WO2006135763A2 - Inhibiteurs d'enzymes de reparation de l'adn et procedes d'utilisation de ceux-ci - Google Patents

Inhibiteurs d'enzymes de reparation de l'adn et procedes d'utilisation de ceux-ci Download PDF

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Publication number
WO2006135763A2
WO2006135763A2 PCT/US2006/022513 US2006022513W WO2006135763A2 WO 2006135763 A2 WO2006135763 A2 WO 2006135763A2 US 2006022513 W US2006022513 W US 2006022513W WO 2006135763 A2 WO2006135763 A2 WO 2006135763A2
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uracil
compound
enzyme
dna
uber
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PCT/US2006/022513
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WO2006135763A3 (fr
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James T. Stivers
Daniel J. Krosky
Yu Lin Jiang
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The Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals

Definitions

  • the present invention relates generally to compositions and methods for treating diseases, especially cancer, viral and bacterial infections or related disorders, through the modification and/or inhibition of the uracil base excision pathway (UBEP).
  • the invention further relates to new chemical compounds for modifying and/or inhibiting UBEP enzymes are provided, screening methods to identify the compounds, pharmaceutical compositions comprising the compounds, and methods of treating cancer, viral and bacterial infections or related disorders by administering the compounds.
  • Other aspects of the invention are described in or are obvious from the following disclosure (and within the ambitof the invention).
  • Uracil arises in DNA by two distinct pathways involving spontaneous or enzymatic cytosine deamination, or alternatively, misincorporation of dUTP in place of TTP during DNA synthesis.
  • the incorporation of uracil into DNA is usually harmful to an organism or genome and the occurrence of resulting DNA damage (e.g. double-strand breaks) should be repaired to preserve genomic operability.
  • the DNA repair enzymes and processes that repair uracil-linked DNA damage are emerging as desirable targets for small molecule inhibitors given the key role of such systems in a wide range of biological processes including the generation of antibody diversity, DNA replication in a number of viruses, and the formation of DNA strand breaks during anticancer drug therapy.
  • the instant invention is based, at least in part, on the discovery of a new class of small-molecule compounds that inhibit DNA repair enzymes of the uracil base excision pathway.
  • This new class of compounds comprises an active-site targeting element, a second binding element, optionally a third binding element, and a linker moiety which covalently tethers the active-site targeting element with the second binding element and the third binding element, if present.
  • the present inventors conceived and discovered these new compounds and methods to identify same by focusing of active-site substrates (or ligands) or portions thereof that target the whole inhibitor compound to the active site of a DNA repair enzyme, while a flexibly tethered secondary binding moiety explores nearby surfaces and binds to a binding pocket adjacent to the active site.
  • the present invention also relates to improved methods for building large libraries of candidate inhibitors and also to improved high- throughput assays that screen the libraries to identify the inventive compounds.
  • the instant invention further provides methods of treating diseases that are affected by the uracil base excision repair pathway, including but not limited to cancers and infections by viruses, bacteria and parasites.
  • Pharmaceutical compositions comprising the new class of compounds for the treatment of diseases that are affected by the uracil base excision repair pathway are also provided by the invention.
  • the compositions may also be used to enhance existing treatments of disease, e.g. cancer, by co-administering a compound of the invention and a known cytotoxic agent.
  • the invention also relates to kits comprising pharmaceutical
  • 211592 1 compositions for the treatment of disease and kits for testing a compound for inhibitory activity against the UBER enzymes or other enzymes of the invention.
  • the specific aspects and embodiments of the invention will be more thoroughly detailed below.
  • UBER uracil base excision repair
  • Another object of the invention is to provide improved methods for synthesizing, screening and identifying the new compounds of the invention which are capable of inhibiting an enzyme of the uracil base excision repair (UBER) pathway and which may be used in the treatment of various diseases and disorders including cancer and infectious diseases.
  • UBER uracil base excision repair
  • a still further object of the invention is to provide advantageous and improved methods for treating diseases and disorders that can be affected by uracil incorporation and/or uracil-linked DNA damage by administering the inhibitor compounds of the invention, that is the compounds identified as inhibitors of enzymes of the UBER pathway.
  • the present invention provides a compound comprising a active- site targeting element, a second binding element, and a linker which links the active-site targeting element and the second binding element.
  • the compound can further comprise a third binding element.
  • Another embodiment of the invention relates to a method for treating a viral infection in a patient in need thereof by inhibiting an enzyme of the uracil base excision repair (UBER) pathway comprising administering a therapeutically effective amount of a compound of the invention to the patient, thereby treating the viral infection in the patient.
  • the viral infection can be a virus selected from the group consisting of HTV-I, HIV-2, herpes simplex virus, cytomegalo virus, smallpox virus, avian flu virus, West Nile virus, Epstein Barr virus, and influenza virus.
  • the viral infection is an HIV infection.
  • a still further embodimet of the invention relates to a method for treating cancer in a patient in need thereof, the method comprising administering to the patient separately or in an admixture a cytotoxic agent and a therapeutically effective amount of a compound of the invention.
  • a method for inhibiting the biological activity of uracil DNA glycosylase, pUTPase, or AP endonuclease I in a subject comprising administering to the subject an effective amount of a compound of the invention.
  • a further embodiment relates to a method for treating an infectious disease in a patient in need thereof by inhibiting an enzyme of the uracil base excision repair (UBER) pathway comprising administering a therapeutically effective amount of a compound of the invention to the patient, thereby treating the infectious disease in the patient.
  • UBER uracil base excision repair
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the invention together with a pharmaceutically acceptable carrier.
  • a cytotoxic agent such as an anti-cancer, anti-bacterial, or an anti-viral compound, may be coadministered.
  • the present invention further relates to a kit for inhibiting an enzyme of the uracil base excision repair (UBER) pathway, the kit comprising a container comprising a compound of the invention, and instructions for using the compound to inhibit the enzyme of the uracil base excision repair (UBER) pathway.
  • UBER uracil base excision repair
  • kits for detecting or identifying inhibitors of an enzyme of the uracil excision repair (UBER) pathway comprising (a) an enzyme of the uracil base excision repair (UBER) pathway; (b) a reagent comprising (i) a first uracil-containing nucleic acid sequence; (ii) a second nucleic acid sequence complementary to the first sequence; (iii) a linker linking the first and second nucleic acid sequences; (iv) a fluorescent moiety, and (v) a quencher of the fluorescent moiety; and (c) instructions for use; and wherein increased fluorescence of the fluorescent moiety indicates that the compound is capable of inhibiting the enzyme of the uracil base excision repair (UBER) pathway.
  • FIG. 1 Origins and consequences of uracil in DNA.
  • Uracil may arise from incorporation of dUTP during DNA replication, or by spontaneous or enzyme-catalyzed DNA cytidine deamination.
  • the drug 5-fluorouracil (5-FU) promotes dUTP incorporation by inhibiting TMP synthesis by thymidylate synthase (TS), and the enzyme dUTPase prevents incorporation by hydrolyzing dUTP to dUMP.
  • Uracil itself may have direct antiviral effects in the context of UA or UG base pairs.
  • Uracil is removed from the DNA sugar-phosphate backbone by the uracil DNA glycosylase activity of UNG, leaving an abasic site ( ⁇ ).
  • This abasic site can be processed to nicked DNA by the abasic site endonuclease APE-I to give rise to a number of different outcomes, or be repaired by the combined action of APE-I , pol ⁇ and DNA ligase.
  • FIG. 2 Role of UDG in removing misincorporated dUTP during HIV-I infections.
  • hUDG is packaged into the HIV-I particle and removes uracil that is incorporated into the virus genome due to the high dUTP levels of resting macrophages.
  • uracil persists in the viral genome and prevents integration.
  • FIG. 3 General Approach of Substrate Fragment Tethering (SFT).
  • SFT involves linking a substrate fragment or known ligand to flexible linkers that can be derivatized on the other end with a library of functional groups with different electronic and geometric properties.
  • the substrate fragment targets the linked molecule to the active site, while the additional substituents can probe other nearby binding sites for increased affinity.
  • FIG. 4 High-throughput (HTP) UDG kinetic assay.
  • A The HTP assay relies on molecular beacon technology. Excision of multiple uracil bases by the enzyme destabilizes the hairpin structure thereby releasing the 5' FAM fluorophore from the quenching effects of the 3' dabsyl group.
  • B Steady-state kinetic analysis of the hUDG reaction using the molecular beacon hairpin substrate.
  • FIG. 5 Inhibition of UNG2 in primary macrophage cell culture. Human SWl 3 cells were treated for 4h with inhibitor and then cell extracts were prepared and incubated with the radiolabeled uracil-containing probe DNA. Lane 1 : uracil containing probe without
  • lane 2 probe incubated with untreated cell extract (negative control); lane 3: probe incubated with extract treated with 4 ⁇ M of recombinant TAT-conjugated uracil DNA glycosylase inhibitor protein (positive control); lane 4: probe incubated with cell extract treated with 1 ⁇ M of compound 1.
  • FIG. 6 Representative HTP screening results using the molecular beacon substrate.
  • A Screen of oxime dimer mixtures derived from uracil aldehyde 1 and aryl aldehydes 13-17. No inhibition was observed for any oxime derived from 1 regardless of linker length (n).
  • B Screen of oxime dimer mixtures derived from uracil aldehyde 3 and aryl aldehydes 13-17. The mixed oxime derived from 3 and 13 shows significant inhibition and this derivative was further optimized. For 14-17, the observed inhibition represents that from the 3-3 homodimers that are present in the mixtures.
  • FIG. 7 Structures of active heterodimers and dependence of inhibition on linker Length ' a Reactions were performed in the presence of 100 ⁇ M oxime mixture and 50 nM substrate concentration.
  • FIG. 8. IC 50 analysis of for 2-(2) ⁇ 3 (Y), 3-(3)-13 (A) and 3-(3)-27 ( ⁇ ).
  • FIG. 9. Mode of inhibition analysis. Double reciprocal plots and secondary slope and intercept replots for inhibition by increasing concentrations of (A) 3 ⁇ (3)-27, (B) 2-(2)-13, and (C) uracil. Slope and intercept effects in the inset to (C) are shown as squares and triangles, respectively.
  • FIG. 10 Possible biochemical linkages between 5-FU toxicity and damage to RNA and DNA.
  • 5-FU may interfere with nucleic acid structure and function through inhibition of thymidylate synthase and disruption of nucleotide pool balance or the direct incorporation of 5-fluoronucleotides into DNA and RNA.
  • B Although it has been hypothesized that uracil base excision repair plays an important role in mediating 5-FU toxicity, the lethal intermediate along this pathway remains to be identified. Possible intermediates moving clockwise around the cycle are the mutagenic 5-FU base itself, the abasic product of the UNGl reaction, or various incised products derived from the intact abasic site.
  • FIG. 11 Time courses for killing of wild type and repair-deficient Saccharomyces cerevisiae by 5-FU. Yeast were shaken in 150 ⁇ M 5-FU for given times and then diluted and plated.
  • A Single mutants lacking enzymes along the uracil base excision repair pathway have distinct 5-FU survival curves. Ungl and rad27 yeast are protected from the effects of 5-FU while apnl yeast are more sensitive to the drug relative to wild type.
  • B
  • Apnlapn2ntglntg2 yeast are more sensitive to 5-FU relative to wild type and the apnl strain.
  • C Homologous recombination deficient radSl yeast are modestly protected against 5-FU as compared to wild-type yeast.
  • D The NER and mismatch repair deficient rad2 and mshl yeast show no increased sensitivity relative to wild type at this concentration of 5-FU.
  • FIG. 12 Cell cycle profiles for Saccharomyces cerevisiae treated with 5-FU. Yeast were incubated in media with or without 150 ⁇ M 5-FU, fixed at given times with ethanol, and stained with propidium iodide.
  • A Wild type yeast treated with 5-FU arrest in the G 1 /S phase of the cell cycle.
  • B Ungl yeast arrest in G 1 ZS phase of the cell cycle.
  • C Apnl yeast arrest in the G 2 ZM phase of the cell cycle.
  • FIG. 13 Uracil, 5-FU, and abasic site accumulation in yeast genomic DNA.
  • DNA from yeast grown in media containing 150 ⁇ M 5-FU or its absence was digested with E. coli UNGl and human APEl and then fractionated by electrophoresis using an agarose gel. Double strand breaks resulting from the enzymatic removal of U and 5-FU and cleavage of abasic sites appear as a smear of lower molecular weight fragments.
  • FIG. 14 GC-MS quantification of U and 5-FU incorporation into the genomic DNA of yeast grown in the absence and presence of 5-FU. Cells were incubated in media containing 5-FU such that 70-90% cell killing was achieved. Genomic DNA was then isolated and digested with E. coli Ung to release U and 5-FU.
  • A Uracil levels detected in the presence and absence of 5-FU.
  • B 5-FU levels detected in the presence and absence of 5-FU.
  • FIG. 15 5-Fluorouracil incorporation into RNA. Wild-type yeast were grown in media in the absence and presence of 5-FU and cellular RNA was isolated and digested to its constituent nucleosides using mung bean nuclease and calf intestinal phosphatase. A small peak corresponding to 5-fluorouridine is visible in the HPLC spectrum of the digested RNA from the drug-treated cells (middle panel, inset). This peak is not seen with yeast grown in the absence of 5-FU (top panel, inset). The lower panel shows the elution pattern for authentic nucleoside standards.
  • FIG. 16 Aldehyde reactive probe slot-blot (ASB) assay for intact and nicked abasic sites.
  • A Wild-type yeast grown in the absence and presence of 5-FU.
  • B ungl yeast, s3ame as (A).
  • C apnl yeast, same as (A).
  • FIG. 17 DNA repair pathways implicated in 5-FU-mediated cell killing.
  • the model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ungl yeast are much less sensitive to 5-FU than the wild-type
  • 211592 1 strain indicating that uracilated DNA is not the mediator of 5-FU toxicity
  • the apnlapn2ntglntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity
  • the rad27 and c ⁇ nlmd.27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction.
  • Several backup pathways for repair of abasic sites and 5'dRp groups are indicated.
  • NER nucleotide excision repair
  • HR homologous recombination
  • FIG. 18 shows a synthetic scheme for preparing nonnucleotide deoxyuridine substrate fragments.
  • FIG. 19 shows synthetic schemes for preparing dUTPase inhibitor compounds according to the invention.
  • FIG. 20 shows a scheme for preparing an imino sugar-aldehyde transition state mimic.
  • the present invention solves the deficiencies and needs of the art by providing new and effective compounds, methods and strategies that are effective in treating various diseases, including cancer, infectious diseases, bacterial infections, and viral infections, through the use of novel compounds that generally providing an inhibitory action against the enzymes of the uracil base excision repair (UBER) pathway, which includes the enzymes uracil DNA glycosylase, pUTPase and AP endonuclease I.
  • the inhibitor compounds of the invention can comprise an active-site targeting element, a second binding element, and a linker which links the active-site targeting element and the second binding element.
  • the activ-site targeting element can be a uracil substrate fragment.
  • the compounds can include a third binding element.
  • the present invention also relates to methods of screening to identify the inhibitory compounds of the invention that inhibit the enzymes of the UBER pathway. Methods for using the inventive compounds to treat subjects having
  • 211592 1 diseases including cancer, bacterial infections, and viral injections are also provided by the present invention, as well as pharmaceutical compositions for the treatment of same.
  • the present invention relates to screening methods and kits for identifying the inhibitory compounds of the invention.
  • the "uracil base excision repair (UBER) pathway” includes, at least, the enzymes uracil DNA glycosylase, pUTPase, and AP endonuclease I. Further information as to the nature of each of the UBER enzymes of the invention are provided herewith.
  • UBER enzymes or "UBER pathway enzymes” refers one or more of enzymes of the uracil base excision repair (UBER) pathway and includes, for example, uracil DNA glycosylase, pUTPase and AP endonuclease.
  • UBER enzymes are not limited to any particular source and can be from, for example, humans, bacteria, microorganisms, and viruses.
  • host uracil base excision repair (UBER) pathway enzymes or similarly “host UBER enzymes,” or any similar expression, is meant to distinguish between the UBER enzymes of a host animal (e.g. a human) from the UBER enzymes that may be provided by an infectious organism or virus (e.g. an HTV virus or pathogenic bacteria).
  • UBER uracil base excision repair
  • VBER viral uracil base excision repair pathway enzymes
  • a host animal e.g. a human
  • a virus e.g. an HTV virus
  • bacterial uracil base excision repair (UBER) pathway enzymes or similarly “bacterial UBER enzymes,” or any similar expression, is meant to distinguish between the UBER enzymes of a host animal (e.g. a human) from the UBER enzymes that may be provided by an infectious bacteria (e.g. pathogenic bacteria).
  • UBER bacterial uracil base excision repair
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a UBER pathway inhibitor of the present invention which is effective for producing some desired therapeutic effect by inhibiting an enzyme of the UBER pathway, including inhibition of uracil DNA glycosylase, pUTPase, and AP endonuclease I, in at least a sub-population of cells in an animal and thereby altering
  • UBER pathway enzymes uracil DNA glycosylase
  • UNG uracil DNA glycosylase
  • UNG2 mitochondriachondrial type
  • AP endonuclease I may be referred to as "APE” or "APE-I.”
  • a “therapeutic effect” results, to some extent, in a measurable response in the treated disease or condition, e.g. bacterial or viral infectious disease or cancer.
  • a therapeutic effect can include a cure, or a lessening of the growth rate or size of a lesion such as a tumor, or an increase in the survival time of treated patients compared to controls, among other possible effects.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject glycosylase inhibitor from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject glycosylase inhibitor from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxyniethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydrox
  • Substrate Fragment Tethering involves linking a substrate fragment or known ligand to flexible linkers that can be derivatized on the other end with a library of functional groups with different electronic and geometric properties.
  • the substrate fragment targets the linked molecule to the active site, while the additional substituents can probe other nearby binding sites for increased affinity.
  • uracil-lmked DNA damage is the damage to DNA that can result from the misincorporation of uracil into DNA, such as, for example double-strand breaks.
  • alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,
  • alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone
  • a straight chain or branched chain alkyl has 10 or fewer carbon atoms in its backbone (e.g., C 1 -C 10 for straight chain, C 3 -C 10 for branched chain), and more preferably 6 or fewer.
  • preferred cycloalkyls have from 4-7 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • alkyl includes both "unsubstituted alkyls" and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
  • Cycloalkyls can be further substituted, e.g., with the substituents described above.
  • An "alkylaryl” or an “aralkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • the term “alkyl” also includes the side chains of natural and unnatural amino acids. Examples of halogenated alkyl groups include fluoromethyl,
  • 211592 1 difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, perfluoromethyl, perchloromethyl, perfluoroethyl, perchloroethyl, etc.
  • aryl or “aromatic” includes groups, including 5- and 6-membered single- ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, phenyl, pyrrole, furan, thiophene, thiazole, isothiaozole, imidazole, triazole, tetrazole, pyrazole, oxazole, isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
  • aryl includes multicyclic aryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole, benzodioxazole, benzothiazole, benzoimidazole, benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole, benzofuran, purine, benzofuran, deazapurine, or indolizine.
  • aryl groups having heteroatoms in the ring structure may also be referred to as “aryl heterocycles", “heterocycles,” “heteroaryls” or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkylaminoacarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyL alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ure
  • amine or "amino” includes compounds where a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • arylamino and “diarylamino” include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively.
  • alkylarylamino alkylaminoaryl or “arylaminoalkyl” refers to an amino group that is bound to at least one alkyl group and at least one aryl group.
  • alkaminoalkyl refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom that is also bound to an alkyl group.
  • amide or "aminocarboxy” includes compounds or moieties that contain a nitrogen atom that is bound to the carbon of a carbonyl or a thiocarbonyl group.
  • alkaminocarboxy groups that include alkyl, alkenyl, or alkynyl groups bound to an amino group bound to a carboxy group. It includes arylaminocarboxy groups that include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group.
  • alkylaminocarboxy include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties, respectively, are bound to a nitrogen atom which is in turn bound to the carbon of a carbonyl group.
  • halogen or halo refers to F, Cl, Br, or I.
  • cyano refers to the group -CN.
  • alcohol includes organic compound in which a hydroxyl group is bound to a carbon atom .
  • Alcohols can be primary, secondary, or tertiary in structure.
  • aldehyde refers to the group -C(O)H, or a compound including such a group.
  • the present invention relates to compounds that have an inhibitory effect on one or more enzymes of the UBER pathway.
  • Methods for screening and identifying the inhibitory compounds of invention are provided as well as methods and kits for administering the compounds to treat diseases including cancers and viral and bacterial infections.
  • the present invention provides a compound comprising a active-site targeting element, a second binding element, and a linker which links the active-site targeting element and the second binding element.
  • the invention provides compounds have two elements which are capable of binding to one or more sites on a UBER enzyme.
  • the bifunctional compounds of the invention can be adapted to provide inhibition of a variety of enzymes.
  • active site targeting element refers to a moiety which is capable of binding to or interacting with the active site of an enzyme.
  • An active site targeting element can be, e.g., a derivative or analog of a natural substrate of the enzyme, or a transition state mimic.
  • the active-site targeting element can be, for example, a substrate or fragment thereof or a ligand or fragment thereof that has affinity for the active site of a UBER enzyme.
  • 211592 1 site targeting element can be a naturally-occurring substrate/ligand or it can be a synthetic molecule, including non-naturally occurring synthetic molecules that have affinity for the active site of the enzymes of the invention.
  • a requirement of the active-site targeting element is that it be capable of targeting the inhibitor compounds of the invention to the active site of the enzyme.
  • the active-site targeting element is a uracil substrate fragment, i.e., a uracil-based moiety, e.g., a derivative or analog of uracil.
  • a "uracil substrate fragment” is a moiety including a uracil portion or a derivative thereof or fragment thereof that is capable of targeting the inhibitor compounds of the invention to one or more UBER enzymes, especially uracil DNA glycosylase and pUTPase.
  • the uracil substrate fragment can include, for example, a uracil ring with functionality suitable for connection to a linker moiety.
  • the uracil substrate fragment can include, for example, a uracil derivative having an aldehyde functionality (a uracil-aldehyde).
  • a "second binding element” is a moiety that is tethered to the active- site targeting element through a flexible linker.
  • the second binding element interacts and binds to a target enzyme, e.g., a UBER enzyme, e.g., by binding to or interacting with a binding pocket outside of (but preferably adjacent to) the active site.
  • a target enzyme e.g., a UBER enzyme
  • Exempalry second binding elements are described in more detail herein, and include moieties such as aryl groups (included substituted phenyl groups, including hydroxylated phenyl groups) and stibonic acids, and derivatives thereof.
  • the present invention relates to inhibitory compounds comprising a third binding element.
  • the present invention relates to inhibitory compounds comprising a third binding element.
  • a "third binding element” is a moiety that is tethered to the active-site targeting element and a second binding element through a flexible linker.
  • the third binding element interacts and binds to a target enzyme, e.g., a UBER enzyme, e.g., by binding to or interacting with a binding pocket outside of (but preferably adjacent to) the active site.
  • the third binding element is the same as the second binding element, i.e., the compound of the invention includes two (or more) identical second binding elements.
  • the invention relates to a compound comprising a active- site targeting element, a second binding element, and a linker which links the active-site targeting element and the second binding element, wherein the compound is represented by the formula (Formula I):
  • the present invention relates to a compound comprising a active-site targeting element, a second binding element, and a linker which links the active- site targeting element and the second binding element, wherein the compound is represented by the formula (Formula II):
  • A is, independently for each occurrence, O, CH 2 , or a single bond;
  • Ar is an aromatic or heteroaromatic group;
  • n is an integer from 2 to 10; and
  • p is 1, 2 or 3.
  • Compounds of the invention also include the salts (especially pharmaceutically acceptable salts), solvates, clathrates, hydrates, polymorphs, or prodrugs of any of the compounds disclosed herein
  • linker is a moiety that is capable of covalently tethering an active-site targeting element to a second binding element.
  • the linker is not limited to any
  • the linker includes a diaminoalkanediol molecule of variable lengths.
  • the linker can also have multiple points of attachment for an active-site targeting moiety and a plurality of second (and third, fourth, etc.) binding moieties.
  • the linker can be a three-prong linker for covalently tethering an active-site targeting element, a second binding element and a third binding element.
  • the three-prong structure can be referred to as a triskelion compound or structure.
  • the linker should provide sufficient flexibility such that the active-site targeting element and second binding element (and the third binding element, if present) are capable of simultaneous binding to or interacting with their respective binding sites.
  • the inhibitor compounds of the invention are small molecules and are suitable for administration to human or animal subjects in need thereof.
  • small molecule or “low molecular weight compound” is meant a molecule having a molecular weight of equal to or less than about 5000 daltons, equal to or less than about 2000 daltons, equal to or less than about 1000 daltons, or equal to or less that about 600 daltons. In other embodiments, the small molecule is still smaller, for example less than about 500, 400, or 300 daltons.
  • such compounds may be found in compound libraries, combinatorial libraries, natural products libraries, and other similar sources, and may further be obtained by chemical modification of compounds found in those libraries, such as by a process of medicinal chemistry as understood by those skilled in the art, which can be used to produce compounds having desired pharmacological properties.
  • the compounds of the invention can be prepared by a variety of methods, some of which are known in the art.
  • compounds of the present invention can be prepared according to the general methods shown in the reaction schemes below (and described in detail in the Examples).
  • Some of the novel compounds of this invention can be prepared using synthetic chemistry techniques well known in the art (see, e.g., Comprehensive Organic Synthesis, Trost, B. M. and Fleming, I. eds., Pergamon Press, Oxford).
  • the compounds of the invention can be prepared by reaction of chemcial reagents corresponding to the uracil moiety, the linker (or tether) moiety, and the second binding element (or third binding element, etc.).
  • chemcial reagents corresponding to the uracil moiety, the linker (or tether) moiety, and the second binding element (or third binding element, etc.).
  • uracil moiety reagents include a uracil derivative having a reactive functionality suitable for reacting with a linker reagent.
  • the uracil moiety is derivatized such that the uracil moiety of the compound of the invention as able to bind to or interact with the enzyme active site.
  • Illustrative examples of uracil reagents include the following:
  • X is a leaving group such as Cl, I, Br, tosylate, mesylate, and the like.
  • a uracil-containing compound e.g., a uracil derivative
  • a linker reagent having an amine (-NH 2 ) or R-O- NH 2 functionality under conditions suitable for formation of the imine or oxime group, as shown in Scheme 1.
  • this reaction is performed in the presence of a catalytic amount of an acid such as acetic acid.
  • the reaction can be performed in a solvent such as dimethyl sulfoxide (DMSO).
  • a uracil-containing compound e.g., a uracil derivative
  • a leaving group functionality such as Cl, I, Br, tosylate, mesylate, and the like
  • a linker reagent having a nucleophilic group such as an amine (-NH 2 ) or R-O-NH 2 functionality, or a hydroxyl or thiol group, under conditions suitable for nucleophilic displacement of the leaving group by the nucleophilic group, thereby linking the uracil-containing compound to the linker.
  • the reaction will generally be performed in the presence of a base.
  • the base can be, e.g., potassium t-butoxide or sodium hydride.
  • polar aprotic solvents such as DMSO, tetrahydrofuran (THF), and acetonitrile may be used.
  • a uracil-contaming compound e.g., a uracil derivative
  • a nucleophilic group such as an amine (-NH 2 ) or oxyamine functionality, or a hydroxyl or thiol group
  • a linker reagent having a leaving group functionality shown below in Scheme 3 as X
  • X a leaving group functionality
  • the uracil moiety is shown as having an amine group for reaction with the linker, but other nucleophilic moieties may also be useful as indicated above.
  • the reaction will generally be performed in the presence of a base.
  • the base can be, e.g., potassium t-butoxide or sodium hydride.
  • polar aprotic solvents such as DMSO, tetrahydrofuran (THF), and acetonitrile may be used.
  • a uracil-containing compound e.g., a uracil derivative
  • a nucleophilic group such as an amine (-NH 2 ) or R-O-NH 2 functionality, or a hydroxyl or thiol group
  • a linker reagent having a carboxylate functionality (either a free carboxylic acid or an ester, e.g., an activated ester) under conditions suitable for formation of an ester, amide, or thioester, thereby linking the uracil- containing compound to the linker.
  • X is a leaving group such as Cl, pentafluorophenyl, and the like.
  • a linker reagent can be any bifunctional reagent capable of reacting with and covalently linking the uracil moiety and the second binding element.
  • the linker can be trifunctional. In certain embodiments, the linker is not trifunctional.
  • a linker reagent can have the formula H 2 N-O-(CH 2 ) n - 0-NH 2 , wherein n is an integer from 2 to 10.
  • a linker reagent can have the formula H 2 N-(CH 2 ) n -NH 2 , wherein n is an integer from 2 to 10.
  • a linker reagent can have the formula HO-(CHa) n -OH, wherein n is an integer from 2 to 10.
  • a linker reagent can have the formula LG-(CH 2 ) n -LG, wherein n is an integer from 2 to 10 and LG is a leaving group such as Cl, Br, mesylate,
  • the linker reagent can have the formula H 2 N- O-(CH 2 ) n -C[-(CH 2 ) n -0-NH 2 J m H 3-OT , wherein n is independently for each occurrence an integer from 2 to 10 and m is 1, 2 or 3.
  • Non-limiting examples of linker reagents include the following:
  • uracil, linker, and second (or third, etc.) binding element reagents for use in preparing the compounds of the invention can be selected by one of ordinary skill in the art in view of the disclosure herein.
  • reagents can be selected, and compounds can be prepared and rapidly screened, as disclosed herein, to determine particular reagents which result in active compounds. This information can be used to select further reagents for synthesis in an iterative fashion.
  • linker reagent can be reacted with the uracil moiety first, and the linker-uracil compound can then be reacted with the second (or third, etc.) binding moiety to form a
  • the linker can be reacted with the second (or third, etc.) binding moiety first, and the linker-binding moiety compound can then be reacted with the uracil moiety to form a compound of the invention.
  • the reaction of a linker with the uracil moiety and the second (or third, etc.) binding moiety can also be carried out simultaneously, in certain embodiments, when the reactions are carried out simultaneously, it may be possible for mixtures of products (e.g., statistical mixtures of condensation products) to form. If desired, these mixtures can be separated by conventional techniques, such as chromatographic purification methods.
  • linkages can be prepared by similar reactions. Sensitive moieties on a uracil or linker moiety can be protected prior to coupling.
  • the chemicals used in the above-described synthetic routes may include, for example, solvents, reagents, catalysts, and protecting group and deprotecting group reagents.
  • the methods described above may also additionally include steps, either before or after the steps described specifically herein, to add or remove suitable protecting groups in order to ultimately allow synthesis of the compounds of the invention. In addition, various synthetic steps may be performed in an alternate sequence or order to give the desired compounds.
  • Synthetic chemistry transformations and protecting group methodologies useful in synthesizing applicable compounds of the invention are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3 rd Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.
  • the linker reagent can also be linked to the second, third, etc. binding elements, in reactions analogous to those shown in Schemes 1-4.
  • a second binding element which contains an aldehyde group can be reacted with a linker reagent having an amine (- NH 2 ) or R-O-NH 2 functionality under conditions suitable for formation of the imine or oxime group.
  • the second (and third, etc. binding elements) can be selected to provide binding affinity and specificity for a target enzyme.
  • reagents corresponding to binding elements include aldehydes, alkyl halides, arylalkyl halides (e.g., benzyl halides), and the like.
  • Illustrative reagents include the following:
  • Additional illustrative reagents include the following:
  • Analogs of the above reagents may also be useful for preparing compounds of the invention.
  • the invention also provides compounds having second and third binding elements.
  • the linker comprises wherein n is independently for each occurrence an integer from 2 to 10 and m is 1, 2 or 3.
  • the second and third (and optionally fourth) binding element are as described herein and may be the same or different.
  • the uracil moiety can be a moiety as described above.
  • exemplary compounds include the following:
  • the uracil- or ligand-oxime products are connected to a library of aldehydes or ketones using the free ONH 2 group on the bivalent linker.
  • the reactions often go to completion, reducing or eliminating the requirement for purification steps.
  • ketones react more slowly and may require 72 hours incubation in the presence of trichloroacetic
  • the compounds can be prepared in microtiter plates for convenience in screening.
  • an imino sugar aldehyde library can be prepared, based on the finding that imino sugars in a DNA context are tight binding glycosyl cation transition state mimics (X 1 ⁇ 10 pM) (see, e.g., Jiang, et al.,. Bioorg Chem 32, 244-62 (2004); Jiang et al., Abstracts of Papers of the American Chemical Society 224, 140-BIOL (2002); and Bianchet, M.A. et al. Biochemistry 42, 12455-60 (2003).
  • the imino sugar-aldehyde maybe synthesized as outlined in Figure 20 for tethering to the library of aldehydes and ketones, e.g., as described above.
  • the invention also provides dUTPase inhibitors.
  • the dUTPase inhibitors include a uracil or dUTP analog or derivative as the active site targeting moiety, a linker, and a second (or third, etc.) binding element.
  • dUTPase inhibitors can have structures similar to the UNG inhibitors already described, and certain of the UNG inhibiotrs are also dUTPase inhibitors (see the Examples).
  • dUTPase inhibitors can include a deoxyuridine analog instead of the uracil analog of the UNG inhibitors.
  • An exemplary scheme for the synthesis of nonnucleotide deoxyuridine substrate fragments is shown in Figure 18.
  • the substrate fragments shown in Figure 18 are linked to aldehyde (RCHO) and ketone (RiCOR 2 ) library elements using diaminooxyalkane linkers as described in detail herein and illustrated in Figure 19 (left).
  • the library elements can be coupled to the 5' aminooxy group of the substrate fragment as shown in the right half of Figure 19.
  • An alternative approach for obtaining suitable active site targeting compounds is to screen commercially available chemical libraries for weak inhibitors and then attach an aldehyde functional group to provide a handle for tethering.
  • an aldehyde- oxyamine tethering approach if the initial hit has a carboxylate, sulfonamide, secondary amine, or hydroxamic acid functional group, these groups may be directly and efficiently tethered to an organic halide library in the presence of a catlyst such as tetrabutylammonium
  • the invention includes additional compounds including pentavalent antimony (-SbO 3 "2 ), a likely DNA phosphate mimic.
  • -SbO 3 "2 pentavalent antimony
  • tethered ligands based on several of the weaker affinity inhibitors identified in the initial screening efforts can be made using the procedure shown below, using a compound identified in Example 4 as an illustration:
  • This method involves tethering off the carboxylate position using a organic halide libarary. Using this procedure there is a possibility that the antimonate group could become alkylated as well.
  • antimonate esters are very labile, and can be selectively hydrolyzed in weakly basic solutions while preserving the carboxylate.
  • the carboxylate position is amenable to modification based on initial SAR information.
  • the active site targeting moiety can be a mimic of the natural substrate of APE-I, e.g., a derivative, analog, or mimic of an abasic site.
  • the invention provides libraries of compounds of the invention.
  • the libraries can have e.g., 10-1000 compounds.
  • the compounds can be provided in solution, or in solid form.
  • the invention relates to methods for screening and identifying inhibitors of UBER pathway enzymes.
  • the invention relates to a method of screening a library of candidate compounds for an inhibitor capable of inhibiting an enzyme of the uracil base excision repair (UBER) pathway, hi one aspect, the method comprises the steps of (a) providing an enzyme of the uracil base excision repair (UBER) pathway, (b) providing a candidate compound, (c) providing a molecular beacon reagent, (d) contacting the enzyme of the uracil base excision repair (UBER) pathway with the candidate compound in the presence of the reagent, wherein increased fluorescence indicates that the compound is capable of inhibiting the enzyme of the uracil base excision repair (UBER) pathway.
  • the reagent comprises a first uracil-containing nucleic acid sequence; (ii) a second nucleic acid sequence complementary to the first sequence; (iii) a linker linking the first and second nucleic acid sequences; (iv) a fluorescent moiety, and (v) a quencher of the fluorescent moiety.
  • the library of candidate inhibitors can be carried as described above.
  • the size (i.e. total number of candidate inhibitors) of the libraries preferably contains about 100 candidate inhibitors. More preferably, the library contains about 1,000 candidate inhibitors. Preferably still, the library contains about 10,000 candidate inhibitors.
  • the library may contain more than 10 5 , more than 10 6 , or more.
  • the present invention relates to a method of determining if a compound is an inhibitor of a UBEP enzyme comprising the steps of (a) creating an admixture comprising a UBEP enzyme, a candidate inhibitor compound, and a duplex nucleic acid molecule comprising a fluorescent moiety covalently attached to one strand and a fluorescence quencher covalently attached to the complimentary strand, wherein UBEP enzyme activity allows for measurable fluorescence emission from the fluorescent moiety, and (b) measuring the fluorescence of the fluorescent moiety, thereby measuring the activity of the UBEP enzyme, wherein, a decrease in the activity of the UBEP enzyme in the presence of the candidate inhibitor compound compared to the activity of the UBEP enzyme in the absence of the candidate inhibitor compound is indicative that the candidate inhibitor compound is an inhibitor of the UBEP enzyme.
  • a high-throughput screening strategy is employed to screen libraries of candidate inhibitors of uracil DNA glycosylase.
  • the strategy takes advantage of the extrahelical uracil recognition mechanism of uracil DNA glycosylase by using the specificity and binding energy of a uracil substrate fragment to target the active site of uracil DNA glycosylase.
  • the candidate inhibitors in the library can be constructed as previously described and comprise a random array of second binding elements and additionally can comprise a random array of third binding elements tethered to the uracil substrate fragment.
  • the uracil substrate fragment targets the inhibitor to the active site of uracil DNA glycosylase and the second binding elements and third binding elements explorate nearby binding pockets.
  • the high-throughput assay uses molecular beacon technology.
  • the molecular beacon probe comprises two separate but complimentary oligonucleotide sequences that contain multiple uracil bases such that the molecular beacon probe becomes a substrate for uracil DNA glycosylase.
  • the molecular beacon probe can comprise a fluorescent tag on one DNA strand and a quenching moiety on the complementary strand with efficiently quenches the fluorescence of the tag and where the two DNA strands can be joined by a hairpin DNA structure to help preserve the quenching by the quenching moiety.
  • the molecular probe In the presence of a functional uracil DNA glycosylase the molecular probe the multiple uracil bases are removed and the two paired strands spontaneously separate, thus removing the quenching moiety from the fluorescent tag and causing the molecular beacon to fluoresce.
  • the reporter is an oligonucleotide that can function as a substrate for the UBER enzyme, e.g. uracil DNA glycosylase or AP endonuclease I, and alter its fluorescence signal.
  • the oligonucleotide is a molecular beacon (Tyagi and Kramer (1996) Nat. Biotechnol. 14:303-308), a luminescence-based reagent whose fluorescence signal is dependent on intermolecular and intramolecular interactions.
  • the fluorescent biosensor is constructed by introducing a fluorescence energy transfer pair of fluorescent dyes such that there is one at each end (5' and 3') of the reagent.
  • the dyes can be of any class that contains a protein reactive moiety and fluorochromes whose excitation and emission spectra overlap sufficiently to provide fluorescence energy transfer between the dyes in the resting state, including, but not limited to, fluorescein and rhodamine (Molecular Probes, Inc.).
  • fluorescein and rhodamine Molecular Probes, Inc.
  • any molecular beacon oligonucleotide is contemplated by the present invention so long as it forms a substrate for the UBER enzymes used in the compound screen and provides a measurable and detectable difference in fluorescence between the stabilized and unstabilized forms.
  • the invention contemplates screening reagents, e.g. molecular beacon reagents, that utilize any known or available fluorophore and quenchers and utilize any known or available method and/or device for screening, detecting and measuring fluorescence signals.
  • screening reagents e.g. molecular beacon reagents, that utilize any known or available fluorophore and quenchers and utilize any known or available method and/or device for screening, detecting and measuring fluorescence signals.
  • fluorophores are used to measure enzymatic activity (UBER enzyme activities) and, thus, detect the presence of a particular inhibitors.
  • any fluorophore may be used, including BODIPY, fluorescein, fluorescein substitutes (Alexa Fluor dye, Oregon green dye), long wavelength dyes, and UV-excited fluorophores. These and additional fluorophores are listed in Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed. W. T. Mason, ed. Academic Press (1999) (incorporated herein by reference).
  • the fluorophore is 6-carboxyfluorescein (FAM).
  • FAM has an excitation range of 460-500 nm.
  • Fluorescent labels have the advantage of requiring few precautions in handling, and being amenable to high-throughput visualization techniques (optical analysis including digitization of the image for analysis in an integrated system comprising a computer).
  • Preferred labels are typically characterized by one or more of the following: high sensitivity, high stability, low background, low environmental sensitivity and high specificity in labeling.
  • Many fluorescent labels are commercially available from the SIGMA chemical company (Saint Louis, Mo.), Molecular Probes (Eugene, Oreg.), R&D systems (Minneapolis, Minn.),
  • a quencher or "quencher moiety” is a molecule that absorbs the energy of the excited fluorophore. Close proximity of a fluorophore and a quencher allow for the energy to be transferred from the fluorophore to the quencher. By absorbing this energy, the quencher prevents the fluorophore from releasing the energy in the form of a photon, thereby preventing fluorescence.
  • Quenchers may be categorized as non-fluorescent and fluorescent quenchers. Non-fluorescent quenchers are capable of quenching the fluorescence of a wide variety of fluorophores. Generally, non-fluorescent quenchers absorb energy from the fluorophore and release the energy as heat.
  • non-fluorescent quenchers examples include 4-(4'-dimethylaminophenylazo)benzoic acid) (DABCYL), QSY-7, and QSY-33.
  • Fluorescent quenchers tend to be specific to fluorophores that emit at a specific wavelength range. Fluorescent quenchers often involve fluorescence resonance energy transfer (FRET). In many instances the fluorescent quencher molecule is also a fluorophore. In such cases, close proximity of the fluorophore and fluorescent quencher is indicated by a decrease in fluorescence of the "fluorophore" and an increase in fluorescence of the fluorescent quencher.
  • FRET fluorescence resonance energy transfer
  • fluorescent fluorophore pairs include fluorescein/tetramethylrhodamine, IAEDANS/fluorescein, fluorescein/fluorescein, and BODIPY FL/BODIPY FL.
  • the fluorophore When choosing a fluorophore, a quencher, or where to position these molecules, it may be important to consider, and preferably to test, the effect of the fluorophore or quencher on the enzymatic activity of the UBER enzyme. Also, it is preferable that the fluorophore display a high quantum yield and energy transfer efficiency. Long- wavelength (excitation and emission) fluorophores are preferred because of less interference from other absorbing species. The fluorophore should also be less sensitive to pH change or to non-specific quenching by metal ions or other species.
  • WO 99/27351 (incorporated herein in its entirety) describes a monolithic bioelectrical device comprising a bioreporter and an optical application specific integrated circuit (OASIC). The device allows remote sampling for the presence of substances in solution.
  • OASIC optical application specific integrated circuit
  • the fluorescent tag of the molecular beacon of the invention is 5'FAM and the quenching moiety a 3'-dabsyl moiety.
  • the fluorescent tags and quenching moieties are not limited to any particular moieties so long as the fluorescent tag and quenching moiety operate properly, i.e.
  • the invention relates to a high-throughput screening of library candidate inhibitors for inhibitors of enzymes the recognize extrahelical bases in DNA or free nucleosides, other that the enzymes of the UBER pathway.
  • the synthesis and screening strategies described above can be adapted for the discovery of inhibitors of other enzymes that recognize extrahelical bases in DNA or free nucleosides.
  • the inhibitor screening strategies of the invention can proceed using high-throughput means.
  • One of skill in the art can perform these assays using high throughput methods such as microtiter plates in combination with automatic plate reading fluorometers to rapidly screen large libraries of compounds.
  • a library of candidate compounds can be rapidly screened by pooling reaction products together into a series of multi-well analytical plates, such as 96- or 384-well microtiter plates, to form mixtures of reaction products in each well. Instrumentation for high-throughput processing of the multi-well plate can be employed to rapidly measure and screen the pooled samples.
  • a high-throughput imaging system for automated detection for use with the present methods can comprise an excitation source, a monochromator (or any device capable of spectrally resolving the image,
  • the excitation source can comprise blue or UV wavelengths shorter than the emission wavelength(s) to be detected.
  • This may be: a broadband UV light source, such as a deuterium lamp with a filter in front; the output of a white light source such as a xenon lamp or a deuterium lamp after passing through a monochromator to extract out the desired wavelengths; or any of a number of continuous wave (cw) gas lasers, including but not limited to any of the Argon Ion laser lines (457, 488, 514, etc.
  • solid state diode lasers in the blue such as GaN and GaAs (doubled) based lasers or the doubled or tripled output of YAG or YLF based lasers; or any of the pulsed lasers with output in the blue.
  • the emitted light can be detected with a device that provides spectral information for the substrate, e.g., grating spectrometer, prism spectrometer, imaging spectrometer, or the like, or use of interference (bandpass) filters.
  • a device that provides spectral information for the substrate e.g., grating spectrometer, prism spectrometer, imaging spectrometer, or the like, or use of interference (bandpass) filters.
  • Spectral information can be generated by collecting more than one image via different bandpass, longpass, or shortpass filters (interference filters, or electronically tunable filters are appropriate). More than one imager may be used to gather data simultaneously through dedicated filters, or the filter may be changed in front of a single imager.
  • Imaging based systems like the Biometric imaging system, scan a surface to find fluorescent signals.
  • the assays require a UBER enzyme, e.g., a human, viral, or bacterial UBER enzyme such as a uracil DNA glycosylase, a pUTPase or a AP endonuclease I, and a DNA substrate comprising: a duplex nucleic acid molecule that has a label that is not detectable when the DNA is a duplex, but is detectable when the DNA duplex is disrupted, e.g., by uracil DNA glycosylase activity.
  • a UBER enzyme e.g., a human, viral, or bacterial UBER enzyme such as a uracil DNA glycosylase, a pUTPase or a AP endonuclease I
  • a DNA substrate comprising: a duplex nucleic acid molecule that has a label that is not detectable when the DNA is a duplex, but is detectable when the DNA duplex is disrupted, e.g.,
  • the assays use molecular beacon technology wherein a fluorescent label is attached to the 3' end of one strand of the duplex and a fluorescence quencher is attached to the 5' end of the complimentary stand of the duplex, or vice versa.
  • a fluorescent label is attached to the 3' end of one strand of the duplex and a fluorescence quencher is attached to the 5' end of the complimentary stand of the duplex, or vice versa.
  • the DNA duplex (or molecular beacon reagent) can comprise two complementary strands of DNA joined by a hairpin linkage and wherein each of the strands contain multiple uracil bases.
  • One of the strands comprises a fluorescent moiety and the other strand comprises a quencher moiety.
  • the duplex is a target for active uracil DNA glycosylase which acts to destabilize the hairpin
  • the instant invention provides methods to determine if a compound is an inhibitor of UBER enzyme by contacting a UBER enzyme with the DNA duplex described above and a candidate inhibitor and determining if the candidate inhibitor is capable of inhibiting the action of the UBER enzyme by comparing the activity of the UBER enzyme in the presence of the candidate inhibitor to the activity in the absence of the inhibitor.
  • the activity of the UBER enzyme is determined by measuring the fluorescence emission of the fluorescent tag.
  • a fluorometer one of skill in the art can measure the fluorescent emission of a fluorescent moiety by exciting the moiety with a wavelength of light that excites the particular fluorescent moiety and measuring the fluorescence emission that results.
  • the assays use uracil DNA glycosylase. In other embodiments, the assays use AP endonuclease I.
  • the UPER enzyme is a human, viral, bacterial, or parasitic, e.g., from malaria or trypanosome.
  • the viral UPER enzymes can be for example from any virus that may provide for its own UPER enzymes, for example HIV, HIV-I, HFV-2, herpes simplex virus, cytomegalo virus, smallpox virus, avian flu virus, West Nile virus, Epstein Barr virus or influenza virus.
  • UBER enzymes for which inhibitors could be screened according to the invention can also include those from Candida albicans, Trypanosomatidae family (including Leishmannia major and Trypanosoma cruzi).
  • the assays described herein are used to identify anticancer, antiviral, antibacterial, or antipatho genie compounds for use in treating subjects having cancer or infectious diseases or disorders.
  • 211592 1 enzymes of the invention can also be determined by several methods known in the art, such as the gel mobility shift assay or such assays as described in Manuel et al. (1995) J Biol Chem 10, 270:2652-2661; Maccubbin et al. (1994) Cancer Biochem Biophys 14:183-191; Morgan et al. (1988) Biochem Cell Biol 66:157-160; Seal et al. (1987) Biochim Biophys Acta 925:226-233; and Evans et al. (1984) Can J Biochem Cell Biol 62:1275-1282, each of which are incorporated herein by reference.
  • a candidate inhibitor compound in one assay format can be assessed for its ability to inhibit a glycosylase activity by combining the compound with the glycosylase and a suitable substrates for the glycosylase activity. The resulting combination is maintained under conditions appropriate for the glycosylase to act upon the substrate. The extent to which the substrate is converted to product in the presence of the compound is compared with the extent of substrate conversion in the absence of the compound.
  • the assay uses a synthetic substrate such as ultraviolet (UV) or chemically modified covalently closed circular (CCC) DNA to score for glycosylase activity.
  • a synthetic substrate such as ultraviolet (UV) or chemically modified covalently closed circular (CCC) DNA
  • UV ultraviolet
  • CCC covalently closed circular
  • ethidium fluorescence can be used as a probe whereby the topological properties of CCC DNA can be used as a reporter for glycosylase activity.
  • the formation of AP sites by the activity of a glycosylase can be scored by monitoring changes in the conformation of the CCC DNA, and the inhibitory potency of each of the subject compounds towards the particular glycosylase being tested can be easily monitored in this assay.
  • DNA labeled in its purine or pyrminidine bases (as appropriate) is admixed with the target glycosylase and the test candidate compound. After some period of time, the reaction is stopped and the DNA precipitated. The label remaining behind in the supernatant, e.g., from soluble bases released from the DNA by the activity of the glycosylase, is quantitated and compared to the same reaction lacking the test compound.
  • Still other assay formats can rely simply on detecting binding of the test compound with the glycosylase.
  • a gel shift assay to detect the binding of an oligonucleotide including the subject pyrrolidine nucleotide analogs may be used, hi other embodiment, simple competitive binding assays can be carried out to screen for potential glycosylase inhibitors.
  • suitable controls for selectivity can be provided.
  • other glycosylases can be used as counter screens.
  • compounds within the scope of the invention include those which inhibit a glycosylase in a specific manner, e.g., as between glycosylases from different organisms and/or between different isoforms.
  • the subject inhibitors can be selected based ' on an ability to inhibit an adenine glycosylase without substantially inhibiting a uracil glycosylase.
  • differential screening assays can be used to select for those compounds of the present invention with specificity for non-human glycosylase enzymes.
  • compounds which act specifically on pathogens e.g., are antibacterial, antifungal or anti-parasitic agents
  • inhibitors of a bacterial glycosylase such as MutY
  • MutY can be used in the treatment of bacterial infections.
  • human adenine glycosylases such as MYH
  • glycosylase inhibitors can be selected which, for example, have Ki's for inhibition of the bacterial enzyme which are orders of magnitude less than for the equivalent human enzyme.
  • DNA encounters various assaults on its native structure and sequence throughout the life span of a cell. Even the simplest of cells and even viruses evolved methods to protect the integrity of the DNA structure and sequence but did not completely destroy the possibility of random mutations that facilitate change. Attacks from both endogenous, often products of cellular metabolism and molecular instability, and exogenous sources, such as environmental toxins, ionizing radiation, and chemotherapy, on the DNA are mainly corrected through four major repair pathways: mismatch repair (MMR), nucleotide excision repair (NER), direct reversal, and base excision repair (BER).
  • MMR mismatch repair
  • NER nucleotide excision repair
  • BER base excision repair
  • the uracil DNA glycosylase is a type of base
  • uracil base excision repair UBER pathway.
  • uracil DNA glycosylase pUTPase
  • AP endonuclease I AP endonuclease I. It is generally thought that in uracil base excision repair the removal of the misincorporated uracil occurs in two steps. First, the uracil base is released by uracil DNA glycosylase. Second, the abasic sugar (AP site) is excised by AP endonuclease I.
  • chemotherapeutic agents are an important component of current cancer chemotherapeutic regimens; however, current cancer treatment is often ineffective because of toxic side effects, ineffective delivery, and cellular resistance.
  • side effects particularly peripheral organ system damage
  • cellular resistance can potentially be alleviated by the use of DNA repair proteins or agents that inhibit DNA repair, respectively. In the latter case, these agents interact with cells leading to cell death or inhibition of cellular growth by inducing nucleotide modifications or DNA structural malformations leading to cytotoxic or mutagenic effects.
  • chemotherapeutic agents act to inhibit or damage the cellular proteins necessary for growth and cell division but will not be addressed in this review.
  • Cell killing is the purpose of chemotherapeutic agents on tumor cells; however, the unfortunate side effect is that normal cells are damaged and sacrificed as well.
  • DNA can accumulate uracil residues in two distinct ways.
  • dUTP deoxyuridine triphosphate
  • reverse transcriptases e.g. HIV reverse transcriptase
  • deoxycytidine can be deaminated to deoxyuridine as a result of various environmental stresses.
  • DNA polymerases and reverse transcriptases are not known to discriminate between dUTP and dTTP and thus, the incorporation of dUTP into DNA is dependent upon the intracellular concentration of dUTP when compared to dTTP. This ratio is referred to as "the dUTP:dTTP ratio" or the dUTP:dTTP ratio
  • the concentration of dUTP in the cell is dependent upon the rate of synthesis of dUTP and the rate of dUTPase-catalyzed hydrolysis of dUTP. And, under certain normal physiological conditions the action of dUTPase result in or maintain a dUTP concentration that is very low (from undetectable to less than about 100 pmole/106 cells) as compared to dTTP concentration (from 10-80 nmole/106 cells).
  • the uracil base plays a much more diverse role in human biology, disease and anticancer therapy.
  • the UBER machinery has been found to participate in the process of generating somatic mutations during antibody maturation in B cells, and uracilincorporation and/or removal is critical in the life cycles of herpes, cytoniegalo, pox and type 1 human immunodeficiency viruses (HIV-I).
  • this pathway also generates the pharmacologically active single and double strand DNA breaks that are the essential tumor killing lesions produced by the widely used anticancer drugs 5- fluorouracil and methotrexate, and generates the characteristic chromosomal translocations found in some B cell lymphomas.
  • pharmacologic agents that inhibit these processes are desirable for both investigational and therapeutic purposes.
  • uracil DNA glycosylase and deoxyuridine triphosphate nucleotidohydrolase are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA.
  • the UBER pathway also includes AP endonuclease I, which catalyzes the removal of the abasic site formed by uracil removal. Given that these enzymes are emerging as interesting pharmacologic targets, the present inventors have sought out methods for the rapid and efficient identification of small molecule ligands that can inhibit their activity.
  • a potential application of uracil DNA glycosylase and dUTPase inhibitors are antiretro viral agents.
  • HIV-I specifically packages uracil DNA glycosylase into virus particles via interaction with the virus encoded integrase protein (Int), or perhaps a ternary complex between uracil DNA glycosylase, hit and the viral Vpr protein.
  • virus encoded integrase protein Int
  • host uracil DNA glycosylase is sequestered by the HIV and is required viable proliferation of the virus, without which replication of the HIV genome would result in excess accumulation of uracil thereby leading to an abortive condition.
  • Additional information regarding DNA repair enzymes and their role in viral lifecylces can be found for example in Chen et al, "Roles of uracil-DNA glycosylase and dUTPase in virus replication," J. Gen. Virology, (2002) 83, 2339-2345, which is incorporated herein by reference.
  • UNG cleaves the N-glycosyl bond between the uracil base
  • UNGl mitochondrial
  • UNG2 nuclear
  • Uracil DNA glycosylase is required for infection of nondividing cells such as macrophages and resting T cells, and virus particles produced from UNG depleted cells are incapable of infecting new target cells. Infection of macrophages helps maintain a viral reservoir in the host that is crucial for virus spread to the lymphoid organs and T-helper lymphocytes, and ultimately, AIDS pathogenesis. Uracil DNA glycosylase is apparently recruited to minimize uracil incorporation into the viral genome in these cells, which have naturally high levels of dUTP, a good substrate for the viral reverse transcriptase.
  • dUTPase Inhibition of dUTPase would be expected to further increase dUTP levels in macrophages, resulting in even more uracil misincorporation into the viral genome.
  • Pharmacologic targeting of a uracil DNA glycosylase and dUTPase is extremely attractive because these targets would not be susceptible to the same high mutagenesis rate and resulting drug resistance as viral encoded proteins.
  • Targeting uracil DNA glycosylase is a viable therapeutic strategy because it is not an essential enzyme.
  • uracil DNA glycosylase knock-out mice display no remarkable phenotype, nor do uracil DNA glycosylase null yeast or human cell lines.
  • dUTPase is an essential enzyme in all organisms, it would be expected that rapidly replicating viruses such as HIV-I would show higher sensitivity than the host, providing a potential therapeutic window.
  • dUTPase is a ubiquitous enzyme that functions in the hydrolysis of dUTP to dUMP and pyrophosphate. This reaction is thought to occur primarily to limit pools of intracellular dUTP in order to prevent significant dUMP incorporation into DNA during replication and repair (Mclntosh et al., 1992).
  • a second role of dUTPase is to provide substrate (dUMP) for the de novo synthesis of thymidylate. The effects of a compromised dUTPase activity have been well documented in prokaryotes (El-Hajj et al., 1988). Mutations in Escherichia coli dUTPase, which lower
  • viruses In addition to prokaryotes and eukaryotes, a number of viruses are known to encode a dUTPase function.
  • a diverse group of viruses including herpesviruses (Caradonna et al., J.Biol. Chem. (1981), 256, 9834-9837) poxviruses (Broyles et al., Virology, (1993), 195, 863-865), and retroviruses (Threadgill et al., J. Virol. (1993), 67 2592-2600) encode a viral dUTPase activity.
  • a specific subset of the lentivirus group encodes dUTPase as part of the pol gene product in addition to the reverse transcriptase, integrase, and protease functions (Threadgill et al., J. Virol. (1993), 67 2592-2600).
  • the human immunodeficiency virus types 1 and 2 do not contain a virus-encoded dUTPase function (Elder et al. J. Virol. (1992), 66, 1791-1794) and may rely on the dUTPase of the host cell.
  • the question of whether dUTPase is essential for viral replication has been addressed in both herpesvirus and retrovirus groups.
  • Null mutants of viral dUTPases demonstrate that this enzyme is required for successful viral replication in nondividing cells in which the cellular levels of dUTPase are exceptionally low.
  • virus-encoded dUTPase is not required for replication in actively growing cultured cells where dUTPase levels are high (Threadgill et al., J. Virol. (1993), 67 2592-2600). It has been postulated that virus-encoded dUTPase
  • 211592 1 expands the tropism of certain viruses by allowing viral replication in nondividing cell types with low cellular dUTPase activity (Threadgill et al., J. Virol. (1993), 67 2592-2600).
  • AP endonuclease I is involved in DNA repair following removal of a damaged nucleotide by a glycosylase to form an AP site (abasic site).
  • AP sites can be formed through ionizing radiation, oxidative agents, and even spontaneous hydrolysis of the N- glycosylic bond (approximately 10,000 bases are lost per cell per day) (Friedberg et al., DNA Repair and Mutagenesis, ASM Press, 1995). Repair of AP sites inhibits both the mutational and cytotoxic effects of nucleic acid loss.
  • DNA polymerase encounters an AP site, progression is usually halted; however, occasionally DNA polymerase will bypass the AP site often incorporating an A opposite the baseless site potentially leading to mutations.
  • AP endonucleases recognize abasic sites and cleave the phosphodiesterDNA backbone.
  • endonuclease IV nfo; endo IV
  • exonuclease III xth; exo III
  • endo IV acts as the minor bacterial endonuclease cleaving the DNA backbone on the 5' side of an AP site similar to all AP endonucleases but lacks 3'- to 5 '-exonuclease activity found in the exo III family (Friedberg et al., 1995+; Yu et al., 1999+).
  • Endo IV also contains 3'-diesterase activity and is capable of removing 3 '-phosphate and 3'-phosphoglycolate adducts, which occur following single strand breaks and oxidative damage-induced AP sites (Friedberg et al., 1995+; Yu et al., 1999+).
  • endo IV family members have been characterized in both unicellular and simple multicellular eukaryotic cells: APNl in Saccharomyces cerevisiae, and CeApnl in Caenorhabditis elegans, respectively, where APNl acts as the major AP endonuclease activity in the cell (Friedberg et al., 1995+; Yu et al., 1999+). Haring et al. (1994)+ demonstrated that 4' oxidized lesions, a common product of bleomycin damage, are repaired by endo IV with a 4-fold greater efficiency than native PJ? sites, whereas exo III requires a 400-fold higher concentration for the same repair rate compared with unmodified AP sites. These data suggest endo IV family members play a role in protecting cells from oxidative damage, strand breaks, and specific AP sites
  • E. coli exo III A second group of AP endonucleases and the major AP endonuclease in E. coli and mammalian cells, the E. coli exo III family, has also been characterized. Again the representative member, exonuclease III ⁇ xth; exo III), was initially described in E. coli and represents approximately 90% ofE.coli's endonuclease activity (Friedberg et al., 1995+; Yu et al., 1999+). Similar to endo IV enzymes, the exo III family cleaves on the 5' side of the AP
  • Exo Ill-like activity has been isolated, identified, and characterized from other bacteria, Drosophila, and mammals including humans.
  • the major AP endonuclease in humans (APEX/hAPE/HAPl /APEl) was identified and characterized in 1991 by three different groups (Demple et al., 1991+; Friedberg et al., 1995+; Kelley and Erickson, 2000+).
  • APE/ref-1 AP endonuclease
  • 3 '-phosphodiesterase AP endonuclease
  • redox function Human APE/ref-1 recombinant protein contains similar AP endonuclease activity as exoIII; however, the 3'- phosphodiesterase activity is relatively low compared with other class members and might not be biologically significant (Winters et al., 1994+).
  • Complementation of E. coli deficient in AP endonuclease activity by APE provides significant protection from MMS and little resistance to H 2 O 2 providing further evidence that 3' blocking group removal activity is limited in APE (Robson and Hickson, 199 U).
  • the present invention relates to inhibitors (and methods of using same, treating with same, and screening for same) of enzymes of the UBER pathway, in particular, uracil DNA glycosylase, pUTPase and AP endonuclease I.
  • enzymes of the UBER pathway in particular, uracil DNA glycosylase, pUTPase and AP endonuclease I.
  • UBER pathway enzymes are nearly ubiquitous in life on Earth, including even viruses, and they are required if not essential for life, the present invention is not intended to be limited to inhibitors of human UBER enzymes.
  • the present invention provides methods that can be used to identify inhibitors that would be effective against the corresponding UBER pathway enzymes in any other organism (or virus) possessing such enzymes. For example, the methods of the invention can be applied to the
  • a further embodiment of the invention relates to methods of using the compounds of the invention to treat diseases and disorders.
  • the inhibitors of UBER pathway enzymes of the present invention are useful in a wide variety of applications due to their ability to alter the DNA repair response of a cell or virus.
  • the inhibitors of the invention can be administered, for example, as pharmaceutical compositions, either alone or in combination with one or more cytotoxic, anti- viral, anti-bacterial, or antiparasitic agents.
  • the present invention provides methods for treating viral infections, for example, infections by retroviruses, herpesvirus, cytomegalo virus, smallpox virus, avian flu virus, flaviruses, West Nile virus, Epstein Barr virus or influenza virus.
  • One preferred embodiment of the present invention provides relates to treating retroviral infections.
  • Host immune cells use two viral uracilation strategies to defend against retroviral infection: maintenance of high levels of dUTP and directed enzymatic deamination of cytosine residues in viral genomes. Macrophages are quiescent, nondividing cells, and represent an important phase in the pathogenesis of the virus in which a viral reservoir within . the host is established. Since macrophages are not undergoing DNA replication, they can maintain high dUTP levels as a barrier to infection, and in fact, the ratio dUTP/TTP is about one in macrophages.
  • RT reverse transcriptase
  • APOBEC-3G DNA cytidine deaminase that selectively deaminates cytidine to deoxyuridine in viral DNA.
  • APOBEC-3G DNA cytidine deaminase
  • Retroviruses have evolved several countermeasures that prevent or repair uracil incorporation in proviral DNA.
  • Non-primate retroviruses such as equine infectious anemia virus and feline immunodeficiency virus, encode a dUTPase enzyme which breaks down the dUTP pools within macrophages.
  • HIV-I utilizes a strategy of molecular piracy to replicate in the presence of dUTP.
  • HIV-I packages the host nuclear UNG2, a potent uracil DNA glycosylase, into virion particles via interactions with the integrase portion of the Gag-Pol precursor protein.
  • UNG2 Upon infection of a target macrophage, the host UNG2 then forms a complex with RT, and excises uracil residues as they are incorporated into the proviral DNA.
  • Producer cells that are deficient in UNG2 produce virus particles that are noninfectious to cells that have high levels of dUTP.
  • the host enzyme UNG2 and/or dUTPase represent a new therapeutic targets in antiretroviral therapy.
  • HIV-I has also evolved countermeasures to APOBEC-3G mediated uracilation of its genome during infection of actively dividing T cells.
  • Vif viral infectivity factor
  • the present invention provides methods for treating a viral infection in a patient in need thereof by inhibiting an enzyme of the uracil base excision repair (UBER) pathway comprising administering a therapeutically effective amount of a compound of the invention to the patient, thereby treating the viral infection in the patient.
  • the compound can comprise an active-site targeting element, a second binding element, and a linker which links the active-site targeting element and the second binding element.
  • the inventive compounds further encompass inhibitors with a third binding element.
  • the enzyme can be any enzyme of the UBER pathway, including, for example uracil DNA glycosylase, pUTPase and AP endonuclease I.
  • the inhibitor of the present invention inhibits a host uracil base excision repair (UBER) pathway enzyme.
  • the host can be, for example, a human.
  • the inhibitor of the invention inhibits a viral uracil base excision repair (UBER) pathway enzyme.
  • the inhibitors can inhibit the viral UBER enzymes.
  • the inhibitors of the viral UBER enzymes may also inhibit, at least to a degree, the host's UBER enzymes.
  • the virus of the viral infection utilizes the host's UBER enzymes, e.g. HIV virus
  • the inhibitors can inhibit the host UBER enzymes.
  • the viral infections treated by the methods of the invention can be caused by any virus known to man that requires UBER enzymes for successful proliferation, and especially can include infections by retroviruses, herpesvirus, cytomegalo virus, smallpox virus, avian flu virus, flaviruses, West Nile virus, Epstein Barr virus or influenza virus.
  • the viral infection to be treated by the inventive methods can especially be HIV.
  • the present invention provides methods for treating cancers by administering the inventive compounds of the invention having inhibitory action against UBER enzymes.
  • the methods for treating cancer can include co-administering either separately or in an admixture at least one inhibitor of the invention and a cytotoxic agent.
  • the subject inhibitors can be combined with other therapeutics, e.g., such as cell cycle inhibitors, agents which promote apoptosis, agents which strengthen the immune response, and/or RxR agonists.
  • Some of the co-administered therapeutics may be given in smaller doses due to an additive, and sometimes synergistic effect with the UPEG enzyme inhibitors of the invention.
  • the subject inhibitor is administered in conjunction with a DNA damaging agent. There are now almost 100 cytotoxic drugs licensed for use in cancer therapy in the United States.
  • 211592 1 inhibitors can be employed to enhance the anti-pro liferative effect of a DNA damaging agent (also referred to herein as a "genotoxic agent").
  • a DNA damaging agent also referred to herein as a "genotoxic agent”
  • chemotherapeutic agents work by initiating DNA damage. Cell death in response to DNA damage in most instances has been shown to result from apoptosis.
  • DNA damaging agents such as radiation or chemotherapeutics (e.g., 5-FU (5-fluorouracil), cisplatin or methotrexate) is often limited by the development of resistance. Biochemistry and tissue culture studies indicate that such resistance is a function of the capacity of cancer cells to repair damaged DNA.
  • the art demonstrates enhanced expression of DNA repair enzymes by DNA resistant phenotypes of certain human carcinoma cell lines. See, e.g., Lai et al., (1988) Biochem. Pharmacol.
  • a method for increasing the sensitivity of a cell to a DNA damaging agent comprising co-administering to the cell, a DNA damaging agent (including radiation) along with a UBER enzyme inhibitor.
  • the inhibitor can have the structure according to Formula I or II above.
  • coadministered and “in combination” in this context means that the drugs/radiation treatments are given substantially contemporaneously, either simultaneously or sequentially. If given sequentially, at the onset of administration of the inhibitor, the DNA damaging agent is preferably still detectable at effective concentrations at the site of treatment, or DNA repair has not been substantially completed by the treated cell.
  • the inventive compound and the DNA damaging agent may be administered "in combination” by preparing an "admixture” comprising both the inventive compound inhibitor and the DNA damaging agent in a single pharmaceutical composition.
  • the present invention relates to compounds and methods for their administration for treating cancer through the inhibition of human dUTPase and AP- Endonuclease (APE-I).
  • APE-I AP- Endonuclease
  • the prodrug 5-fluorouracil (5-FU) is "co-administered" or administered "in combination," e.g. in an admixture, together with an inhibitor of the invention.
  • 5-fluorouracil (5-FU) has historically been used to treat varied types of malignancies including colorectal, breast, and head and neck cancers. In the year 2002, 5-FU was given to over 2 million patients worldwide making it one of the most widely used anticancer drugs.
  • the mechanism of 5-FU involves covalent inhibition of thymidylate synthase, an essential enzyme responsible for synthesizing TMP from dUMP. This is the major de novo pathway by which the cell produces thymidine precursors for DNA replication.
  • Inhibition of TS by 5-FU not only depletes the dTTP pool within the cell, but also increases the pools of dUTP and dFUTP that can be incorporated during DNA replication.
  • Uracil is normally excluded from DNA by maintaining a low cellular dUTP pool through the action of deoxyuridine triphosphate nucleotidohydrolase (dUTPase).
  • dUTPase deoxyuridine triphosphate nucleotidohydrolase
  • dUMP and FdUMP may be incorporated into genomic DNA, resulting in 'futile cycling' of UBER because of the depleted dTTP pool.
  • SSBs single-strand breaks
  • DSBs double-strand breaks
  • the subject inhibitors can be used to inhibit proliferation of cells, such as by causing cell-cycle arrest or cell death (including apoptosis in eukaryotic cells).
  • the subject inhibitors can be used to inhibit proliferation of mammalian cells, especially human cells, e.g., in the treatment of disorders marked by unwanted cell proliferation.
  • the subject inhibitors can be selected for use in the treatment of septicemias or fungicemias.
  • the inhibitors can be used in the treatment of viral infection. They can also be used in agricultural applications, e.g., for defoliation and other crop control, as well as in insecticidal preparations.
  • Embodiments relating to methods and compounds for treating cancer or more
  • the subject inhibitors can be used alone, or in conjunction with other agents, in the treatment of unwanted cell proliferation, such as maybe due to transformation of the cells, e.g., neoplastic or hyperplastic, or for purposes of wound healing, treatment of restenosis and other unwanted smooth muscle proliferation, cosmetic applications, etc.
  • the subject method can be used in the treatment of sarcomas, carcinomas and/or leukemias.
  • Exemplary disorders for which the subject method may be used as part of a treatment regimen include: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinom
  • kits for carrying out the administeration of the subject inhibitors with DNA damaging compounds comprises a glycosylase inhibitor formulated in a pharmaceutical carrier, and at least one DNA damaging agent, formulated with the glycosylase inhibitor or, as appropriate, in one or more separate pharmaceutical preparations.
  • the subject method can also be used to selectively kill cells in a heterogenous cell population, e.g., comprised of cells of two or more phylogenetically different species of organisms.
  • DNA damaging agents or "genotoxic” agents
  • Many of these drugs currently are used to treat infections and neoplastic diseases in mammals, e.g., humans.
  • two general classes of compounds that are suitable for use as the genotoxic agent in the present method include DNA alkylating agents and DNA intercalating agents.
  • the genotoxic agent can be a precursor that becomes reactive
  • the genotoxic agent can be photoactivated.
  • One class of photoactivatable compounds are the psoralens, derivatives of the tricyclic furocournarin that produce pyrimidine base adducts.
  • trimethylpsoralen can be used in the subject method.
  • Another class of photoactivatable genotoxic agents is represented by the dacarbazines.
  • antibiotics include synthetic and naturally occurring antibiotics.
  • antineoplastic antibiotics include but are not limited to the following classes of compounds: amsacrine; actinornycin A, C, D or F; azaserine; bleomycin; carminomycin; daunomycin or 14-hydroxydaunomycin; mitomycin A, B or C; mitoxantrone; plicamycin; and the like.
  • Neoplasias currently managed by the foregoing include leukemias, lymphomas, myelomas, neuroblastomas, neoplasias of bladder, testicular, edometrial, gastric, or lung origin.
  • Still another general class of genotoxic agents members of which alkylate DNA, includes the haloethyhiitrosoureas, especially the chloroethyhiitrosoureas.
  • Representative members of this class include carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine and streptozotocin.
  • Neoplasias currently managed by the foregoing and particularly amenableto the subject method include Hodgkin's, non-Hodgkin's and Burkitt's lymphomas, myelomas, glioblastomas and medulloblastomas, pancreatic islet cell carcinomas, small cell lung carcinomas and the like.
  • Yet another general class of genotoxic agents members of which alkylate DNA, includes the sulfer and nitrogen mustards. These compounds damage DNA primarily by forming covalent adducts at the N7 atom of guanine.
  • Representative members inlcude chlorambucil, cyclophosphamide, ifosfamide, melphalan, mechloroethamine, novembicine, trofosfamide and the like.
  • the subject glycosylase inhibitors are combined with sulfer and nitrogen mustards in the treatment of, preferably, Hodgkin's, non-Hodgkin's and Burkitt's and other lymphomas, leukemias, myelomas, medullomas, neuroblastomas, small cell lung carcinomas, osteogenic sarcoma, neoplasias of breast, endometrial and testicular tissue, and the like.
  • Another class of genotoxic agents include heavy metal coordination compounds, including platinum compounds.
  • 211592 1 these compounds form covalent interactions to DNA to form, e.g., cis-1,2- intrastrand dinucleotide adducts.
  • exemplary members of this group include cis- diamminedichloro-platmum(Ii) (cisp latin), cis ⁇ diammino-(l,l- cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-diammino-(l,2, -cyclohexyl)- dichloroplatinum(II), and cis-(l,2-ethylene-diammine)dichloroplatinum(II).
  • Neoplastic conditions treated with such genotoxic agents include testicular, endometrial, cervical, gastric, squamous cell, adrenocortical and small cell lung carcinomas, as well as medulloblastomas and neuroblastomas.
  • DNA alkylating agents related to ethylenimines and methylmelamines. These compounds include altretamine, triethylenephosphoramide, triethylenethiophoramide and triethylenemelamine.
  • Additional classes of DNA alkylating agents include the alkyl sulfonates, e.g., busulfan; the azinidines, such as benzodepa; as well as mitoguazone, mitoxantone and procarbazine.
  • the subject inhibitors can also be used for radiosensitization, e.g., to augment radiation therapy and/or photodynamic tumor therapy.
  • the subject inhibitors by interfering with DNA repair, can be used to enhance radiation damage.
  • the present invention provides a method for augmenting treatment with ionizing raidiation, e.g., including but not limited to x-rays, and internal and external gamma emitting radioisotopes. In this mode, the subject method can enhance the localization of cytotoxicity of radiation therapy.
  • Ionizing radiation has been postulated to induce multiple biological effects by direct interaction with DNA or through the formation of free radical species leading to DNA damage. These effects include gene mutations, malignant transformation, and cell killing.
  • Radiation therapy relies of two types of ionizing radiation: (1) subatomic particle radiation, which consists of alpha particles, beta particles (electrons), neutrons, protons, mesons, and heavy ions, and (2) electromagnetic radiation, which exists as a family of waves of varying frequency, including high-frequency x-rays.
  • Electromagnetic radiation in the form of x-rays is most commonly used in megavoltage radiation therapy to treat common malignant tumors.
  • Gamma rays a form of electromagnetic radiation similar to x-rays but emitted by radioactive isotopes of radium,
  • the present invention provides a method of radiation therapy for a host harboring a tumor.
  • the method includes the steps of administering to the host a DNA glycosylase inhibitor of the present invention and administering ionizing radiation to the host in proximity to the tumor.
  • the subject inhibitors can also be used to generate cells, e.g., in culture, that can be used to ascertain the carcinogenic/mutagenic activity of an agent or environmental condition (such as radiation), or the ability of a test agent to protect against DNA damage.
  • the most widely used cell-based carcmogen/mutagen screening assay is the Ames test.
  • the Ames test utilizes several unique strains of Salmonella typhimurium that are histidine-dependent for growth and that lack the usual DNA repair enzymes. The frequency of normal mutations that render the bacteria independent of histidine (i.e., the frequency of spontaneous revertants) is low. The test allows one to evaluate the impact of a compound on this revertant frequency.
  • the cells of the present invention can be generated from any organism, and particularly from human cells. Thus, the test cells of the present invention can be better models for the carcinogenic/mutagenic activity of an agent on human cells, removing the need to extrapolate data from a bacterial cell.
  • the test cell is generated by contacting it with one or more of the subject inhibitors in order to render it at least partially defective for DNA repair.
  • the repair-defective test cell is then contacted with the test agent, or placed under the test conditions.
  • a change in phenotype of the cell can be used to score for mutation, with the frequency of that phenotypic change in the presence of the test agent or condition being compared to its absence.
  • the cell is contacted with a known carcingoen, and a second agent can be added and its ability to protect against DNA damage is assessed.
  • the subject inhibitors can be used to isolate UBER enzymes from cell preparations.
  • the method comprises contacting a cytoplasmic preparation of a cell with one or more glycosylase inhibitors which has been derivatized to a solid support, e.g., for a period of time sufficient for DNA glycosylases present in the cytoplasmic preparation to bind to the solid support.
  • the support can removed from contact with the cytoplasmic preparation, and accordingly isolates bound DNA glycosylase from the cell preparation.
  • Microsequencing can be carried out on the protein, or antibodies can be raised against the protein, and ultimately used to clone a gene encoding the isolated glycosylase.
  • treatment includes the diminishment or alleviation of at least one symptom of the diseases or disorders of the invention.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • subject include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from the diseases or disorders or the invention.
  • the method can also include the step of identifying a subject in need of treatment of diseases or disorders described above.
  • the identification can be in the judgment of a subject or a health professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or a diagnostic method).
  • infection includes the pathological state resulting from the invasion of a subject by pathogenic microorganisms, e.g., fungus, bacteria or viruses.
  • exemplary infections include those caused by the vaccinia family of viruses, malaria, and trypanosomes or by retroviruses such as HIV.
  • the invention also relates to inhibitors of bacterial UBER enzymes and methods of controlling, mitigating or treating bacterial infections by inhibiting essential bacterial UBER enzymes.
  • Exemplarly bacteria for which infections thereby in a subject may be treated by the present invention include, for example, Mycobacterium paratuberculosis, Listeria monocytogenes, pathogenic Escherichia coli, Enterobacteriacea, urinary pathogens, and Helicobacter pylori.
  • the invention may also relate to inhibitors of fungal or parasitic UBER enzymes.
  • Exlempary fungal and/or parasites include, for example, Candida albicans, Trypanosomatidae family, Leishmania major, and Trypanosoma cruzi.
  • the inhibitors can be administered either alone or in combination with (e.g. as an admixture) or in sequential order with an anti-fungal, anti-bacterial, or anti-parasitic agent.
  • Enterobacteriacae is meant the groups of gram negative rods, motile by perichichous flagella or nonmotile, which do not form endospores or microcysts, and, are not acid-fast. These group of bacteria can grow in the presence or absence of oxygen, use D- glucose as sole carbon source, and produce acid and visible gas. These include but are not limited to, the following genera of microorganisms: Escherichia, Shigella, Klebsiella, Enterobacter, Citrobacter, Proteus, Salmonella, Providencia, Morganella, Yersinia, Erwinia,
  • urinary pathogen or "uropathogen” is meant bacteria that cause the human and veterinary urinary tract infections.
  • Such bacteria include, but are not limited to, the group of enteric gram negative bacilli (Enterobacteriacae) which normally reside in the intestinal tract, which when found in the urinary tract often produce clinical symptoms of urinary tract infection.
  • enteric gram negative bacteria such as Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia retteri, and Acinetobacter spp.
  • enteric gram negative bacteria such as Escherichia coli, Klebsiella spp., Enterobacter spp., Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia retteri, and Acinetobacter spp.
  • cancer includes malignancies characterized by deregulated or uncontrolled cell growth, for instance carcinomas, sarcomas, leukemias, and lymphomas.
  • cancer includes primary malignant tumors, e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original tumor, and secondary malignant tumors, e.g., those arising from metastasis, the migration of tumor cells to secondary sites that are different from the site of the original tumor.
  • carcinoma includes malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostate carcinomas, endocrine system carcinomas, melanomas, choriocarcinoma, and carcinomas of the cervix, lung, head and neck, colon, and ovary.
  • carcinoma also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • sarcoma includes malignant tumors of mesodermal connective tissue, e.g., tumors of bone, fat, and cartilage.
  • leukemia and “lymphoma” include malignancies of the hematopoietic cells of the bone marrow. Leukemias tend to proliferate as single cells, whereas lymphomas tend to proliferate as solid tumor masses. Examples of leukemias include acute myeloid leukemia (AML), acute promyelocyte leukemia, chronic myelogenous leukemia, mixed-
  • 211592 1 lineage leukemia, acute monoblastic leukemia, acute lymphoblastic leukemia, acute non- lymphoblastic leukemia, blastic mantle cell leukemia, myelodyplastic syndrome, T cell leukemia, B cell leukemia, and chronic lymphocytic leukemia.
  • lymphomas examples include Hodgkin's disease, non-Hodgkh ⁇ s lymphoma, B cell lymphoma, epitheliotropic lymphoma, composite lymphoma, anaplastic large cell lymphoma, gastric and non-gastric mucosa-associated lymphoid tissue lymphoma, lymphoproliferative disease, T cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, lymphoplasmacytoid lymphoma, and multiple myeloma.
  • the therapeutic methods of the present invention can be applied to cancerous cells of mesenchymal origin, such as those producing sarcomas (e.g., fibrosarcoma, myxosarcoma, liosarcoma, chondrosarcoma, osteogenic sarcoma or chordosarcoma, angiosarcoma, endotheliosardcoma, lympangiosarcoma, synoviosarcoma or mesothelisosarcoma); leukemias and lymphomas such as granulocytic leukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease; sarcomas such as leiomysarcoma or rhabdomysarcoma, tumors of epithelial origin such as squamous cell carcinoma, basal cell carcinoma,
  • Additional cell types amenable to treatment according to the methods described herein include those giving rise to mammary carcinomas, gastrointestinal carcinoma, such as colonic carcinomas, bladder carcinoma, prostate carcinoma, and squamous cell carcinoma of the neck and head region.
  • Examples of cancers amenable to treatment according to the methods described herein include vaginal, cervical, and breast cancers.in which the tumor cells form recognizable glandular structures.
  • chemotherapeutic agent includes chemical reagents that inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents are well known in the art (see e.g., Gilman A. G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202-1263 (1990)), and are typically used to treat neoplastic diseases. The chemotherapeutic agents generally employed
  • chemotherapeutic agents include: bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar), goserelin acetate (Zoladex), granisetron (Kytril), irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), tretinoin (Retin-A), Triapine, vincristine,
  • an effective amount of a compound is that amount necessary or sufficient to treat or prevent the diseases of concern in the present invention, e.g. cancer and infections by bacteria, viruses, and parasites.
  • an effective amount of a compound is the amount sufficient to inhibit undesirable cell growth in a subject, or eliminate a viral or bacterial infection in a subject.
  • the effective amount can vary depending on such factors as the size and weight of the subject, the type of illness, or the particular compound. For example, the choice of the compound can affect what constitutes an "effective amount” or "therapeutically effective amount.”
  • One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the compound without undue experimentation.
  • the present invention provides a pharmaceutical composition comprising the inhibitor compounds of the invention.
  • composition includes preparations suitable for administration to mammals, e.g., humans.
  • pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals.
  • the carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • lozenges using a flavored basis, usually sucrose and acacia or tragacanth
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalciuni phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds;
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and e
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or
  • 211592 1 more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile
  • 211592 1 injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms maybe ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion,
  • 211592 1 ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • the compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention maybe varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • intravenous and subcutaneous doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day.
  • An effective amount is that amount treats the diseases or disorders of the invention.
  • the effective daily dose of the active compound maybe administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • compositions described herein may be administered with one or more other active ingredients that would aid in treating a subject having a disease or disorder of the invention.
  • pharmaceutical compositions of the invention may be formulated to contain one or more additional active ingredients that would aid in treating a subject having the disease or disorder of the invention, e.g., anticancer compounds or antimalarial compounds.
  • a kit for inhibiting an enzyme of the UBER pathway.
  • the kit can comprise a container comprising any compound of the invention as described herein previously or within the ambit of the invention.
  • the kit may also include instructions for using the compound to inhibit the enzyme of the UBER pathway.
  • the kit can also include the pharmaceutical compositions of the invention and can include instructions and any devices necessary or advantageous or useful for the administration of the pharmaceutical compositions, e.g. a syringe or delivery implement, for administering the
  • 211592 1 pharmaceutical composition to a subject for the treatment of the diseases of the invention.
  • diseases contemplated by the invention are those diseases that are or have the potential to be affected by the UBER pathway.
  • kits containing reagents for testing a compound for the ability to inhibit the UBER pathway enzymes of the invention e.g. uracil DNA glycosylase, pUTPase, and AP endonuclease I.
  • the invention provides a kit for determining if a compound is an inhibitor of an enzyme of the uracil excision repair (UBER) pathway comprising an enzyme of the uracil base excision repair (UBER) pathway.
  • the kit can further comprise a reagent for detecting testing the inhibitory activity of a compound.
  • the reagent can comprise (i) a first uracil-containing nucleic acid sequence; (ii) a second nucleic acid sequence complementary to the first sequence; (iii) a linker linking the first and second nucleic acid sequences; (iv) a fluorescent moiety, and (v) a quencher of the fluorescent moiety.
  • the kit can also contain instructions for use.
  • the increased fluorescence of the fluorescent moiety indicates that the compound is capable of inhibiting the enzyme of the uracil base excision repair (UBER) pathway.
  • the kit can be used to test a single inhibitor or a plurality of inhibitors, for example, a library of inhibitors.
  • kits All the essential materials and reagents required for inhibiting an enzyme of the UBER pathway can be assembled together in a kit.
  • the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the reagent of the inventive kits can be a molecular beacon, which are also described in more detail above.
  • molecular beacon technology was developed for uses in detecting amplification products in PCR reactions.
  • the molecular "beacon probe” method was described by Tyagi et al. (Nature Biotech., 14:303 309 (1996)) and which is also the subject of U.S. Pat. No. 5,312,728 to Lizardi et al., which is incorporated herein by reference in its entirety.
  • Molecular beacons are oligonucleotide molecules that can form hairpin structures. On one end of the oligonucleotide (either the 5'-
  • the acceptor moiety is a quencher, absorbing energy from the donor.
  • the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched.
  • the molecular beacon probe which hybridizes to one of the strands of the PCR product, is in "open conformation," and fluorescence is detected, while those that remain unhybridized will not fluoresce.
  • Molecular beacons have also been used in other application including following the activity of DNases, the restriction endonuclease BamHI, small non-enzyme DNA cleavage agents, and ribonuclease H.
  • kits may contain materials for PCR analyses, such primers, buffers and appropriate solvents.
  • the kit may contain antibodies, secondary antibodies that binding primary antibodies, labels or signal generating compounds (either conjugated or unconjugated) and various reagents for the generation and detection of signals.
  • an inhibitor of the UBER pathway may be formulated into a single or separate pharmaceutically acceptable syringeable composition.
  • the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.
  • kits of these kits may also be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means.
  • the kits of the invention may also include an instruction sheet defining administration of the inhibitor compounds of the invention and for explaining the assays for determining inhibition of the UBER enzymes in samples.
  • kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal.
  • an instrument may be an inhalant, syringe,
  • 211592 1 pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
  • Other instrumentation includes devices that permit the reading or monitoring of reactions in vitro.
  • an integrated high-throughput (HTP) platform for discovering small molecule ligands that inhibit UNG is presented.
  • the strategy takes advantage of the extrahelical uracil recognition mechanism of UNG by using the specificity and binding energy of a uracil ligand to target the UNG active site and then covalent tethering of random functional groups for exploration of nearby binding pockets.
  • Library members can be rapidly screened using a robust HTP activity assay, and initial hits are quickly optimized using subsequent structure-activity studies.
  • This tethering approach which uses efficient oxime chemistry ( Figure 2), is related to the "combinatorial target-guided ligand assembly" method of Ellman et al, but differs in that the uracil ligand specifically targets the active site rather than irrelevant regions of the enzyme.
  • the hit-rate and binding affinities of early hits are higher than the more random approach of Ellman and colleagues.
  • This synthetic and screening strategy should be easily adaptable for the discovery of inhibitors of other enzymes that recognize extrahelical bases in DNA or free nucleosides.
  • Example 1 Methods and materials used in Example 1 are as follows.
  • O O'-Diaminoalkanediol linkers of variable length (ethyl, propyl, butyl, pentyl, hexyl) were prepared from the corresponding dibromoalkanes in two steps according to literature procedures.
  • Tethered Oxime Dimers A set of 14 aryl aldehydes was selected for library synthesis for coupling to the three uracil containing aldehydes using the O, O'-diaminoalkanediol linkers as follows. To each 0.5-ml well of a Matrix microtiter plate was added a DMSO stock solution of AcOH (20 ⁇ l, 150 niM, 3 ⁇ mol), uracil aldehyde 1, 2 or 3 (20 ⁇ l, 150 mM, 3 ⁇ mol) and a single aryl aldehyde (20 ⁇ l, 150 niM, 3 ⁇ mol). The plate was carefully agitated to make the solutions homogenous.
  • the second set of oxime dimers based on the 3-(3)-l3 hit discovered in the first screening round were synthesized in an identical fashion as described above using uracil aldehyde 3 and hydroxybenzaldehydes 18 through 42, and the O, O'-diaminopropanediol linker.
  • the most potent inhibitor identified from this second round of screening (3-(3)-27) was synthesized in larger scale and thoroughly characterized.
  • the oximes all eluted with baseline resolution in the order: U-U homodimer, U-R heterodimer, followed by the R-R homodimer.
  • This HPLC method was also used to confirm the expected 1:2:1 stoichiometrics of homodimer and heterodimer oxime formation, using ten representative uracil and aryl aldehydes from the library.
  • Aldehyde 43 was synthesized by removing the methyl groups of the commercially available 3, 4-dimethoxy-6-fluorobenzaldehyde using BBr 3 in CH 2 Cl 2 .
  • the aldehydes 44 and 45 were synthesized by removing the methylene group of the corresponding 2-halogenated piperonal using AlCl 3 and 6N HCl.
  • Aldehyde 46 was commercially available.
  • O-methyloximes 48-51 were made using a similar method.
  • the substrate in this HTS assay was synthesized using standard phosphoramidite DNA solid phase chemistry using reagents purchased from Glen Research. The DNA was purified using anion exchange chromatography followed by desalting using reversed phase methods. The sequence and size was confirmed using analytical denaturing polyacrylamide gel electrophoresis and MALDI- MS.
  • the substrate is a double-stranded 14-mer DNA containing nine U-A base pairs (5'- FAM-GCA CUU AAG AAU UG (SEQ ID No. 1) : 3 '-DABS YL-CA AUU CUU AAG UGC (SEQ ID No. 2)).
  • the UNG HTS assay is performed as follows.
  • reaction buffer 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 7.5 mM MgCl 2 , 0.002% brij-35.
  • the reactions were initiated by the addition of 20 ⁇ L 250 nM molecular beacon substrate in reaction buffer.
  • the plates are incubated at ambient temperature in a fluorescence plate reader for 30 minutes, and the progress of the reaction was monitored every five minutes (Ex. 485 nm/ Em. 520 nm).
  • the final concentrations of the reagents in the assay are 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 7.5 mM MgCl 2 , 0.002% Brij-35, 25 pM human UNG, 50 nM molecular beacon substrate, 100 ⁇ M total compound, 5% DMSO.
  • the MgCl 2 is essential to increase the stability of the double-stranded DNA substrate, and thus decrease the initial fluorescence of the molecular beacon and increase the maximum signal of the assay.
  • Addition of Brij-35, a non-ionic detergent is essential to stabilize human UNG at the low concentration used in this assay.
  • a similar assay has been described by Maksimenko et al.
  • the substrate used in mechanism of inhibition studies was a modified DNA hairpin where the two strands described above are connected by a hexa- polyethylene glycol linker (PEG-U9). This substrate was easier to synthesize and purify than an all DNA hairpin, and unlike the double stranded DNA substrate, does not require MgCl 2 to achieve minimum fluorescence.
  • PEG-U9 hexa- polyethylene glycol linker
  • reaction buffer (20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.2 mM MgCl 2 , 0.002% Brij-35, 1 mM DTT). Eight different DNA concentrations were used in the range of 62.5 to 2000 nM. Reactions were initiated by the addition of 20 ⁇ L 0.5 nM human UNG in reaction buffer.
  • the final concentrations of reagents in the assay are 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.2 mM MgCl 2 , 0.002% Brij-35, 1 mM DTT, 5% DMSO, 0.1 nM human UNG, 62.5-2000 nM PEG-U9 hairpin DNA, and variable amounts of inhibitor.
  • the plates were incubated at ambient temperature in a fluorescence plate reader for 60 minutes, and the progress of each reaction was monitored every five minutes (Ex. 485 nm/ Em. 520 nm). Afterwards, E, coli UNG was added to each well to drive the reactions to completion, and the overall change in fluorescence values were measured.
  • the reactions typically proceed to 85-99 % completion after overnight incubation (DMSO solvent, 37 0 C), and produce a 1:2:1 statistical mixture of the homodimeric (U ⁇ U, R ⁇ R) and heterodimeric (U ⁇ R) oximes for each of the five linker lengths present.
  • oxime derivatives with bulky substituents are generally found to be >95 % in the trans configuration 3 .
  • the unpurified oxime mixtures were directly screened for inhibition of UNG at -100 ⁇ M total oxime concentration to ensure that each component in the mixture is present at a concentration in the range 5 to 10 ⁇ M. If significant inhibition is observed by any mixture, the linker length and RCHO binding element that gave rise to the inhibition can be identified by resynthesis of the individual oximes using a single linker length in each reaction (see below).
  • the uracil homodimers present in some reaction mixtures are inhibitory even in the absence of any active heterodimer.
  • the purified homodimers of various lengths that are based on 6-formyluracil (3) give rise to about 22 % inhibition in all the mixtures based on 3 under the screening conditions (not shown).
  • the homodimers of 1 and 2 show no detectable inhibition under the same conditions.
  • the screening assay must be robust enough to detect any additional inhibition resulting from an active heterodimer in the mixture.
  • one DNA strand is labeled with a fluorescent 5'- FAM, and the complementary strand is modified with a 3'-dabsyl moiety that serves to efficiently quench the fluorescence of the FAM group through contact quenching.
  • the two DNA strands are linked in a hairpin configuration using an 18 atom
  • K 0 and K n represent dissociation constants for inhibitor binding sites that are competitive and noncompetitive with substrate, respectively.
  • K n c and K n s represent the dissociation constants for inhibitor binding to the noncompetitive site when the active site is occupied by the competitively bound inhibitor or substrate, respectively.
  • the Michaelis-Menten parameters for the substrate were fixed using values from nonlinear regression fits (Fig. 6). Other parameters were obtained from simulations to the data using the program Dynafit (cf. Supplemental Information)
  • uracil congeners with small substituents at the five position would favor binding to the active site
  • uracil derivatives with bulkier substituents such as the dihydroxybenzaldoxime of 2-(2)-l3
  • the active site of UNG uses the bulky side chain of a tyrosine to exclude thymidine (5-methyluracil)
  • 6-substituted uracil derivatives such as 3-(3)-21 have been generally observed to bind to the active site.
  • the uracil-based inhibitors found here have revealed a possible pyrimidine discrimination site that may be employed during the multistep extrahelical uracil recognition mechanism.
  • the noncompetitive inhibition mode for 2-(2)- 13 requires that the final extrahelical state can be attained, albeit inefficiently, even when the transient uracil binding site is occupied by the inhibitor, hi contrast, the partial uncompetitive mechanism for binding of 3-( ⁇ )-21 to its second site does not present the same apparent discrepancy, because for uncompetitive inhibition, the compound binds after the substrate is fully inside the active site pocket (see above).
  • the sum of the binding energies of each element equals the entire binding free energy of the whole tethered molecule, then it may be concluded that (i) the tether is energetically inert with respect to binding, and (ii) the binding of one element does not affect the other by induced strain or forcing a tighter fit. If the whole tethered molecule binds much more weakly or tightly than expected from the summation of the binding free energies of the two
  • nonadditive energetic effects are present. Such effects would indicate either an energetic penalty for tethering (antagonistic binding of the parts), or alternatively, a nonadditive energetic benefit (synergistic binding of the parts).
  • the above provides an efficient strategy to develop small molecule inhibitors of UNG that have the potential for activity in cell culture or in vivo.
  • the method is quite general and could be adapted to target other enzymes that bind extrahelical bases or free nucleosides.
  • Two future targets of the current uracil mixed oxime library would be the essential bacterial enzyme deoxyuridine nucleotidylhydrolase which converts dUTP to dUMP, and human thymidine phosphorylase, an enzyme implicated in vascularization of
  • 211592 1 tumors Such inhibitors could serve as useful tools to study the life cycle of pathogenic human viruses, the biology of uracil base excision repair in normal cell lines and tissues, and mechanisms of tumor vascularization.
  • EXAMPLE 2 hi this example, an integrated high-throughput (HTP) platform for the synthesis and evaluation of uracil-directed small molecule libraries based upon triskelion oxyamine scaffolds.
  • the strategy is to attach a uracil-aldehyde ligand to one or two arms of the triskelion scaffold and then derivatize the vacant position(s) with a random library of aldehydes (RCHO).
  • RCHO aldehydes
  • the uracil moiety is expected to weakly target the fully functionalized compound to the active site rather than irrelevant regions of the enzyme, and the random functional groups can then explore nearby binding pockets resulting in increased affinity over that of the uracil alone.
  • Library compounds are rapidly screened using robust HTP activity assays, from which several inhibitors of uracil DNA glycosylase and dUTPase have been identified.
  • Example 2 Methods and materials used in Example 2 are as follows.
  • the plate was carefully agitated to make the solutions homogenous, and 22 ⁇ L of a DMSO solution of the triskelion oxyamine was then added (150 mM, 3.3 ⁇ mol). The plate was sealed and further agitated and incubated in an oven for I2h at 37 0 C.
  • the oximes all eluted with baseline resolution in the order (1) the homotrimer oxime derived from 4 or 5, (2) the heterotrimer oxime derived from 4 or 5 and either 8, 15 or 19, and (3) the homotrimer oxime derived from 8, 15 or 19.
  • the final concentrations of the reagents in the assay are 10 mM Tris-HCl, pH 8.0, 20 mM NaCl, 7.5 mM MgCl 2 , 0.002% Brij-35, 25 or 100 pM human UNG, 50 nM molecular beacon substrate, 100 ⁇ M total compound, 5% DMSO.
  • IC 5O analysis was performed using the same conditions except that the concentration of compound was varied in the range 0.01 to 100 micromolar.
  • a triskelion oxyamine scaffold is synthesized in two steps from tris(hydroxymethyl)methane.
  • the three oxyamine groups are derivatized with a uracil-aldehyde and a library of 215 aldehyde binding elements (RCHO) via the formation of stable oxime linkages.
  • RCHO 215 aldehyde binding elements
  • Each linking reaction is carried out in one well of a 96-well microtiter plate that contains one molar equivalent uracil aldehyde, two molar equivalents RCHO library member, and one molar equivalent oxyamine triskelion scaffold.
  • the reactions typically proceed to 85-99 % completion after overnight incubation.
  • 51 and 52 are the most potent nonnucleotide inhibitors of human dUTPase yet reported.
  • tethering 15 to 5 produced an increase in binding affinity of 75 -fold relative to uracil alone, and tethering 5 to 15 brought about at least a 1, 000-fold increase in binding affinity relative to 15 alone.
  • tethering of the two parts has brought about increases in binding affinity of at least 700- fold as compared to the separate components.
  • a trivial but potentially useful modification of the tethering approach would be to incorporate two different R groups into the triskelion scaffold. This is easily accomplished by first synthesizing and isolating the monoderivatized uracil compound, and then reacting the remaining two oxyamine positions with a mixture of two aldehydes (unpublished).
  • oxime libraries there are a number of currently used drugs with oxime functional groups: the selective serotonin reuptake inhibitor, fluvoxamine, the monobactam antibiotic, aztreonam, and several preclinical antimicrobial drugs.
  • the activity of these drugs indicates that oxime linkages are stable and useful in real clinical applications.
  • oximes are susceptible to reduction in metabolic reactions involving cytochrome P450 mediated transformations. Depending on the pharmacokinetic and pharmacodynamic properties of the individual oximes, this may or may not pose a problem.
  • fluvoxamine although extensively processed in first-pass metabolism, has a reasonable serum half-life of 12 hours.
  • triskelion libraries based on substrate fragments will be useful for rapid inhibitor development against a variety of enzymes.
  • inhibitors of UBER could also target one mechanism of drug resistance.
  • the same inhibition strategy could be used to target pathogenic microorganisms such as the fungus C. albicans, herpesviruses, and parasites from the Trypanosomatidae family (such as Leishmania major and Trypanosoma cruzi), all of which depend on stringent pyrimidine nucleotide pool balance for viability.
  • the budding yeast Saccharomyces cerevisiae is an idealorganism to investigate the importance of uracil BER in 5-FU toxicity because deletion yeast strains in most steps of this process are available, as well as other DNA repair pathways, and the yeast pathway is
  • Example 3 Methods and materials used in Example 3 are as follows.
  • Yeast media and growth conditions Wild type yeast were grown in YEPD (1% yeast extract, 2% peptone (BD Biosciences), 2% dextrose (J.T. Baker)); mutant strains in YEPD supplemented with 200 ⁇ g/ml geneticin (Gibco). Selection for fresh MATa single mutants was performed as described (29) using sporulation buffer (1% KOAc, 0.005% ZnOAc, Sigma) and MATa. selective "magic medium" (SC-Leu-His-Arg+L-canavanine (60 ⁇ g /ml)+geneticin; all amino acids and L-canavanine from Sigma, yeast nitrogenous base without amino acids and ammonium sulfate from Difco). The ⁇ pnl and quadruple mutants derived from FFl 8733 and r ⁇ d27 yeast prepared using a URA3 deletion cassette were plated on the appropriate selective media (Table 1).
  • Mutant Strain Missing enzyme activity Potential effect of mutation after 5-FU treatment ung1 Uracil DNA glycosylase. Removes Accumulation of uracil and 5- uracil and 5-FU from DNA. FU in DNA apnl Abasic endonuclease. Cleaves DNA Accumulation of abasic sites in backbone 5' to abasic sites; also 3' DNA phosphodiesterase activity. rad27 Flap endonuclease. Removes 5' Accumulation of 5' dRP- deoxyribose phosphate (dRP) blocked sites in DNA blocking groups.
  • dRP dRP- deoxyribose phosphate
  • apn1apn2 ntg1 ntg2 Abasic endonucleases (apnl , apn2) Accumulation of intact abasic and 3' AP lyases (ntg1 and ntg2). sites in DNA rad51 ssDNA binding protein. Critical for Diminished capacity for homologous recombination. recombinational repair of
  • DSB's and SSB's generated from cleaved abasic sites rad2 Endonuclease required for incision Ablation of alternative pathway on the 3 1 side of a DNA lesion during for repair of 5-FU induced NER.
  • DNA lesions msh2 Dimerizes with Msh3 or Msh6 to bind Ablation of alternative pathway
  • DNA lesions apn1rad27 Deficient in abasic site endonuclease Increased levels of abasic and flap endonuclease sites and/or 5' dRP-blocked sites in DNA
  • 5-FU survival timecourses Yeast were grown to logarithmic phase and diluted to 5 x 10 6 cells/ml into YEPD +/- 150 ⁇ M 5-FU(Sigma) based on a preliminary estimate of the EC5 0 of 5-FU for the parental BY4741 strain. At given times, cells were diluted, plated, and incubated at 30 degrees Celsius until countable colonies had formed. Colony counting was assisted with the use of a Gel Doc imaging system (Bio-Rad), and data was plotted using GraphPad Prism.
  • 5-FU dose-response studies Yeast were grown to logarithmic phase and diluted to 5 x 10 6 cells/ml into media containing varying concentrations of 5-FU. After 4 h of shaking at 30 degrees Celsius, the cells were diluted, plated, and incubated until counting. Data was plotted and EC 50 values were determined using GraphPad Prism.
  • Yeast were grown to logarithmic phase, diluted to 5 x 10 6 cells/ml into media +/-150 ⁇ M 5-FU, and prepared for flow cytometry as described previously (31). Briefly, at given time intervals, 5 x 10 6 cells were pelleted, washed, and resuspended in 250 ⁇ l sterile water. Cold 100% ethanol was added to 70%, with vortexing. The cells were allowed to fix overnight at 4 degrees Celsius before being washed and resuspended in 100 ⁇ l 50 mM sodium citrate, pH 7.0 (Fisher), and digested with 100 ⁇ g/ml RNase A at 37 degrees Celsius for 2 h.
  • yeast cells were grown to logarithmic phase and diluted to 5 x 10 6 cells/ml into 5-FU to achieve 70-90% cell killing across all samples (wt, 150OM 5-FU for 6 h, ungl, 3 mM 5-FU for 4 h, and apnl, 150 ⁇ M 5-FU for 3 h). Aliquots were diluted and plated, and the genomic DNA was purified and digested with 10 nM Ungovernight at room temperature in buffer containing 10 mM Tris-HCl, pH 7.5, 2.5 mM MgCl 2 and 25 mM NaCl.
  • uracil- 13 C4, 15 N2 and 5-FU- 13 C4, 15 N2 were added as internal standards (stable isotope labeled U and 5-FU were purchased from Cambridge Isotope Laboratories). Thenthe DNA was precipitated with 70% EtOH, centrifuged, and the supernatant and pellet fractions were separated. Ethanol was removed from supernatant fractions under vacuum in a SpeedVac at room temperature. Aqueous supernatant fractions were frozen in liquid nitrogen, lyophilized to dryness for 18 h, and then trimethylsilylated and analyzed by GS/MS as described (32-34).
  • selected-ion monitoring was used to monitor the characteristic ions of the trimethylsilyl derivatives of uracil (m/z 256 and m/z 24I) 5 uracil- 14 C 4 , 15 N 2 (m/z 262 and m/z 247), 5-FU (m/z 274 and m/z 259) and 5-FU- 13 C 45 15 N 2 (m/z 280 and m/z 265) during GC/MS analysis (In each case, the first ion is the molecular ion and the second one is the ion that results from the loss of methyl radical from the molecular ion).
  • RNA 5-FU incorporation analysis wt yeast were grown to logarithmic phase, diluted to 5 x 10 6 cells/ml into YEPD +/- one EC 50 of 5-FU, and shaken at 30 degrees Celsius for 6 hours. Cellular RNA was then isolated using the RNeasy Kit (Qiagen). Ten Dg of RNA was digested to nucleosides using mung bean nuclease (10 U) and calf intestinal phosphatase (10 U) overnight at 37 degrees Celsius in buffer containing 10 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 50 mM NaCl 5 andl mM dithiothreitol (all reagents from New England Biolabs).
  • HPLC HPLC was carried out using an analytical Aqua reversed phase Cl 8 column (Phenomenex) and isocratic elution with 3% acetonitrile in aqueous 0.1 M TEAA, pH 7.0 at a flow rate of 1 ml/min.
  • Aldehyde reactive probe-slot-blot (ASB) assay Yeast were grown to logarithmic phase, diluted to 5 x 10 6 cells/ml into YEPD +/- one EC 50 of 5-FU, and shaken for 6 h at 30 degrees Celsius. The DNA was isolated using Genomic-tips (Qiagen) and subjected to the aldehyde reactive probe slot-blot assay as described. Briefly, 4 ⁇ g of each DNA sample was digestedwith E.
  • coli exonuclease III (145 U, New England Biolabs) for 1 min at 37 degrees Celsius, 100 mM putrescine at 37 degrees Celsius for 30 min (Acros Organics), both exonuclease III and putrescine, or left undigested. All samples were precipitated with EtOH, resuspended in PBS, and incubated with 1 mM aldehyde reactive probe (ARP, Dojindo Laboratories) for 10 min at 37 degrees Celsius.
  • ARP aldehyde reactive probe
  • the DNA was resuspended in TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), quantified by ethidium bromide spotting, and 0.5 ⁇ g of each sample was diluted to a volume of 100 ⁇ l in TE, heat denatured, and mixed with an equal volume of 2OX SSC (3 M NaCl, 0.3 M sodium citrate).
  • 2OX SSC 3 M NaCl, 0.3 M sodium citrate.
  • the denatured DNA was slot-blotted onto Hybond-C Super nitrocellulose (Amersham Biosciences) using a Minifold II slot blot manifold (Schleicher & Schuell).
  • the membrane was soaked in 5X SSC for 15 min at 37 degrees Celsius, dried, baked at 75 degrees Celsius under vacuum for 2 h, and then rewet and blocked in 10 ml of preincubation buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 0.5% casein, 0.25% BSA, 0.1% Tween 20) for 45 min at room temperature, with shaking.
  • preincubation buffer (20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 0.5% casein, 0.25% BSA, 0.1% Tween 20
  • 5-FU survival time-courses forwild-type and BER deficient yeast were measured in the absence and presence of the drug.
  • all uracil BER deficient strains exhibited indistinguishable doubling times over a 6 h growth period (not shown). Exceptions were the rad.51 and rad27 strains that had doubling times about 1.5 times longer.
  • 150 ⁇ M 5-FU a concentration similar to the EC 50 for the wild-type parent yeast strain (see below), marked differences in survival were observed (FIG. 1 IA).
  • the ungl strain which is deficient in the removal of uracil and 5- FU,showed marked protection against 5-FU as compared to the wild-type strain, and it was not possible to achieve any greater than about 60% killing even after 54 h of treatment using this concentration of 5-FU.
  • the apnl strain that is deficient in cleavage of the 5' phosphodiester linkage of the abasic site product of the Ungl reaction showed rapid and complete cell killing (FIG. 1 IA).
  • the log linear survival time course without lags for this strain suggests the rapid formation of a toxic species from 5-FU.
  • this quadruple deletion mutant was constructed using a different wild-type strain that is less sensitive to 5-FU (FFl 8733, FIG. HB),we also measured the survival curve for the apnl mutant in this genetic background.
  • the FFl 8733 wild-type strain showed 70 % survival at 24 h incubation with 150 ⁇ M 5-FU, while the apnl and apnlapn2ntglntg2 strains showed increasing sensitivities (40 % and 2 % survival, respectively).
  • Rad51 is the yeast homologue for the bacterial RecA protein, which is critical for homologous recombination, but this deletion dido effect on the time dependence and only a smalleffect on 5-FU
  • Rad2 is a single-stranded DNA endonuclease that nicks the DNA backbone 3' to a damaged site, and is the yeast homologue of the human XPG protein.
  • rad2 showed an indistinguishable survival curve from that of the wild-type strain BY4741 as did the mismatch repair deficient strain msJi2 (FIG. 1 ID).
  • RNA was isolated and subjected to extensive enzymatic digestion with mung bean nuclease and calfmtestinal phosphatase followed by reversed-phase HPLC analysis of the resulting nucleosides (FIG. 15).
  • a very small amount 5-FUrd was detected in RNA corresponding to about 3% of the uridine that was present. In other words, about 1 in every 30 uridine nucleotides in RNA is replaced with 5-FUrd. This much higher level of 5-FU in RNA as compared to DNA may reflect the higher pool of cellular 5-FUTP as compared to FdUTP.
  • 211592 1 afforded by chemiluminescence methods after covalently derivatizing the DNA, fixing it to a nitrocellulose membrane, and then probing with strepavidin-conjugated horseradish peroxidase.
  • This assay may also be combined with enzymatic analyses to ascertain the fraction of the total abasic sites which are nicked on the 5' and 3' phosphodiester linkages. For instance, 3' nicks can be detected using a 5' AP endonuclease (ExoIII) which completely severs the site from the DNA, precluding its binding to nitrocellulose and chemiluminescence detection.
  • ExoIII 5' AP endonuclease
  • 5' nicks can be inferred by chemical cleavage of the 3' phosphodiester linkage using putrescine. It should be noted that if rapid processing of sites occurs, then this assay will grossly underestimate the flux of total abasic sites. In other words, the assay is most informative if the rate-limiting process is a step or steps involved in endonucleolytic turnover of these sites.
  • ASB analysis of genomic DNA isolated from the BY4741 wild-type strain showed about 12 abasic sites per 10 6 base pairs in the absence of 5-FU. Essentially all of these sites were nicked at either the 5' or 3' sides, indicating that the initial abasic site product of the Ungl reaction is efficiently processed to these nicked forms (top panel, FIG. 16A).
  • the genomic DNA of the ungl strain showed about 5-fold lower levels of abasic sites as compared to the wild-type strain in the absence of 5-FU, and essentially all of these were in the 5' nicked form (top panel, FIG. 16B).
  • 2H592 1 targets thymidylate synthase. Nevertheless, processing of abasic sites by Apnl was found to be important both in our work and this previous study.
  • the following example demonstrates synthesis of a library of candidate inhibitors of UBER enzymes.
  • the approach demonstrated here involves tethering a chemical library of molecules to pieces of substrates (or ligands) that already bind weakly to an enzyme active site.
  • the substrate (or ligand) fragment targets the entire tethered molecule to the active site where the library pieces can then explore adjacent binding pockets.
  • the instant approach has the following strengths: (1) library synthesis is economical and very rapid and can be performed in microtiter plate format, (2) the reactions are extremely efficient and no purification of any products is required, (3) mixtures of flexible linkers are used in each reaction which allows multiple tethering lengths to be probed simultaneously in activity screens, and (4) the method is easily adaptable to any desired target.
  • This approach also takes advantage of the very efficient reaction of oxyamines with aldehydes to form stable oxime linkages.
  • a suitable substrate fragment (S) is identified and derivatized at a nonperturbing position with an aldehyde functional group. Then the substrate fragment is tethered to one end of a bifunctional alkyloxyamine linker, which is then derivatized on the other end with a library of aldehyde compounds (RCHO).
  • RCHO aldehyde compounds
  • dUTPase Developing and characterizing small molecule inhibitors that target human dUTPase, UNG and APE-I.
  • dUTPase Several targeted libraries against human dUTPase (we have cloned and overexpressed human dUTPase in large amounts) were prepared.
  • One such 215 compound library is based on the uracil fragment of the substrate dUTP, which was derivatized with an aldehyde at the Nl nitrogen. The libraries were screened using a high-throughput dUTPase assay that detects inorganic phosphate produced from the cleavage of dUTP into dUMP and PPi.
  • UNG Preliminary SFT libraries against UNG2 have been constructed and have been evaluated using the high-throughput (HTP) screen shown in FIG 4. This robust assay may be implemented for use in 96-well and 384-well plate formats.
  • HTP high-throughput
  • a uracil can be tethered to the best RCHO fragment to increase binding affinity by over 300-fold over the uracil fragment alone.
  • the data show the identification of an inhibitor based on uracil smdp- carboxybenzaldehyde, which has favorable chemical properties.
  • this cell permeable small molecule inhibitor behaves as a substrate DNA mimic.
  • glycosyl cation transition state mimics based on cationic imino sugar scaffolds that will also be used as targeting fragments for tethering.
  • the data further show an inhibitor that is active against UNG2 in primary macrophage cell culture as shown in FIG. 5.
  • APE-I Human abasic site endonuclease (APE-I) (also identified herein as “AP endonuclease I”) was purified and overexpressed and a high throughput screen was developed for this enzyme that is in principle similar to the screen shown in FIG. 4 for UNG, except that the uracil sites are substituted with stable tetrahyrofuran abasic sites which are good abasic site substrate mimics for APE-I . Thus when APE-I cleaves the phosphodiester backbone the strands dissociate yielding an 8-fold fluorescence increase (not shown).
  • yeast AP-endonulease deletion mutants in were found to be hypersensitive to high dUTP levels brought about by treatment with the drug 5-fluorouracil (5-FU). This effect was found to arise from depletion of the TTP pool, and concomitant increases in dUTP levels, leading to incorporation of enormous amounts of uracil into the genome during DNA replication. In fact, treatment with 5 -FU leads to one in four thymines being replaced by uracil, which ultimately leads to enhanced cell killing in the absence of a functional UBER pathway. These results provide excellent proof-of-principle that deletion or inhibition of UBER components can increase the toxicity of 5-FU.
  • HIV- 1 associated uracil DNA glycosylase activity controls dUTP misincorporation in viral DNA and is essential to the HIV-I life cycle. MoI Cell 17, 479-90.
  • Jjtnai, K. Slupphaug, G.; Lee, W. L; Revy, P.; Nonoyama, S.; Catalan, N.; YeI, L.; Forveille, M.; Kavli, B.; Krokan, H. E.; Ochs, H. D.; Fischer, A.; Durandy, A., Nat Immunol 2003, 4, 1023-1028.
  • Segel I. H., Enzyme Kinetics, ed.; John Wiley & Sons, Inc.: New York, 1993; 'Vol. 1 p. ) Seiple, L.; Jaruga, P.; Dizdaroglu, M.; Stivers, J. T., Nucleic Acids Res 2006, 34, 140-151. Sekino, Y.; Bruner, S. D.; Verdine, G. L., J Biol Chem 2000, 275, 36506-36508.
  • the uracil Nl-acetaldehyde O-methyl oxime (49) showed undetectable inhibition (Kc > 10 mM.

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Abstract

L'invention concerne des composés inhibant des enzymes de la voie de réparation par excision de base de l'uracile (UBER). L'invention concerne, en outre, des inhibiteurs de l'uracile ADN glycosylase, de la pUTPase et de l'AP endonucléase I. Les composés peuvent comprendre un fragment de substrat d'uracile et un second élément de liaison liés par covalence au moyen d'une molécule de liant. L'invention concerne également les composés comprenant un fragment de substrat d'uracile, un deuxième élément de liaison et un troisième élément de liaison liés ensemble par covalence au moyen d'une molécule de liant. L'invention concerne également des méthodes de traitement de maladies et de troubles chez un sujet nécessitant un tel traitement, le cancer et/ou des infections bactériennes, virales et parasitaires faisant partie de tels trouble et/ou maladies et les méthodes comprenant l'étape consistant à administrer les inhibiteurs selon l'invention. Les inhibiteurs administrés inhibent les enzymes de la voie UBER, traitant ainsi les maladies et les troubles selon l'invention.
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