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WO2006015369A9 - Dosage d'identification d'inhibiteurs de topoisomerase - Google Patents

Dosage d'identification d'inhibiteurs de topoisomerase

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Publication number
WO2006015369A9
WO2006015369A9 PCT/US2005/027605 US2005027605W WO2006015369A9 WO 2006015369 A9 WO2006015369 A9 WO 2006015369A9 US 2005027605 W US2005027605 W US 2005027605W WO 2006015369 A9 WO2006015369 A9 WO 2006015369A9
Authority
WO
WIPO (PCT)
Prior art keywords
topoisomerase
acid molecule
activity
nucleic acid
candidate
Prior art date
Application number
PCT/US2005/027605
Other languages
English (en)
Other versions
WO2006015369A2 (fr
WO2006015369A3 (fr
Inventor
James T Stivers
Keehwan Kwon
Original Assignee
Univ Johns Hopkins
James T Stivers
Keehwan Kwon
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Univ Johns Hopkins, James T Stivers, Keehwan Kwon filed Critical Univ Johns Hopkins
Publication of WO2006015369A2 publication Critical patent/WO2006015369A2/fr
Publication of WO2006015369A9 publication Critical patent/WO2006015369A9/fr
Priority to US11/701,981 priority Critical patent/US20080020973A1/en
Publication of WO2006015369A3 publication Critical patent/WO2006015369A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • topo I Type IB DNA topoisomerases
  • topo I catalyze the reversible cleavage of the phosphodiester backbone of DNA using a nucleophilic active site tyrosine residue (i).
  • the DNA 5 '-hydroxyl either serves as the leaving group during nucleophilic attack by the tyrosine, or as the nucleophile in the reverse reaction in which the tyrosine is expelled, and the DNA backbone is religated (Figure IA).
  • the active site of topo I exhibits plasticity and allows alternative reactions to occur with remarkable catalytic promiscuity (2).
  • These reactions include DNA strand exchange (3), attack of the phosphotyrosine intermediate by exogenous small molecule nucleophiles such as hydrogen peroxide and glycerol (4), and a ribonuclease (RNase) activity in which the tyrosine attacks the 3' phosphodiester linkage of a single ribonucleotide in an otherwise DNA context (5).
  • RNase ribonuclease
  • the 5' hydroxyl of the DNA and the 2' hydroxyl of the ribonucleotide compete for attack at the phosphotyrosine linkage, leading to either religation of DNA, or irreversible formation of a 2', 3' cyclic phosphodiester ( Figure IB).
  • the type IB enzyme isolated from vaccinia virus has a unique and robust site- Attorney Docket No.:63858-PCT (71699)
  • the vaccinia topo I cleaves DNA at the 3' phosphodiester of the consensus sequence CCCTT ⁇ X in duplex DNA.
  • the site-specific RNase activity is synthetically quite useful, and has been used to release isotopically labeled DNA generated by DNA polymerase catalyzed primer extension from the 3 ' hydroxyl using labeled nucleotide triphosphate precursors ((5).
  • the instant invention is based, at least in part, on the discovery of a continuous spectroscopic assay for DNA topoisomerase activity.
  • the inventors for the first time, have demonstrated a multiple turnover assay for DNA topoisomerase using a DNA substrate having one or more ribonucleotide substitutions. These assays allow for high throughput screening methods to identify inhibitors of topoisomerase.
  • the instant invention provides screening methods, methods of treating topoisomerase associated diseases and disorders, compositions for the treatment of topoisomerase associated diseases and disorders, kits to screen for inhibitors of topoisomerase, pharmaceutical compositions for the treatment of topoisomerase associated diseases and disorders, and kits comprising pharmaceutical compositions for the treatment of topoisomerase associated diseases and disorders.
  • the specific aspects and embodiments of the invention will be more thoroughly detailed below.
  • the instant invention provides a method for measuring the activity of a topoisomerase by contacting a topoisomerase with a duplex nucleic acid molecule that allows for multiple turnover of the topoisomerase comprising a fluorescent moiety covalently attached to one strand of the duplex nucleic acid molecule and a fluorescence quencher covalently attached to the complimentary strand of the duplex nucleic acid molecule, wherein topoisomerase activity results in measurable fluorescence from the fluorescent moiety, and measuring the fluorescence of the fluorescent moiety, thereby measuring the acivity of the topoisomerase.
  • the duplex nucleic acid molecule is a deoxyribonucleic acid molecule.
  • the multiple turnover of the topoisomerase is due to the duplex nucleic acid molecule further comprising one or more ribonucleotides, or analogs thereof, e.g., a uridine ribonucleotide.
  • the fluorescence is measured spectroscopically, e.g., using a fluorometer.
  • the topoisomerase is a type I topoisomerase, e.g., type IA or IB.
  • the topoisomerase is a vaccinia virus topoisomerase IB or a human topoisomerase IB.
  • the topoisomerase is a type II topoisomerase.
  • the duplex nucleic acid molecule is comprised of SEQ ID NO: 1 and SEQ ID NO:2.
  • SEQ ID NO: 1 contains a fluorescent moiety, e.g., FAM
  • SEQ ID NO:2 contains a fluorescence quencher, e.g., DAB.
  • the duplex nucleic acid molecule is a comprised of SEQ ID NO:1 and SEQ ID NO:2 and the topoisomerase is vaccinia virus topoisomerase IB.
  • the duplex nucleic acid molecule is comprised of SEQ ID NO:3 and SEQ ID NO:4.
  • the duplex nucleic acid molecule is a comprised of SEQ ID NO:3 and SEQ ID NO:4 and the topoisomerase is human topoisomerase IB.
  • the topoisomerase is a pathogen topoisomerase, e.g., a malaria or trypanosome topoisomerase.
  • the method further involves contacting the topoisomerase with a candidate topoisomerase inhibitor.
  • the method further Attorney Docket No.:63858-PCT (71699)
  • the instant invention provides a method of determining if a compound is an antiviral, antimalarial, antitrypanosome or antibacterial agent by creating an admixture comprising topoisomerase, a candidate antiviral, antimalarial, antitrypanosome or antibacterial agent, and a duplex nucleic acid molecule that allows for multiple turnover of the topoisomerase comprising a fluorescent moiety covalently attached to one strand and a fluorescence quencher covalently attached to the complimentary strand, wherein topoisomerase activity allows for measurable fluorescence emission from the fluorescent moiety; measuring the fluorescence of the fluorescent moiety, thereby measuring the activity of the topoisomerase, wherein, a decrease in the activity of the topoisomerase in the presence of
  • the duplex nucleic acid molecule is a deoxyribonucleic acid molecule.
  • the multiple turnover of the topoisomerase is due to the duplex nucleic acid molecule further comprising one or more ribonucleotides, or analogs thereof, e.g., a uridine ribonucleotide.
  • the fluorescence is measured spectroscopically, e.g., using a fluorometer.
  • the topoisomerase is a type I topoisomerase, e.g., type IA or IB.
  • the topoisomerase is a viral, bacterial, malarial, or trypanosome topoisomerase IB.
  • the topoisomerase is a type II topoisomerase.
  • the duplex nucleic acid molecule is comprised of SEQ ID NO:1 and SEQ ID NO:2. In a related embodiment, the duplex nucleic acid molecule is a Attorney Docket No.:63858-PCT (71699)
  • topoisomerase is vaccinia virus topoisomerase IB.
  • the invention provides a method for testing a library of candidate antiviral, antimalarial, antitrypanosome or antibacterial agents for the ability to inhibit topoisomerase activity.
  • the instant invention provides a method of determining if a compound is an anticancer agent by creating an admixture comprising topoisomerase, a candidate anticancer agent, and a duplex nucleic acid molecule that allows for multiple turnover of the topoisomerase comprising a fluorescent moiety covalently attached to one strand and a fluorescence quencher covalently attached to the complimentary strand, wherein topoisomerase activity allows for measurable fluorescence emission from the fluorescent moiety, measuring the fluorescence of the fluorescent moiety, thereby measuring the activity of the topoisomerase, wherein, a decrease in the activity of the topoisomerase in the presence of the candidate anticancer agent compared to the level of activity of the topoisomerase in the absence of the candidate anticancer agent is indicative that the candidate inhibitor is
  • the duplex nucleic acid molecule is a deoxyribonucleic acid molecule.
  • the multiple turnover of the topoisomerase is due to the duplex nucleic acid molecule further comprising one or more ribonucleotides, or analogs thereof, e.g., a uridine ribonucleotide.
  • the fluorescence is measured spectroscopically, e.g., using a fluorometer.
  • the duplex nucleic acid molecule is comprised of SEQ ID NO:3 and SEQ ID NO:4.
  • the duplex nucleic acid molecule is a comprised of SEQ ID NO:3 and SEQ ID NO:4 and the topoisomerase is human topoisomerase IB.
  • the invention provides a method for testing a library of candidate anticancer agents for the ability to inhibit topoisomerase activity.
  • the invention provides a method for treating a subject having a topoisomerase associate disease or disorder by administering to the subject an effective amount of a compound of Formula 1 : Attorney Docket No.:63858-PCT (71699)
  • R 2 -R 5 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; and R 1 is H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; a compound of Formula 2:
  • Ri is H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide
  • Ri-R 4 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; a compound of Formula 4: Attorney Docket No.:63858-PCT (71699)
  • Ri-R 7 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; a compound of Formula 5:
  • R 1 -R 3 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; or a compound of Formula 6:
  • Ri-R 3 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • the topoisomerase associated disease or disorder is selected from the group consisting of cancer, viral infection or bacterial infection.
  • the invention provides a method of treating a subject having a topoisomerase associated disease or disorder by administering to the subject an effective amount of a compound identified in Table 2, 3 or 4, thereby treating the subject.
  • the compound is selected from the compounds listed in Table 4.
  • the topoisomerase associated disease or disorder is selected from the group consisting of cancer, viral infection or bacterial infection.
  • the instant invention provides a pharmaceutical composition for the treatment of a topoisomerase associated disease or disorder comprising a compound identified as Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, or Formula 6, or a compound identified in Tables 2, 3, or 4, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is useful in the treatment of viral infection, pathogen infection or bacterial infection.
  • the invention provides a kit comprising a pharmaceutical composition described herein and instructions for use.
  • the kit is useful for the topoisomerase associated disease or disorder, e.g., cancer, viral infection or bacterial infection.
  • the invention provides a kit for determining if a compound is a topoisomerase inhibitor comprising, a duplex nucleic acid molecule that allows for multiple turnover of the topoisomerase comprising a fluorescent moiety covalently attached to one strand of the duplex nucleic acid molecule and a fluorescence quencher covalently attached to the complimentary strand of the duplex nucleic acid molecule, wherein topoisomerase activity results in measurable fluorescence from the fluorescent moiety, and instructions for use.
  • the kit may further comprise a topoisomerase, e.g., a human, viral, pathogenic, or bacterial topoisomerase.
  • Figure 1 is a schematic representation of Topo I catalyzed reactions.
  • Figure IA depicts the reversible site-specific cleavage and ligation reaction in which the nucleophilic tyrosine attacks a DNA phosphodiester linkage, expelling a 5' hydroxyl leaving group.
  • Figure IB depicts the irreversible ribonuclease reaction in which the enzyme attacks the 3' phosphodiester linkage of uridine within the consensus sequence 5'-CCCTU-3'.
  • the phosphotyrosine intermediate is then attacked by the 2' hydroxyl group resulting in turnover of the enzyme and formation of a cyclic 2', 3' phosphodiester product.
  • Figures 2A-B depict the Molecular beacon assay for topo I and fluorescence emission spectra for the free molecular beacon substrate and product of the topo I RNase reaction.
  • Figure 2 A demonstrates that the assay relies on the strong quenching of the 3' 6-carboxyfluorescein fluorophore (FAM) in the substrate by a dabcyl group (DAB) that is covalently attached to the 5' end of the complementary strand.
  • DAB dabcyl group
  • Figure 2B depicts the fluorescein emission spectra for the 18U-FAM/18-DAB duplex (50 nM) before (lower spectrum) and after (upper spectrum) 30 min incubation with vaccinia DNA topoisomerase IB (vTopo). The fluorescence intensity increases by 9-fold when the reaction is completed. The final fluorescence is similar to that of the free 18U-FAM single stranded DNA (middle spectrum).
  • Figure 3 depicts presteady-state kinetic analysis of the topo I RNase reaction.
  • Ten nanomolar enzyme was incubated with the molecular beacon substrate at the following concentrations: 5OnM, 10OnM, 20OnM, 40OnM and 80OnM.
  • the solid curves were obtained from global analysis of the time courses using computer simulation and the mechanism shown in Scheme 1.
  • the global best-fit kinetic parameters are reported in Table 1.
  • the kinetic constants in the individual fits that are shown were allowed to float within ⁇ 15% of the global values.
  • Figure 4 depicts representative inhibitor screening data obtained using the molecular beacon assay described herein in a 96-well microtiter plate format. The figure shows the first three rows of plate 3846 of the NCI Diversity Library Collection. One of the most potent inhibitors detected in our partial screen of this library is compound
  • Figures 5A-B depict the mechanism of inhibition of the topo I RNase activity by compound 112983.
  • the reactions included 10 nM topo I, and either 50 nM ( Figure 5A) Attorney Docket No.:63858-PCT (71699)
  • Figures 6A-B depict inhibition of DNA supercoil relaxation catalyzed by vTopo and human topoisomerase type IB (hTopo).
  • Figure 6A depicts the results of an experiment in which vTopo and pUC19/AID supercoiled DNA were incubated with increasing concentrations of 112983 for nine minutes before quenching the reactions with 0.4 % SDS. The supercoiled and relaxed DNAs were then resolved on a 1% agarose gel and detected with ethidium bromide staining. The relative intensities of the substrate and product bands were determined by fluorescence imaging to calculate the fraction of the DNA in each lane that was in the relaxed form.
  • FIGS. 7A-B indicate that compound 112983 does not inhibit DNA binding of
  • FIG. 7A depicts noncovalent binding of Y274F vTopo to FAM-18AP/24 duplex as monitored by the increase in the steady-state fluorescein anisotropy of the DNA (FAM, filled circles), or increase in 2-aminopurine fluorescence (2AP, open circles).
  • Figure 7B depicts steady-state anisotropy of (1) the free FAM-18AP/24 mer DNA, (2) the Y274F-FAM-18AP/24 complex, (3-5) the complex in the presence of 112983, with Attorney Docket No.:63858-PCT (71699)
  • Figures 8A-C indicate that compound 112983 does not inhibit the single turnover
  • FIG. 8 A depicts suicide DNA cleavage reaction with fluorescence detection.
  • vTopo was rapidly mixed with a suicide substrate DNA containing a 2-aminopurine fluorescent label in the 6 mer leaving strand (13) and the fluorescence increase was monitored using stopped-flow fluorescence.
  • Figure 8B depicts the results of a single-turnover religation reaction.
  • a covalent complex between vTopo and a 5'- 32 P-labeled 12/24 suicide DNA substrate was formed and rapidly mixed with a complementary 12 mer DNA strand that was provided in large molar excess.
  • the 5'-OH of the 12 mer attacks the covalent phophotyrosine linkage resulting in the formation of a labeled 24 mer duplex that can be separated from the covalent complex electrophoretically using a 15 % SDS- polyacrylamide gel.
  • the ligation reaction was performed in the absence and presence of compound 112983 (32 ⁇ M). The reactions were quenched at 10 s, and the percent of the DNA that was covalently bound was determined by phosphorimaging.
  • FIG. 8C depicts equilibrium DNA cleavage.
  • vTopo 360 nM was added to a solution of 5'- 32 P-labeled 40/40 mer (300 nM) and incubated for five minutes before quenching the equilibrium cleavage-religation reaction by the addition of 5 % SDS.
  • the fraction covalent complex was determined after separating the free and covalently bound DNA by gel electrophoresis.
  • the percent covalently bound DNA decreased by 50 % in the presence of 100 ⁇ M compound (lane 3), further confirming that the compound decreases the cleavage equilibrium (K c ⁇ ).
  • the instant invention is based, at least in part, on the discovery of a continuous spectroscopic assay for DNA topoisomerase activity.
  • the inventors for the first time, Attorney Docket No.:63858-PCT (71699)
  • Topoisomerases have been identified as effective drug targets for the treatment of cancer and infection, e.g., viral and bacterial infection. Accordingly, the instant invention provides screening methods, methods of treating topoisomerase associated diseases and disorders, compositions for the treatment of topoisomerase associated diseases and disorders, kits to screen for inhibitors of topoisomerase, pharmaceutical compositions for the treatment of topoisomerase associated diseases and disorders; and kits comprising pharmaceutical compositions for the treatment of topoisomerase associated diseases and disorders.
  • the instant invention provides for the first time continuous topoisomerase activity assays. These assays provided allow for monitoring of topoisomerase activity under multiple turnover conditions. In preferred embodiments of the invention, the assays are used to identify inhibitors of topoisomerase activity.
  • the assays require a topoisomerase, e.g., a human, viral, or bacterial topoisomerase, and a DNA substrate comprising: a duplex nucleic acid molecule that allows for multiple turnover of the topoisomerase that has a label that is not detectable when the DNA is a duplex, but is detectable when the DNA duplex is disrupted, i.e., by topoisomerase activity.
  • 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. Using this technology, when acted upon by topoisomerase, the fluorescent moieties released from the quencher and produces a measurable fluorescent signal.
  • the assays utilize a mutant DNA duplex that allows for turnover of the enzyme, hi one embodiment, the duplex is:
  • U is undine ribonucleotide
  • FAM is the fluorescent label 6-carboxyfluorescein
  • DAB is the fluorescence quencher dabcyl.
  • This substrate is optimized for vaccinia virus topoisomerase type I, but one of skill in the art will realize that modifications of the DNA sequence can be made to tailor the substrate to different topoisomerases, i.e., topoisomerases from different species. Accordingly, in another embodiment, the substrate is optimized for human topoisomerase IB and has the sequence:
  • FAM is the fluorescent label 6-carboxyfluorescein
  • DAB is the fluorescence quencher dabyl
  • U is undine ribonucleotide
  • ⁇ is tetrahydrofuran abasic analog
  • the inventors believe that the substitution of the ribonucleotide uridine for the 3' thymidine of the consensus cleavage sequence allows for multiple turn over of the enzyme. See the Examples.
  • the exemplary assays of the invention use fluorescent labels to measure the activity of topoisomerase, any label that can emit a different signal when attached to duplex DNA as opposed to single stranded DNA will be useful in the methods of the invention.
  • the instant invention provides methods to determine if a compound is an inhibitor of topoisomerase by contacting a topoisomerase with the DNA duplex described above and a candidate inhibitor and determining if the candidate inhibitor is capable of inhibiting the action of the topoisomerase by comparing the activity of the topoisomerase in the presence of the candidate inhibitor to the activity in the absence of the inhibitor.
  • the activity of the topoisomerase is determined by measuring the fluorescence emission of the fluorescent tag. Using 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. For further details see the Examples.
  • the assays use type I topoisomerases, e.g., type IA or IB. In other embodiments, the assays use type II topoisomerases.
  • type I topoisomerases e.g., type IA or IB.
  • type II topoisomerases e.g., type II topoisomerases.
  • the topoisomerase is a human, viral, e.g., vaccinia virus, bacterial, or parasitic, e.g., from malaria or trypanosome.
  • the assays described herein allow for the screening of libraries of compounds for inhibition of topoisomerase.
  • One of skill in the art can perform these assays using high throughput methods such as microtiter plates and plate reading fluorometers to rapidly screen large libraries of compounds.
  • the assays described herein are used to identify anticancer, antiviral, antibacterial, or antipathogenic compounds for use in treating subjects having a topoisomerase associated disease or disorder.
  • topoisomerase assays described herein can be used to identify compounds that are capable of inhibiting the activity of topoisomerase. Accordingly, using the assays described herein, it has been demonstrated that the compounds in Tables 2, 3, and 4 are inhibitors of topoisomerase activity.
  • topoisomerase inhibitors having the structure of Formula 1:
  • R 2 -R 5 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide; and R 1 is H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • the instant invention provides topoisomerase inhibitors having the structure of Formula 2: Attorney Docket No.:63858-PCT (71699)
  • Ri is H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide
  • the instant invention provides topoisomerase inhibitors having the structure of Formula 3:
  • Ri, R 2 , R 3 , and R 4 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • the instant invention provides topoisomerase inhibitors having the structure of Formula 4:
  • Ri ; R 2; R 3j R 4 , R 5 , and R 6 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • R 3j R 4 , R 5 , and R 6 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • the instant invention provides topoisomerase inhibitors having the structure of Formula 5:
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • the instant invention provides topoisomerase inhibitors having the structure of Formula 5:
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are each independently H, a halide, an alkyl, an aryl, a cyano, an alcohol, an amine or an amide.
  • 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., Ci-C 1O for straight chain, C 3 -Ci 0 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, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, perfluoromethyl, perchloromethyl, perfluoroethyl, perchloroethyl, etc.
  • aryl 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, Attorney Docket No.: 63858-PCT (71699)
  • 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 ureido), amidino, imino, sulfhydryl, alkylthio, ary
  • 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.
  • halide includes compounds comprising a halogen with a more electropositive element or group.
  • exemplary halides include NaCl, KI, LiCl, CuCl 2 , ClF, CH 3 Br, and CHI 3 .
  • cyano includes compounds having an monovalent 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.
  • the compounds identified in Tables 2, 3, 4, and as Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, and Formula 6 are useful in the treatment of topoisomerase associated diseases and disorders. Accordingly, the instant invention provides compounds having the structure identified as Formula 1, Formula 2, Formula 3, Formula 4, Formula 5, or Formula 6 that are inhibitors of topoisomerase activity.
  • phrases "pharmaceutically acceptable carrier” is art recognized and 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, corn 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 magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'
  • 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), le
  • 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 more accessory ingredients, hi general, 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 Attorney Docket No.:63858-PCT (71699)
  • 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 dicalcium 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; wetting agents, such as, for example, cetyl alcohol and glycerol monostea
  • 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 Attorney Docket No.:63858-PCT (71699)
  • 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 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.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline 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 more compounds of the invention with one or more suitable nonirritating Attorney Docket No.:63858-PCT (71699)
  • 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.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one Attorney Docket No.:63858-PCT (71699)
  • sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile 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 may be 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 may be 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 maybe 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 Attorney Docket No.:63858-PCT (71699)
  • 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.
  • the 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, 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.
  • routes 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 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. For example, 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. Generally, 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 an topoisomerase associated disease or disorder. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a compound of the present invention While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition. Moreover, the pharmaceutical compositions described herein may be administered with one or more other active ingredients that would aid in treating a subject having a topoisomerase associated disease or disorder.
  • the Attorney Docket No.:63858-PCT (71699) the Attorney Docket No.:63858-PCT (71699)
  • compositions of the invention may be formulated to contain one or more additional active ingredients that would aid in treating a subject having a topoisomerase associated disease or disorder, e.g., anticancer compounds or antimalarial compounds.
  • compositions can be included in a container, pack, kit or dispenser together with instructions, e.g., written instructions, for administration, particularly such instructions for use of the active agent to treat against a topoisomerase associated disease or disorder.
  • instructions e.g., written instructions
  • the container, pack, kit or dispenser may also contain, for example, one or more additional active ingredients that would aid in treating a subject having a topoisomerase associated disease or disorder, e.g., anticancer compounds or antimalarial compounds.
  • kits containing reagents for testing a compound for the ability to inhibit topoisomerase activity along with instructions, e.g., written instructions, for using the kit.
  • the kit may contain a duplex DNA substrate such as those described herein and instructions for use.
  • the kit may contain one or more topoisomerases to be screened against one or more compounds.
  • topoisomerase associated diseases and disorders is intended to include diseases and disorders characterized by aberrant expression or activity, or a disease or disorder that relies on the activity of a topoisomerase to progress.
  • exemplary topoisomerase associated diseases and disorders are cancer and infection, e.g., bacterial and viral infection.
  • Viral infections such as vaccinia family viral infections can be treated with topoisomerase inhibitors.
  • pathogenic infections such as malaria and trypanosome infection can be treated with topoisomerase inhibitors.
  • treatment includes the diminishment or alleviation of at least one symptom associated or caused by a topoisomerase associated disease or disorder.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.
  • subject is intended to include organisms, e.g., prokaryotes and eukaryotes, which are capable of suffering from or afflicted with a topoisomerase associated disease or disorder.
  • subjects 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 a topoisomerase associated disease or disorder.
  • infection includes the pathological state resulting from the invasion of a subject by pathogenic microorganisms, e.g., fungus, bacteria or viruses.
  • pathogenic microorganisms e.g., fungus, bacteria or viruses.
  • Exemplary infections include those caused by the vaccinia family of viruses, malaria, and trypanosomes.
  • 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 Attorney Docket No.:63858-PCT (71699)
  • AML acute myeloid leukemia
  • AML acute myeloid leukemia
  • chronic myelogenous leukemia mixed-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-Hodgkin'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 in chemotherapy treatments are listed below in Table 6.
  • 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, and vinorelbine tartrate (Navelbine
  • an effective amount of a compound is that amount necessary or sufficient to treat or prevent a topoisomerase associated disease or disorder, hi an example, 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”.
  • pharmaceutical composition includes preparations suitable for administration to mammals, e.g., humans.
  • the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a 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.
  • a 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.
  • Enzymes and Plasmid DNA The cloning and purification of wild-type and Y274F vaccinia topoisomerase has been previously described (13). The enzyme concentration was determined by UV absorbance using an extinction coefficient of 28,140 M -1 Cm "1 in a buffer containing 20 mM sodium phosphate pH 6.0 and 6 M guanidinium hydrochloride (13). Human topoisomerase was obtained from Sigma. The plasmid pUC19/AID was constructed from pUC19 by inserting the 600 bp gene encoding the enzyme activation induced cytidine deaminase (AID) into the restriction sites Nde ⁇ and Hindlll.
  • RNA and DNA Substrates with Fluorescent Tags The sequence of the 18 mer DNA/RNA substrate containing a single uridine ribonucleotide substitution for the 3' thymidine residue of the consensus cleavage sequence is shown below, where FAM is 6- carboxyfluorescein, 1 and DAB is the universal fluorescence quencher, dabcyl.
  • FAM 6- carboxyfluorescein
  • DAB the universal fluorescence quencher
  • nucleotide just 3' of the ultimate T of the pentameric concensus sequence was labeled with the fluorescent adenine analogue, 2-aminopurine (P).
  • P fluorescent adenine analogue
  • These two probes allowed (i) measurement of protein binding by monitoring the increase in fluorescein anisotropy of the DNA, and (ii) DNA cleavage by the increase in 2-aminopurine fluorescence when the 6 mer leaving group rapidly dissociates after strand scission (13).
  • the oligonucleotide strands were synthesized using an ABI 394 synthesizer using nucleoside phosphoramidites obtained from Glen Research.
  • the oligonucleotides were purified using anion exchange HPLC and desalted using disposable gel filtration columns (PD-IO, Pharmacia). The purity of oligonucleotides was confirmed using electrophoresis through a 20 % polyacrylamide gel containing 7 M urea and MALDI-TOF analysis.
  • the DNA duplexes were prepared in buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 9.0) by mixing the two strands in a molar ratio of 1.1 : 1 (the dabcyl-labeled strand was in slight molar excess).
  • Fluorescein fluorescence emission spectra of 18U-F AM/ 18-DAB were collected using a Fluoromax-3 fluorimeter (Instruments S. A. Inc.) at 37 0 C. The measurements were performed using an excitation wavelength of 492 nm, and the emission was monitored from 510 to 620 nm. The excitation and emission slit widths were set at 0.5 and 5 nm, respectively. Further experimental details are reported in the legend to Figure 2.
  • the equilibrium dissociation constant was calculated by nonlinear-least squares fitting of the data to eq 1, where A is the fluorescence anisotropy at a given concentration of vTopo, A 0 and A f are the anisotropics of the free DNA and the vTopo-DNA complex, and [E] tot Attorney Docket No.:63858-PCT (71699)
  • A A 0 - ((A 0 - ⁇ f )/2[DNA] tot ⁇ b - Jb 2 - 4[E] tot [DNA] tot ⁇ )
  • 112983 displaced the bound DNA from Y274F, a complex was formed using 0.1 ⁇ M FAM-18AP/24 and 2 ⁇ M Y274F, and the anisotropy was measured as increasing concentrations of 112983 were added to the solution (0 to 28 ⁇ M).
  • excitation at 492 nm were followed by taking three fluorescence readings in each well over a 30 minute time period.
  • aminopurine fluorescence was followed using excitation at 315 ran with monitoring at emission wavelengths greater than 360 run using a cut off filter (13).
  • a Continuous Multiple-Turnover Kinetic Assay for Topo I A molecular beacon substrate was constructed that contained a 6-carboxyfluorescein group (FAM) on the 3' end of the scissile strand and a dabcyl (DAB) quench attached to the 5' end of the complementary strand ( Figure 2). Cleavage of the scissile strand by topo I was expected to rapidly release the FAM labeled 6 mer strand to solution, thereby relieving the strong fluorescence quench provided by the DAB group. For this kinetic assay to be mechanistically useful, the release of the 6 mer FAM strand needs to be fast compared to strand cleavage and 2', 3' cyclic phosphodiester formation.
  • FAM 6-carboxyfluorescein group
  • DAB dabcyl
  • topo I Ribonuclease Activity The ribonuclease reaction of topo I occurs by a minimum of three steps involving substrate binding and dissociation to form an ES complex with the rate constants k on and k Oft , attack of the tyrosine nucleophile to generate a phosphotyrosine intermediate E-I with a rate Attorney Docket No.:63858-PCT (71699)
  • the substrate concentration dependence and amplitude of the exponential burst phase provide information on the binding and dissociation rate constants to form ES, as well as the rate constant for formation of the covalent intermediate, E-I.
  • the subsequent linear rate provides information on the rate-limiting turnover of E-I to form free enzyme and product.
  • the rate of intermediate formation is comparable to the rate of the subsequent steps the burst will be attenuated, and if product inhibition is pronounced, a linear steady state region will be difficult to measure.
  • vTopo DNA cleavage and religation
  • a "suicide" DNA substrate is employed in which the strand containing the 5' OH leaving group is sufficiently small that it rapidly dissociates upon cleavage of the phosphodiester linkage (75), resulting in irreversible formation of the covalent complex. If the leaving strand contains a 2- aminopurine fluorescent label on the 5' end, rapid strand dissociation leads to an increase in the 2-aminopurine fluorescence that is a measure of the preceding rate-limiting cleavage reaction (13).
  • the covalent complex is first formed using a suicide substrate and then the ligation reaction is initiated by the addition of a large excess of a complementary DNA strand containing a 5' OH nucleophile.
  • the extent of ligation may be assessed by electrophoretically separating the covalent complex and ligated DNA using SDS-PAGE (75).
  • 112983 follows a novel inhibitory mechanism for type IB topoisomerases. This small molecule selectively inhibits the steady-state RNase and supercoil unwinding reactions of vTopo but not hTopo, and yet remarkably, exhibits no detectable inhibition of DNA binding, or the single-turnover half reactions of cleavage and religation (Figs. 7 and 8). These observations eliminate two simple mechanisms of inhibition such as competition with substrate binding, or targeting of the cleavage chemistry via the formation of an inhibitory ESN complex (where N is 112983).
  • a viable mechanism that is consistent with all of the data is one in which 112983 targets the E-I covalent complex and inhibits attack of the 2' OH in the ribonuclease reaction, and supercoil unwinding with the plasmid substrate (Scheme 3). According to this mechanism, at infinite concentration
  • Topo I Inhibitors can be targeted at multiple points along their reaction pathways ranging from the noncovalent DNA binding step (18, 20), the attack of the tyrosine nucleophile (9, 21, 22), the reverse attack of the 5'-OH (23), and the supercoiled unwinding step (9).
  • vTopo DNA binding by vTopo is competitively inhibited by two structurally related antibiotic molecules, novobiocin and coumermycin (18). The inhibition by these molecules is surprising given that they are potent inhibitors of bacterial DNA gyrase enzymes, where they act by binding to the ATP binding sites of these enzymes (24, 25).
  • Other natural product inhibitors of vaccinia virus type IB topoisomerases have been identified (20, 23, 26, 27), but compound 112983 is the simplest yet identified.
  • Clinically useful drugs that target topoisomerase IB such as the camptothecin derivatives (28), act by stabilizing the covalent complex and promoting arrest of replication and transcription forks (29). These large five ring aromatic molecules impose a physical block to ligation by intercalating directly at the cleavage site (30), and are referred to as "poisons" rather than inhibitors of these enzymes.

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  • General Engineering & Computer Science (AREA)
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  • Biomedical Technology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un dosage spectroscopique continu de l'activité topoisomérase d'ADN. Cette invention concerne aussi des inhibiteurs de topoisomérase et des compositions pharmaceutiques destinées au traitement des maladies et ses troubles associés à la topoisomérase.
PCT/US2005/027605 2004-08-03 2005-08-03 Dosage d'identification d'inhibiteurs de topoisomerase WO2006015369A2 (fr)

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US11/701,981 US20080020973A1 (en) 2004-08-03 2007-02-01 Assays for identification of topoisomerase inhibitors

Applications Claiming Priority (6)

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US59839504P 2004-08-03 2004-08-03
US60/598,395 2004-08-03
US59839804P 2004-08-08 2004-08-08
US60/598,398 2004-08-08
US69325205P 2005-06-23 2005-06-23
US60/693,252 2005-06-23

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WO2007146375A2 (fr) * 2006-06-14 2007-12-21 Government Of The United States Of America, As Represented By The Secretary Department Of Health And Human Services Composés de pyrimido-dione-quinoléine hautement solubles et procédés de traitement du cancer
WO2008124145A1 (fr) * 2007-04-05 2008-10-16 Government Of The U.S.A., As Represented By The Secretary, Department Of Health And Human Services Composés stéroïdes, compositions et procédés de traitement
WO2012036573A2 (fr) * 2010-09-14 2012-03-22 Instytut Biochemii I Biofizyki Pan Composés modulateurs d'une protéine cftr mutante et leur utilisation pour le traitement de maladies associées à un dysfonctionnement de la protéine cftr
US10016402B2 (en) 2011-02-08 2018-07-10 Children's Medical Center Corporation Methods for treatment of melanoma
WO2020248075A1 (fr) * 2019-06-12 2020-12-17 The Governors Of The University Of Alberta Ciblage de la réparation de l'adn dans des cellules tumorales par inhibition d'ercc1-xpf

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US6620588B1 (en) * 2000-05-31 2003-09-16 Salk Institute For Biological Studies Method of identifying inhibitors of topoisomerase DNA religation

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