimental design follows this paradigm.

Intranasal inoculation of mice with W has been proposed to model the normal path of variola inoculation in humans. Intranasal W infection at an moi of 10³ to 10⁶ of 8 week old female BALB/c mice leads to dramatic weight loss, reduced activity, and ultimately death within 10 days (Reading et al. (2003) J. Immunol, 170: 1435-1442).

The effect of ONO 12380 on lesion size (for intradermal inoculation) or mortality

(for intranasal inoculation) in W WR-infected mice is assessed. Half the mice are treated with ONO 12380 (administered via pump), and the control mice are treated equally with PBS or the drug formulation. Initially, the highest dose of ONO 12380 achievable without toxic effects is used. For mice inoculated intradermally, lesion size is measured daily. For mice infected intranasally, weight is measured daily.

At day 10 mice are sacrificed and brains and lungs are harvested. Mice losing greater than 30% of their body weight are sarcrificed immediately. Tissues are frozen and thawed tree times and sonicated, and the viral titre determined by plaque assay on 3T3 cells (Reading et al. (2003) J. Immunol, 170: 1435-1442). Data are analyzed statistically by the nonparametric Mann- Whitney t test, and if ON012380-treated mice harbor significantly different plaque forming units compared to control mice (p < 0.01) then it is concluded that the drug influences viral burden in infected mice. To rule out the possibility that viral invasion and proliferation is blocked by the drug formulation, or by some non-specific means, the effects of the formulation alone will be measured.

To assess the health of mice inoculated intranasally, appearance of mice are graded by a blinded observer: one point is assigned to each condition: listlessness, ruffled coat, (maximum score = 2; minimum score (robust health) = 0). In addition, body weight results are expressed as average values +/- one standard error. Treatment groups include at least five mice. Statistical analysis is calculated by the Mann-Whitney t test, with p < 0.01 considered significant. If drug treated groups yield reduced pathology scores, it is concluded that ONO 12380 therapy positively affects W disease outcome. Assessment of acquisition of immunity to W. This study assesses whether

ONO 12380 treatment allows effective vaccination. The drug or the carrier is administered via inoculation as described above. When the animal recovers and drug delivery has been discontinued, the animal is reinoculated. Inoculation is carried out either intradermally and the size of the ensuing scab determined, or intranasally at a dose lethal to animals not previously exposed to the virus. Scar size or mortality rates are assessed and are similar to animals not previously exposed if ONO 12380 interfere with acquisition of immunity. Alternatively, measurement of serum titres against known W proteins and carefully dosing the drugs to avoid complications can be utilized.

Reduction of infectivity in immunocompromised patients. This study assesses whether ON012380 is useful in limiting W disease in immunocompromised individuals. Ragl^"/7Rag2^"/" mice have no capacity to mount an adaptive immune response and develop severe infections. Whether intradermal inoculation with W produces a more severe disease in these animals compared to matched wild-type animals is assessed. If so, whether administration of ONO 12380 alone or in combination serves to protect the animal from a more severe infection will be analyzed.

Experiment 2 - Effects of Non-ATP Competitive Tyrosine Kinase Inhibitors on Pathogenesis of TB

This experiment is designed to study the effect of non-ATP competitive tyrosine kinase inhibitors such as ON012380 on the pathogenesis of Mycobacterium tuberculosis (TB), the etiologic agent of tuberculosis. Invasion of TB into a cultured human macrophages (line THP-I) is carried out essentially as described in Miller and Shinnick (2001), BMC Microbiol, 1:26. Briefly, TB cultures are added to the cells for between 30 minutes and two hours. Actinomycin D is then be added to the cultures to kill any bacteria remaining extracellularly. The actinomycin D is then washed away, and the cells are lysed to release invaginated bacteria. The lysate is then plated on bacterial plates, and the number of recovered colonies are counted. The experiments are performed with or without addition of ONO 12380 at concentrations ranging from 100 nM to 10 μM, concentrations that have proven effective in other EPEC and W assays for other tyrosine kinase inhibitors.

Colony counts are an indication of whether invasion is inhibited. Cell growth assays and trypan blue exclusion are used to verify that the macrophages are not adversely affected by the drugs. Results are expected to show that ONO 12380 increases the intercellular survival of M. tuberculosis, thus demonstrating that non-ATP competitive tyrosine kinase inhibitors are effective in inhibiting TB infection.

Experiment 3 - ONO 12380 Inhibits Vaccinia EEV Release

Extracellular enveloped virus (EEV) from supernatants of vaccinia (strain IHD-J)- infected BSC-40 cells or BSC-40 cells treated with 10 μM of both ATP competitive and non-ATP competitive tyrosine kinase inhibitors were quantified. Drugs used were the ATP competitive tyrosine kinase inhibitors PD 166326, AMN- 107, and BMS-354825 and the non-ATP competitive inhibitor ON012380. Supernatants were treated with 2D5 monoclonal antibody to reduce contamination from intracellular virus release by cell lysis. Results demonstrated that the non-ATP competitive inhibitor ONO 12380 blocked formation of EEV comets compared to controls (Figure 1). For measurements of secreted EEV, cells were incubated in 2% FBS DMEM, media was removed 24 hours after infection centrifuged for 15 minutes at 400 x g, dilutions of 1:1000 made and added to uninfected BSC-40 cell monolayers, and the number of plaques assessed 2-4 days subsequently. Drugs were present throughout the infection at a concentration of lOμM. Prior to addition, the supernatant collected from infected cells was incubated with anti- IMV mAb (2D5; at a dilution of ascites of 1 : 100), which has been shown to inactivate IMV but not EEV. Similar results were obtained with vaccinia strain WR. Next, BSC-40 monolayers were used to assess and compare the ability of ATP competitive tyrosine kinase inhibitors PD166326, STI-571, BMS354825, and AMN107 and the non-ATP competitive inhibitor ONO 12380 to inhibit plaque formation by variola. The monolayers were pretreated or mock-treated in the presence or absence of the various compounds in RPMI plus 2% FBS (RPMI-2%) at varying concentrations in triplicate for 30 minutes at room temperature. Three concentrations of each compound were evaluated; 5OnM, 500 nM, 5 μM and mock-treated controls per each concentration of each compound were used on duplicate 6-well plates.

Monolayers were infected with a suspension of variola strain Solaimen in the presence or absence of each compound, such that approximately 50 PFU per well of virus were observed. Plates were incubated at 35°C, 6% CO2 for 1 hour and rocked at 15 minutes intervals to ensure an even infection of the monolayer. The inoculum was removed, and the monolayer was rinsed 1 time with RPMI-2%. The monolayers were overlaid with medium ± the appropriate compound at the appropriate concentrations and incubated at 35⁰C, 6% CO2 for 4 days. The plates were then -irradiated at the kill dose (4.4 x 106 rad) and removed from the Biosafety Level 4 laboratory for analysis via immunohistochemical staining with an orthopoxvirus specific polyclonal antibody.

All tyrosine kinase inhibitors tested, including the non-ATP competitive inhibitor ON012380, blocked formation of EEV comets compared to controls (see Figure 2). Similar results were achieved with monkeypox. The presence of "comets" associated with the major plaques in the control cells are due to EEV released from those plaques.

In a further study, monolayers were overlayed with CMC agar to restrict formation of EEV comets. In that study, plaque size was used as an indicator of actin motility, which mediates spread of virus to an apposing uninfected cell. Under these conditions PD166326, ON012380, and BMS354825 all reduced plaque size to

"pinpoints" indicating that the inhibitors block Src and AbI family kinases associated with actin motility (see Figure 3).

Collectively, the results of Experiment 3 demonstrate that the non-ATP competitive inhibitor ONO 12380 inhibits vaccinia EEV release and thus is effective in the treatment of Vaccinia viral infection.

Experiment 4 - Effects of Non-ATP Competitive Tyrosine Kinase Inhibitors on Aspects of Polyoma Virus Infection

In this set of studies, the effects of both ATP competitive and non-ATP competitive tyrosine kinase inhibitors on polyoma virus (PyV) infection and replication were assessed and compared. In addition, to determine whether the Abl-family kinases AbI and Arg participate in PyV replication, cell lines derived from transgenic mice lacking either or both of these kinases were used, together with both ATP competitive and non-ATP competitive inhibitors that specifically target these kinases.

Tyrosine kinases are involved in early steps of polyomavirus entry into host cells. After binding to host cell surface sialylated gangliosides and sialylated glycoproteins, individual polyoma virions are internalized via monopinocytotic vesicles derived from cell membrane invaginations (Smith and Helenius (2004) Science 304:237-242). Although endocytic pathways differ among polyomavirus family members, virions of all polyomaviruses examined to date induce actin-dependent endocytosis, followed by microtubule-mediated trafficking of single virion-containing vesicles to smooth ER tubules (Pelkmans (2005) Curr. Opin. Microbiol, 8:331-337; Marsh and Helenius (2006) Cell, 124:729-740). Virion uncoating occurs in the nucleus or possibly upon exit from the smooth ER; the host cell nucleus is the site of viral gene transcription, viral DNA replication, and progeny assembly. Virus uptake activates protein tyrosine kinase(s) and induces a transient reorganization of the actin network Pelkmans et al. (2002) Science, 296:535-539; Gilbert et al. (2003) J. Virol., 77: 2615-2622; Gilbert and Benjamin (2004) J. Virol. , 78: 12259-12267. It has been shown that tyrosine kinases (defined by the relatively non-specific tyrosine kinase inhibitor genistein) are required for SV40 (monkey polyomavirus) internalization and development of "actin tails", to which tyrosine kinases and individual virions colocalize (Pelkmans et al. (2002) Science, 296:535-539). Actin tails formed by vaccinia virus likewise require tyrosine kinases of the AbI- and Src- families. Importantly, the tyrosine kinase inhibitor genistein has also been shown to inhibit JCV and BKV cell entry (Querbes et al. (2004) J. Virol., 78:250-256; Eash et al. (2004) J. Virol., 78: 11583-11590). Taken together, these studies indicate a common role for tyrosine kinases in providing a molecular signal for cell entry by polyomaviruses from different species. The data described below demonstrate involvement of Abl-family tyrosine kinases (i.e., AbI and Arg) in PyV infection and that non-ATP competitive inhibitors can specifically target these kinases.

Tyrosine kinases also participate at other stages of PyV infection. Middle T (MT), a 421 -amino acid type II integral membrane protein, provides a scaffold for cellular tyrosine kinases, lipid kinases and tyrosine phosphatases, as well as several adaptor proteins (Dilworth (2002) Nat. Rev. Cancer, 2:951-956). Cellular transformation and tumor induction by PyV require constitutive expression of the MT oncoprotein (Raptis et al. (1985) MoI Cell. Biol, 5:2476-2485), and transformation-competent MT is required to enable PyV to establish persistent infection (Freund et al. (1992) Virology, 191:716-723). Among the most proximal signaling events orchestrated by MT is the binding of Src family members (c-Src, and to lesser extents c-Fyn and c-Yes), which dramatically augments their kinase activity and, by phosphorylating specific tyrosines on MT, creates docking sites for SH2 domain-containing enzymes and adaptor proteins (Gottlieb and Villarreal (2001) Microbiol. MoI. Biol. Rev., 65:288-318). Mutations in MT that prevent c-Src binding invariably render PyV transformation-incompetent. MT also contributes to cellular transformation by coordinately forcing cells into cycle and blocking apoptosis ( Dahl et al. (1998) J. Virol, 72:3221-3226), and promotes viral assembly by indirectly inducing threonine phosphorylation of VPl (Garcea et «/.(1989) Virology, 168:312-319). Inhibition of Src family kinases, then, is not only expected to prevent PyV tumorigenesis, but would also be expected to limit persistent virus infection as well. The data described below suggests that inhibitors that target both AbI- or AbI- and Src-family kinases, such as the non-ATP competitive inhibitor ONO 12380 are also effective after entry of the virus. These data demonstrate that this dual kinase inhibitor operates at multiple stages of the PyV lifecycle.

Effects of tyrosine kinases and their inhibitors on PyV T protein expression. 3T3 cells were infected by PyV for 24 h in the absence or presence of STI-571 (10 μM), BMS354825 (10 μM), AMN107 (10 μM), or ON012380 (10 μM). Because the free ATP concentration inside cells is 2-5 mM, μM concentrations were needed in cells for those compounds that compete with ATP. 25 μg protein/lane was resolved by SDS-PAGE, and immunoblotted using the F4 pan-PyV T protein mAb. Infection of 3T3 cells lacking either AbI or Arg kinase (Koleske et al. (1998) Neuron, 21 : 1259-1272) produced lower levels of the viral T proteins (Large T, Middle T and Small T) compared to matched 3T3 cells derived from wild-type animals (negative and positive lanes, respectively, Figure 4A and Figure 4B). Gleevec substantially reduced early viral protein expression in 3T3 cells (Figure 4A). BMS-354825, AMN-107, and ON012380 each reduced PyV replication, as measured by production of viral T proteins (Figure 4B).

Wild type 3T3 cells or 3T3 cells derived from AbIl^"7", Arg^"7", or Abl^^'Arg^"7" mice were PyV infected for 24 h; 25 μg protein/lane was resolved by SDS-PAGE, and immunoblotted using the F4 pan-PyV T protein mAb. Surprisingly, no viral T proteins were detectable in 3T3 cells derived from mice deficient in both AbI and Arg (Figure 4C). Note that F4 cross-reacts with actin, the -40 kD band running just ahead of Middle T. The band immediately below the actin band in the infected samples is a LT degradation product.

Together, these data demonstrate that Abl-family kinases, acting in a redundant fashion, mediate PyV infection, and that both ATP competitive and non-ATP competitive tyrosine kinase inhibitors inhibit PyV infection.

Immunofluorescence assay for PyV infection. An immunofluorescence assay for PyV infection was developed using the pan-T niAb F4 (Pallas et al. (1986) J. Virol, 60: 1075-1084), which detects T antigens in the nucleus of infected cells. Primary macrophages or 3T3 cells were infected with PyV or left uninfected. After 2 hrs, extracellular virions were neutralized and left an additional 24 hrs. Cells were then fixed and stained with T antigen mAb F4-Cy3 to recognize infected cells, and with DAPI and FITC-phalloidin to recognized DNA and actin respectively. Cells were treated with Gleevec or ONO 12380 (10 μM) before and during infection. To quantitate data, the number of T-antigen positive cells was scored as a fraction of the number of D API+ nuclei in at least 15 images, and averaged. The experiment was repeated three times. Representative images and quantitation from one experiment are shown (Figure 5).

As seen in Figure 5A, T antigen was evident in infected but not uninfected cells. Notably, these effects were observed in both primary macrophages and 3T3 fibroblast cell lines. Quantitation of these data as shown in Figure 5B and Figure 5C demonstrated that the production of T antigen was blocked by both Gleevec and ONO 12380, and none was evident in Abl^VArg^"7" cells. Taken together, these data demonstrate that PyV infection requires Abl-family tyrosine kinases and that both ATP competitive and non-ATP competitive tyrosine kinase inhibitors inhibit PyV infection. Effect of ON012380 on PyV replication. To study the effect of non-ATP competitive tyrosine kinase inhibitors on PyV replication at a step after virion uncoating, 10 μM ONO 12380 was added to 3T3 cells 2 hours after PyV infection. Cells were allowed to incubate an additional 24 hours, and then fixed and stained with F4 mAb and counted as described above. Addition of ONO 12380 2 hours after virus adsorption markedly reduced the number of infected cells compared to controls as measured by the % of T antigen positive cells (Figure 6; the minus sign on the left corresponds to the control condition without administration of ON012380). This result demonstrates that non-ATP competitive tyrosine kinase inhibitors inhibit PyV replication. Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Further, it must be noted that as used in this specification and the appended embodiments, the singular forms "a," an" and "the" include plural referents unless the context clearly dictates otherwise.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for preventing or treating a bacterial infection or a viral infection comprising administering to a subject in need thereof a therapeutically effective amount of a non-ATP competitive tyrosine kinase inhibitor.

2. The method of claim 1, wherein said non-ATP competitive tyrosine kinase inhibitor is the compound:

or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

3. The method of claim 1, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I:

wherein:

X is selected from the group consisting of (i) and (ii) below :

X₁ is selected from the group consisting of (i), (ii) and (iii) below:

wherein X] is optionally protected with one or more chemical protecting groups; g is 0 or 1; each M is a bivalent connecting group independently selected from the group consisting of -(C₁-C₆) alkylene-, -(CH₂)_a-V-(CH₂)_b-, -(CH₂)_d-W-(CH₂)_e-, and -Z-; each y is independently selected from the group consisting of 0 and 1; each V is independently selected from the group consisting of arylene, heteroarylene, -C(=O)-, -C(=S)-, -S(=O)-, -SO₂-, -C(=O))-; -C(=O)(C₁- C₆)ρerfluoroalkylene-, -Ct=O)NR₄-, -Ct=S)NR₄-, and-SO₂NR₄-; each W is independently selected from the group consisting Of -NR₄-, -0-, and -S-

each a is independently selected from the group consisting of O, 1, 2 and 3; each b is independently selected from the group consisting of O, 1, 2 and 3; each d is independently selected from the group consisting of 1, 2 and 3; each e is independently selected from the group consisting of O, 1, 2 and 3;

wherein the absolute stereochemistry of -Z- is D or L or a mixture of D and L; each R_a is independently selected from the group consisting of -H, -(C₁ -C₆))alkyl, -(CH₂)₃-NH-C(NH₂)(=NH), -CH₂C(=O)NH₂, -CH₂COOH, -CH₂SH, -(CH₂)₂C(=O)-NH₂, -(CH₂)₂COOH, -CH₂-(2-imidazolyl), -CH(CH₃)-CH₂-CH₃, -CH₂CH(CH₃)₂, -(CH₂)₄-NH₂, -( CH₂)₂-S-CH₃, phenyl, CH₂-phenyl,-CH₂-OH, -CH(OH)-CH₃,-CH₂-(3-indolyl), -CH₂-(4- hydroxyphenyl), -CH(CH₃)₂, and -CH₂-CH₃; and includes compounds wherein R_a and Ri combine to form a 5-, 6-, or 7-membered heterocyclic ring; each R₁ is independently selected from the group consisting of -H, unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₅, - C(=O)N(R₄)₂, -CR₄R₅R₇, -C(=NH)-N(R₄)₂, -(C,-C₆)perfluoroalkyl, -CF₂Cl, - P(=O)(OR₄)₂, -OP(=O)(OR₄)₂ and a monovalent peptidyl moiety with a molecular weight of less than 1000; provided that when y is 0 and R₁ is -CO₂R₅, R₅ is not -H; each R₂ is independently selected from the group consisting of -H, -(C₁ -C₆) alkyl, and aryl(Ci-C₃)alkyl, wherein -R₂ and -(M)y-Ri may optionally be linked covalently to form a 5-, 6-, or 7-membered substituted or unsubstituted heterocycle; each R₃ is independently selected from -(C₁ -C₆)alkyl; each R₄ is independently selected from the group consisting of -H, and -(C₁ -C₆) alkyl; wherein: when R₄ and Ri are bonded to the same nitrogen atom, Ri and R₄ may combine to form a heterocycle; and when two R₄ groups are geminally bonded to the same nitrogen, the two R₄ groups may combine to form a heterocycle; each R₅ is independently selected from the group consisting of -H, -(C₁ -C₆)alkyl and -(C₁ -C₆)acyl; each R₆ is independently selected from the group consisting of -H, -(C₁ -C₆)alkyl, -CO₂R₅, -C(=O)R₇, -OR₅, -OC(=O)(CH₂)₂CO₂R₅, -SR₄, guanidino, -N(R₄)₂,-N⁺(R₄)₃, -N⁺ (CH₂CH₂OH)₃, phenyl, substituted phenyl, heterocyclic, substituted heterocyclic and halogen; each R₇ is independently selected from the group consisting of -H, -R_a, halogen, -(C₁ -C₆)alkyl, -N(R₄):. and heterocycles containing two nitrogen atoms; and

Q is selected from the group consisting of -H, -(C₁ -C₆)alkoxy, halogen, -(Ci- C₆)alkyl and -N(Rt)₂; wherein the substituents for the substituted aryl and substituted heterocyclic groups comprising or included within R₁, R_a, R₂, R₆, and R₇, are independently selected from the group consisting of halogen, (C₁-C₆)alkyl, (Ci- C₆)alkoxy, -NO₂, -ON, -CO₂R₅, -C(=O)O(d-C₃)alkyl, -OR₅, -(C₂-C₆)-OH, phosphonato, -N(R₄);,, -NHC(=O)(C,-C₆)alkyl, sulfamyl, -OC(=O)(C₁-C₃)alkyl, -0(C₂- C₆)-N((C,-C₆)alkyl)₂, and -CF₃; provided (1) when R₁ is a monovalent peptidyl moiety of molecular weight less than 1000 and V is -C (=0)-, -C(=S)-, -S(=O)-, or -SO₂-, and b is 0; then said peptidyl moiety is coupled to M through the amino terminus of the peptidyl moiety or through a side chain amino group to form an amide, thioamide, sulfonamide, or sulfonamide respectively; (2) when R] is a monovalent peptidyl moiety of molecular weight less than 1000 and V is -C(=O)NR₃-, -SO₂NR₃-, or -NR₄-, and b is 0, then said peptidyl moiety is coupled to M through the carboxy terminus of the peptidyl moiety or through a sidechain carboxyl group to form an imide, sulfonimide, or carboxamide respectively; and

(3) when Ri is a monovalent peptidyl moiety of molecular weight less than 1000 and W is -S- or -O-, and d is 0, then said peptidyl moiety is coupled to M through the carboxy terminus of the peptidyl moiety or through a sidechain carboxyl group to form a carbothioic acid ester or the carboxylic ester respectively; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

4. The method of claim 3, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

5. The method of claim 3, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

6. The method of claim 3, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound selected from the group consisting of: (E)-2,4,6-trimethoxystyryl-3-[4-(4-methylpiperazin-l-yl]benzamido)-4-methoxy- benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(acetoxyacetamido)-4-methoxy-benzylsulfone;

(E)-2₅4,6-trimethoxystyryl-3-(triethylammoniumacetamido)-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-[tri- (2-hydroxyethylammomum)acetamido]-4- methoxy-benzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(2-methyl-2-hydroxypropionamido)-4- methoxybenzyl-sulfone; (E)-2,4,6-trimethoxystyryl-3-(2-methyl-2-acetoxypropionamido)-4- methoxybenzyl-sulfone;

(E)-2,4,6-trimethoxystyryl-3-(2-acetoxypropionamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(trifluoroacetamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(trifluoromethanesulfonamido)-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-(phosphonatoacetamido)-4-methoxybenzylsulfone, disodium salt; (E)-2,4,6-trimethoxystyryl-3-(methylcarbamoyl)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(2,2-difluoromalonamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(pentafluoropropionamido)-4- methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(methyl-2,2-difluoromalonamido-4-methoxybenzyl- sulfone;

(E)-2,4,6-trimethoxystyryl-3-(2,2-difluoromalonamido)-4-methoxybenzylsulfone;

(E)-2,4,6-trimethoxystyryl-3-(dimethylamino-α, α-difluoroacetamido)-4-methoxy- ben2ylsulfone; and

(E)-2,4,6-trimethoxystyryl-3-(2, 2,3, 3, tetrafluorosuccinamido)-4- methoxybenzyl-sulfone; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

7. The method ot claim 3, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein: each V is independently selected from the group consisting of -C(=O)-, - C(=S)-, -S(=O)-, -SO₂-; -CC=O)NR₄-, -CC=S)NR₄-, and -SO₂NR₄-;

wherein the absolute stereochemistry of -Z- is either D or L each R_a is independently selected from the group consisting of -H, -CH₃, -(CH₂)₃- NH-C(NH₂)(=NH), -CH₂CC=O)NH₂, -CH₂COOH, -CH₂SH, -(CH₂)₂C(=O)-NH₂, - (CH₂)₂COOH, -CH₂-(2-imidazolyl), -CH(CH₃)-CH₂-CH₃, -CH₂CH(CH₃)₂, -(CH₂)₄-NH₂, - (CH₂)2-S-CH₃, phenyl, CH₂-phenyl, -CH₂-OH, -CH(OH)-CH₃, -CH₂-(3-indolyl), -CH₂- (4-hydroxyphenyl), -CH(CH₃)₂, and- CH₂-CH₃; and includes compounds wherein R_a and R] combine to form a 5-, 6-, or 7-membered heterocyclic ring; each Ri is independently selected from the group consisting of -H, unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₅, - C(=O)N(R₄)₂, -CHR₆R₇, -C(=NH)-N(R₄)₂, and a monovalentpeptidyl moiety with a molecular weight of less than 1000; provided that when y is O and Ri is -CO₂R₅, R₅ is not -H; each R₆ is independently selected from the group consisting of -H, -(Ci-C₅)alkyl, -CO₂R₅, -C(=O)R₇, -OH, -SR₄, -(C₁-C₃)alkoxy, -(Ci-C₃)alkylthio, guanidino, -N(R₄)₂, phenyl, substituted phenyl, heterocyclic, substituted heterocyclic and halogen; and each R₇ is independently selected from the group consisting of -H, halogen,-(C₁ -C₆)alkyl, N(R₄)₂, and heterocycles containing two nitrogen atoms; wherein the substituents for the substituted aryl and substituted heterocyclic groups comprising or included within R₁, R_a, R₂, R₆, and R₇, are independently selected from the group consisting of halogen, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, -NO₂, -C≡N,

CO₂R₅, -C(=O)O(Ci-C₃)alkyl, -OH, -(C₂-C₆)-OH, phosphonato, -N(R₄);., -NHC (O)(Ci- C₆)alkyl, sulfamyl, -OC(=O) (C,-C₃)alkyl, -O(C₂-C₆)-N((Ci-C₆)alkyl)₂, and -CF₃; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

8. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 0; and R₂ is -H; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

9. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

R₂ is -H, y is 0; and

Ri is selected from the group consisting of unsubstituted aryl, substituted aryl, substituted heterocyclic, unsubstituted heterocyclic, -CO₂R₃; -C(=O)N(R₄)₂,-CHR₆R₇, -C (=NH)-N(R4)₂ and a monovalent peptidyl moiety with a molecular weight of less than 1000; provided that when y is 0 and Ri is -CO₂R₅, R₅ is not -H; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

10. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 1; M is -(CH₂)_a-V-(CH₂)_b-; and V is -C(O)-; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

11. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 1 ; and M is -Z-; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

12. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 1; and M is -(CH₂)_a-V-(CH₂)_b-; and V is -SO₂-; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

13. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein: X is

and y is 0; and R, is -C(=NH)-N(R₄)₂; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

14. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 1; and M is -(C₁ -C₆)alkylene-; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

15. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein:

X is

and y is 1; and M is -(CH₂)_a-V-(CH₂)_b-; and V is -C(=O)NR₄-; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

16. The method of claim 7, wherein said non-ATP competitive tyrosine kinase inhibitor is a compound according to Formula I wherein: X is

and y is 0; R₁ is -CHR₆R₇; R₆ is CO₂R₅ and R₇ is R_a; or a pharmaceutically acceptable salt, enantiomer, analog, ester, amide, prodrug, metabolite, or derivative thereof.

17. The method of any one of claims 1 to 16, wherein said non-ATP competitive tyrosine kinase inhibitor inhibits at least one Abl-family tyrosine kinase or Src-family tyrosine kinase.

18. The method of any one of claims 1 to 16, wherein said viral infection is caused by a Vaccinia virus, a variola virus, a JC, a BK, a herpes, a human immunodeficiency virus, or Pseudomonas aeruginosa.

19. The method of any one of claims 1 to 16, wherein said bacterial infection is caused by Escherichia coli, Helicobacter pylori, Listeria monocytogenes, Salmonella typhimurium, Shigella Flexneri, or Mycobacterium tuberculosis.

20. The method of any one of claims 1 to 19, wherein said non-ATP competitive tyrosine kinase inhibitor is administered in combination with an ATP competitive tyrosine kinase inhibitor selected from the group consisting of STI-571, STI- X, PD166326, BMS-354825, and AMN107.