# Patent

## Title
Methods of inhibiting poxvirus growth

## Abstract

This disclosure ascribes new functions to derivatives of tetralin, anthraquinone, naphthylamine, tri-amino-pyrimidine, xanthen-3-one, and/or cinnamic acid (including, for example, NSC270718R, NSC117285R, NSC170008Y, NSC306711P, NSC119913X, NSC119915Z, NSC119911V, NSC119910U, NSC128437O, NSC125908P, NSC9600Q, or NSC13778J, each obtained from the Structure Diversity Set, National Institutes of Health, National Cancer Institute, Developmental Therapeutics Program). These compounds are shown to be effective inhibitors of viral essential protein kinases (such as poxvirus B1 and/or F10 protein kinases). Exemplary chemical structures for viral protein kinase (VPK) inhibitors are provided, as are methods of using such compounds, for instance, to inhibit VPK activity and/or poxvirus growth, and/or for the treatment of poxvirus infection. Also provided are pharmaceutical compositions including disclosed VPK inhibitors.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is the U.S. National Stage of International Application No. PCT/US2006/039836, filed Oct. 10, 2006 (published in English under PCT Article 21(2)), which claims the benefit of U.S. Provisional Application No. 60/727,001, filed Oct. 13, 2005, both of which applications are incorporated herein in their entirety.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with United States government support pursuant to grant no. R01 CA 42350 from the National Institutes of Health; the United States government has certain rights in the invention.


FIELD
This disclosure concerns the use of derivatives , of tetralin, anthraquinone, naphthylamine, tri-amino-pyrimidine, xanthen-3-one, and/or cinnamic acid to inhibit viral protein kinases and poxvirus growth, and to treat poxvirus infection.
BACKGROUND
Poxviruses are the largest known animal viruses with approximately 200 distinct genes (Moss, In: Fields Virology, ed. by Knipe and Howley, Philadelphia: Lippincott Williams & Wilkins, 2001, pp. 2849-2883). They are DNA viruses that replicate entirely in the cytoplasm. Thus, a subset of their gene products carries out the functions that are essential for the viruses to be independent of the host-cell nucleus. The other viral gene products use or modulate a wide array of host-cell and immune-system processes.
Poxviruses infect most vertebrates and invertebrates causing a variety of diseases of veterinary and medical importance. The Poxviridae family has two main subfamilies, the chordopoxvirinae, which infect vertebrates, and the entomopoxvirinae, which infect insects. The chordopoxviruses include the genera Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Mollusciposvirus, and Yatapoxvirus. Each of the chordopoxviruses has a restricted and specific host array. Humans are the sole hosts of two poxviruses, variola virus (smallpox virus) and molluscum contagiosum virus; however many members of Orthopoxvirus, Parapoxvirus, and Yatapoxvirus are zoonotic, i.e., can infect both animals and humans. Vaccinia virus is the virus used in the variola virus vaccine, and it is widely used as a model poxvirus in the laboratory. Variola virus and vaccinia virus are members of the Orthopoxvirus genus.
Smallpox virus (variola major) has the potential to be used as a weapon. The virus is contagious, easy to store, and infection is fatal in 30-40% of unimmunized individuals (Davis et al., Microbiology, Hagerstown, Md.: Harper & Row, 1980, pp. 1077-1093; Harrison et al., Proc. Natl. Acad. Sci. USA, 101(31): 11178-11192, 2004). Importantly, more than half the world's population has never been vaccinated against this virus and essentially no one has been re-immunized for at least 20 years. Efforts to resume immunization and re-immunization have foundered. A drug that inhibited the growth of variola should prevent and cure acute smallpox infection even in unimmunized individuals. To this end, efforts have been made to develop nucleoside analogs as anti-poxvirus drugs (Bray et al., J. Infect. Dis., 181: 10-19, 2000; De Clercq, Clin. Microbiol. Rev., 14: 382-397, 2001; Keith et al., Antimicrob. Agents Chemother., 47: 2193-2198, 2003). One promising drug currently available is Cidofovir, a cytosine analog (Keith et al., Antimicrob. Agents Chemother., 47: 2193-2198, 2003). It shows considerable anti-poxvirus activity in animals but cannot be administered orally.
The poxviruses, including variola and vaccinia viruses, encode two protein kinases, B1 and F10 (Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91: 7653-7657, 1994; Lin et al., J. Virol., 66: 2717-2723, 1992; Traktman et al., J. Biol. Chem., 264: 21458-21461, 1989). Both kinases are essential for vaccinia virus growth. Temperature-sensitive mutations in either the B1 or F10 genes prevent vaccinia virus multiplication at the non-permissive temperature (Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91: 7653-7657, 1994; Traktman et al., J. Biol. Chem., 264: 21458-21461, 1989). Additionally, vaccinia virus F10 catalytic activity has been shown to be required for virus growth (Szajner et al., J. Virol., 78: 257-265, 2004). Substances that inhibit the enzymatic activity of either or both poxvirus protein kinases are needed to increase and diversify the arsenal of anti-poxviral drugs.
One consideration in the discovery of viral protein kinase inhibitors is cross-reactivity with protein kinases normally expressed in host cells. Human cells, for example, express hundreds of protein kinases that often have important functions in the regulation of cell growth and metabolism. An anti-viral drug that coincidentally inhibits important cellular protein kinases may have adverse side effects for the host cell and/or organism; however, this is certainly not always the case. Several high-affinity inhibitors of cellular protein kinases have proved to be both useful in treatment of a number of human malignancies and to exhibit acceptable toxicity. Gleevec (Novartis), an inhibitor of Abl, the platelet-derived growth factor receptor, and the Kit receptor is efficacious in chronic myeloid leukemia (Druker et al., N. Engl. J. Med., 344: 1031-1037, 2001; Joensuu et al., N. Engl. J. Med., 344: 1052-1056, 2001), gastrointestinal stromal tumor (Joensuu et al., N. Engl. J. Med., 344: 1052-1056, 2001), and hypereosinophilia syndrome (Schaller and Burkland, Med. Gen. Med., 3: 9, 2001). Iressa (AstraZeneca), an inhibitor of the epidermal growth factor receptor, is valuable in treatment of a subset of small cell lung tumors (Han et al., J. Clin. Oncol., 23: 2493-2501, 2005; Mitsudomi et al., J. Clin. Oncol., 23: 2513-2520, 2005). The foregoing drugs are tolerated well even when used chronically.
Advantageously, a poxvirus protein kinase inhibitor would only need to be used acutely to treat or prevent variola infection. The virus life cycle is less than 24 hours and infection with smallpox virus lasts approximately two weeks in humans (Davis et al., Microbiology, Hagerstown, Md.: Harper & Row, 1980, pp. 1077-1093). Therefore, even if a viral protein kinase inhibitor cross-reacted to some extent with host cell protein kinases, any side effects of the inhibitor may be short lived and clinically acceptable.
New drugs are needed to combat the multitude of diseases caused by poxviruses, especially those poxviruses that could be used as bioterrorism agents, such as variola virus.
SUMMARY
This disclosure concerns the discovery that poxvirus protein kinases (such as the B1 and/or F10 kinases) and poxvirus growth are inhibited by certain derivatives of tetralin, anthraquinone, xanthen-3-one, naphthylamine, cinnamic acid, and/or tri-amino-pyrimidine. This important discovery enables, for instance, methods of inhibiting poxvirus protein kinases and poxvirus growth, and methods of treating poxvirus infection.
Exemplary viral protein kinase inhibitors are provided throughout the disclosure and, by way of example, include compounds having one of the following general structures:




Other exemplary viral protein kinase inhibitors include compounds, such as NSC270718R; NSC117285R (2-hydroxy-4-(2,4,6-triaminopyrimidin-5-yl)diazenyl-benzoic acid); NSC170008Y (2-acetyl-7,8-bis(dihydroxymethylidene)-3-ethyl-9-hydroxy-anthracene-1,4,6,10-tetrone); NSC306711P; NSC119913X (7-methyl-6-(4,5,6,-trihydroxy-3-oxo-xanthen-9-yl)-bicyclo[2.2.1]hetp-2-ene-5-carboxylic acid); NSC119915Z (3-(4,5,6-trihydroxy-3-oxo-xanthen-9-yl)propanoic acid); NSC119911V (3-(4,5,6-trihydroxy-3-oxo-xanthen-9-yl)prop-2-enoic acid); NSC119910U (2-(4,5,6-trihydroxy-3-oxo-xanthen-9-yl)cyclohexane-1-carboxylic acid); NSC128437O (4-(4-cyclohexylamino-9,10-dioxo-anthracen-1-yl)aminobenzenesulfonic acid); NSC125908P (3-(9,10-dioxo-2-sulfo-anthracen-1-yl)diazenyl-2-hydroxy-benzoic acid); NSC9600Q (4-[N′-(2-hydroxynaphthalen-1-yl)-N′-sulfo-hydrazino]benzenesulfonic acid); or NSC13778J (3-(3-stibonophenyl)prop-2-enoic acid).
Advantageously, poxvirus F10 and B1 protein kinases are involved in poxvirus growth; thus, poxvirus growth can be inhibited by interfering with the function of such kinases. Moreover, the F10 and B1 kinases have no, or only distantly related, homologs in the human genome; thus, inhibitors of such kinases are less likely to interfere with the functions of cellular kinases in the host cell or organism.
The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a series of bar graphs showing virus yield (plaque-forming units per infected cell) of ts2, ts25, or wild-type (wt) vaccinia virus, or vesicular stomatitis virus (VSV) grown in HeLa cells in the presence of 1% DMSO (control) or 75 μM or 100 μM NSC270718-R or its “2”-methyl derivative. NSC270718-R inhibited the growth of each vaccinia virus, but not the growth of VSV. The NSC270718-R derivative did not inhibit the growth of ts2 or ts25 vaccinia virus at the permissive temperature.
FIG. 2 shows a time course of human Ramos B cell growth in the absence (no treatment) or presence of 1% DMSO, 50 μM NSC270718-R, or 100 μM NSC270718-R. The cells were counted with a hemacytometer after growth for 24 and 48 hours at 37° C. NSC270718-R had no effect on the growth of this human cell line.
FIG. 3 is an alignment of the amino acid sequences of the F10 kinases from vaccinia (SEQ ID NO: 4) and variola major (SEQ ID NO: 12) viruses.
FIG. 4 is an alignment of the amino acid sequences of the B1 kinases from vaccinia (SEQ ID NO: 2) and variola major (SEQ ID NO: 8) viruses.
FIG. 5 is an alignment of F10 kinase homologs from the indicated Orthopoxviruses. The alignment was generated with CLUSTALW (publicly available at the website ebi.ac.uk/clustalw) using default parameters. An asterisk (*) indicates identity among the residues at the indicated location, a colon (:) indicates conservative substitutions among the residues at the indicated location, and a period (.) indicates semi-conservative substitutions among the residues at the indicated location. SEQ ID NOs: 4, 18, 20, 22, 24, 26, 28 and 30 from the top to bottom.
FIG. 6 is an alignment of F10 kinase homologs from the indicated poxviruses (Lumpy_skin=lumpy skin disease virus, Mule_deer_pox=mule deer poxvirus, Yaba_monkey=Yaba monkey tumor virus, Yaba_like_disease=Yaba-like disease virus). The alignment was generated with CLUSTALW (publicly available at the website ebi.ac.uk/clustalw) using default parameters. An asterisk (*) indicates identity among the residues at the indicated location, a colon (:) indicates conservative substitutions among the residues at the indicated location, and a period (.) indicates semi-conservative substitutions among the residues at the indicated location. SEQ ID NOs: 4, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56 from the top to bottom.
FIG. 7 is an alignment of B1 kinase homologs from the indicated Orthopoxviruses. The particular viral strain or isolate is shown after the virus name (WR=Western Reserve, Acambis=Acambis 3000 Modified Virus Ankara (MVA), TT=Tian Tan, Variola_maj_India=Variola major (India-1967, isolate=Ind3), Variola_maj_BD=Variola major (Bangladesh-1975), Variola_min_Garcia=Variola minor (Garcia-1966), BR=Brighton Red, MPXV=MPXV-WRAIR7-61; Walter Reed 267). The alignment was generated with CLUSTALW (publicly available at the website ebi.ac.uk/clustalw) using default parameters. An asterisk (*) indicates identity among the residues at the indicated location, a colon (:) indicates conservative substitutions among the residues at the indicated location, and a period (.) indicates semi-conservative substitutions among the residues at the indicated location. SEQ ID NOs: 2, 58, 60, 62, 64, 66, 68, 8, 70, 72, 74, 76, 78, 80, 82, 84, 86, and 88 from top to bottom.
FIG. 8 is an alignment of B1 kinase homologs from the indicated poxviruses (Lumpy_skin=lumpy skin disease virus, Mule_deer_pox=mule deer poxvirus, Yaba_monkey=Yaba monkey tumor virus, Yaba_like_disease=Yaba-like disease virus). The alignment was generated with CLUSTALW (publicly available at the website ebi.ac.uk/clustalw) using default parameters. An asterisk (*) indicates identity among the residues at the indicated location, a colon (:) indicates conservative substitutions among the residues at the indicated location, and a period (.) indicates semi-conservative substitutions among the residues at the indicated location. SEQ ID NOs: 90, 92, 94, 102, 104, 100, 96, 98, 8, 72, 80, 74, 78, 2, 84, 86, 106, and 108 from top to bottom.
FIG. 9 is a graph illustrating that compound 270718-R does not inhibit the growth of human HeLa cells. HeLa cells were seeded at a density of 1.5×104 cells per well in a 48 well dish and allowed to adhere and grow for 18 hours. The growth medium was then replaced with medium containing 0.5% DMSO, or medium containing 0.5% DMSO and either 75 μM 270718-R or 100 μM 270718-R. The cells were counted with a Coulter counter 24 and 48 hours after addition of the drug. Each point represents the average of the three wells counted at each time point. The data are from a single representative experiment.
FIG. 10 shows virus yields (pfu/cell) from HeLa cells grown in 2% bovine calf serum and the indicated concentrations of 270718-R. Values are shown as a percentage of the yield from HeLa cells grown in the same medium containing vehicle (0.5% DMSO) only. The data represent the averages of three experiments. The error bars indicate the standard deviations of the data.



SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. All sequence database accession numbers referenced herein are understood to refer to the version of the sequence identified by that accession number as it was available on the filing date of U.S. Provisional Application No. 60/727,001. In the accompanying sequence listing:
SEQ ID NO: 1 is a nucleic acid sequence encoding vaccinia virus (Copenhagen strain) B1 kinase (see also, GenBank Accession No. M35027, B1R gene, CDS=162884 . . . 163786; or GenBank Accession No. NC—001559, CDS=162884 . . . 163786).
SEQ ID NO: 2 is an amino acid sequence of vaccinia virus (Copenhagen strain) B1 kinase (see also, GenBank Accession No. AAA48194 or NP—063857).
SEQ ID NO: 3 is a nucleic acid sequence encoding vaccinia virus (Copenhagen strain) F10 kinase (see also, GenBank Accession No. M35027, F10L gene, CDS=complement (39563 . . . 40882); or GenBank Accession No. NC—001559, CDS=complement (39563 . . . 40882)).
SEQ ID NO: 4 is an amino acid sequence of vaccinia virus (Copenhagen strain) F10 kinase (see also, GenBank Accession No. AAA48026 or NP—063689).
SEQ ID NO: 5 is a nucleic acid sequence encoding vaccinia virus (Ankara strain) F10 kinase (see also, GenBank Accession No. U94848, MVA039L gene, CDS=complement (31416 . . . 32735)).
SEQ ID NO: 6 is an amino acid sequence of vaccinia virus (Ankara strain) F10 kinase (see also, GenBank Accession No. AAB96420).
SEQ ID NO: 7 is a nucleic acid sequence encoding variola major virus (India-1967, ssp. major strain, isolate Ind3) B1 kinase (see also, GenBank Accession No. NC—001611, B1R gene, CDS=152700 . . . 153602; or GenBank Accession No. X69198, BIR gene, CDS=152700 . . . 153602).
SEQ ID NO: 8 is an amino acid sequence of variola major (India-1967, ssp. major strain, isolate Ind3) B1 kinase (see also, GenBank Accession No. NP—042213 or CAA491110).
SEQ ID NO: 9 is a nucleic acid sequence encoding variola major virus (India-1967, ssp. major strain, isolate Ind3) F10 kinase (see also, GenBank Accession No. NC—001611, C14L gene, CDS=complement (27297 . . . 28616)).
SEQ ID NO: 10 is an amino acid sequence of variola major virus (India-1967, ssp. major strain, isolate Ind3) F10 kinase (see also, GenBank Accession No. NP—042078).
SEQ ID NO: 11 is a nucleic acid sequence encoding variola major virus (strain Congo-1965) F10 kinase (see also, GenBank Accession No. U18337, C14L gene, CDS=complement (27303 . . . 28622)).
SEQ ID NO: 12 is an amino acid sequence of variola major virus (strain Congo-1965) F10 kinase (see also, GenBank Accession No. AAA69339).
SEQ ID NOs: 13 and 14 are a primer pair useful, at least, for amplifying the gene encoding vaccinia virus B1 kinase. Such primers also will amplify genes encoding B1 kinase homologs from at least some of the respective viral genomes (in particular, those B1-encoding homologs having a relatively high percentage of sequence identity (e.g., at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity) to the vaccinia virus B1-encoding gene, such as variola virus and others).
SEQ ID NOs: 15 and 16 are a primer pair useful, at least, for amplifying the gene encoding vaccinia virus F10 kinase. Such primers also will amplify at least some of the genes encoding F10 kinase homologs from the respective viral genomes (in particular, those F10-encoding homologs having a relatively high percentage of sequence identity (e.g., at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% sequence identity) to the vaccinia virus F10-encoding gene, such as variola virus and others).
SEQ ID NOs: 17-56 are nucleic acid and corresponding amino acid sequences of exemplary F10 VPK homologs (see, e.g., FIGS. 5 and 6).
SEQ ID NOs: 57-108 are nucleic acid and corresponding amino acid sequences of exemplary B1 kinases or B1 VPK homologs (see, e.g., FIGS. 7 and 8).
DETAILED DESCRIPTION
I. Abbreviations and Terms
















PK
protein kinase



PFU
plaque forming unit



PVT
polyvinyl toluene



SPA
scintillation proximity assay



VPK
viral protein kinase



VSV
vesicular stomatitis virus










Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in chemistry may be found in The McGraw-Hill Dictionary of Chemical Terms, Second Edition, New York: McGraw-Hill, 2003, and Dean, Lange's Handbook of Chemistry, Fifteen Edition, New York: McGraw-Hill, 1999. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. “Comprising” means “including”; hence, “comprising A or B” means “including A or B” or “including A and B.”
In order to facilitate review of the various embodiments of the invention, the following explanations of specific terms are provided:
“Acyl” refers to a group having the structure —C(O)R, where R may be alkyl, or substituted alkyl. “Lower acyl” groups are those that contain from 1 to 10 (such as from 1 to 6) carbon atoms.
“Acyloxy” refers to a group having the structure RCOO—, where R may be alkyl or substituted alkyl. “Lower acyloxy” groups contain from 1 to 10 (such as from 1 to 6) carbon atoms.
The term “aliphatic” refers to a straight-chain, branched-chain, or cyclic alkane, alkene, or alkyne. In some examples, an aliphatic group contains from 1 to 25 carbon atoms; for example, from 1 to 15, from 1 to 10, or from 1 to 6 carbon atoms. An aliphatic group having 10 or fewer carbon atoms (such as from 1 to 10 or from 1 to 6 carbon atoms) may be referred to as a “lower aliphatic” group. Unless expressly referred to as an “unsubstituted aliphatic,” aliphatic groups can either be unsubstituted or substituted. An aliphatic group can be substituted with one or more substituents (for instance, up to two substituents for each methylene carbon in an aliphatic chain, or up to one substituent for each carbon of a —C═C— double bond in an aliphatic chain, or up to one substituent for a carbon of a terminal methine group). Exemplary aliphatic substituents include, for instance, amine, amide, sulfonamide, halogen, cyano, carboxy, hydroxy, mercapto, trifluoromethyl, alkyl, alkoxy, alkylthio, thioalkoxy, arylalkyl, heteroaryl, alkylamino, dialkylamino, other functionality known to those of skill in the art and combinations thereof.
The term “alkoxy” refers to a group having the formula —OR, wherein R is an alkyl group. “Lower alkoxy” refers to an —OR group in which the R group has from 1 to 10 carbon atoms, such as from 1 to 6 carbon atoms. Examples of lower alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy and butoxy groups.
“Alkenyl” refers to a straight-chain, branched-chain, or cyclic hydrocarbon group including one or more double bonds that may or may not be conjugated with each other. Alkenyl groups may be unsubstituted or substituted, so the term “alkenyl” is to be understood to include both unsubstituted alkenyl groups and substituted alkenyl groups unless clearly indicated otherwise. In some examples, an alkenyl group has from 2 to 25 carbon atoms. “Lower alkenyl” groups contain from 2 to 10 (such as from 2 to 6) carbon atoms.
The term “alkyl” refers to a straight-chain, branched-chain, or cyclic hydrocarbon, which is saturated. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
In some examples, an alkyl group contains from 1 to 25 carbon atoms, unless a different number of carbon atoms is expressly stated. The term “lower alkyl” refers to an alkyl group having from 1 to 10 carbon atoms (such as from 1 to 6 carbon atoms). This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, and n-amyl. Lower alkyl groups can also be unsubstituted or substituted, where a specific example of a substituted alkyl is 1,1-dimethyl propyl. Particular examples of lower alkyls are methyl, butyl and propyl (including isopropyl).
The term “alkylthio” refers to a group having the structure —SR, where R is alkyl.
“Alkynyl” refers to a straight-chain, branched-chain, or cyclic hydrocarbon group including one or more triple bonds. Alkynyl groups may be unsubstituted or substituted, so the term “alkynyl” is to be understood to include both unsubstituted and substituted alkynyl groups unless clearly indicated otherwise. In some examples, an alkynyl group contains from 2 to 25 carbon atoms. “Lower alkynyl” groups are those that contain from 2 to 10 (such as from 2 to 6) carbon atoms.
The term “amino” refers to a substituent having the structure —NRR′, wherein R and R′ each are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy or thioalkoxy groups. Particular examples of amino groups include “alkylamino” groups (—NHR, where R is alkyl), “dialkylamino” groups (—NRR′, where R and R′ are both alkyl), and “arylamino groups (—NHR, where R is aryl, e.g., substituted or unsubstituted phenyl). Where used alone in reference to a specific compound (such as in the name of a compound or a particular substituent at a particular position in a structure) the term “amino” refers to the group —NH2.
The term “amino-substituted alkyl” refers to an alkyl group substituted with at least one amino group.
“Animal” refers to living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes all animals, including humans, simians, dogs, cats, horses, cows, rodents, etc. Likewise, the term “mammal” includes both human and non-human mammals.
The term “aryl” refers to a polyunsaturated, aromatic moiety that can be a single ring or multiple rings (for example, from 1 to 3 rings), which are fused together or linked covalently. Aryl groups can be unsubstituted or substituted. The term “aryl” also includes “heteroaryl” groups (or rings) that contain at least one heteroatom (such as from 1 to 4) independently selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Aryl and heteroaryl groups also can be fused to a ring of a molecule, typically at adjacent atoms in the ring of the molecule. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, tetrazolyl, benzo[b]furanyl, benzo[b]thienyl, 2,3-dihydrobenzo[1,4]dioxin-6-yl, benzo[1,3]dioxol-5-yl, and 6-quinolyl.
“Azo” refers to a substituent having the structure —N═N—R where R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl (such as substitute or unsubstituted phenyl), or substituted or unsubstituted alkoxy or thioalkoxy groups.
“Carbocycle” (or “carbocyclic”) means a saturated or unsaturated cyclic radical of 3 to 8 ring atoms in which each of the ring atoms are carbon. The carbocyclic ring may be optionally substituted independently with one, two or three substituents selected from alkyl, heteroalkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aliphatic, heteroaliphatic, aryl, aralkyl, heteroaryl, heteroaralkyl, halo, cyano, acyl, acylamino, amino, monosubstituted amino, disubstituted amino, —COOR (where R is hydrogen or alkyl), —XR (where X is O or S(O)n, where n is an integer from 0 to 2, and R is hydrogen, alkyl, haloalkyl, cycloalkyl, aralkyl, aryl, heteroaryl or heteroaralkyl), and —C(O)N(R′)R′′ (where R′ and R′′ are independently selected from hydrogen and alkyl). Representative examples include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl, cycloheptenyl, or cycloheptyl-2, 3, or 4-one, and the like.
“Carbonyl-containing” refers to any substituent containing a carbon-oxygen double bond (C═O), including substituents based on —COR or —RCHO where R is an aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine. Carbonyl-containing groups include, for example, aldehydes, ketones, carboxylic acids, and esters. Alternatively, “carbonyl-containing group” refers to —RC(O)R′ groups wherein R and R′ are independently aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or secondary, tertiary, or quaternary amine. Examples include —COOH, —CH2COOH, —CH2COOCH3, —CH2CONH2, —CH2CON(CH3)2.
“Carboxyl” refers to —COOH substituent or its conjugate base —COO−.
“Carboxylate” refers to any salt, ester, or conjugate base of a carboxylic acid.
A “derivative” is a chemical substance that differs from another chemical substance by one or more functional groups. Preferably, a derivative retains a biological activity of a molecule from which it was derived (such as B1 and/or F10 protein kinase inhibitory activity or poxvirus growth inhibitory activity).
The term “diamine” refers to a substituent having the structure —NR′—(CH2)n—NR′′R′′′, wherein n=1-5 and R′, R′′, and R′′′ each are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy or thioalkoxy groups, for example, each are independently H or substituted or unsubstituted alkyl, such as each being independently H or unsubstituted or substituted lower alkyl. A diamine group can be further substituted, for example, with additional lower alkyl groups.
The term “halogen” refers to fluoro, bromo, chloro, and iodo substituents.
Hybridization: Nucleic acid sequences (such as, oligonucleotides and their analogs) hybridize by hydrogen bonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary bases. Generally, nucleic acid consists of nitrogenous bases that are either pyrimidines (cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between a pyrimidine and a purine, and the bonding of the pyrimidine to the purine is referred to as “base pairing.” More specifically, A will hydrogen bond to T or U, and G will bond to C. “Complementary” refers to the base pairing that occurs between two distinct nucleic acid sequences or two distinct regions of the same nucleic acid sequence. For example, an oligonucleotide (or other nucleic acid sequence) can be complementary to a VPK-encoding nucleic acid.
“Specifically hybridizable” and “specifically complementary” are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between a nucleic acid sequence (such as, an oligonucleotide) and the DNA or RNA target. The oligonucleotide or oligonucleotide analog need not be 100% complementary to its target sequence to be specifically hybridizable. An oligonucleotide or analog is specifically hybridizable when binding of the oligonucleotide or analog to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide or analog to non-target sequences under conditions where specific binding is desired, for example under physiological conditions in the case of in vivo assays or systems. Such binding is referred to as specific hybridization.
Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ and/or Mg++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.
For purposes of the present disclosure, “stringent conditions” encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. “Stringent conditions” may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, “moderate stringency” conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of “medium stringency” are those under which molecules with more than 15% mismatch will not hybridize, and conditions of “high stringency” are those under which sequences with more than 10% mismatch will not hybridize. Conditions of “very high stringency” are those under which sequences with more than 6% mismatch will not hybridize. Exemplary hybridization conditions are provided elsewhere in this specification.
The term “nitro” refers to a substituent having the structure —NO2.
Nucleic acid molecule: This term refers to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A polynucleotide may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
Nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendent moieties (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations.
The term “phosphonate” refers to phosphonates of the formula —PO3R wherein R represents hydrogen, a cationic counterion, or an aliphatic (such as a lower aliphatic or an alkyl, e.g., lower alkyl) or aromatic moiety.
A “poxvirus” is any virus belonging to the family Poxyiridae. Poxyiridae are characterized by, at least, a relatively large, double-stranded DNA genome (ranging from approximately 130 to 400 kbp). Virions are enveloped, slightly pleomorphic, ovoid, or brick shaped (approximately 140-260 nm in diameter and 220-450 nm long). Virions are composed of an external coat containing lipid and tubular or globular protein structures enclosing one or two lateral bodies and a core, which contains the genome. Particular poxviruses may belong to the chordopoxvirinae or entomopoxvirinae subfamily, which infect vertebrate or insect hosts, respectively. A poxvirus of the chordopoxvirinae subfamily may further belong to the genus Orthopoxvirus (including, e.g., monkeypox virus, vaccinia virus, buffalopoxvirus, camelpox virus, cowpox virus, elephantpox virus, variola virus (such as variola major and/or variola minor viruses), volepox virus, ectromelia virus, raccoonpox virus, skunkpox virus, or taterapox virus), Parapoxvirus (including, e.g., bovine papular stomatitis virus, Orf virus, psuedocowpox virus, sealpox virus, or Auzduk disease virus), Avipoxvirus (including, e.g., fowlpox virus), Capripoxvirus (including, e.g., sheeppox virus, lumpy skin disease virus, or goatpox virus), Leporipoxvirus (including, e.g., myxoma virus, or Shope fibroma virus), Suipoxvirus (including, e.g., swinepox virus), Mollusciposvirus (including, e.g., molluscum contagiosum virus), or Yatapoxvirus (including, e.g., tanapox virus or Yaba monkey tumor virus). Viruses of the Othropoxvirus and Parapoxvirus genera can be further characterized as zoonotic (including, e.g., monkeypox virus, vaccinia virus, buffalopoxvirus, camelpox virus, cowpox virus, elephantpox virus, bovine papular stomatitis virus, Orf virus, psuedocowpox virus, or sealpox virus) or nonzoonotic (including, e.g., variola virus, volepox virus, ectromelia virus, raccoonpox virus, skunkpox virus, taterapox virus, or Auzduk disease virus). Zoonotic viruses can infect multiple species of hosts (e.g., humans and animals), while nonzoonotic viruses are believed to infect only a single host species (e.g., humans, fowl, or monkey, etc.). In some examples, a poxvirus is an Orthopoxvirus. In more specific examples, a poxvirus is vaccinia virus or variola virus. The complete genomes (including cross-references to individual genes included therein and proteins encoded thereby) of over 25 poxviruses are known and publicly available (see, for instance, GenBank Accession Nos. M35027, U94848, AF095689, L22579, X69198, Y16780, AF380138, AF012825, AY009089, AF438165, AF482758, AF170726, AF170722, AF198100, AF325528, AY077835, AY077836, AY077832, AY077833, AY077834, AF410153, U60315, AJ293568, AF063866, and/or AF250284).
The term “purified” does not require absolute or even substantial purity; rather, it is intended as a relative term. Thus, for example, a purified preparation is one in which a desired component (e.g., VPK inhibitor or VPK) is more enriched than it was in a preceding environment (e.g., when in a laboratory production vessel). A desired component (e.g., VPK inhibitor or VPK) is purified, for example, when at least about 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% of a sample by weight is composed of the desired component. Purity of a compound may be determined, for example, by high pressure liquid chromatography (HPLC) or other conventional methods.
Compounds described herein may be obtained in a purified form or purified by any of the means known in the art, including silica gel and/or alumina chromatography. See, e.g., Introduction to Modern Liquid Chromatography, 2nd Edition, ed. by Snyder and Kirkland, New York: John Wiley and Sons, 1979; and Thin Layer Chromatography, ed. by Stahl, New York: Springer-Verlag, 1969.
Sequence identity: The similarity between two nucleic acid sequences or between two amino acid sequences is expressed in terms of the level of sequence identity shared between the sequences. Sequence identity is typically expressed in terms of percentage identity; the higher the percentage, the more similar the two sequences.
Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444, 1988; Higgins and Sharp, Gene 73: 237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucleic Acids Research 16: 10881-10890, 1988; Huang, et al., Computer Applications in the Biosciences 8: 155-165, 1992; Pearson et al., Methods in Molecular Biology 24: 307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett., 174: 247-250, 1999. Altschul et al. present a detailed consideration of sequence-alignment methods and homology calculations (J. Mol. Biol. 215: 403-410, 1990).
The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST, Altschul et al., J. Mol. Biol. 215: 403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence-analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the internet under the help section for BLAST.
For comparisons of amino acid sequences of greater than about 30 amino acids, the “Blast 2 sequences” function of the BLAST (Blastp) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=5]; cost to extend a gap [default=2]; penalty for a mismatch [default=−3]; reward for a match [default=1]; expectation value (E) [default=10.0]; word size [default=3]; number of one-line descriptions (V) [default=100]; number of alignments to show (B) [default=100]). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties).
For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST (Blastn) program is employed using the default BLOSUM62 matrix set to default parameters (cost to open a gap [default=11]; cost to extend a gap [default=1]; expectation value (E) [default=10.0]; word size [default=11]; number of one-line descriptions. (V) [default=100]; number of alignments to show (B) [default=100]).
A “sulfonate” is a salt, ester, or anion of a sulfonic acid (RSO2OH).
The term “sulfonyl” refers to a substituent including the bivalent group —SO2— or, more typically, —SO2R, where R is substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy or thioalkoxy. Sulfonyl groups include “alkylsulfonyl” groups having the structure —SO2R, where R is unsubstituted or substituted alkyl. Similarly, “lower alkylsulfonyl” refers to —SO2R, wherein R is unsubstituted or substituted lower alkyl. “Arylsulfonyl” refers to groups having the structure —SO2R, where R is unsubstituted or substituted aryl (such as phenyl). A sulfonyl group optionally can be substituted with a variety of substituents to form different sulfonyl groups, including, for example, sulfonic acids, sulfonamides, sulfonate esters and sulfones.
Treating or treatment: With respect to disease (such as smallpox), either term includes (i) preventing the disease, e.g., causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (ii) inhibiting the disease, e.g., arresting the development of the disease or its clinical symptoms, or (iii) relieving the disease, e.g., causing regression of the disease or its clinical symptoms.
A “viral protein kinase” is a protein kinase encoded by a viral nucleic acid (such as a viral genome or a gene isolated from a viral genome). In particular examples, a viral protein kinase is expressed by a poxvirus genome (such as an Orthopoxvirus, like variola virus or vaccinia virus). As is commonly known, a protein kinase is an enzyme that modifies (e.g., phosphorylates) a target protein (also referred to as a substrate) by chemically adding one or more phosphate groups to the target protein. The chemical activity of a kinase involves removing a phosphate group from ATP and covalently attaching the phosphate group to free hydroxyl groups of serine(s), threonine(s) and/or tyrosine(s) present in the target protein. Kinases that phosphorylate both serine and threonine are referred to as “serine/threonine protein kinases”; other kinases act on tyrosine and are referred to as “tyrosine kinases.” Some kinases can phosphorylate serine, threonine and tyrosine (on the same or different substrates) and are referred to as “dual-specificity kinases.” Certain examples herein involve dual-specificity kinases viral protein kinases (such as, B1 kinase and/or F10 kinase and/or variants (such as homologs) of either). In particular examples (e.g., with particular substrates), a viral protein kinase (such as, B1 kinase and/or F10 kinase and/or variants (such as homologs) of either) can act as either a serine/threonine kinase or a tyrosine kinase. Kinase-dependent phosphorylation often results in a functional change of the target protein, for example, by changing enzyme activity, cellular location or association with other proteins.
Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed “isomers”. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers”. Stereoisomers that are not mirror images of one another are termed “diastereomers” and those that are non-superimposable mirror images of each other are termed “enantiomers.” When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (−) isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a “racemic mixture.”
The compounds described herein may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see, e.g., March, Advanced Organic Chemistry, 4th edition, New York: John Wiley and Sons, 1992, Chapter 4). The symbol  in a chemical structure represents a double bond having either a cis or trans orientation.
Some exemplary compounds described herein can be obtained from the chemical repository of the National Institutes of Health, National Cancer Institute, Developmental Therapeutics Program, Structural Diversity Set. The compounds in the Structural Diversity Set are each assigned an identifier of 3 to 7 numbers and a letter, all of which is sometimes preceded by “NSC”; thus, compounds herein named NSC#####A, NSC####-A, ####A, or ####-A (where “####” can be from 3 to 7 numbers and “A” is any letter) should be understood to be compounds from the Structural Diversity Set.
It is further to be understood that any molecular weight or molecular mass values are approximate and are provided only for description. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control.
Except as otherwise noted, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978.
II. Viral Protein Kinases
Virus genomes range in size from approximately 3.2 kilobase pairs (kbp) to approximately 800 kbp. The larger viral genomes encode a multiplicity of viral proteins. Some such proteins are essential for viral replication and, therefore, provide useful targets for identification and/or design of anti-viral drugs.
Protein kinases are among the proteins encoded by some viruses, such as poxviruses, herpes viruses (e.g., cytomegalovirus), or baculoviruses. As a class of proteins, protein kinases often have profound functional effects, and viral protein kinases are no exception. Normal viral function is dependent upon the activity(ies) of various viral protein kinases (see, e.g., Prichard et al., J. Virol., 73: 5663-5670, 1999; Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91(16): 7653-7657, 1994; Rempel et al., J. Virol., 64(2): 574-583, 1990). For example, poxviruses have two essential protein kinases, which are most studied in vaccinia virus (an Orthopoxvirus). The vaccinia virus F10L kinase (an exemplary F10 kinase) is encapsidated in the virion and plays an important role in virion morphogenesis (Traktman et al., J. Virol., 69: 6581-6587, 1995; Wang and Shuman, J. Virol., 69: 6376-6388, 1995). The vaccinia virus B1R protein kinase (an exemplary B1 kinase) is expressed early in infection, is found in the virosomes, and is also packaged into virions. Temperature-sensitive mutations that map to the B1R gene produce virus that cannot replicate its DNA at the restrictive temperature (Rempel and Traktman, J. Virol., 66: 4413-4426, 1992).
An exemplary B1 kinase, the vaccinia virus B1R gene product, phosphorylates casein and histones in vitro (Banham and Smith, Virol., 191: 803-812, 1992; Lin et al., J. Virol., 66: 2717-2723, 1992; Rempel and Traktman, J. Virol., 66: 4413-4426, 1992), ribosomal proteins Sa and S2 in vivo and in vitro (Banham et al., FEBS Lett., 321: 27-31, 1993), and vaccinia virus protein H5R in vivo and in vitro (Beaud et al., J. Virol., 69: 1819-1826, 1995). Typically, the foregoing phosphorylations are on serine and/or threonine. Casein and histones are readily available commercially and, at least for that reason, are advantageous substrates for use in in vitro kinase activity assays. The phosphorylation of the ribosomal proteins and H5R has been implicated in normal poxvirus (e.g., vaccinia virus) growth and/or infectivity (Punjabi and Traktman, J. Virol., 79: 2171-2190, 2005).
An exemplary F10 kinase, the vaccinia virus F10 kinase, phosphorylates casein, phosvitin and myelin basic protein in vitro (Lin and Broyles, Proc. Natl. Acad. Sci. USA, 91: 7653-7657, 1994). Due to the commercial availability of these substrates, they are advantageous substrates for use in in vitro kinase activity assays. An exemplary vaccinia F10 kinase also phosphorylates vaccinia proteins A30 (in infected cells and in vitro) and G7 (in infected cells) (Mercer and Traktman, J. Virol., 79: 7146-7161, 2005). Phosphorylations of casein, phosvitin, myelin basic protein, A30 and G7 substrates by a F10 kinase, typically, are on serine and threonine. As an example, a vaccinia virus F10 kinase also phosphorylates vaccinia protein A17 on tyrosine, at least, during infection in cells (Derrien et al., J. Virol., 73: 7287-7296, 1999). Phosphorylation of A17 has been implicated in normal poxvirus (e.g., vaccinia virus) growth.
The disclosure herein of small molecules that inhibit B1 and/or F10 kinases makes possible, for example, methods of inhibiting viral protein kinases (including, B1 and/or F10 kinases and/or homologs and/or variants of either) and methods of inhibiting the growth of viruses expressing such viral protein kinases (including, poxviruses, like variola virus).
A. VPK Variants and Homologs
This disclosure provides methods of inhibiting viral protein kinases (VPKs), including variants (such as homologs) of a B1 protein kinase or a F10 protein kinase. Amino acid sequences of representative B1 and F10 protein kinases are shown in SEQ ID NO: 2 and 8 (also in FIGS. 7 and 8), and SEQ ID NOs: 4, 6, and 10 (also in FIGS. 5 and 6), respectively. Variants of a B1 protein kinase or a F10 protein kinase include polypeptides that differ in amino acid sequence from described (or otherwise publicly known) B1 or F10 protein kinase sequences, but that substantially retain a wild-type function of a prototypical B1 or F10 kinase.
VPK variants (including VPK homologs) contemplated by this disclosure have at least one function of a prototypical B1 kinase or F10 kinase. For example, a VPK variant will phosphorylate (e.g., serine and threonine and/or tyrosine residues of) a target protein (such as, casein, histones, ribosomal proteins Sa or S2, vaccinia virus protein H5R, phosvitin, myelin basic protein, and/or vaccinia proteins A30 or G7) under conditions well known in the art or such as those described in Example 1. A virus having a knock-out or knock-down mutation of a VPK (e.g., B1 or F10 kinase) variant will not replicate as efficiently as a virus carrying a non-mutated form of the VPK variant; provided that the viral genome does not also include one or more genes having redundant VPK (e.g., B1 and/or F10 kinase) function(s). In some examples, a B1 kinase variant will have the ability to in vivo phosphorylate (e.g., serine or threonine residues of) at least one of ribosomal proteins Sa or S2 and/or vaccinia virus protein H5R. In other examples, a F10 kinase variant will have the ability to in vivo phosphorylate (e.g., serine or threonine residues of) at least one of vaccinia proteins A30 or G7; and/or the ability to in vivo phosphorylate vaccinia virus protein A17 (e.g., on a tyrosine residue). Functional assays useful for characterizing a VPK (such as B1 or F10 kinase) variant are provided below.
FIGS. 5, 6, 7 and 8 provide structural information from which functional correlates can be derived. FIG. 5 is an alignment of F10 kinases and its homologs from a variety of Orthopoxviruses. As shown in FIG. 5, Orthopoxvirus F10 homologs are highly conserved and are likely to have similar function and be similarly inhibited by disclosed VPK inhibitors. FIG. 6 is an alignment of F10 kinases and its homologs from a variety of poxviruses. This alignment teaches that F10 kinases and its homologs are also conserved across poxviruses. Moreover, FIG. 6 shows identical (*) and conservatively substituted (:) residues across the range of poxvirus species presented. Modification of residues that are conserved or conservatively substituted across related species has a higher probability of adversely affecting a function of the protein; hence, in the making of functional F10 variants, such residues indicated in FIG. 6 are preferably avoided. FIGS. 6 and 7 teach analogous information with respect to B1 kinase and its homologs (and variants).
In some embodiments, VPK (such as B1 or F10 kinase) variants include polypeptides that share at least 40% amino acid sequence identity with a B1 or F10 kinase polypeptide sequence provided herein (e.g., SEQ ID NO: 2, 4, 6, 8, or 10); for example, some VPK (such as B1 or F10 kinase) variants will share at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with a sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
With the description herein of prototypical B1 and F10 kinase amino acid sequences and corresponding nucleic acid sequences, VPK (e.g., B1 or F10 kinase) variants are easily obtained by conventional molecular methods. VPK (e.g., B1 or F10 kinase) variants can be naturally occurring (e.g., homologs) or produced by any method known in the art for making polypeptide variants. In some embodiments, a VPK (e.g., B1 or F10 kinase) variant is produced by manipulation of a described (or other publicly available) VPK-encoding nucleotide sequence (e.g., SEQ ID NOs: 1, 3, 5, 7, or 9) using standard procedures, including without limitation the commonly known techniques of site-directed mutagenesis or PCR. Naturally occurring VPK (e.g., B1 or F10 kinase) variants can be isolated using any of a myriad of protein purification techniques known in the art (for example, Scopes, Protein Purification. Principles and Practice, 3rd Edition, New York: Springer-Verlag, 1994; Protein Purification Techniques, 2nd Edition, ed. by Simon Roe, New York: Oxford University Press, 2001; Membrane Protein Purification and Crystallization, 2nd Edition, ed. by Hunte et al., San Diego: Academic Press, 2003). A nucleic acid sequence that encodes all or part of a VPK (e.g., B1 or F10 kinase) variant can be readily determined simply by applying a genetic code (such as a poxvirus genetic code) to the respective portion of the variant's amino acid sequence.
In some embodiments, VPK (e.g., B1 or F10 kinase) variants involve the substitution of one or several amino acids for amino acids having similar biochemical properties (so-called conservative substitutions). Conservative amino acid substitutions are likely to have minimal impact on the activity of the resultant protein. Further information about conservative substitutions can be found, for instance, in Ben Bassat et al. (J. Bacteriol., 169: 751-757, 1987), O'Regan et al. (Gene, 77: 237-251, 1989), Sahin-Toth et al. (Protein Sci., 3: 240-247, 1994), Hochuli et al. (Bio/Technology, 6: 1321-1325, 1988) and in widely used textbooks of genetics and molecular biology. In some examples, VPK (e.g., B1 or F10 kinase) variants can have no more than 3, 5, 10, 15, 20, 25, 30, 40, or 50 conservative amino acid changes compared to SEQ ID NO: 2, 4, 6, 8, or 10, as applicable. The following table shows exemplary conservative amino acid substitutions:











Original Residue
Conservative Substitutions







Ala
Ser


Arg
Lys


Asn
Gln; His


Asp
Glu


Cys
Ser


Gln
Asn


Glu
Asp


Gly
Pro


His
Asn; Gln


Ile
Leu; Val


Leu
Ile; Val


Lys
Arg; Gln; Glu


Met
Leu; Ile


Phe
Met; Leu; Tyr


Ser
Thr


Thr
Ser


Trp
Tyr


Tyr
Trp; Phe


Val
Ile; Leu









Nucleotide sequences encoding VPK (e.g., B1 or F10 kinase) variants are comprehended by this disclosure; for example, to express a corresponding variant for use in a disclosed method. Such nucleotide variants may be naturally occurring (such as orthologs from other organism) or produced using commonly known techniques, including without limitation site-directed mutagenesis. Standard techniques for DNA mutagenesis are provided, for instance, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, Ch. 15). In addition, numerous commercially available kits are available to perform DNA mutagenesis (see, for example, QuikchangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneTailorTM Site-Directed Mutagenesis System (Invitrogen); GPSTM-M Mutagenesis System (New England Biolabs, DiversifyTM PCR Random Mutagenesis Kit (BD Biosciences Clontech); Mutation Generation System (MJ Research); ExsiteTM PCR-Based Site-Directed Mutagenesis Kit (Stratagene); GeneMorphTM PCR Mutagenesis Kit (Stratagene); or LA PCR Mutagenesis Kit (Takara Mirus Bio)).
Other conventional methods for isolating a nucleic acid sequence encoding a VPK (e.g., B1 or F10 kinase) variant include library screening (including nucleic acid libraries or expression libraries) or PCR. Direct PCR amplification may be performed on genomic libraries prepared from a virus having a B1 or F10 kinase homolog (such as a poxvirus, an Orthopoxvirus, or other vaccinia or variola virus strains), or RT-PCR may be performed using RNA extracted from cells infected with such viruses using standard methods. Conventional hybridization techniques (like nucleic acid library screening) involve the use of a labeled probe derived from a B1 kinase- or F10 kinase-encoding nucleic acid sequence, which probe is hybridized to a nucleic acid (e.g., genomic DNA) library prepared using a virus having a B1 or F10 kinase homolog. A hybridizing colony or plaque (depending on the type of library used) is purified and the cloned sequence contained in that colony or plaque isolated and characterized.
In some embodiments, a nucleotide sequence encoding a VPK (e.g., B1 or F10 kinase) variant (including VPK homologs) shares at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% nucleotide sequence identity with a described (or otherwise publicly available) VPK-encoding (e.g., B1 kinase- or F10 kinase-encoding) nucleic acid sequence (including SEQ ID NO: 1, 3, 5, 7, or 9). Alternatively, related nucleic acid molecules can have no more than 3, 5, 10, 20, 50, 75, or 100 nucleic acid changes compared to SEQ ID NO: 1, 3, 5, 7, or 9.
In one embodiment, VPK (e.g., B1 or F10 kinase) nucleic acid variants may differ from the disclosed sequences by alteration of the coding region to fit the codon usage bias of a particular organism, for example, an organism into which the nucleic acid molecule is to be introduced. In other embodiments, VPK (e.g., B1 or F10 kinase) variants are derived by taking advantage of the degeneracy of the genetic code to alter the VPK (e.g., B1 kinase or F10 kinase) coding sequence. In these embodiments, the variant nucleotide sequence may be substantially different from a prototypic B1 kinase- or F10 kinase-encoding nucleic acid sequence (e.g., SEQ ID NO: 1, 3, 5, 7, or 9) and, nevertheless, encode a protein having an amino acid sequence substantially similar (if not identical) to a disclosed B1 kinase or F10 kinase. For example, because of redundancy in the genetic code, any one of four nucleotide codons encode alanine (i.e., GCT, GCG, GCC or GCA); accordingly, the sequence encoding any alanine residue within a VPK (e.g., B1 or F10 kinase) polypeptide could be changed to any of these alternative codons without affecting the amino acid composition or characteristics of the encoded protein. Analogous redundancies are well known for each amino acid. The genetic codes for a variety of organisms are publicly available on the National Center for Biotechnology Information (NCBI) Taxonomy website.
An alternative indication that two nucleic acid molecules are closely related is that the two molecules hybridize to each other. In certain embodiments, VPK (e.g., B1 or F10 kinase) nucleic acid variants hybridize to a disclosed VPK (e.g., B1 or F10 kinase) nucleic acid sequence (or fragments thereof), for example, under low stringency, high stringency, or very high stringency conditions. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization, although wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, chapters 9 and 11.
The following are representative hybridization conditions and are not meant to be limiting.












Very High Stringency (detects sequences that share


about 90% sequence identity)








Hybridization:
5x SSC at 65° C. for 16 hours


Wash twice:
2x SSC at room temperature (RT) for 15 minutes



each


Wash twice:
0.5x SSC at 65° C. for 20 minutes each







High Stringency (detects sequences that share


about 80% sequence identity or greater)








Hybridization:
5x-6x SSC at 65° C.-70° C. for 16-20 hours


Wash twice:
2x SSC at RT for 5-20 minutes each


Wash twice:
1x SSC at 55° C.-70° C. for 30 minutes each







Low Stringency (detects sequences that share


greater than about 50% sequence identity)








Hybridization:
6x SSC at RT to 55° C. for 16-20 hours


Wash at least twice:
2x-3x SSC at RT to 55° C. for 20-30 minutes



each.









The nucleic acid sequence of a variant, then, can be isolated using conventional methods, such as library screening (including nucleic acid libraries or expression libraries) or PCR. Direct PCR amplification may be performed on cDNA or genomic libraries prepared from a virus having a B1 or F10 kinase homolog (such as a poxvirus, an Orthopoxvirus, or other vaccinia or variola virus strains), or RT-PCR may be performed using RNA extracted from cells infected with such viruses using standard methods. For conventional hybridization techniques, a labeled probe derived from a B1 kinase- or F10 kinase-encoding nucleic acid sequence may be hybridized to a cDNA or genomic library prepared from a virus having a B1 or F10 kinase homolog. The hybridization probe is preferably conjugated with a detectable label such as a radioactive label. A hybridization signal may be detected using methods known in the art. The hybridizing colony or plaque (depending on the type of library used) is purified and the cloned sequence contained in that colony or plaque isolated and characterized.
B. B1 Viral Protein Kinase
The amino acid sequences of prototypical B1 (or B1-like) VPKs and the nucleic acid sequences encoding the same are well known. Exemplary sequences for vaccinia virus (Copenhagen strain) B1 kinase are provided in SEQ ID NOs: 1 (nucleic acid sequence) and 2 (amino acid sequence). Exemplary sequences for variola major virus (India-1967 strain) B1 kinase are provided in SEQ ID NOs: 7 (nucleic acid sequence) and 8 (amino acid sequence). Routine comparison (e.g., using BLASTP software, for amino acid sequence, or BLASTN software, for nucleic acid sequences) of these (or other) prototypical B1 (or B1-like) sequences against publicly available databases (such as GenBank non-redundant and/or patent databases) reveals numerous B1 protein kinase homologs. Several exemplary B1 protein kinase homologs and information for obtaining the known sequences are provided in the following table:







TABLE 1







B1 Protein Kinase Homologs1








Source
GenBank Accession No. (Protein Name; Viral Strain (as applicable))





Vaccinia virus
NP_063857.1 (B1R; strain Copenhagen; SEQ ID NO: 2), AAA48194.1



(B1R; strain Copenhagen), AAA47963.1 (B1 30 kDa protein; strain



Western Reserve; SEQ ID NO: 58); AAB96545.1 (protein kinase, similar



to vaccinia Copenhagen B1R; strain Ankara; SEQ ID NO: 60);



AAT10565.1 (serine/threonine kinase, similar to Vaccinia Copenhagen



B1R; strain Acambis 3000 Modified Virus Ankara (MVA); SEQ ID



NO: 62); AAF34067.1 (TB1R; strain Tian Tan; SEQ ID NO: 64);



AAO89462.1 (ser/thr kinase, similar to VACCP-B1R; strain Western



Reserve); BAA00519.1 (B1R; strain Western Reserve); BAA01831.1



(34.2K protein; strain Western Reserve); AAW23907.1 (putative ser/thr



protein kinase; strain LC16mO; SEQ ID NO: 66); AAW23625.1 (putative



ser/thr protein kinase; strain LC16m8; SEQ ID NO: 68)


Variola major
NP_042213.1 (B1R; SEQ ID NO: 8); CAA49110.1 (B1R), AAA60910.1


virus
(homolog of vaccinia virus CDS B1R; SEQ ID NO: 70)


Variola minor
CAB54770.1 (H1R protein; SEQ ID NO: 72); H72171 (H1R protein -


virus
strain Garcia-1966);


Cowpox virus
CAD90727.1 (B1R protein; SEQ ID NO: 74); AAM13635.1 (CPXV196



protein; SEQ ID NO: 76); NP_619977.1 (CPXV196 protein)


Rabbitpox virus
AAS49878.1(RPXV165; SEQ ID NO: 78)


Camelpox virus
AAL73885.1 (CMLV178; SEQ ID NO: 80); AAG37677.1 (CMP175R;



SEQ ID NO: 82); NP_570568.1 (CMLV178)


Ectromelia virus
AAM92457.1 (EVM152; SEQ ID NO: 84); NP_671671.1 (EVM152);



AAC99565.1 (C6R); NP_671679.1 (C6R)


Monkeypox virus
AAU01366.1 (MPXV-WRAIR156; SEQ ID NO: 86); NP_536591.1



(B3R; SEQ ID NO: 88); AAL40622.1 (B3R)


lumpy skin disease
AAN02707.1 (SEQ ID NO: 90); AAK85100.1 (LSDV139); NP_150573.1


virus
(LSDV139); AAN02865.1 (putative Ser/Thr protein kinase)


Sheeppox virus
NP_659709.1 (SEQ ID NO: 92)


Mule deer poxvirus
YP_227529.1 (SEQ ID NO: 94)


Yaba monkey tumor
AAR07494.1 (142R; SEQ ID NO: 96); NP_938393.1 (142R)


virus


Yaba-like disease
CAC21380.1 (142R protein; SEQ ID NO: 98); NP_073527.1 (142R


virus
protein)


Swinepox virus
AAL69876.1 (SPV137; SEQ ID NO: 100); NP_570297.1 (SPV137)


Myxoma virus
AAF15030.1 (m142R; SEQ ID NO: 102); NP_051856.1 (m142R)


Rabbit fibroma
AAF18022.1 (gp142R; SEQ ID NO: 104); NP_052028.1 (gp142R)


virus


Fowlpox virus
CAE52750.1 (B1R-like protein; SEQ ID NO: 106); AAF44556.1



(FPV212); NP_039175.1 (FPV212); CAE52761.1 (B1R-like protein);



AAF44570.1 (FPV226); NP_039189.1 (FPV226)


Canarypox virus
NP_955309.1 (CNPV286; SEQ ID NO: 108); AAR83632.1 (CNPV286);



NP_955322.1 (CNPV299); AAR83645.1 (CNPV299)


Mus musculus
AAH16676.1 (Vaccinia related kinase 1, isoform c); NP_001025015.1



(vaccinia related kinase 1 isoform c); NP_035835.1 (vaccinia related



kinase 1 isoform a); NP_001025014.1 (vaccinia related kinase 1 isoform



b); AAC29496.1 (serine/threonine protein kinase 51PK(S)); CAI52021.1



(vaccinia related kinase 2); AAN64922.1 (VRK2); NP_081536.1



(vaccinia related kinase 2)


Gallus gallus
NP_001006485.1 (vaccinia related kinase 1)


Rattus norvegicus
XP_576097.1 (vaccinia related kinase 1 (predicted)); NP_001012194.1



(vaccinia related kinase 1 (predicted))


Homo sapiens
NP_003375.1 (vaccinia related kinase 1); AAH36434.1 (VRK2 protein);



NP_006287.2 (vaccinia related kinase 2); AAO73052.1 (vaccinia related



kinase 2 isoform 6); CAD54446.2 (vaccinia-related kinase 2);



AAO73048.1 (vaccinia related kinase 2 isoform 2); AAH27854.1



(Vaccinia related kinase 2); AAO73047.1 (vaccinia related kinase 2



isoform 1); AAO73049.1 (vaccinia related kinase 2 isoform 3)


Pan troglodytes
XP_510157.1 (Vaccinia-related kinase-1)





1Results are selected from “Sequences producing significant alignments” obtained by BLASTP 2.2.12 (Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997) search of GenBank non-redundant database using default parameters and the amino acid sequence of vaccinia virus (Copenhagen strain) B1 kinase as set forth in SEQ ID NO: 2. A nucleic acid sequence corresponding to any of the amino acid sequences referenced above can be obtained by following the “CDS” link provided in each of the amino acid sequence records.






C. F10 Viral Protein Kinase

The amino acid sequences of prototypical F10 (or F10-like) VPKs and the nucleic acid sequences encoding the same are well known. Exemplary sequences for vaccinia virus (Copenhagen and Ankara strains) F10 kinase are provided in SEQ ID NOs: 3 and 5 (nucleic acid sequence) and SEQ ID NOs: 4 and 6 (amino acid sequence). Exemplary sequences for variola major virus (India-1967 strain) F10 kinase are provided in SEQ ID NO: 9 (nucleic acid sequence) and 10 (amino acid sequence). Routine comparison (e.g., using BLASTP software, for amino acid sequence, or BLASTN software, for nucleic acid sequences) of these (or other) prototypical F10 (or F10-like) sequences against publicly available databases (such as GenBank non-redundant and/or patent databases) reveals numerous F10 protein kinase homologs. Several exemplary F10 kinase homologs and information for obtaining the known sequences are provided in the following table:







TABLE 2







F10 Viral Protein Kinase Homologs1








Source
GenBank Accession No. (Protein Name; Viral Strain (as applicable))





Vaccinia virus
NP_063689.1 (putative ser/thr protein kinase; strain Copenhagen; SEQ ID



NO: 4); AAA48026.1 (F10L; putative; strain Copenhagen); AAB96420.1



(serine/threonine protein kinase 2; strain Ankara); AAT10437.1



(serine/threonine kinase; strain Acambis 3000 Modified Virus Ankara



(MVA)); AAW23730.1 (putative ser/thr protein kinase; strain LC16mO);



AAW23448.1 (putative ser/thr protein kinase; strain LC16m8);



AAO89328.1 (ser/thr kinase; strain Western Reserve); AAA83263.1



(protein kinase 2; strain Western Reserve); AAA48288.1 (F8; strain



LIVP); AAF33901.1 (TF10L; strain Tian Tan)


Rabbitpox virus
AAS49751.1 (RPXV038; SEQ ID NO: 18)


Cowpox virus
CAD90598.1 (G10L protein; SEQ ID NO: 20); AAM13503.1 (CPXV057



protein); NP_619845.1 (CPXV057 protein)


Ectromelia virus
AAM92337.1 (EVM033; SEQ ID NO: 24); NP_671551.1 (EVM033)


Monkeypox virus
AAU01246.1 (MPXV-WRAIR036; SEQ ID NO: 22); NP_536469.1



(C16L); AAL40500.1 (C16L)


Camelpox virus
AAL73752.1 (CMLV045; SEQ ID NO: 26); AAG37505.1 (CMP45L);



NP_570435.1 (CMLV045)


Variola major
AAA69445.1 (C14L; SEQ ID NO: 28); AAA69339.1 (C14L);


virus
AAA60782.1 (homolog of vaccinia virus CDS F10L); NP_042078.1



(C14L); CAA48975.1 (C14L); AAB29628.1 (C14L product; variola virus



VAR, India-1967)


Variola minor
CAB54634.1 (E10L protein; SEQ ID NO: 30); AAA69380.1 (E10L)


virus


Mule deer poxvirus
YP_227408.1 (Serine/threonine protein kinase; SEQ ID NO: 32)


Yaba monkey tumor
AAR07382.1 (25L; SEQ ID NO: 34); NP_938281.1 (25L)


virus


Yaba-like disease virus
CAC21263.1 (25L protein; SEQ ID NO: 36); NP_073410.1 (25L protein)


Swinepox virus
AAL69761.1 (SPV022 putative serine/threonine protein kinase; SEQ ID



NO: 38); AAC37851.1 (C20L); NP_570182.1 (SPV022 putative



serine/threonine protein kinase)


lumpy skin disease
AAN02750.1 (putative Ser/Thr protein kinase; SEQ ID NO: 40);


virus
AAN02592.1 putative Ser/Thr protein kinase; AAK84986.1 (LSDV025



putative ser/thr protein kinase; NP_150459.1 (LSDV025 putative ser/thr



protein kinase); AAK43565.1 (protein kinase)


Sheeppox virus
NP_659598.1 (Ser/Thr protein kinase; SEQ ID NO: 42)


Rabbit fibroma
AAF17904.1 (gp020L; SEQ ID NO: 44); NP_051909.1 (gp020L)


virus


Myxoma virus
AAF14908.1 (m20L; SEQ ID NO: 46); NP_051734.1 (m20L)


Molluscum
AAC55145.1 (MC017L; SEQ ID NO: 48); AAB57937.1 (similar to


contagiosum
variola C14L and vaccinia F10L); NP_043968.1 (MC017L); AAB49658.1



(similar to Vaccina virus protein kinase F10L); T30619 (probable



serine/threonine-specific protein kinase 17L); AAA97934.1 (protein



kinase 2 homolog)


Orf virus
AAR98225.1 (ORF130 putative serine/threonine protein kinase; SEQ ID



NO: 50); AAO31700.1 (vaccinia virus F10L-like protein); NP_957907.1



(ORF130 putative serine/threonine protein kinase); AAR98355.1



(ORF130 putative serine/threonine protein kinase)


Bovine papular
NP_958038.1 (ORF130 protein kinase; SEQ ID NO: 52); AAR98486.1


stomatitis virus
(ORF130 protein kinase); AAO31708.1 (vaccinia virus F10L-like protein)


Fowlpox virus
CAE52652.1 (virus assembly protein F10L orthologue; SEQ ID NO: 54);



AAF44455.1 (ORF FPV111 Serine/threonine protein kinase);



NP_039074.1 (ORF FPV111 Serine/threonine protein kinase)


Canarypox virus
NP_955161.1 (CNPV138 putative serine/threonine protein kinase; SEQ



ID NO: 56); AAR83484.1 (CNPV138 putative serine/threonine protein



kinase)


Vertebrates
No significant homology to protein sequences in GenBank non-redundant



database.





1Results selected from “Sequences producing significant alignments” obtained by BLASTP 2.2.12 (Altschul et al., Nucleic Acids Res., 25: 3389-3402, 1997) search of GenBank non-redundant database using default parameters and the amino acid sequence of vaccinia virus (Copenhagen strain) F10 kinase as set forth in SEQ ID NO: 4. A nucleic acid sequence corresponding to any of the amino acid sequences referenced above can be obtained by following the “CDS” link provided in each of the amino acid sequence records.






III. VPK Inhibitors

One aspect of the disclosure pertains to compounds that herein are identified as inhibitors of VPK activity and/or viral (e.g., poxvirus) growth. Among other things, these compounds can be used to treat viral (such as poxvirus) infection, such as smallpox and a variety of other human and veterinary diseases. Pharmaceutically acceptable salts and stereoisomers of the compounds also are contemplated in some embodiments.
In the structures that follow, all valency requirements are understood to be satisfied. Thus, for example, carbon atoms have four bonds to other atoms, even if all such bonds are not shown. Where all four bonds to a carbon atom are not shown, additional bonds to hydrogen atoms are implied.
Disclosed viral protein kinase inhibitors are derivatives of tetralin, anthraquinone, naphthylamine, tri-amino-pyrimidine, xanthen-3-one, and/or cinnamic acid. Exemplary compounds are provided throughout the disclosure. Some representative examples are shown in the following table.











Structure
Name(s)







Exemplary B1 Inhibitors1







NSC270718R


 






NSC117285R IUPAC: 2-hydroxy- 4-(2,4,6-triaminopyrimidin-5-yl)- diazenyl-benzoic acid


 






NSC170008Y IUPAC: 2-acetyl- 7,8-bis(dihydroxymethylidene)- 3-ethyl-9-hydroxy-anthracene- 1,4,6,10-tetrone (Depositor-supplied Name: 7-acetyl-6-ethyl-9,10-dihydro- 3,5,8-trihydroxy-9,10-dioxo- 1,2-anthracenedicarboxylic acid)


 






NSC306711P


 






NSC119913X IUPAC: 7-methyl-6-(4,5,6,-trihydroxy- 3-oxo-xanthen-9-yl)- bicyclo[2.2.1]hetp-2-ene- 5-carboxylic acid (Depositor-supplied name: 7-methyl-3-(4,5,6-trihydroxy-3-oxo- 3H-xanthen-9-yl)- bicyclo[2.2.1]hept-5-ene- 2-carboxylic acid)


 






NSC119915Z IUPAC: 3-(4,5,6-trihydroxy-3-oxo-xanthen- 9-yl)propanoic acid (Depositor-supplied name: 4,5,6-trihydroxy-3-oxo- 3H-xanthene-9-propanoic acid)


 






NSC119911V IUPAC: 3-(4,5,6-trihydroxy-3-oxo-xanthen- 9-yl)prop-2-enoic acid


 






NSC119910U IUPAC: 2-(4,5,6-trihydroxy-3-oxo-xanthen- 9-yl)cyclohexane-1-carboxylic acid (Depositor-supplied name: 2-(4,5,6-trihydroxy-3-oxo- 3H-xanthen-9-yl)- cyclohexanecarboxylic acid


 


Exemplary F10 Inhibitors:






NSC128437O IUPAC: 4-(4-cyclohexylamino-9,10-dioxo- anthracen-1-yl)aminobenzene- sulfonic acid


 






NSC125908P IUPAC: 3-(9,10-dioxo-2-sulfo-anthracen- 1-yl)diazenyl-2-hydroxy-benzoic acid


 






NSC9600Q IUPAC: 4-[N′-(2-hydroxynaphthalen-1-yl)- N′-sulfo-hydrazino]benzenesulfonic acid


 






NSC13778J IUPAC: 3-(3-stibonophenyl)prop-2-enoic acid





1NSC119110-U, NSC119111-V, NSC119913-X, NSC119915-Z, NSC170008-Y, and NSC306711-P also inhibited the F10 protein kinase by at least 88% (as compared to control).






A. Tetralin Derivatives

In some embodiments, a disclosed protein kinase inhibitor (such as a B1 and/or F10 protein kinase inhibitor) conforms to the chemical structure of Formula I:




wherein R1-4 are independently hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), alkoxy, nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); R5, R6, R7 and R8 are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)); X1-X12 are independently hydrogen or halogen (such as chloro, or fluoro); and bonds between C1-C2 and C3-C4 are independently a single bond or a double bond. Typically, X4 and X5 and X10 and X11 are oriented cis relative to each other; however, the C1-C2 and C3-C4 double bonds can be trans. In some examples, at least two of R1-4 are independently sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester).

In other embodiments, exemplified by Formula IA (below), R1-4 and C1-C2 and C3-C4 are as described for Formula I, and X is a halogen, such as chloro or fluoro. In particular examples, X is chloro. In other examples, X is fluoro.




In some embodiments, exemplified by Formula IB (below), C1-C2 and C3-C4 and X are as described for Formula IA, and R1 and R2 are independently aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), alkoxy, nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester).




In other examples, R1 and/or R2 comprise an anionic group or an ester thereof, such as a carboxylate, phosphonate, sulfonate, or the like.
In more particular examples, R1 and R2 are independently sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), alkoxy, nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester). In other examples, R1 and R2 are independently sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl) or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester). In still other particular examples, R1 is carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester) and R2 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl). In other examples, R1 and R2 are independently —COOH (or its conjugate base), —COOR, or —SO3H (or its conjugate base).
Particular embodiments of compounds having the structure of Formula IB include compounds exemplified by Formula IB1:




wherein R1 and R2 are as described for Formula IB.

Even more particular embodiments include compounds exemplified by Formula IB2:




wherein R9 is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)). With continued reference to Formula IB2, particular examples of R9 groups include, without limitation, hydrogen, methyl, ethyl and benzyl. Throughout this disclosure, unless otherwise expressly mentioned, embodiments including —SO3H substituents implicitly include the corresponding conjugate base (—SO3−), carboxylic acid groups (—COOH) implicitly include the corresponding conjugate base (—COO−), and —PO3H2 substituents implicitly include the corresponding conjugate bases (—PO3H− and —PO3−2).

Other representative VPK inhibitors are provided in the following table:
















 











R1
R2
X
R5-8
C1—C2 and C3—C4





—COOR (where
—COOR (where
Cl or F
H or lower alkyl
independently a


R = lower alkyl),
R = lower alkyl),


double bond or a


—COOH, —COO−,
—COOH, —COO−,


single bond


—SO3H, —SO3−,
—SO3H, —SO3−,


—NO2, —PO3−2,
—NO2, —PO3−2,


—R—SO3H (where
—R—SO3H (where


R = lower alkyl),
R = lower alkyl),


—R—SO3− (where
—R—SO3− (where


R = lower alkyl)
R = lower alkyl)


same as above
same as above
Cl
same as above
same as above


same as above
same as above
F
same as above
same as above


same as above
same as above
Cl or F
H
same as above


same as above
same as above
Cl or F
lower alkyl
same as above


same as above
same as above
Cl or F
H or lower alkyl
each a double






bond


same as above
same as above
Cl or F
H or lower alkyl
each a single bond


same as above
same as above
Cl or F
H or lower alkyl
C1-C2 a double






bond and C3-C4 a






single bond


same as above
same as above
Cl or F
H or lower alkyl
C1-C2 a single






bond and C3-C4 a






double bond


same as above
same as above
Cl
H
each a double






bond


same as above
—SO3H, —SO3−,
Cl
H
each a double



—R—SO3H (where


bond



R = lower alkyl),



—R—SO3− (where



R = lower alkyl)


—COOR (where
—COOR (where
Cl
H
each a double


R = lower alkyl),
R = lower alkyl),


bond


—COOH, —COO−
—COOH, —COO−,



—SO3H, —SO3−,



—NO2, —PO3−2,



—R—SO3H (where



R = lower alkyl),



—R—SO3− (where



R = lower alkyl)


—COOR (where
—SO3H, —SO3−,
Cl
H
each a double


R = lower alkyl),
—R—SO3H (where


bond


—COOH, —COO−
R = lower alkyl),



—R—SO3− (where



R = lower alkyl)


—COOR (where
—SO3H, —SO3−,
Cl or F
H or lower alkyl
independently a


R = lower alkyl),
—R—SO3H (where


double bond or a


—COOH, —COO−
R = lower alkyl),


single bond



—R—SO3− (where



R = lower alkyl)


—COOR (where
—SO3H, —SO3−,
Cl or F
H
each a double


R = lower alkyl),
—R—SO3H (where


bond


—COOH, —COO−
R = lower alkyl),



—R—SO3− (where



R = lower alkyl)









Disclosed tetralin derivatives inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, a disclosed tetralin derivative (such as a compound exemplified by Formula I, Formula IA, Formula IB, Formula IB1, or Formula IB2) inhibits a B1 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In other examples, a disclosed tetralin derivative (such as a compound exemplified by Formula I, Formula IA, Formula IB, Formula IB1, or Formula IB2) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
B. Derivatives of Anthraquinone
In some embodiments, disclosed protein kinase inhibitors (such as a B1 and/or F10 protein kinase inhibitor) conform to the chemical structure of Formula II:




wherein Y1 and Y2 are independently O, S, or N—R (where R is hydrogen or aliphatic, such as lower aliphatic or alkyl (e.g., lower alkyl)); R1 is hydroxyl, alkoxy (such as lower alkoxy, e.g., methoxy), amino (such as arylamino) or azo; R2 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); R3 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or carbonyl-containing (such as acyl, lower acyl, acyloxy, carboxyl, or ester); R4 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), hydroxyl, alkoxy (such as lower alkoxy), or amino (such as alkylamino); R5, R6 and R7 are independently hydrogen, hydroxyl, alkoxy, carbonyl-containing, sulfonyl, or phosphonate; and R8 is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)).

Other exemplary VPK inhibitors, which are derived from anthraquinone, have the following structure:




wherein Y1 and Y2, and R1, R2, and R4 are as described for compounds having the structure of Formula II. In more particular embodiments, Y1 and Y2 are O; R1 is amino (such as arylamino) or azo; R2 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); and R4 is hydrogen, lower alkyl, or amino (such as alkylamino).

Particular embodiments of compounds having the structure of Formula IIA include compounds exemplified by Formula IIA1:




wherein R2 and R4 are as described for compounds having the structure of Formula II; R15 is hydrogen or lower alkyl; and R16 is substituted or unsubstituted aryl (such as substituted or unsubstituted phenyl). In another embodiment, R2 and R4 are as described for compounds having the structure of Formula II; R15 is absent; and R16 has the structure ═N—R, where R is substituted or unsubstituted aryl (such as substituted or unsubstituted phenyl, or —N—R20 (as described below)).

Further examples include compounds having one of the following structures:




With regard to exemplary compounds having the structure of Formula IIA1a, (R17)n represents five substituents independently selected from hydrogen, carbonyl-containing, phosphonate, and sulfonyl; and R18 and R19 are independently hydrogen, or aliphatic (for example, lower aliphatic or alkyl (such as lower alkyl or carbocylic (for instance, cyclohexane))). In more particular examples, (R17)n represents five substituents wherein four substituents are hydrogen and the fifth substituent is sulfonyl (such as —SO3H or SO3−); R18 is hydrogen or lower alkyl; and R19 is alkyl (such as lower alkyl or carbocylic (for instance, cyclohexane)).
With regard to exemplary compounds having the structure of Formula IIA1b, R2 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); and R20 is aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or aryl (such as substituted or unsubstituted phenyl). In more particular examples, R2 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl) and R20 is a di-substituted phenyl; for example, independently substituted with carboxyl and/or hydroxyl groups.
Other exemplary VPK inhibitors derived from anthraquinone have the following structure:




wherein Y1 and Y2 are independently O, S, or N—R (where R is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl))); R1, R4, and R7 are independently hydroxyl or alkoxy (such as lower alkoxy, e.g., methoxy); R2 is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)); R3 is carbonyl-containing (such as acyl, lower acyl, acyloxy, carboxyl, or ester); R4 is hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), hydroxyl, alkoxy (such as lower alkoxy); and R5, and R6 are independently carbonyl-containing, sulfonyl, or phosphonate. In more particular embodiments, Y1 and Y2 are O; and R1-R7 are as previously described in this paragraph.

Particular embodiments of compounds having the structure of Formula IIB include compounds exemplified by Formula IIB1:




wherein R2 and R9-R14 are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)). In some examples, R2 and R9-R14 are independently hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl). In other examples, R2 and R10 are independently lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl); and R9 and R11-R14 are hydrogen.

Disclosed derivatives of anthraquinone inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, disclosed anthraquinone derivatives (such as a compound exemplified by Formula II, Formula IIB or Formula IIB1) inhibit a B1 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In other examples, a disclosed anthraquinone derivative (such as a compound exemplified by Formula II, Formula IIA, Formula IIA1, Formula IIA1a, or Formula IIA1b) inhibits a F10 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In still other examples, a disclosed anthraquinone derivative (such as a compound exemplified by Formula II, Formula IIA, Formula IIA1, Formula IIA1a, Formula IIA1b, Formula IIB or Formula IIB1) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
C. Xanthen-3-One Derivatives
In some embodiments, disclosed protein kinase inhibitors (such as a B1 and/or F10 protein kinase inhibitor) conform to the chemical structure of Formula III:




wherein R1 is aliphatic (for example, alkyl (e.g., carbocyclic, carbobicyclic) or alkene) substituted with one or more (such as one, two, three, four, five or six) carbonyl-containing and/or sulfonyl groups; and R2-R4 are independently hydrogen or aliphatic, for example, lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl). In some examples, R1 is a substituted aliphatic group; for example, substituted with a carboxylic acid or carboxylic ester moiety (such as a straight-chain or branched-chain, saturated or unsaturated carboxylic acid; or a saturated or partially unsaturated carbocyclic group substituted with a carboxylic acid); and R2-R4 are independently hydrogen, lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl), or acyl. In other examples, R1 is a lower alkyl carboxylic acid (such as —(CH2)n—CO2R, e.g., —CH2—CH2—COOH), a lower alkenyl carboxylic acid (such as —CH═CH—CO2R, e.g., —CH═CH—COOH), a carbocycle (such as a cyclohexyl moiety) substituted with a carboxylic acid, or a bicyclic, unsaturated carbocycle (such as a bicycloheptenyl moiety) substituted with a carboxylic acid; and R2-R4 are independently hydrogen, lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl) or acyl, such as —C(O)R, wherein R represents a lower alkyl group.

Disclosed xanthen-3-one derivatives inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, a disclosed xanthen-3-one derivative (such as a compound exemplified by Formula III) inhibits a B1 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In other examples, a disclosed xanthen-3-one derivative (such as a compound exemplified by Formula III) inhibits a F10 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In still other examples, a disclosed xanthen-3-one derivative (such as a compound exemplified by Formula III) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
D. Naphthylamine Derivatives
In some embodiments, a disclosed protein kinase inhibitor (such as a B1 and/or F10 protein kinase inhibitor) conforms to the chemical structure of Formula IV:




wherein (R1)n represents four substituents independently selected from hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), or aryl; R2, R4, and R5 are independently hydrogen, aliphatic (for example, lower aliphatic or alkyl (such as lower alkyl)), sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), hydroxyl, alkoxy, nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); R3 is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)); and R6 is hydrogen or amino (such as substituted or unsubstituted arylamino).

More particular examples include compounds conforming to the following structure:




wherein R2 and R4 are independently sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), hydroxyl, alkoxy, nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); and (R1)′ and (R1)′′ form a polycyclic ring system having the following structure:





In even more particular examples, (R1)′ and (R1)′′ form the immediately preceding polycyclic ring system; and R2 and R4 are independently sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester).

Other embodiments of compounds having the structure of Formula IV include compounds exemplified by Formula IVB:




wherein R4 is hydroxyl or alkoxy (such as methoxy or ethoxy); R5 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); R7 is hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)); and R8 is aryl (such as substituted or unsubstituted phenyl). In more particular embodiments, R4 is hydroxyl or alkoxy (such as methoxy or ethoxy); R5 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); R7 is hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl); and R8 is substituted phenyl.

Some specific examples conform to the structure shown below in Formula IVB1:




wherein R5 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), nitro, phosphonate, or carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); (R9)m represents five substituents independently selected from hydrogen, sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), nitro, phosphonate, and carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester); and R10 is hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl). In other examples conforming to Formula IVB1, R5 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); (R9)m represents five substituents independently selected from hydrogen and sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); and R10 is hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl). In still other examples conforming to Formula IVB1, R5 is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); (R9)m represents five substituents wherein four substituents are hydrogen and one substituent is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl); and R10 is hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl).

Disclosed naphthylamine derivatives inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, disclosed naphthylamine derivatives (such as a compound exemplified by Formula IV or Formula IVA) inhibit a B1 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In other examples, a disclosed naphthylamine derivative (such as a compound exemplified by Formula IV, Formula IVA, Formula IVB or Formula IVB1) inhibits a F10 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In still other examples, a disclosed naphthylamine derivative (such as a compound exemplified by Formula IV, Formula IVA, Formula IVB or Formula IVB1) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
E. Cinnamic Acid Derivatives
In some embodiments, a disclosed protein kinase inhibitor (such as a B1 and/or F10 protein kinase inhibitor) conforms to the chemical structure of Formula V:




wherein X is sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), phosphonate (such as —P(O)(OH)2 or its conjugate base), carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester), or —Sb(O)(OH)2 (or its conjugate base); and R1, R2 and R3 are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)). As indicated in Formula V, substituents around the double bond in the side chain may be oriented in cis or trans. In particular examples, X is —SO3H (or its conjugate base, SO3−), —PO3H2 or its conjugate base), —COOH (or its conjugate base), —COOR (wherein R is lower alkyl), or —Sb(O)(OH)2 (or its conjugate base); and R1, R2 and R3 are independently hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl). In other particular examples, X is —Sb(O)(OH)2 (or its conjugate base); and R1, R2 and R3 are independently hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl).

Disclosed cinnamic acid derivatives inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, disclosed cinnamic acid derivatives (such as a compound exemplified by Formula V) inhibit a F10 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus F10 kinase). In other examples, a disclosed cinnamic acid derivative (such as a compound exemplified by Formula V) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
F. Tri-Amino-Pyrimidine Derivatives
In some embodiments, a disclosed protein kinase inhibitor (such as a B1 and/or F10 protein kinase inhibitor) conforms to the chemical structure of Formula VI:




wherein R1, R2, and R4-R7 are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl); and (R3)n represents five substituents independently selected from hydrogen, aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl)), carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester), sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), phosphonate, hydroxyl, and alkoxy. In particular examples, two of the (R3)n substituents are independently carbonyl-containing (such as acyl, acyloxy, carboxyl, or ester), sulfonyl (such as sulfonic acid, alkylsulfonyl (e.g., lower alkylsulfonyl) or arylsulfonyl), phosphonate, hydroxyl, or alkoxy, and the other three (R3)n substituents are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl).

In other compound embodiments having the structure of Formula IV, R1, R2, and R4-R7 are independently hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl); and (R3)n represents five substituents, three of which are independently hydrogen, or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl), and two of which are independently carboxyl (such as —COOH or —COOR, where R is lower alkyl), —SO3H (or its conjugate base, SO3−), —PO3H2 or its conjugate base), hydroxyl, or alkoxy (such as methoxy or ethoxy).
Some specific examples conform to the structure shown below in Formula VIA:




wherein R1, R2, and R4-9 are independently hydrogen or aliphatic (such as lower aliphatic or alkyl (e.g., lower alkyl). In particular examples, R1, R2, and R4-9 are independently hydrogen or lower alkyl (such as methyl, ethyl, propyl, i-propyl, butyl or i-butyl).

Disclosed tri-amino-pyrimidine derivatives inhibit, at least, (i) an activity (e.g., kinase activity) of a B1 kinase and/or a F10 kinase, and/or a homolog or variant of either, and/or (ii) viral (e.g., poxvirus) growth. In particular examples, disclosed tri-amino-pyrimidine derivatives (such as a compound exemplified by Formula VI or Formula VIA) inhibit a B1 kinase and/or a homolog or variant thereof (such as a vaccinia virus and/or variola virus B1 kinase). In other examples, a disclosed tri-amino-pyrimidine derivative (such as a compound exemplified by Formula VI, Formula VIA) inhibits virus growth (such as poxvirus growth, for example, chordopoxvirus growth or Orthopoxvirus growth, each including vaccinia virus and/or variola virus growth).
G. Considerations for Viral Protein Kinase Inhibitors
Prodrug derivatives of disclosed VPK inhibitors can be prepared. In one example, a prodrug derivative includes one or more substituents that are converted in vivo into one or more different substituents. In several instances, the prodrugs also fall within the scope of the range of compounds described above. For example, prodrugs can be prepared by reacting a compound with an acylating or carbamylating agent, such as 1,1-acyloxyalkylcarbonochloridate, p-nitrophenyl carbonate or the like, as is known to those of skill in the art of medicinal chemistry. Further examples of methods suitable for preparing prodrug derivatives of viral protein kinase inhibitors are described by Saulnier et al., Biorg Med. Chem. Lett., 4: 1985, 1994.
Protected derivatives of the viral protein kinase inhibitors disclosed herein also can be made. Exemplary techniques for preparing such derivatives are provided by Greene, Protecting Groups in Organic Synthesis, 3rd Edition, New York: John Wiley & Sons, Inc., 1999.
In some embodiments a disclosed viral protein kinase inhibitor is included in a pharmaceutical composition and/or administered to a subject for the treatment of poxvirus (such as variola virus) infection. Accordingly, it is advantageous for some VPK inhibitor embodiments to have characteristics suitable for in vivo administration. One rule of thumb for drug design is Lipinski's “Rule of 5” (Lipinski et al., Adv. Drug Deliv. Rev., 23(1-3): 3-25, 1997), which suggests that absorption and permeation of a drug candidate (which is not a biological transporter substrate) is likely to be increased if at least three of the following parameters are satisfied: (i) there are no more than about 5H-bond donors (expressed as the sum of hydroxyl and amine groups); (ii) the molecular weight is not substantially greater than 500 g/mole (but can be anything less); (iii) the logP (logarithm of the octanol-water partition coefficient of the drug candidate) is no more than about 5; and (iv) there are no more than about 10H-bond acceptors (the sum of nitrogens and oxygens). Some VPK inhibitor embodiments may satisfy one, two, three or all four Rule of 5 parameters.
H. Obtaining VPK Inhibitors
Certain of the disclosed VPK inhibitors are available from the Developmental Therapeutics Program, National Cancer Institutes, National Institutes of Health, Bethesda, Md., U.S.A. Other disclosed VPK inhibitors are structurally related to the publicly available compounds and can be synthesized from such compounds using syntheses commonly known in the art. Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing the disclosed compounds are available (see, e.g., Smith and March, March's Advanced Organic Chemistry Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001; Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978; or Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002).
I. Functional Assays for VPK Inhibitors
The disclosed VPK inhibitors are useful, at least, in the treatment of poxvirus infection (such as smallpox) and to inhibit virus (such as poxvirus growth). Such VPK inhibitors are believed to exert an inhibitory effect on poxviruses (and thereby treat poxvirus-related disease) by interfering with the activity of one or more essential protein kinases (such as the B1 and/or F10 kinase). Accordingly, if it is desirable to do so, non-limiting methods useful to functionally characterize VPK inhibitors are (i) in vitro protein kinase assays, and (ii) virus growth assays (e.g., plaque formation assays in cultured cells and/or virus growth assays in cultured cells. A disclosed VPK inhibitor is expected to reduce viral protein kinase activity and/or virus growth as compared to such activity or growth measured in the absence of such inhibitor. Numerous assays suitable for the measurement of viral protein kinase activity and/or virus (such as poxvirus) growth in the presence and absence of disclosed VPK inhibitors are known (see, e.g., Protein Kinase Protocols, First Edition, ed. by A. Reith, New York: Humana Press, 2001; Methods in Enzymology, Volume 200, “Protein Phosphorylation, Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression,” ed. by Hunter and Sefton, New York: Academic Press, 1991; Methods in Enzymology, Volume 201, “Protein Phosphorylation, Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases,” ed. by Hunter and Sefton, New York: Academic Press, 1991; Vaccinia Virus and Poxyirology: Methods and Protocols, ed. by Issacs, Clifton, N.J.: Humana Press, 2004). Exemplary methods are provided below and in the Examples.
1. Viral Protein Kinase Assays
A traditional method for assaying the activity of protein kinase inhibitors is a radiometric assay in which the gamma phosphate of ATP is labeled with either 32P or 33P. When a viral protein kinase (such as a B1 or F10 kinase), a protein substrate (such as casein) and labeled ATP are mixed under suitable conditions (see, e.g., Example 1), the VPK transfers the gamma phosphate to a hydroxyl (e.g., serine and/or threonine) of the protein substrate. As a result, the substrate protein (i.e., target) becomes covalently labeled with the radioisotope. The target protein is removed from the free radiolabeled ATP (for example, by immunoprecipitation) and the amount of radioactive protein is determined. In the presence of a VPK inhibitor, the amount of radioactive target protein will be reduced as compared to control.
An alternative radiometric assay, which is more amenable to higher throughput, is the SPA or scintillation proximity assay (see e.g., Hart et al., Mol. Immunol., 16: 265, 1979; and U.S. Pat. Nos. 4,271,139; 4,382,074; and 4,568,649). In this assay, a target protein (such as casein) is directly or indirectly immobilized on scintillant-impregnated beads. The VPK, target protein, and radioactive ATP are placed in a reaction mixture as described above. Radio-labeled target protein is captured on the scintillant-impregnated beads, which causes the scintillant to emit light. The amount of emitted light is proportional to the amount of phosphorylation of the target protein (and, hence, to the activity of the VPK). A decrease in the amount of emitted light in the presence of a compound identifies that compound as a VPK inhibitor.
Protein kinase activity (and inhibition thereof) can also be determined using fluorescence polarization techniques. In these assays, the enzyme transfers the gamma phosphate of ATP to a protein or peptide substrate. This activity is monitored by detecting the phosphor-peptide by such means as an antibody specific for the phosphorylated protein. The binding of the antibody to the phosphor-peptide will slow the free rotation of the peptide in solution and, therefore, a polarization signal from the product of the catalytic reaction can be detected (see, e.g., U.S. Pat. App. Pub. No. 2001/0004522; Turek et al., Anal. Biochem., 299(1): 25-53, 2001). A VPK inhibitor will affect the amount of polarization signal measured in the reaction.
As yet another alternative, protein kinase activity can be assayed using the AlphascreenTM technique (PerkinElmer). This assay involves a phospho-specific antibody that recognizes the phosphorylated substrate of the kinase in question. A biotinylated substrate peptide is bound to streptavidin-coated “donor” beads. The kinase reaction is carried out in the presence of “acceptor” beads to which the phospho-specific antibody is bound. Laser excitation causes the “donor” beads to release singlet state oxygen. When released in close proximity to the “acceptor” bead, the singlet state oxygen generates a chemiluminescent signal that is then amplified. Light emission is proportional to substrate phosphorylation.
The fluorescence resonance energy transfer (FRET)-based Z′-LyteTM technique (PanVera/Invitrogen) also can be used to measure protein kinase activity. This assay measures FRET in a protein kinase substrate peptide labeled at either end with coumarin and fluorescein. A high level of FRET occurs in the intact peptide. The assay measures the protease sensitivity of the peptide. Once cleaved by a protease, FRET no longer occurs. The peptide is designed so that the site phosphorylation by the kinase of interest is within the protease cleavage site. Phosphorylation inhibits cleavage and therefore prevents the inhibition of FRET by proteolytic degradation. A high level of residual FRET is indicative of protein kinase activity and, consequently, a protein kinase inhibitor would decrease the FRET.
2. Virus Growth Assays
Virus growth assays detect, for example, to what extent (or whether) a putative VPK inhibitor can slow virus growth. Two exemplary virus growth assays are in vitro cell culture assays and plaque reduction assays.
In a representative in vitro cell culture assay (see, e.g., Example 1), cultured cells susceptible to infection by the virus of interest (such as a poxvirus, like vaccinia virus) are grown to a desired cell density (such as 4×105 cells per well in a 12-well plate) under culture conditions (e.g., 37° C., 95% O2, 5% CO2 and 98% relative humidity) suitable to the particular cell type. The cultured cells, then, are infected (either with a single or multiple inoculum(s)) with a known amount of virus (such as, about 0.03 plaque-forming units per cell). The virus inoculum remains in contact with the cells for a sufficient time (such as 30 minutes) to permit virus to adsorb to the cells. Unbound virus is removed and medium containing inhibitor or vehicle (control) is then added. Infection is permitted to proceed for a sufficient time (e.g., about 18-24 hours) for the virus to replicate (at least under control conditions) to a comfortably measurable level. Virus is harvested, for example, by removal of the medium (e.g., for viruses that shed from the cells) and/or by collection of infected cells and isolation of virus from such cells.
Virus can be isolated from infected cells by any method known in the art. In one exemplary method, cells are disrupted (e.g., by cycles of freezing and thawing and/or shearing with a syringe needle (such as 1.5 inch 22 gauge needle)), and debris is removed by centrifugation (e.g., 750×g for 1 minute). Virus are collected in the supernatant, which, optionally, is mixed with protease (e.g., 0.25% trypsin for 30 minutes at 37° C.). Debris, again, can be removed by centrifugation (e.g., 750×g for 1 minute). Then, supernatant containing virus is serially diluted for plaque assay.
In the plaque assay, cultured cells (e.g., in a series of culture dishes) are contacted with virus (e.g., serial dilutions) for a sufficient time to permit virus to adsorb to the cells. The infected monolayers of cells, then, are overlaid with medium containing agarose (and no inhibitor). Incubation of the cells is continued for a period of time (e.g., from about 18 to about 40 hours), after which time, the number of plaques are counted (e.g., by staining cells with 1% crystal violet in 20% ethanol). A VPK inhibitor would be expected to reduce the number of plaques relative to control.
A plaque reduction assay also is useful to determine the ability of a compound (such as a VPK inhibitor) to inhibit virus growth (such as, poxvirus growth). In this assay, monolayers of cultured cells susceptible to infection by the virus of interest (e.g., poxvirus, such as vaccinia virus) are exposed to a viral inoculum. After a period of time for adsorption of the viral particles to the cells, the culture medium is removed and replaced with a nutrient agarose containing the test compound (e.g., putative VPK inhibitor). After a period of incubation (e.g., several days), plaques, which are areas where cells have died as a result of viral infection, are counted. In the case of vaccinia virus, plaques can be recognized after about 48 hours by staining with crystal violet or neutral red. An effective VPK inhibitor would be expected to reduce the number of plaques as compared to control. Particular conditions for an exemplary plaque reduction assay are described, for instance, in Landry et al. (Antimicrob. Agents Chemother., 44(3): 688-692, 2000).
IV. Methods of Use
The present disclosure includes methods of inhibiting a viral protein kinase (such as, a B1 kinase, a F10 kinase or a variant (including homologs) thereof), and methods of treating poxvirus infection and/or inhibiting poxvirus growth.
In some examples, a viral protein kinase or a virus recited in a disclosed method is (or is from) a poxvirus, such as a chordopoxvirus (for example an Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Mollusciposvirus, or Yatapoxvirus, or a combination thereof). Specific method embodiments involve Orthopoxviruses (or VPKs therefrom), including without limitation variola virus, vaccinia virus, monkeypox virus, buffalopox virus, camelpox virus, elephantpox virus, volepox virus, ectromelia virus, raccoonpox viruse, skunkpox viruse, Uasin Gishu disease virus, or taterapox virus, or a combination thereof. Some method embodiments involve the treatment or growth inhibition of variola virus (such as variola major or variola minor virus), or inhibition of variola virus VPKs. Other method embodiments involve poxviruses capable of infecting human hosts (such as monkeypox virus, vaccinia virus, buffalopox virus, cowpox virus, elephantpox virus, variola virus, bovine papular stomatitis virus, orf virus, pseudocowpox virus, sealpox virus, tanapox virus, or Yaba monkey tumor virus, or combinations thereof), or inhibition of VPKs from such human pathogens. Some methods of treatment involve the treatment of smallpox, human monkeypox, parapoxvirus infection, molluscum contagiosum virus infection, or human cowpox.
A. Treatment Methods
Disclosed methods of treating a poxvirus infection or an associated disease include administering a disclosed VPK inhibitor (and, optionally, one or more other pharmaceutical agents) to a subject in a pharmaceutically acceptable carrier and in an amount effective to treat poxvirus (such as variola virus) infection or an associated disease (such as smallpox). The treatment can be used prophylactically in any subject in a demographic group at substantial risk for such diseases; for example, children in Central Africa (who are at particular risk for monkeypox infection), or persons who have not previously been immunized with a vaccine against poxvirus infection (such as the smallpox vaccine). Alternatively, subjects can be selected using more specific criteria, such as a probable or definitive diagnosis of poxvirus infection or smallpox or other poxvirus-based disease based on, for example, clinical signs and symptoms and/or laboratory evidence of poxvirus infection. For example, smallpox (or variola virus infection) may present clinically with abrupt onset of fever and prostration with a macular rash (on the head, limbs, hands (including palms) and feet (including soles) and inside the mouth), which rash progresses to vesicles which become pustular, ulcerated, scabbed, and healed with scarring; provided that the subject recovers in the face of an approximately 40% mortality rate. Other poxvirus infections may be clinically identified based on localised pustules with scar formation (e.g., vaccinia virus), ulcerative lesions (sometimes called “milkers nodules”; e.g., cowpox); non-ulcerative milker's nodules (e.g., pseudocowpox virus); single painless, papulo-vesicular lesion on the hand, forearm or face (e.g., ORF virus); or other known symptoms of poxvirus infection. Laboratory tests useful for identifying poxvirus infection include histological examination of a curetted or biopsied lesion, electron microscopy, immunohistochemistry using antibodies specific for poxvirus proteins, in situ hybridization or PCR using poxvirus-specific nucleic acid probes or primers, respectively, antigen detecting agar gel immune precipitation test, or other commonly known diagnostic tests (see, e.g., Mangana-Vougiouka et al., Mol. Cell. Probes, 14(5): 305-10, 2000).
Routes of administration useful in the disclosed methods include but are not limited to oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic, nasal, and transdermal. Formulations for these dosage forms are described elsewhere herein.
An effective amount of a VPK inhibitor will depend, at least, on the particular method of use, the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. A “therapeutically effective amount” of a composition is a quantity of a specified compound sufficient to achieve a desired effect in a subject (host) being treated. For example, this may be the amount of a VPK inhibitor necessary to prevent, inhibit, reduce or relieve poxvirus infection and/or one or more symptoms of poxvirus-related disease (such as smallpox) in a subject, preferably without causing a substantial cytotoxic effect on host cells. It is anticipated that disclosed VPK inhibitors will be well tolerated in mammalian subjects (such as humans) because the toxicity of some VPK inhibitors (for instance, NSC270718-R, NSC306711-P, NSC119913-X, NSC119915-Z, NSC119911-V, and NSC119910-U) have been tested in mammals and no toxic effects were observed at the tested doses (information available at the website dtp.nci.nih.gov/dtpstandard/servlet/InvivoScreen?testshortname=Tumor+PS+%28ip%29+in+06&sear chtype=NSC&searchlist=#####, where “#####” are the numbers only from the NSC identifier for the compound).
Therapeutically effective doses (or growth inhibitory amounts) of a disclosed VPK inhibitor or pharmaceutical composition can be determined by one of skill in the art, with a goal of achieving local (e.g., tissue) concentrations that are at least as high as the IC50 of the applicable compound disclosed in the examples herein. An example of a dosage range is from about 0.1 to about 200 mg/kg body weight orally in single or divided doses (such as from about 5 mg/kg to about 200 mg/kg). In particular examples, a dosage range is from about 1.0 to about 100 mg/kg body weight orally in single or divided doses, including from about 1.0 to about 50 mg/kg body weight, from about 1.0 to about 25 mg/kg body weight, from about 1.0 to about 10 mg/kg body weight (assuming an average body weight of approximately 70 kg; values adjusted accordingly for persons weighing more or less than average). For oral administration, the compositions are, for example, provided in the form of a tablet containing from about 50 to about 1000 mg of the active ingredient, particularly about 75 mg, about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg, about 750 mg, or about 1000 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject being treated. In one exemplar oral dosage regimen, a tablet containing from about 500 mg to about 1000 mg active ingredient is administered once (e.g., a loading dose) followed by administration of 1⁄2 dosage tablets (e.g., from about 250 to about 500 mg) each 6 to 24 hours for at least 3 days.
The specific dose level and frequency of dosage for any particular subject may be varied and will depend upon a variety of factors, including the activity of the specific VPK inhibitor, the metabolic stability and length of action of that compound, the age, body weight, general health, sex and diet of the subject, mode and time of administration, rate of excretion, drug combination, and severity of the condition of the host undergoing therapy.
The present disclosure also contemplates combinations of one or more disclosed VPK inhibitors with one or more other agents or therapies useful in the treatment of poxvirus infection or poxvirus-related disease. For example, one or more disclosed VPK inhibitors may be administered in combination with effective doses of other medicinal and pharmaceutical agents, or in combination other non-medicinal therapies, such as hormone or radiation therapy. The term “administration in combination with” refers to both concurrent and sequential administration of the active agents. In some examples, the one or more other anti-viral agents or therapies include cidofovir, hexadecyloxypropyll-cidofovir, and the diacetate ester prodrug of 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (HOE961) (Smee et al., Int. J. Antimicrobial Agents, 23: 430-437, 2004).
B. Methods of Inhibiting Virus Growth
Other disclosed methods involve inhibiting the growth of a virus (such as a poxvirus, like variola virus) by contacting the virus with a growth inhibitory amount of a disclosed VPK inhibitor. The phrase “inhibiting virus growth” (and analogous phrases, such as inhibition of virus growth) conveys a wide-range of inhibitory effects that an agent (e.g., VPK inhibitors) may have on the normal (i.e., control) rate of virus growth. The terms “virus growth,” “virus multiplication,” and “virus replication” are intended to be synonymous. In some instances, duplication of a viral genome could be used as a measure of virus replication; however, duplication of a viral genome is only one possible (and optional) indicator of viral growth.
The phrase “inhibiting virus growth” (or like terminology) may be considered relative to the normal (i.e., uninhibited or control) rate of growth of a particular virus or viruses of interest (e.g., poxvirus, such as variola virus or vaccinia virus). Thus, inhibiting virus growth includes situations wherein the normal growth rate of a virus has slowed (i.e., virus number (or plaque-forming units (pfus)) increases over time, but not as rapidly as control), equals zero (i.e., there is substantially no change in virus number (or pfus) in the population over time, e.g., virus growth is approximately equal to inhibition of virus growth), or becomes negative (i.e., virus number (or pfus) decrease over time). A negative rate of virus growth can (but need not) result in clearance of substantially all viruses from a host cell or organism. In particular instances, a VPK inhibitor can reduce virus growth by at least about 60% (as compared to untreated control); for example, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, or even up to by about 95% or nearly 100%.
Contact between a VPK inhibitor and a poxvirus may occur in vitro (such as in culture conditions) or in vivo (such as in a subject infected with at least one poxvirus). In certain method embodiments, growth inhibitory amounts include amounts described in the Examples (for example, IC50 concentrations). In some examples, a growth inhibitory amount is from about 10 nm to about 1 μM, from about 0.1 μM to about 10 μM, from about 1 μM to about 100 μM, from about 2.5 μM to about 250 μM, or from about 10 μM to about 500 μM (such as from about 25 μM to about 400 μM, from about 50 μM to about 300 μM, or from about 100 μM to about 250 μM).
Methods for determining inhibition of viral growth are provided in Example 1 and elsewhere in this specification.
C. Methods of Inhibiting VPK Activity
Disclosed herein are methods of inhibiting an activity of a viral protein kinase by contacting a viral protein kinase with an inhibitory amount of a disclosed VPK inhibitor. Contact between a VPK inhibitor and a viral protein kinase may occur in vitro (such as in a reaction vessel, or in a cell culture) or in vivo (such as in a subject infected with a virus (e.g., poxvirus) that expresses a viral protein kinase). Viral protein kinases have been described in detail elsewhere in this specification, but, by way of example, include the B1 kinase, F10 kinase and/or variants (such as homologs thereof) thereof. An activity of a viral protein kinase that can be inhibited by a disclosed VPK inhibitor includes kinase activity (e.g., the ability to transfer a γ-phosphate group from ATP to a substrate protein), and/or ATP and/or protein substrate affinities. Exemplary assays for measuring kinase activity (and inhibition thereof) have been provided elsewhere herein. Assays for determining substrate (e.g., ATP and/or protein substrate) binding affinities are well known in the art; for example, the reaction rate can be measured at varying concentrations of substrate, either ATP and/or peptide/protein, and substrate affinities calculated by plotting reaction rates according to Lineweaver-Burk (as described in biochemistry textbooks).
An inhibitory amount can be any amount that reduces an activity of a viral protein kinase (such as, B1 kinase and/or F10 kinase or a variant thereof). In particular instances, a VPK inhibitor can reduce an activity (such as, kinase activity) of a viral protein kinase by at least about 60% (as compared to untreated control); for example, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, or even up to by about 95% or nearly 100%. In certain method embodiments, inhibitory amounts include amounts described in the Examples (for example, IC50 concentrations). In other embodiments, an inhibitory amount is from about 10 nM to about 25 μM of a disclosed VPK inhibitor (such as from about 50 nM to about 15 μM, from about 0.1 μM to about 10 μM, from about 0.5 μM to about 7 μM, or from about 1 μM to about 5 μM).
V. Pharmaceutical Compositions
The disclosed VPK inhibitors are useful, at least, for the treatment of poxvirus infection inhibiting the growth of viruses, such as poxviruses. Accordingly, pharmaceutical compositions comprising at least one disclosed VPK inhibitor are also described herein.
Formulations for pharmaceutical compositions are well known in the art. For example, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975, describes exemplary formulations (and components thereof) suitable for pharmaceutical delivery of disclosed VPK inhibitors. Pharmaceutical compositions comprising at least one of the disclosed VPK inhibitors can be formulated for use in human or veterinary medicine. Particular formulations of a disclosed pharmaceutical composition may depend, for example, on the mode of administration (e.g., oral or parenteral) and/or on the location of the infection to be treated. In some embodiments, formulations include a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a VPK inhibitor. In other embodiments, other medicinal or pharmaceutical agents, for example, with similar, related or complementary effects on the affliction being treated (such as poxvirus infection or smallpox), can also be included as active ingredients in a pharmaceutical composition.
Pharmaceutically acceptable carriers useful for the disclosed methods and compositions are conventional in the art. The nature of a pharmaceutical carrier will depend on the particular mode of administration being employed. For example, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can optionally contain minor amounts of non-toxic auxiliary substances (e.g., excipients), such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate. Other non-limiting excipients include, nonionic solubilizers, such as cremophor, or proteins, such as human serum albumin or plasma preparations.
The disclosed pharmaceutical compositions may be formulated as a pharmaceutically acceptable salt of a disclosed VPK inhibitor. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydriodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. Lists of other suitable pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., 1985. A pharmaceutically acceptable salt may also serve to adjust the osmotic pressure of the composition.
The dosage form of a disclosed pharmaceutical composition will be determined by the mode of administration chosen. For example, in addition to injectable fluids, topical or oral dosage forms may be employed. Topical preparations may include eye drops, ointments, sprays and the like. Oral formulations may be liquid (e.g., syrups, solutions or suspensions), or solid (e.g., powders, pills, tablets, or capsules). Methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.
Certain embodiments of the pharmaceutical compositions comprising a disclosed VPK inhibitor may be formulated in unit dosage form suitable for individual administration of precise dosages. The amount of active ingredient (e.g., VPK inhibitor) administered will depend on the subject being treated, the severity of the affliction, and the manner of administration, and is known to those skilled in the art. Within these bounds, the formulation to be administered will contain a quantity of the extracts or compounds disclosed herein in an amount effective to achieve the desired effect in the subject being treated.
EXAMPLES
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.
Example 1
Representative Materials and Methods
This Example describes materials and methods used in the following Examples 2-7.
A. Chemicals
Nineteen commercially available protein kinase inhibitors were tested for activity against poxvirus B1 or F10 kinase. ML-7, Emodin, H-89, Indirubin-3′-monoxime-5-sulphonic acid, 5-iodo-indirubin-3′-monoxime, staurosporine, KT 5720, and Ro-31-8220 were obtained from Calbiochem. Y-27632, Indirubin-3′-monoxime, SP600125, and bisindolylmaleimides I, IV and V were obtained from Alexis. IC261 was from VWR. CKI-7 was from United States Biological Corp. Epigallocatechin and apigenin were from Sigma. Gleevec/STI571 can be obtained from Novartis.
Naphthalene-bis(hexachlorocyclopentadiene) adduct, 2-nitronaphthalene-bis(hexachlorocyclopentadiene) adduct, and 3-nitro-2-naphthoic acid-bis(hexachlorocyclopentadiene) adduct were obtained from Fisher. 2-methyl-naphthalene-bis(hexachlorocyclopentadiene) adduct and 2-bromonaphthalene-bis(hexachlorocyclopentadiene) adduct were obtained from Aldrich. The foregoing chemical names are those provided by the respective supplier.
B. Viruses
Wild-type vaccinia virus and Condit's ts2 and ts25 (Condit and Motyczka, Virology, 113: 224-241, 1981; Traktman et al., J. Biol. Chem., 264: 21458-21461, 1989) mutants of vaccinia encoding temperature-sensitive B1 protein kinase were obtained from Paula Traktman. Wild-type vaccinia virus also is commercially available from American Type Culture Collection.
C. Protein Purification
DNA sequences encoding the B1 and F10 kinases of vaccinia virus were isolated by PCR using vaccinia VTF7-3 virus (Fuerst et al., Proc. Natl. Acad. Sci. USA, 83: 8122-8126, 1986) as a template. The B1-encoding nucleotide sequence was amplified with the primers:








(SEQ ID NO: 13)











5′-ATAGAATTCACATTATGAACTTTCAAGGACTT-3′;




and



 








(SEQ ID NO: 14)











5′-TATCTCGAGCACCACACTTAATAATATACACC-3′.







The F10-encoding nucleotide sequence was amplified using the primers:










5′-ATACTCGAGGAAATGGGTGTTGCCAAT-3′;
(SEQ ID NO: 15)



and


 


5′-GCGAAGCTTTTAGTTTCCGCCATTTAT-3′.
(SEQ ID NO: 16)






The amplified sequences were ligated into pGEX-KG (Amersham Pharmacia) to create GST fusion proteins (see, e.g., Guan and Dixon, Anal. Biochem., 192: 262-267, 1991). Four mutations (changing Trp150 to Leu, His170 to Asn, Lys 364 to Glu, and Gly438 to Arg) were introduced into the gene encoding vaccinia F10 by PCR-mediated mutagenesis to convert it to a gene encoding variola F10. E. coli strain BL21 (DE3)pLysS (Invitrogen) was transformed with plasmids encoding GST-fusion proteins containing vaccinia B1 or variola F10. To isolate the kinases, the transformed bacteria were grown to an OD600 of 0.6 at 37° C. and placed on ice for 30 minutes. Ethanol was added to a concentration of 2% and isopropylthiogalactoside was added to a concentration of 500 μM. Cultures were then incubated for 48 hours at 18° C. with shaking (Derrien et al., J. Virol., 73: 7287-7296, 1999). Bacteria were lysed in 20 mM Tris-HCl, pH 8.4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.2% lysozyme with freeze-thawing and sonication. GST-proteins were isolated with glutathione-Sepharose (Amersham).
D. Scintillation Proximity Assay
The Structural Diversity Set of compounds from the Developmental Therapeutics Program at NCI/NIH was screened to identify small molecules exhibiting inhibitory activity toward vaccinia virus B1 or variola F10 using a scintillation proximity assay (SPA). Briefly, this method involves the capture of a 33P-labeled peptide or protein onto streptavidin-coated polyvinyl toluene (PVT) SPA beads. β-particles emitted from the captured 33P-labeled substrate in close proximity to the beads are able to excite the scintillant, resulting in the generation of light which can be quantified. To efficiently harvest the greater energy associated with 33P and to reduce non-specific signal resulting from free [33P]ATP in solution, the beads are settled or centrifuged before counting.
The assays were carried out in 96 well Corning 3600 plates. Each reaction contained either 50 ng of GST-B1 or 150 ng of GST F10, 250 ng biotinylated casein, 0.5 μCi [γ-33P]ATP (PerkinElmer), inhibitor (10 μM in DMSO) or DMSO in 100 μl of 10 mM sodium PIPES, pH 7.0, 5 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol. EDTA (5 mM) was used as a positive control inhibitor in each dish. The reaction was allowed to proceed for 30 minutes at room temperature and was terminated by the addition of 150 μl of 5 mM EDTA, 0.1% Triton X-100 containing 200-300 ng of SPA streptavidin-coated PVT beads (Amersham). The plates were incubated for 15 minutes at room temperature, centrifuged at 1700×g for 15 minutes and then counted in a Packard Top Count microplate scintillation counter. To exclude the possibility of inhibition due to interference with the scintillant, all colored candidate inhibitors were re-analyzed by SPA following aspiration of the supernatant. Additionally, if a colored compound showed inhibitory activity, the pellet of SPA beads was dissolved in SDS polyacrylamide gel electrophoresis sample buffer and the phosphorylation of biotinylated casein was quantified by SDS polyacrylamide gel electrophoresis and PhosphorImaging.
E. In Vitro Protein Kinase Assays
SPA (described above) and gel electrophoresis were used to quantify inhibitor activity with B1 and F10. B1 and F10 kinase reactions for analysis by SDS polyacrylamide gel electrophoresis were carried out in 100 μl of a buffer containing 10 mM sodium PIPES, pH 7.0, 5 mM MnCl2, 5 mM MgCl2, 1 mM dithiothreitol, 50 ng of kinase, 500 ng of casein, 2.5 μCi/ml of [γ-32P]ATP (Amersham), and 1% DMSO or 10 mM inhibitor in DMSO, as appropriate. The reaction was carried out for 30 minutes at room temperature and was terminated by the addition of 25 μl of five-fold concentrated Tris-buffered SDS sample buffer. To assay Lck, the activated F505 form of Lck was obtained from transiently transfected human 293 cells by immunoprecipitation as described by Hurley and Sefton (Oncogene, 4: 265-272, 1989). Lck autophosphorylation kinase assays were carried out as described above except that the reactions were terminated by the addition of 0.5 ml of 150 mM NaCl, 50 mM Tris-HCl, pH 7.2, 0.01% Triton X-100. The immunoprecipitated Lck was recovered by centrifugation and resuspended in SDS gel sample buffer. The immunoprecipitates were subjected to SDS polyacrylamide gel electrophoresis and incorporation of 32P into casein or into Lck was quantified by PhosphorImaging using a Molecular Dynamics Typhoon 8600TM variable mode imager.
F. Virus Growth
HeLa cells were seeded at a density of 4×105 cells per well in a 12 well plate (Corning) in Dulbecco-Vogt-modified Eagle Medium (DMEM) containing 10% bovine calf serum 18-24 hours prior to use. Infection was with a multiplicity of infection of 0.03 plaque-forming units per cell. The virus inoculum was allowed to adsorb for 30 minutes in a volume of 0.075 ml. Unbound virus was removed and medium containing inhibitor or vehicle (DMSO) was then added. The infection was allowed to proceed at 37° C. for 18 hours, in the case of vesicular stomatitis virus (VSV), or 24 hours, in the case of wild-type vaccinia virus. In most examples, the medium contained 10% serum. Example 7 describes results obtained using medium containing 2% serum. Growth of ts2 or ts25 was for 24 hours at 32° C. VSV was harvested by removal of the medium. To harvest vaccinia virus, the cells were scraped off the dish into their growth medium and collected by centrifugation at 1000×g for 5 minutes. The cells were washed once in phosphate-buffered physiological saline, resuspended in DMEM containing 2.5% bovine fetal calf serum and then subjected to three cycles of freezing and thawing. Finally, the virus preparation was subjected to 6 rounds of shearing with a 1.5 inch 22 gauge syringe needle. Debris was removed by centrifugation at 750×g for 1 minute. For analysis, virus was mixed with an equal volume of 0.25% trypsin (Difco) and incubated for 30 minutes at 37° C. Debris was removed by centrifugation at 750×g for 1 minute and the virus was diluted in growth medium containing 10% bovine calf serum.
G. Plaque Assays
BSC-1 cells were seeded at a density of 5×105 cells per well in a 6 well plate (Corning) in DMEM supplemented with 10% bovine calf serum 18-24 hours prior to use. For the plaque assays, the medium was aspirated and diluted virus in 0.2 ml was allowed to adsorb for 30 minutes at 37° C., or 32° C. in the case of temperature-sensitive mutants. Following adsorption, 3 ml of medium was added to each culture. For assays of VSV, the infected monolayers were overlaid with 3.0 ml of medium containing 0.4% agarose. The dishes were incubated at 32° C. or 37° C. as appropriate. Cell monolayers were stained 18 to 40 hours later with 1% crystal violet in 20% ethanol.
Example 2
Twelve Inhibitors of Poxvirus B1 and/or F10 Protein Kinases Identified in Screening Assay
Nineteen commercially available protein kinase inhibitors with diverse specificities (see Example 1) were tested for their abilities to inhibit poxvirus B1 or F10 in vitro. Of these, only staurosporine, a microbial alkaloid, inhibited B1 or F10 kinase. Because of its toxicity (Bertrand et al., Exp. Cell Res., 211: 314-321, 1994), staurosporine was not consider an optimum candidate for an anti-poxvirus drug and was not further examined.
A scintillation proximity assay was then used to screen the Structural Diversity Set of compounds from the Developmental Therapeutics Program of NIH/NCI (information located at the website, dtp.nci.nih.gov/branches/dscb/diversity_explanation.html). The screen was carried out in a 96-well format, and each reaction contained GST-B1 or GST-F10, biotinylated casein (as a substrate), and [γ-33P]ATP. The compound being tested was present at a concentration of 10 μM. Nineteen hundred and ninety compounds were screened with each kinase.
As shown in Table 3, eight compounds (117285-R, 119110-U, 119111-V, 119913-X, 119915-Z, 170008-Y, 270718-R, and 306711-P) inhibited GST-B1 by at least 87% of the control value. Seven of the 8 compounds identified as B1 inhibitors (119110-U, 119111-V, 119913-X, 119915-Z, 170008-Y, 270718-R, and 306711-P) also inhibited F10 by at least 81% (as compared to control). An additional four compounds (9600-Q, 13778-J, 125908-P, and 128437-O) specifically inhibited GST-F10. Two of the F10 inhibitors (9600-Q and 13778-J) exhibited considerable specificity, inhibiting B1 almost not at all at a concentration of 10 μM. In fact, 13778-J stimulated B1 in vitro.







TABLE 3







Inhibition of B1 or F10 kinase activity by 10 μM inhibitor.










Percent activity remaining at 10 μM












Compound
B1
F10







117285-R
4 ± 2
48 ± 20



119910-U
0
6



119911-V
0
2



119913-X
1
2



119915-Z
1
4



170008-Y
12 
2



270718-R
12 ± 14
19 ± 18



306711-P
6
4



9600-Q
93 ± 2 
8 ± 5



13778-J
152 ± 11 
4 ± 4



125908-P
23 
8



128437-O
47 
12 










Four of the B1 inhibitors, 119110-U, 119111-V, 119913-X, and 119915-Z, had similar structures (derivatives of xanthen-3-one) and were related in structure to flourescein.
The potency of each inhibitory compound was measured in vitro. As shown in Table 4, 117285-R, a derivative of 2,6-diaminopurine, was the most potent inhibitor of B1, reducing the activity of the kinase by 50% at a concentration (IC50) of 1 μM. 13778-J, a compound containing pentavalent antimony, was the most potent inhibitor of F10, exhibiting an IC50 of 0.3 μM.







TABLE 4







Concentrations of inhibitors required


for 50% inhibition of kinase activity.









Compound















IC50 (μM) for B1



117285-R
1.0



119910-U
3.5



119911-V
3.0



119913-X
2.5



119915-Z
3.0



170008-Y
2.5



270718-R
2.5



306711-P
2.5




IC50 (μM) for F10



9600-Q
4.0



13778-J
0.3



125908-P
1.0



128437-O
1.0










Example 3
Poxvirus Protein Kinase Inhibitors do not Inhibit a Cellular Protein Kinase
This Example demonstrates that the VPK inhibitors identified in Example 2 do not inhibit a cellular tyrosine protein kinase (Lck) that is expressed in a variety of vertebrate species, including humans. This result indicates that the disclosed VPK inhibitors may not coincidentally inhibit protein kinases of the host cell or organism and, therefore, may be well tolerated when administered to a subject.
Table 5 shows that five of the VPK inhibitors (119910-U, 119913-X, 306711-P, 9600-Q, and 125908-P) minimally inhibited Lck when used at a concentration of 10 μM. The other seven compounds had little effect on the activity of Lck at this concentration.







TABLE 5







Inhibition of Lck kinase activity by 10 μM inhibitor.










Compound
Percent Lck activity remaining







117285-R
76 ± 25



119910-U
59



119911-V
70



119913-X
56



119915-Z
78



170008-Y
81



270718-R
86 ± 46



306711-P
45



9600-Q
53 ± 18



13778-J
79 ± 9 



125908-P
53



128437-O
88










Example 4
VPK Inhibitors Also Inhibit Poxvirus Growth In Vivo
This Example demonstrates that several of the VPK inhibitors identified in Example 2 can also inhibit poxvirus replication in living human cells. In particular, two B1 inhibitors (270718-R and 119913-X) and two F10 inhibitors (13778-J and 128437-O) inhibited the growth of wild-type vaccinia virus and, in the case of the B1 inhibitors, two vaccinia virus mutants (ts2 and ts25) in a human (HeLa) cell culture system containing 10% bovine calf serum.
The ts2 and ts25 vaccinia virus mutants each encode a temperature-sensitive B1 kinase (e.g., Rempel and Traktman, J. Virol., 66: 4413-4426, 1992). It was reasoned that the level of enzymatically active viral kinase in cells infected with the mutant viruses might be limiting, even at the permissive temperature. If so, inhibition of the less active mutant kinase by a compound should be reflected in a measurable reduction in virus yield. Accordingly, the B1 inhibitors were first tested against HeLa cells infected with the ts2 and ts25 mutants. Compounds 270718-R (see FIG. 1) and 119913-X reproducibly inhibited the growth of both the ts2 and ts25 mutant viruses in HeLa cells. Compound 270718-R had the greatest effect. It reduced virus yield by an average of 86%±13% (in seven experiments) at a concentration of 100 μM (FIG. 1 shows a single experiment where 99% inhibition was observed). Compound 119913-X reduced virus yield approximately 84%±5% at a concentration of 200 μM.
The virus growth inhibitory activity of compound 270718-R was also apparent when it was tested with HeLa cells infected with wild-type vaccinia virus. Compound 270718-R inhibited wild-type vaccinia virus by 84%±15% at a concentration of 100 μM. FIG. 1 shows an example where compound 270718-R inhibited the growth of wild-type vaccinia virus by 93% at a concentration of 100 μM.
The other B1 and/or F10 inhibitors from Example 2 (117285-R, 119110-U, 119111-V, 119915-Z, 170008-Y, 306711-P, 9600-Q, and 125908-P) had no apparent effect on virus growth in HeLa cell culture. The low activity of these particular VPK inhibitors in this system may be due to an inability of the compounds to enter the cells. Accordingly, modifying such compounds to enhance cell permeability (e.g., by making the compounds more lipophilic) may markedly improve the abilities of these compounds to inhibit poxvirus growth in vivo.
For safety reasons, vaccinia viruses were used in this Example. Nonetheless, the result presented here are equally applicable to variola virus and other poxviruses. The B1 and F10 protein kinases from smallpox virus are almost identical in sequence to those in vaccinia virus. F10 differs at 4 positions out of 400 (see FIG. 3), B1 differs at 11 positions out of 300 (see FIG. 4). The number of positions that differ will vary slightly depending upon the particular vaccinia virus and variola virus strains from which the respective B1- or F10-encoding sequences are obtained. The high degree of conservation suggests that the two protein kinases are essential for smallpox virus as well as for vaccinia virus. Additionally, the Example 2 screen for F10 inhibitors was performed with variola F10; thus, it is evident that the identified compounds have activity against the smallpox virus protein kinase. It is equally straightforward to mutate vaccinia B1 to smallpox virus B1 using commonly known techniques (e.g., site-directed or PCR-based mutagenesis) to test the B1 inhibitors against the variola B1 kinase homolog.
Example 5
The 270718-R PVK Inhibitor is Non-Toxic to Human Cells
This Example demonstrates that the inhibition of vaccinia virus growth by 270718-R was specific to viruses expressing a protein kinase and was not the result of host cell toxicity.
Using methods analogous to those described in Example 4, the ability of 270718-R to inhibit the growth of vesicular stomatitis virus (VSV), a rhabdovirus that does not encode a protein kinase, was tested. As shown in FIG. 1, compound 270718-R had no effect on the growth of VSV in HeLa cells, even when used at a concentration of 200 μM. Moreover, up to 100 μM 270718-R had no effect on the growth of human Ramos B cells (see FIG. 2) or HeLa cells (see FIG. 9) over a period of 48 hours.
This and the preceding Examples illustrate that the inhibition of poxvirus growth by 270718-R is due to inhibition of the viral protein kinases, rather than to some non-specific effect. At a concentration of 100 μM, 270718-R had no effect on the growth of human Ramos B cells over 48 hours. At this same concentration, 270718-R inhibited the growth of vaccinia virus over 24 hours by 86-99%. Additionally, even when used at a concentration of 200 μM, 270718-R had essentially no effect on the growth of vesicular stomatitis virus, an unrelated virus that does not encode a protein kinase. Additionally, the toxicity of this compound has been studied previously at NCI/NIH in three cell-based assays (information available at the website, dtp.nci.nih.gov/dtpstandard/servlet/dwindex?searchtype=NSC&chemnameboolean=and&outputform at=html&searchlist=270718&Submit=Submit). No growth inhibition was observed in any of the NCI/NIH-conducted cell assay when the drug was used at a concentration of 50 μM. Finally, 270718-R has been administered to mice (information available at the website, dtp.nci.nih.gov/dtpstandard/servlet/InvivoScreen?testshortname=Tumor+PS+%28ip%29+in+06&sear chtype=NSC&searchlist=270718). No toxicity in mice was noted when the compound was injected at a concentration of 200 mg/kg body weight.
Example 6
Negatively Charged Substituents May Enhance PKV Inhibitor Activity
Compound 270718-R is a tetralin (1,2,3,4-tetrahydro-naphthalene) derivative having the structure:




Commercial suppliers name compound 270718-R a “3-sulfo-2-naphthoic acid-bis(hexachlorocyclopentadiene) adduct.” This nomenclature (including the numbering of the substituents) may not correspond with formal naming conventions; however, the locations of the “3” (sulfonyl group) and “2” (carboxyl group) positions in accordance with the suppliers' nomenclature can be inferred from the above structure. Several related compounds (having the same substituent numbering conventions) are commercially available, including naphthalene-bis(hexachlorocyclopentadiene) adduct, 2-methylnaphthalene-bis(hexachlorocyclopentadiene) adduct, 2-bromonaphthalene-bis(hexachlorocyclopentadiene) adduct, and 2-nitronaphthalene-bis(hexachlorocyclopentadiene) adduct. The substituents at positions “2” and “3” in each of these 270718-R-related compounds are shown in the following table:















Substituent













Position
Position



Compound
“2”
“3”







naphthalene-bis(hexachlorocyclo-
—H
—H



pentadiene) adduct



2-methylnaphthalene-bis(hexachloro-
—CH3
—H



cyclopentadiene) adduct



2-bromonaphthalene-bis(hexachloro-
—Br
—H



cyclopentadiene) adduct



2-nitronaphthalene-bis(hexachloro-
—NO2
—H



cyclopentadiene) adduct










Using methods analogous to those described in Examples 1 and 2, none of the above-listed related compounds inhibited poxvirus B1 protein kinase activity in vitro. In addition, as shown in FIG. 1, 2-methylnaphthalene-bis(hexachlorocyclopentadiene) adduct had no effect on the growth of ts2 or ts25 vaccinia virus in HeLa cells at a concentration of 100 μM. At this same concentration, 270718-R reduced the growth of both ts2 and ts25 in HeLa cells by 99% (see Example 4).
This Example suggests that (i) substituents that are negatively charged under physiological conditions (such as carbonate, carboxylate, sulfonate, phosphonate, alkoxycarbonyl, acyloxy, alkoxy, or acyl) at the “2” and/or “3” positions, or (ii) a non-hydrogen substituent at position “3” are likely to facilitate the activity of 270718-R as a protein kinase inhibitor in vitro and poxvirus growth inhibitor in vivo.
Example 7
The Effective Concentration of 270718-R IS Reduced by Serum
The results described in Example 4 were obtained under conditions including 10% bovine calf serum. To test whether serum reduced the effective concentration of 270718-R, the potency of the compound in medium containing only 2% bovine calf serum was determined. Wild-type vaccinia virus and VSV were grown in HeLa cells in medium containing 2% bovine calf serum, 0.5% DMSO, and 10 μM, 25 μM or 50 μM 270718-R. Virus was harvested and assayed as described in Example 1.
As shown in FIG. 10, 270718-R was approximately four-fold more active under reduced-serum conditions. The compound inhibited the growth of wild-type vaccinia virus by 90% at a concentration of approximately 25 μM and by 99% at a concentration of approximately 35 μM. The inhibition of the growth of VSV was slight under these conditions (see FIG. 10). Reduced serum did not increase the activity of the other compounds examined in the foregoing Examples.
While this disclosure has been described with an emphasis upon particular embodiments, it will be obvious to those of ordinary skill in the art that variations of the particular embodiments may be used and it is intended that the disclosure may be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims:

## Claims

1. A method of inhibiting a poxviral protein kinase comprising contacting the poxviral protein kinase with an inhibitory amount of at least one compound having the formula:



wherein R1 and R2 are independently sulfonyl or carbonyl-containing; X is chloro or fluoro; and C1-C2 and C3-C4 are independently a single bond or a double bond.

2. The method of claim 1, wherein the poxviral protein kinase is a B1 protein kinase or F10 protein kinase or a variant of either.

3. The method of claim 1, wherein sulfonyl is sulfonic acid, alkylsulfonyl, or arylsulfonyl; and carbonyl-containing is carboxylic acid or carboxylic acid ester.

4. The method of claim 1, wherein the at least one compound is a 3-sulfo-2-napthoic acid-bis(hexachlorocyclopentadiene) adduct.

5. The method of claim 1, wherein X is chloro.

6. The method of claim 1, wherein the at least one compound is NSC270718R.

7. A method of inhibiting poxvirus growth comprising contacting the poxvirus with a growth inhibitory amount of at least one compound having the formula:



wherein R1 and R2 are independently sulfonyl or carbonyl-containing; X is chloro or fluoro; and C1-C2 and C3-C4 are independently a single bond or a double bond.

8. The method of claim 7, wherein the poxvirus is a member of the chordopoxvirinae subfamily.

9. The method of claim 7, wherein sulfonyl is sulfonic acid, alkylsulfonyl, or arylsulfonyl; and carbonyl-containing is carboxylic acid or carboxylic acid ester.

10. The method of claim 7, wherein the at least one compound is a 3-sulfo-2-napthoic acid-bis(hexachlorocyclopentadiene) adduct.

11. The method of claim 7, wherein the poxvirus is variola virus or vaccinia virus.

12. The method of claim 7, wherein the growth inhibitory amount is no more than about 250 μM.

13. The method of claim 7, wherein the at least one compound is NSC270718R.

14. The method of claim 7, wherein the poxvirus of the poxvirus is a vaccinia virus, variola virus, monkeypox virus, buffalopox virus, cowpox virus, elephantpox virus, bovine papular stomatitis virus, orf virus, pseudocowpox virus, sealpox virus, tanapox virus, or Yaba monkey tumor virus, or combinations thereof.

15. The method of claim 8, wherein the member of the chordopoxvirinae subfamily is an Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Mollusciposvirus, or Yatapoxvirus.

16. A method for treating poxvirus infection in a subject, comprising administering to a subject a therapeutically effective amount of at least one compound having the formula:



wherein R1 and R2 are independently sulfonyl or carbonyl-containing; X is chloro or fluoro; and C1-C2 and C3-C4 are independently a single bond or a double bond; and
wherein administering the at least one compound treats poxvirus infection in the subject.

17. The method of claim 16, wherein the poxvirus of the poxvirus infection is vaccinia virus, variola virus, monkeypox virus, buffalopox virus, cowpox virus, elephantpox virus, bovine papular stomatitis virus, orf virus, pseudocowpox virus, sealpox virus, tanapox virus, or Yaba monkey tumor virus, or combinations thereof.

18. The method of claim 16, wherein sulfonyl is sulfonic acid, alkylsulfonyl, or arylsulfonyl; and carbonyl-containing is carboxylic acid or carboxylic acid ester.

19. The method of claim 16, wherein the at least one compound is a 3-sulfo-2-napthoic acid-bis(hexachlorocyclopentadiene) adduct.

20. The method of claim 16, wherein the poxvirus is a member of the chordopoxvirinae subfamily.

21. The method of claim 16, wherein the poxvirus is vaccinia virus or variola virus.

22. The method of claim 16, wherein the subject is a human.

23. The method of claim 16, wherein the therapeutically effective amount is from about 5 mg/kg to about 200 mg/kg per day.

24. The method of claim 16, wherein the at least one compound is NSC270718R.

25. The method of claim 20, wherein the member of the chordopoxvirinae subfamily is an Orthopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Mollusciposvirus, or Yatapoxvirus.